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The Janus face of immunity : how anti-tumor immunity leads to autoimmunity in paraneoplastic neurological diseases Christina Gebauer To cite this version: Christina Gebauer. The Janus face of immunity : how anti-tumor immunity leads to autoimmunity in paraneoplastic neurological diseases. Immunology. Université Paul Sabatier - Toulouse III, 2016. English. <NNT : 2016TOU30139>. <tel-01492922> HAL Id: tel-01492922 https://tel.archives-ouvertes.fr/tel-01492922 Submitted on 20 Mar 2017 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. 5)µ4& &OWVFEFMPCUFOUJPOEV %0$503"5%&-6/*7&34*5²%&506-064& %ÏMJWSÏQBS Université Toulouse 3 Paul Sabatier (UT3 Paul Sabatier) 1SÏTFOUÏFFUTPVUFOVFQBS Christina GEBAUER le 15. Novembre 2016 5JUSF The Janus Face of Immunity How anti-tumor immunity leads to autoimmunity in paraneoplastic neurological diseases ²DPMF EPDUPSBMF et discipline ou spécialité ED BSB : Immunologie 6OJUÏEFSFDIFSDIF UMR 1043 Centre de Physiopathologie Toulouse Purpan %JSFDUFVSUSJDFT EFʾÒTF Prof. Roland LIBLAU Jury : Prof. Prof. Prof. Prof. Prof. Eliane PIAGGIO Doron MERKLER Francois GHIRINGHELLI Joost VAN MEERWJIK Jacqueline MARVEL Rapportrice Rapporteur Rapporteur Président Examinatrice 5)µ4& &OWVFEFMPCUFOUJPOEV %0$503"5%&-6/*7&34*5²%&506-064& %ÏMJWSÏQBS Université Toulouse 3 Paul Sabatier (UT3 Paul Sabatier) 1SÏTFOUÏFFUTPVUFOVFQBS Christina GEBAUER le 15. Novembre 2016 5JUSF The Janus Face of Immunity How anti-tumor immunity leads to autoimmunity in paraneoplastic neurological diseases ²DPMF EPDUPSBMF et discipline ou spécialité ED BSB : Immunologie 6OJUÏEFSFDIFSDIF UMR 1043 Centre de Physiopathologie Toulouse Purpan %JSFDUFVSUSJDFT EFʾÒTF Prof. Roland LIBLAU Jury : Prof. Prof. Prof. Prof. Prof. Eliane PIAGGIO Doron MERKLER Francois GHIRINGHELLI Joost VAN MEERWJIK Jacqueline MARVEL Rapportrice Rapporteur Rapporteur Président Examinatrice ! _$ /.# ` _Q**.1 '/` _2&$).` _0-&($N!&*)/# #*- ` _0-&($Ngnj` "%&.#$%+* ! #1 /* .-$ /# './ !*0- 4 -. 2*0' .4 $/ 2. ) (5$)" #$& Q +./ 1'' 4.N#$''.)(*0)/$).N .2(5$)"/#$)".N#+ -! /2 /# -N"*//#-*0"# .('')$"./*-(.N#!0)N2.*- N2.))*4 N#(4*2).)0+.0/ "*/'24.(*/$1/ 2# ) - # )*/# -/*+*!(*0)/$)Q 2.#++4N.N # *0/.N ! '/ ./-*)" ) $) ./-0/$' Q *2 1 -N 2*0' )*/ .* !- . ( -$"#/ )*2 $) (4 #$& *! '$! 2$/#*0/ '' /# + *+' -*0) ( ) 2. 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"$#$ 9:/6?7:0;7 ? 9? % 65< &$ !& 230%& 0%" 9:/6?;7 ? =? % &! )0&+" 23 $ &$ & 960 !$ & 96 &'!$/ & + ;- %" $ +" ! % )$ %&'& )& 1! !+ !$ 9 !'$%/ +%% )% "$!$ &! %%%% "$!$&! 1'!$% & + '&! "$!'&! ! 0γ 0α + & &$ %$$ 9:/6? %/ "$% &&( "!&% ! %" !+&% $! !'% $$+ &$ & 960 2&3 !$ 96 2$&3 &'!$/ $#' + ! 0γ0"$!' 9:/6? 9? !$ 9:/6? =? % & %" / !! & $! 8 " & *"$ &%-&$"$% && @!=)&960 <)&96 &'!$%/ 0& +-44"B5/56/0$ &960&'!$$(&$ ! %- ( 0%" 9:/6?7:0;7 ? 9? % 265<3- ( 0%" 9:/6?;7 ?=?%265<3-!$!&&+"%!%265<3/!!&$!8 " & *"$ &% $ %!) / &. &'!$ %,- (' $"$% &% & @ !&$!'"/)!0)+-4444"B5/5556/&."$ &!&'!$0$ %/ !0$ 2 &0!*3&%&- %A !&% &-4444"B5/5556/ >6 **** 10 5 5 0 -5 -10 ns -15 -20 0 -5 -10 -20 -25 -30 -30 -35 -35 ! ! ! ns -15 -25 **** 15 10 % weight loss % weight loss 15 ! ! ! ! "# !# ! "!'!#+*+.( !&.202& .+&.+$.+&% '3 (-"!%&$4 $))))5*%***+%"!!.+&& & $#&.2'+*/($#& 02'+*/(#'+*/(% !'(%'3 (-" !%&$4$))))5*%***+% 1, " #! $ %#) & &( %# ) $ %# ) &! "! " & %# ) $ & '$ +*0/8+#$ %$!%$7 " +*0/8+#$ ,2"! + * 4/ 8+ #$ 5 " $# $ + * 4/ 8+ #$ ! $ $ ,2 , $ + * 1/ 8+ #$ ",2 , +*1/8+ 10 10 10 5 10 4 10 3 10 0 -10 -10 -10 3 0 10 3 10 4 10 4 10 10 3 10 **** 4 3 3 5 -10 3 0 10 3 10 4 -10 10 3 5 -10 3 0 10 3 10 4 10 5 8 6 4 ns 2 10 5 10 4 10 98.4 10 3 10 4 10 1.14 10 10 21.4 3 -10 -10 3 0 10 3 10 4 10 ns 2 0 0 3 4 4 78.1 3 6 5 3.80 95.9 0 -10 5 8 0 0 10 **** 10 10 5 0 0 3 5 # CD45highCD11bhigh cells (x104) # Thy1.2+CD45high cells (x104) 3 5 -10 -10 3 0 10 3 10 4 10 5 3 -10 3 0 10 3 10 4 10 5 31.2% 150 100 18.1% 46.8% CD45.1+ CD4+ T cells CD45.1+ CD8+ T cells CD45.1- CD4+ T cells CD45.1- CD8+ T cells 81.4 50 0 -10 3 0 10 3 10 4 10 5 relative fold increase of MHCII 3.9% 25 20 15 10 5 0 CamK-HA (n=8) WT (n=4) &!&%"" " ! ! &! , 34+07 37 34+07 57,30,30 ) 30, + $ " " $ 0/ 03+#$$00340034,! $ - ". $0+1734 - ". !$ -34+07. -34+0,.! +#$ $0+17340034 63 / $"!"#$#&%-"#$$>/ $ " 7 !$ (!"$#- "%#0#- #? $ #$- 4444!@5/5556/ #$"%$ 0$"$# "$ 8$ "#.$"#""8=#- $"#"";=#- %#8=# " %#;=#2?< 0 3/ $. ## (!"## " 26689$3 "!"#$$& 0 %#/ $. $& "# ## (!"## 02-?;32")-?83/ transferred CD4 transferred CD8 75 100 ns CNS (n=10) Tumor (n=5) Spleen (n=10) 50 25 ** 10 ** ns 5 % among Thy1.2+ CD45.1+ CD4+ T cells % among Thy1.2+ CD45.1+ CD8+ T cells 100 *** 75 ** 50 ** 25 10 5 0 0 IFNγ+TNFα+ GM-CSF+ IFNγ+TNFα+ IL-17A+ 100 CNS (n=10) Tumor (n=5) Spleen (n=10) 50 *** * 10 *** * 5 % among Thy1.2+ CD45.1- CD4+ T cells % among Thy1.2+ CD45.1- CD8+ T cells ** 25 GM-CSF+ IL-17A+ endogenous CD4 endogenous CD8 100 75 ** * 75 *** * 50 ** **** 25 10 *** 5 0 0 IFNγ+TNFα+ GM-CSF+ IL-17A+ IFNγ+TNFα+ GM-CSF+ IL-17A+ !$&$"#" *&0#!89/6=8=#;=#$ 0 "$860$% "/)##'#!" " #" -$% "- #! '1 )#$%$ $')6568/!" ! "$ # $"#"" 89/6= ;= 2%!!" $3- $"#"" 89/6= 8= 2%!!" "$3- %#89/60;=2 '"$3 %#89/608=2 '""$3# $-$% "#!$$(!"##0γ0α-0 "06:"! $$/ $" 7!$(!"$#- &%#" ! 8 " <9 !* # ! !"# # < , !$"-"* 0 >1,16* 00 >1,12* 000 >1,112* 0000 >1,1112 transferred CD8 transferred CD4 * CNS (n=8) TUMOR (n=3) SPLEEN (n=8) % among Thy1.2+ CD45.1+ CD8 +T cells 80 60 ** * ns 40 20 ** 100 % among Thy1.2+ CD45.1+ CD4 +T cells * 100 80 60 40 *** 20 20 * 10 0 0 CD44highCD62Llow KLRG1highCD127low CD44highCD62Llow CD44highCD62Lhigh endogenous CD8 80 * ** 40 * * 20 * 100 % among Thy1.2+ CD45.1- CD4+ T cells % among Thy1.2+ CD45.1- CD8 +T cells CNS (n=8) TUMOR (n=3) SPLEEN (n=8) * 60 CD44highCD62Lhigh endogenous CD4 ** 100 KLRG1highCD127low 80 60 ** 40 ** 20 20 ns 10 0 0 CD44highCD62Llow KLRG1highCD127low CD44highCD62Lhigh CD44highCD62Llow KLRG1highCD127low CD44highCD62Lhigh - &! $#% ""!$!"5)6,(""#.=9/*#$! .=4/" .=9/## #"", !"# ! !# 56,2; 9; .$ ! #/* 56,2; 5; .$ ! !#/* $" 56,2- 9;.&!#/$"56,2-5;.&!!#/"#*#$! " , !! 55* 73 * 2 238 &! """" # #( #! ".5573 &/*#!!(".5573 /!9;"+ "!#-%#!"!5;"+#!(!##'$"#" . 2238&/, # . < / ! 3 # ' !#"* %$!%"! 5!!,!$"-"*0 >1,16*00 >1,12 :7 15 10 % weight loss 5 0 -5 -10 -15 -20 -25 ns IgG (n=16) PS/2 (n=15) ns 3.0 # T cells in the CNS 2.5 2.0 ns 1.5 1.0 0.5 -30 -35 0.0 Thy1.2+CD45.1- Thy1.2+CD45.1+ $ α # )#! " ! . 0( )α0 +*., # ("( "%!% 2 ( "!' 6 ( / $ ' ) %'7 !" $# #! %-.(!" -.' ) # %& % # % %( ! ! %-(.501(-)%-(.501(-5#( 43 400 1.5K 300 98.2 93.5 1.0K 200 100 0 500 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 tumor surface (mm2) 150 4T1-HA (n=6) 4T1 (n=6) 100 50 0 0 5 10 15 20 25 days post-injection ! ! $ +)& +) % $ +) +)&% +) '! # /,( +)& ' # /,( &! ! % *) !% " '*( !% .- 0.8 ΔΔCp 0.6 0.4 0.2 S m C N m nu ju je Tg um nu en ju W T je od du Tg um ac en om od T du st W Tg T W Tg st om ac h h 0.0 # $ ) 0.) $ )01(.40401(.424! # .- #( !" ) ) ( !" " ' #*' +5/, ) +50, ! "( !" # ) !( 33 1.2 # Thy1.2+CD45.1- T cells (x10^4) # Thy1.2+CD45.1+ T cells (x10^4) ** 1.0 0.8 0.6 0.4 0.2 0.0 D10-14 (n=15) n.s. 1.2 1.0 0.8 0.6 0.4 0.2 0.0 D21 (n=5) D10-14 (n=15) D21 (n=5) reinjection of 4T1-HA + CD4 and CD8 T cells reinjection of 4T1-HA reinjection of 4T1-HA 1.2 # Thy1.2+CD45.1- T cells (x104) # Thy1.2+CD45.1+ T cells (x104) reinjection of 4T1-HA + CD4 and CD8 T cells 1.0 n.s. 0.8 0.6 0.4 0.2 0.0 D39 (n=7) D39 (n=7) 1.2 1.0 n.s. 0.8 0.6 0.4 0.2 0.0 D39 (n=7) D39 (n=7) " ('-&*+%(/)+&,)/*/('-& *+%(/,)/./ & *(& !% ##! )($%# #$(' (* )(& # #$ $%! #"!$!!*%! ! $(%)/*+%(/ )(%! !!$(%)/*+%(& )(% ('' &#&!!-##25咔-#$!"&#! &#$# 218 -" 56,2;36-73; 5; " 218 -" 56,2;73;9;",-#!#"&!()#4:$"& (##!( "$# $!" (2,3;56,2; " # 4:, "$#$!"$"(2,3;56,2-"#4:,#! !"#" %$ + !" # # < + -#( #"#+ "=# "#+00 >1,12 % CD25+ cells among CD44+CD62L- cells 100 ** ** * *** * * CD4 CD8 CNS (n=8) TUMOR (n=3) SPLEEN (n=8) 80 60 40 20 0 CD4 CD8 Thy1.2+CD45.1+ Thy1.2+CD45.1- " !#-&!$#% ""!$!"5*6 ("" # .=9/+ #$! .=4/ " .=9/ &! !! #& ( 21 25, ! !#" 56,2; 9;+ 56,2; 5;+$"56,2-9;$"56,2-5;"#+#$! " ##' !""36! ##,#.< /!3 # ' !#"+ %$ !%" ! 5 ! !, !$"-"+ 0 >1,16+00 >1,12+000 >1,112 212 # ' (" %& ! &$)" " &" %$& '& '!&$ &!$&$ !$! & " !& ! & ! !#!& !( #&!! !&+)*- *), Brain Advance Access published September 6, 2016 doi:10.1093/brain/aww225 BRAIN 2016: Page 1 of 12 | 1 CTLA4 blockade elicits paraneoplastic neurological disease in a mouse model Lidia M. Yshii,1,2 Christina M. Gebauer,1,* Béatrice Pignolet,1,3,* Emilie Mauré,1 Clémence Quériault,1 Mandy Pierau,4 Hiromitsu Saito,5 Noboru Suzuki,5 Monika Brunner-Weinzierl,4 Jan Bauer6 and Roland Liblau1 These authors contributed equally to this work. CTLA4 is an inhibitory regulator of immune responses. Therapeutic CTLA4 blockade enhances T cell responses against cancer and provides striking clinical results against advanced melanoma. However, this therapy is associated with immune-related adverse events. Paraneoplastic neurologic disorders are immune-mediated neurological diseases that develop in the setting of malignancy. The target onconeural antigens are expressed physiologically by neurons, and aberrantly by certain tumour cells. These tumourassociated antigens can be presented to T cells, generating an antigen-specific immune response that leads to autoimmunity within the nervous system. To investigate the risk to develop paraneoplastic neurologic disorder after CTLA4 blockade, we generated a mouse model of paraneoplastic neurologic disorder that expresses a neo-self antigen both in Purkinje neurons and in implanted breast tumour cells. Immune checkpoint therapy with anti-CTLA4 monoclonal antibody in this mouse model elicited antigenspecific T cell migration into the cerebellum, and significant neuroinflammation and paraneoplastic neurologic disorder developed only after anti-CTLA4 monoclonal antibody treatment. Moreover, our data strongly suggest that CD8 + T cells play a final effector role by killing the Purkinje neurons. Taken together, we recommend heightened caution when using CTLA4 blockade in patients with gynaecological cancers, or malignancies of neuroectodermal origin, such as small cell lung cancer, as such treatment may promote paraneoplastic neurologic disorders. 1 INSERM UMR U1043 - CNRS U5282, Université de Toulouse, UPS, Centre de Physiopathologie de Toulouse Purpan, Toulouse, 31300, France 2 Department of Pharmacology, Institute of Biomedical Sciences I, University of São Paulo, 05508-900, Brazil 3 Department of Clinical Neurosciences, Toulouse University Hospital, 31059, France 4 Department of Experimental Paediatrics, University Hospital, Otto-von-Guericke University Magdeburg, 39120, Germany 5 Department of Animal Genomics, Functional Genomics Institute, Mie University Life Science Research Center, 2-174 Edobashi, Tsu, Mie 514-8507, Japan 6 Department of Neuroimmunology, Center for Brain Research, Medical University of Vienna, A-1090, Austria Correspondence to: Roland Liblau, MD, PhD, Centre de Physiopathologie de Toulouse, Purpan Hospital, Place du Docteur Baylac TSA 40031, 31059 Toulouse Cedex 9, France E-mail: [email protected] Keywords: paraneoplastic neurological disorder; CTLA4; immunotherapy; Purkinje cells; neuroinflammation Abbreviations: HA = haemagglutinin; mAb = monoclonal antibody Received April 15, 2016. Revised July 21, 2016. Accepted July 21, 2016. ß The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: [email protected] Downloaded from http://brain.oxfordjournals.org/ by guest on September 7, 2016 * 2 | BRAIN 2016: Page 2 of 12 Introduction in cerebellar Purkinje cells and in an implanted breast tumour. Using this in vivo setting, we assessed the impact of anti-CTLA4 therapy on development of paraneoplastic neurological disorder. Materials and methods Mice 6.5-TCR mice, CL4-TCR mice, L7-Cre, Mog-HA, and the Rosa-Stop-HA (Rosa26rtm(HA)1Lib) mice have been described elsewhere (Kirberg et al., 1994; Morgan et al., 1996; Saito et al., 2005; Saxena et al., 2008). L7-Cre mice were backcrossed at least six times on the BALB/c background. We obtained the L7-HA mice by crossing L7-Cre with Rosa-Stop-HA mice. Mice were kept in specific pathogen-free conditions within the UMS006 animal facility, Toulouse, France. All procedures involving animals were in accordance with the European Union guidelines following approval by the local ethics committee. Reverse transcription polymerase chain reaction To assess influenza virus haemagglutinin (HA) expression, Õ total RNA was extracted with TRIzol (Life), followed by reÕ verse transcription (SuperScript III, Invitrogen). The cDNA Õ was used as template for quantitative PCR using SYBR Green I master mix (Roche) on a LightCycler 480 thermocycler (Roche). The following primers were used to amplify HA (forward 5’AAACTCTTCGCGGTCTTTCCA 3’, reverse 5’ GATAAGGTAGCTTGGGCTGC 3’), Ctla4 (forward 5’GCT TCCTAGATTA CCCCTTCTGC 3’, reverse 5’ CGGGCAT GGTTCTGGATCA 3’), and Hprt (forward 5’ TTGCTCGAG ATGTCATGAAGGA 3’, reverse 5’ TGAGAGATCATCT CCA CCAATAACTT 3’). HA mRNA expression was normalized to Hprt mRNA. Cell line and tumour implantation The BALB/c mouse 4T1 breast cancer cell line and its 4T1-HA derivative that expresses HA (a gift from D. Klatzman) were cultured as described (Aslakson and Miller, 1992). 4T1 or 4T1-HA cells (105) were injected subcutaneously into the left flank of mice. Tumours were measured with a Vernier calliper daily for 20 days, and the tumour size (w l) is expressed as mm2. Haemagglutinin-specific T cells Naive HA-specific CD4 + and CD8 + T cells were purified from 6.5-TCR and CL4-TCR mice, respectively. Spleens and lymph nodes were collected and T cells were purified (Dynabeads Untouched Mouse CD4 + or CD8 + Cells kit; Invitrogen). CD4 + T cells were incubated with anti-CD25 (clone PC61) monoclonal antibody (mAb) for Treg depletion. Naive T cells were further purified based on expression of CD62L (Miltenyi Biotech). Naive CD4 + and CD8 + T cells (107 each), depleted of Tregs, were injected intravenously into the Downloaded from http://brain.oxfordjournals.org/ by guest on September 7, 2016 Paraneoplastic neurological disorders are immune-mediated diseases that develop in the setting of malignancy and offer a unique prospect to analyse the interplay between tumour immunity and autoimmune disease (Albert and Darnell, 2004; Steinman, 2014). In paraneoplastic neurological disorders, the pathogenic adaptive immune response targets proteins, so-called onconeural antigens, normally expressed by neurons and aberrantly expressed by tumour cells (Furneaux et al., 1990). Highly specific anti-neuronal autoantibodies in the serum and CSF represent key diagnostic biomarkers. When these autoantibodies recognize cell surface antigens, they most likely contribute to pathogenicity (Tuzun et al., 2009; Graus et al., 2010). In contrast, when onconeural antigens are localized intracellularly, T cells provide a beneficial anti-tumoural response but are implicated in inducing the deleterious autoimmune neurological reaction (Pellkofer et al., 2004; Pignolet et al., 2013). Paraneoplastic cerebellar degeneration, characterized by the selective loss of Purkinje neurons in the cerebellum, is an illustrative example of paraneoplastic neurological disorder targeting intracellular neuronal antigens (Albert et al., 1998). Paraneoplastic cerebellar degeneration develops, in particular, in patients with gynaecologic carcinomas that express the Purkinje neuron-specific CDR2 protein, with small cell lung cancer that expresses the neuronal HuD protein, or with testicular cancer that expresses the Ma2 antigen (Mason et al., 1997; Albert et al., 1998; Dalmau et al., 2004). Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) is expressed transiently following activation of conventional T cells, and constitutively by regulatory T cells (Tregs). This receptor has a crucial role in limiting T cell responses (Allison, 2015). Therefore, therapeutic blockade of CTLA4 with antibodies has been tested to enhance antitumour immunity, and proved to be efficient in murine cancer models (Leach et al., 1996). This has led to the clinical development of anti-CTLA4 antibody (ipilimumab) as immunotherapy in patients with advanced melanoma or other carcinomas (Hodi et al., 2010; Robert et al., 2011). The benefit of this therapy for tumour control and survival has since been confirmed. Collectively, immune checkpoint inhibitors, and their combination, have revolutionized the field of cancer immunotherapy (Allison, 2015). However, anti-CTLA4 therapy is associated with a high frequency of immune-related adverse events, notably autoimmune hypophysitis (Bertrand et al., 2015). Patients affected with gynaecological cancers, or malignancies of neuroectodermal origin, such as small cell lung cancer, are particularly prone to develop paraneoplastic neurological disorders. We, therefore, sought to investigate whether the risk to develop paraneoplastic neurological disorder of autoimmune nature could be enhanced by antiCTLA4 therapy. To test this hypothesis, we generated a mouse system in which the same model antigen is expressed L. M. Yshii et al. Paraneoplastic neurological disorder mice. Mice were assessed daily for weight and neurological signs. Anti-CTLA4 and anti-CD8 treatment For in vivo antibody therapy, the hamster anti-mouse CTLA4 mAb (UC10-4F10-11) was produced by culturing hybridoma cell lines in RPMI 1640 supplemented with low IgG foetal bovine serum. Antibodies were purified from supernatants using protein G purification (GE Healthcare). Mice were treated through intraperitoneal injections of anti-CTLA4 mAb (100 mg/mouse) at Days 1, 0, 2, 4, 6, 8, 10, 12, 14 and 16. In one experiment, depleting anti-CD8 mAb (53.6.7) or control IgG (200 mg/mouse) were injected intraperitoneally at Days 7, 9, 11, 13, 15, 17 and 19. Motor behavioural tests Purification and activation of cerebellum-infiltrating mononuclear cells Brain-infiltrating mononuclear cell purification was described previously (Martin-Blondel et al., 2015). Briefly, 15 days after tumour implantation, mice were perfused with phosphate-buffered saline (PBS) and their cerebellum was dissected. Mononuclear cells were collected and used for flow cytometry staining or in vitro stimulation with peptides. For the latter, cerebellum-infiltrating mononuclear cells were stimulated in vitro for 16 h with HA512-520 or control H-2-Kd-binding peptide (Cw3170-179) for CD8 + T cells, and HA110-119 or control H-2-IAd-binding peptide (OVA323-339) for CD4 + T cells. TM The cells were incubated with GolgiPlug (1:1000; BD) and TM GolgiStop (1:1000; BD) for 16 h. | 3 2012), we found no detectable CD80 and CD86 expression. Data were recorded on an LSRII Fortessa (BD Bioscience) and analysed with the FlowJo software (Tree Star). Immunohistochemistry The light microscopical stainings were performed as described (Bien et al., 2012; Bauer and Lassmann, 2016). The primary antibodies used were: anti-Calbindin D28K (1:2000, Swant), anti-CD3 (1:200, SP7, Zytomed), anti-CD8 (1:100, 4SM15, eBioscience), anti-Iba-1 (1:1000, Wako Chemicals), antiGranzyme B (1:25, Santa Cruz), anti-activated caspase 3 (1:3000, BD), anti-Mac3 (1:200, BD), anti-b2-microglobulin (1:1000, Santa Cruz), and anti-C9neo (a kind gift by Dr Paul Morgan, Cardiff, UK). Staining for immunoglobulin (Ig) was performed using biotinylated donkey-anti-mouse Ig (Jackson). Quantification of immunohistochemistry data was performed by manual counting of Purkinje cells per mm2 of Purkinje cell layer. In brief, for each mouse, the number of Purkinje cells was determined from five equally sized cerebellar cortex regions (central lobules II, III, simple lobule, ansiform lobule, paramedian lobule) and normalized for the length of the Purkinje cell layer. Immunofluorescence The fluorescent stainings were performed as previously described (Bauer and Lassmann, 2016). The primary antibodies used were: rabbit anti-Calbindin D28K (1:2000, Swant), mouse anti-Calbindin D28K (1:2000, Sigma), mouse antiHA37-38 hybridoma supernatant (1:10), rabbit anti-IP3R1 (1:500, ThermoFisher), mouse anti-H-2Kd (1:500, ThermoFisher), anti-Granzyme B (1:25, Santa Cruz) and rat anti-CD8 (1:50). Images were analysed using ImageJ software (NIH). Statistical analysis Statistical analyses for two sets of data with normal distribution and for paired data were performed using the Student’s ttest. Comparisons between multiple groups were performed using the one-way ANOVA (post-test Tukey). For tumour size and weight loss, data were analysed using the two-way ANOVA (post-test Sidak). The analyses were performed on Prism 6 (GraphPad Software). Groups were considered statistically different at a P-value 5 0.05. The data are represented as mean SEM (standard error of the mean). Flow cytometry analysis Lymph node cells, splenocytes, and tumour-infiltrating cells were stained with anti-Thy1.2 (53-2.1, BD), anti-CD45 (30F11, Biolegend), anti-CD8 (53-6.7, BD), anti-CD4 (RM4-5, BD), and anti-CD152 (UC10-4B9, eBioscience) mAbs. Cerebellum-infiltrating cells were stained with the following antibodies: anti-Thy1.2, anti-CD45, anti-B220 (RA3-6B2, BD), anti-CD138 (281-2, BD), anti-CD8, anti-CD4, antiCD11b (M1/70, eBioscience), anti-IFN- (XMG1.2, BD), anti-TNF- (MP6-XT22, BD), and anti-IL-17A (TC1118H10, BD). 4T1 and 4T1-HA tumour cells were stained with anti-CD80 (16-10A1, eBioscience) and anti-CD86 (GL1, BD) mAbs and in agreement with previous data (Ruocco et al., Results Model of paraneoplastic neurological degeneration To model experimentally paraneoplastic cerebellar degeneration, we designed a mouse model expressing the influenza virus haemagglutinin (HA) specifically in Purkinje cells. We crossed the Rosa-Stop-HA mice with the L7-Cre mice, which express the Cre recombinase specifically in Purkinje cells. In the resulting L7-HA mice, the stop cassette is Downloaded from http://brain.oxfordjournals.org/ by guest on September 7, 2016 Motor coordination was assessed with a Rotarod device (Bioseb) with acceleration from 4 to 40 rpm over a 600 s period. For training, mice were tested on seven consecutive days, thrice daily. During the investigation period (Day 0 to Day 20), the latency to fall was measured for each mouse twice daily, and the longest time was recorded. The 100% represents the Day 0 value. To test the spontaneous motor activity an automated actimeter (ActiTrack) was used. This apparatus consists of four 22.5 cm2 surface areas with 16 surrounding infrared beams coupled to a control unit. The activity was recorded for 10 min every day, and total distance travelled (m) was recorded. BRAIN 2016: Page 3 of 12 4 | BRAIN 2016: Page 4 of 12 L. M. Yshii et al. Downloaded from http://brain.oxfordjournals.org/ by guest on September 7, 2016 Figure 1 Generation and characterization of the L7-HA mice. (A) Scheme of the L7-HA double knock-in mice. Top: In the Rosa-StopHA mice, a LoxP-flanked Stop cassette followed by the haemagglutinin (HA) sequence was introduced in the Rosa26 locus. Middle: In the L7-Cre mice, the Cre sequence was inserted in the Purkinje cell-specific L7/pcp2 gene. Bottom: In L7-HA mice, resulting from the crossing of the RosaStop-HA and L7-Cre mice, the Cre-mediated recombination within the Rosa26 locus permits transcription of HA. (B) HA mRNA expression assessed by quantitative RT-PCR in different organs of L7-HA mice (black bar) or cerebellum of MOG-HA mice (open bar). One experiment representative of three is shown. n.d. = not detected. (C) Expression of HA protein in Purkinje cells from L7-HA mice. Double immunostaining for HA (green) and calbindin (Calb, red). Top row: Littermate control mouse (WT), bottom row: L7-HA mouse. Nuclear staining (DAPI). Scale bar = 50 mm. (D–G) CTLA4 expression was assessed by flow cytometry on Day 5 in transferred HA-specific Thy1.2 + CD4 + (D) or Thy1.2 + CD8 + (E) T cells in the lymph nodes of Thy1.1 congenic mice bearing 4T1 or 4T1-HA tumour, and in tumour-infiltrating total CD4 + (F) and CD8 + (G) T cells. excised due to L7-controlled Cre expression, leading to selective expression of HA in Purkinje cells (Fig. 1A). Transcription of HA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in the cerebellum but not in other organs of L7-HA mice, including the rest of the CNS. Cerebellum of MOG-HA mice, which express HA in oligodendrocytes, was used as a positive control (Saxena et al., 2008) (Fig. 1B). Furthermore, by immunohistofluorescence, we confirmed that HA protein expression was confined to Purkinje cells in L7-HA mice as shown by the co-staining for HA and calbindin (Fig. 1C). As expected, HA expression was not detected in littermate control mice (wild-type). Paraneoplastic neurological disorder To further model paraneoplastic neurological disorder, L7-HA or wild-type mice were challenged with the 4T1HA breast cancer cells that express the HA antigen. Given the key role of T cells as effectors of tumour control and paraneoplastic neurological disorder pathogenesis (Albert and Darnell, 2004; Dalmau and Rosenfeld, 2008; Pignolet et al., 2013), on the day of tumour implantation, mice were transferred with HA-specific naive CD4 + and CD8 + (CD4/CD8) T cells. CD4/CD8 T cells were capable of partially controlling tumour growth as compared to mice injected with tumour only (Fig. 2A), but they elicited no patent neurological manifestations. We next addressed whether treatment with a mAb blocking CTLA4 would increase anti-tumour immunity since a high | 5 proportion of T cells activated by the HA-expressing tumour expressed CTLA4/CD152 (Fig. 1D–G). AntiCTLA4 mAb therapy protected mice almost completely from tumour outgrowth, regardless of their expression of HA in the cerebellum (Fig. 2A). Strikingly, L7-HA mice bearing 4T1-HA tumour and treated with anti-CTLA4 mAb lost weight significantly, from Day 15 onwards (Fig. 2B; P 5 0.0001). To investigate whether mice also developed neurological signs, we evaluated motor activity. L7-HA mice bearing 4T1-HA tumour and treated with anti-CTLA4 mAb displayed a significant reduction in forced motor activity on the Rotarod during the diseased period (Fig. 2C; P 5 0.0001). Using the actimeter test, we also revealed reduced spontaneous locomotion in 4T1-HA tumour-bearing L7-HA mice that received anti-CTLA4 mAb (Fig. 2D). Importantly, no such clinical manifestations developed in 4T1-HA tumour-bearing wild-type mice treated with antiCTLA4 mAb or in 4T1-HA tumour-bearing L7-HA mice not treated with anti-CTLA4 mAb (Fig. 2B–D). Figure 2 CTLA4 blockade allows tumour control but causes weight loss and motor impairment in L7-HA mice. (A) Tumour size was determined in L7-HA or wild-type (WT) mice implanted with 4T1-HA tumour cells (105), injected with no T cells or naive HA-specific CD4 + and CD8 + T cells (107 each), with or without treatment with anti-CTLA4 mAb. The values are from the subgroup of mice shown in (B), in which tumour size was assessed daily (5 to 20 mice per group). (B) Weight of 4T1-HA tumour-bearing L7-HA or wild-type mice described in A. The values are from 45 pooled experiments, involving 12 to 32 mice per group. (C) Rotarod performance in wild-type versus L7-HA mice treated as described in A. D0-D5 = sum of the percentage of time (normalized to Day 0) spent by a mouse on the Rotarod from Day 0 to 5, i.e. before disease onset. No significant differences between groups exist before disease onset. D15-D20 = sum of the time spent by a mouse on the Rotarod from Day 15 to 20, i.e. after onset of weight loss. The data are from 45 pooled experiments. (D) Distance (m) travelled during a 10 min cycle. D0-D5 = sum of the distance travelled from Day 0 to 5, i.e. before disease onset. D15-D20 = sum of the distance travelled from Day 15 to 20, i.e. after disease onset. No significant differences between groups exist before disease onset. Pooled data from two independent experiments. *P 5 0.05; **P 5 0.01; and ****P 5 0.0001. Downloaded from http://brain.oxfordjournals.org/ by guest on September 7, 2016 Anti-CTLA4 antibody triggers weight loss and reduced locomotion in L7HA mice BRAIN 2016: Page 5 of 12 6 | BRAIN 2016: Page 6 of 12 Collectively, these results show that clinical signs resembling paraneoplastic neurological disorder occur upon CTLA4 blockade, only when the antigen is expressed both in the CNS and tumour. Therapeutic CTLA4 blockage induces cerebellar inflammation and Purkinje cell loss in L7-HA mice Immune mechanisms of Purkinje cell death in L7-HA mice with paraneoplastic neurological disorder To gain insight into the immune cells responsible for Purkinje cell loss the cerebellum of L7-HA mice with paraneoplastic neurological disorder was further studied by flow cytometry. Contrasting with control mice, the cerebellum of L7-HA mice with paraneoplastic neurological disorder harboured CD45high CD11blow lymphocytes as well as CD45high CD11bhigh myeloid cells (Fig. 4A). Some B cells (13.2 3.4% of blood-derived CD45high cells) including a small number of CD138 + plasma cells (range 24 to 90 cells/cerebellum, n = 14) were present on Day 15 after disease induction. T cells were the dominant population with a mean of 2000 (range 3 to 9219 cells, n = 12) CD4 + and 1300 (range 2 to 7405, n = 12) CD8 + T cells. We then assessed the antigen specificity of these cerebelluminfiltrating T cells. Few CD4 + T cells reacted ex vivo to HA peptide by production of either IL-17 or IFN- (Fig. 4B). By contrast, cerebellum-infiltrating CD8 + T cells exhibited a marked HA-specific production of IFN- and TNF- (Fig. 4C). These data indicate that HA-specific CD8 + T cells are enriched in the cerebellum of L7-HA mice with paraneoplastic neurological disorder. To further dissect the interaction between CD8 + T cells and Purkinje cells, immunohistological investigations were conducted in L7-HA mice exhibiting marked ongoing loss of Purkinje cells and their neighbouring dendritic tree (Fig. 5A and B). Numerous CD8 + T cells were localized in close contact with Purkinje soma or along their dendrites (Fig. 5C and D). In some instances, intimate interactions between CD8 + T cells and Purkinje cells were evidenced, with indentation of the neuronal surface and polarization of the cytolytic granules towards the CD8 + T cell/Purkinje cell interface (Fig. 5E and F). Possibility of direct targeting of Purkinje cells by HA-specific cytotoxic CD8 + T cells was further strengthened by in situ detection of MHC class Iexpression in Purkinje neurons (Fig. 5G). In L7-HA mice with paraneoplastic neurological disorder, some Purkinje cells exhibited DNA fragmentation (Fig. 5H) and caspase 3 activation (Fig. 5I), indicating ongoing apoptosis. Neuronophagia of cells with Purkinje-like morphology by macrophages/microglia was also detected (Fig. 5J). We also assessed Ig deposition and complement activation in the cerebellum of these mice. Whereas Ig leaked into the cerebellum due to blood–brain barrier disruption (Supplementary Fig. 1A), there was neither Ig binding (Supplementary Fig. 1B) nor C9neo deposition (Supplementary Fig. 1C) on Purkinje cells. Therefore, our data suggest that the humoral immune response does not play a main role in this model. Together, these findings indicate that cerebellar inflammation is associated with MHC class I expression on Purkinje cells, physical contact with CD8 + T cells, which polarize their lytic granules towards the neurons, strongly suggesting that cytotoxic T cells deliver a lethal hit to Purkinje cells in an antigen-specific manner. To further explore the in vivo role of CD8 + T cells in our model, we treated mice with a depleting antiCD8 mAb. Whereas mice treated or not with control IgG exhibited loss of Purkinje cells (mean loss of 36 and 44%, respectively), the five mice treated with the depleting antiCD8 mAb did not (data not shown). Downloaded from http://brain.oxfordjournals.org/ by guest on September 7, 2016 To uncover the structural defects underlying the phenotype caused by anti-CTLA4 mAb therapy in L7-HA mice, we performed neuropathological analyses. L7-HA mice implanted with 4T1-HA tumour and treated with antiCTLA4 mAb showed a massive infiltration of T cells in the cerebellum (Fig. 3C). In addition, clear local microglial activation was present in these mice (Fig. 3F). Other brain regions were spared from inflammation (data not shown). Neither T cell infiltration nor microglial activation was detected in the cerebellum of wild-type mice treated with antiCTLA4 mAb (Fig. 3A and D), or L7-HA mice not treated with anti-CTLA4 mAb (Fig. 3B and E). CTLA4 blockade also resulted in loss of Purkinje cell soma and dendritic arborization in L7-HA mice (Fig. 3I), but not in control mice (Fig. 3G and H), as detected by calbindin immunostaining. This Purkinje cell loss was further confirmed by IP3R1 immunoreactivity (Fig. 3J), another characteristic marker of Purkinje cells (Miyata et al., 1999). No detectable Ctla4 mRNA expression was detected in the cerebellum of L7-HA mice, whereas a strong signal was evidenced in the spleen (data not shown). We then quantified the number of Purkinje cells by counting Calbindin + cell bodies in 20 to 40 mm of Purkinje cell layer. A very significant reduction in Purkinje cells was observed in L7-HA mice treated with antiCTLA4 mAb, in comparison to the other groups (Fig. 3K). The magnitude of Purkinje cell loss was similar between Days 20 and 45, underlining the acute nature of the paraneoplastic autoimmune process (data not shown). Overall, in the 32 L7HA mice treated with anti-CTLA4 mAb, the mean loss of Purkinje cells was 40%, with marked interindividual variability ranging from 0 to 81% (Fig. 3K). Six L7-HA mice treated with anti-CTLA4 mAb presented normal Purkinje cell density, including five without cerebellar microglia activation and T cell infiltration. Collectively, these data suggest that the penetrance of disease is not 100% in our model. Thus, in L7-HA mice treated with anti-CTLA4 mAb, paraneoplastic neurological disorder is the consequence of massive cerebellar inflammation associated with the destruction of onconeural antigen-expressing Purkinje neurons. L. M. Yshii et al. Paraneoplastic neurological disorder BRAIN 2016: Page 7 of 12 | 7 Downloaded from http://brain.oxfordjournals.org/ by guest on September 7, 2016 Figure 3 Treatment with anti-CTLA4 antibody induces marked cerebellar inflammation and Purkinje cell loss selectively in L7-HA mice. (A–I) Histological analysis 20 days after transfer of naive HA-specific CD4 + and CD8 + T cells in L7-HA mice (B, C, E, F, H and I) or littermate controls (A, D and G) implanted with 4T1-HA, with (A, C, D, F, G and I) or without (B, E and H) anti-CTLA4 mAb treatment. Representative staining in brown for CD3 + cells (A–C; top row), microglia (Iba-1, D–F; middle row), and Purkinje cells (calbindin, G–I; bottom row); nuclear counterstaining: haematoxylin (blue). Arrows in (I) point to loss of Purkinje cells. Scale bars = 100 mm. (J) Representative images of immunofluorescence confocal microscopy using co-staining with anti-calbindin and anti-IP3R1 antibodies to label Purkinje cells with independent markers. Nuclear staining was performed with DAPI. Scale bar = 50 mm. (K) Quantitative evaluation of the density of Purkinje cells in 4T1-HA tumour-bearing wild-type (WT) and L7-HA mice, treated or not with anti-CTLA4 mAb. Results are expressed as mean SEM of 6–32 mice per group from six independent experiments analysed at either Day 20 or 45 (****P 5 0.0001). 8 | BRAIN 2016: Page 8 of 12 L. M. Yshii et al. infiltrating cells in wild-type (WT) and L7-HA mice implanted with 4T1-HA, 15 days after transfer of naive HA-specific CD4 + and CD8 + T cells and treatment with anti-CTLA4 mAb. (A) Cerebellum-infiltrating cells were stained with anti-CD45 (blood-derived cells), anti-CD11b (myeloid cells), anti-Thy1.2 (T cells), anti-B220 (B cells), anti-CD138 cells (plasma cells), anti-CD4 and anti-CD8 mAbs. (B) Cerebellum-infiltrating CD4 + T cells were stimulated in vitro for 16 h with HA110-119 or a control H-2-IAd-binding peptide and IFN- and IL-17 A production was assessed by intracellular staining. The absolute number of CD4 + T cells that produce IFN- or IL-17 A is shown in the right panels. The mouse indicated by a plus sign is depicted on the flow cytometry plot presented on the left. (C) Cerebellum-infiltrating CD8 + T cells were stimulated in vitro for 16 h with HA512-520 or a control H-2-Kd-binding peptide and IFN- and TNF- production was assessed. The absolute number of CD8 + T cells that produce IFN- or TNF- is plotted on the right panels (*P 5 0.05). The mouse indicated by a plus sign is depicted on the flow cytometry plot presented on the left. Finally, we wondered whether analogous immunological events could contribute to Purkinje cell targeting in human paraneoplastic neurological disorder in which autoantibodies have no demonstrated pathogenic effect. Confirming a previous report (Aboul-Enein et al., 2008), close apposition between CD8 + T cells and remaining calbindin + Purkinje cells was detected in patients with anti-Hu or anti-Ma2 antibody-associated encephalitis with prominent cerebellar involvement (Fig. 5K and L). Discussion Using a mouse model prone to develop paraneoplastic neurological disorder, we explored the delicate balance between tumour immunity and autoimmunity. As expected, the anti-tumour T cell response was sufficient to reduce tumour growth. Similar to the human situation, antiCTLA4 mAb, which antagonizes an inhibitory pathway in T cells, increased the efficacy of this anti-tumour immunity. Strikingly, however, this enhanced tumour control was obtained at the expense of autoimmune paraneoplastic neurological disorder. Actually, without anti-CTLA4 therapy, none of the studied animals developed cerebellar inflammation, whereas the use of anti-CTLA4 mAb led to cerebellar inflammation in 27/32 mice (84%). Anti-CTLA4 therapy promotes anti-tumour immunity through several converging mechanisms in both mice and humans (Allison, 2015). In mice, anti-CTLA4 treatment Downloaded from http://brain.oxfordjournals.org/ by guest on September 7, 2016 Figure 4 Characterization of immune cells within the cerebellum of L7-HA mice. Flow cytometry analysis of the cerebellum- Paraneoplastic neurological disorder BRAIN 2016: Page 9 of 12 | 9 Cerebellum of wild-type (A) and L7-HA mice (B–J) 20 days after transfer of anti-HA specific T cells and anti-CTLA4mAb therapy. (A and B) Calbindin staining shows marked loss of Purkinje cells in the L7-HA mice. Scale bars = 100 mm. (C) Double staining for CD8 (orange-brown) and calbindin (dark blue) shows the presence of large numbers of CD8 + T cells in the molecular layer of the cerebellum. Scale bar = 50 mm. (D) Double staining for CD8 and calbindin reveals cytotoxic T cells in contact with Purkinje cells. Scale bar = 20 mm. (E and F) Confocal fluorescence triple staining for Granzyme-B (red), CD8 (green) and calbindin (blue) shows CD8 + T cells in close apposition to Purkinje cells. Note the position of cytotoxic granules all facing the plasma membrane of the Purkinje cells. Scale bars = 10 mm. (G) Double staining for calbindin (blue) and b2microglobulin (brown) showing MHC class I upregulation in the Purkinje cell. Scale bar = 25 mm. (H) Double staining for TUNEL (blue) and calbindin reveals degeneration of a Purkinje cell (arrowhead). Scale bar = 50 mm. (I) Presence of a caspase-3-positive cell with condensed nucleus in the Purkinje cell layer. Scale bar = 10 mm. (J) Microglial cells stained for Mac-3 enclosing a Purkinje cell. Scale bar = 25 mm. (K) Double staining for calbindin (blue) and CD8 (brown) shows severe loss of Purkinje cells and dendritic arborization in a patient with anti-Hu antibody-associated paraneoplastic neurological disorder. Scale bar = 200 mm. Arrowheads point at some remaining Purkinje cells. The inset shows a higher magnification of a CD8 + T cell (arrowhead) in tight apposition to a Purkinje cell. (L) Loss of Purkinje cells (arrowheads) can also be seen in a patient with anti-Ma2 antibody-associated paraneoplastic neurological disorder. Scale bar = 200 mm. Here the insets reveal multiple CD8 + T cells attacking Purkinje cells. depletes Treg cells selectively from tumour via Fc receptor-expressing local macrophages, while the absolute numbers of effector T cells are increased (Simpson et al., 2013). In addition, CTLA4 blockade on effector T cells, notably on cytotoxic CD8 + T cells, appears essential for the enhanced anti-tumour response following anti-CTLA4 therapy. In that respect, human studies have shown that CTLA4 blockade therapy increases the amplitude of Downloaded from http://brain.oxfordjournals.org/ by guest on September 7, 2016 Figure 5 Immune mechanisms of Purkinje cell loss in experimental and human paraneoplastic cerebellar degeneration. 10 | BRAIN 2016: Page 10 of 12 simultaneously by two or more CD8 + T cells. This feature was recently associated with high probability of target cell death using dynamic two-photon imaging in experimental models of viral infection (Halle et al., 2016). Our data highlight the induction of paraneoplastic neurological disorder after therapeutic blockade of CTLA4, raising the concern that checkpoint inhibitors, by enhancing anti-tumour immunity, may inadvertently increase the likelihood for paraneoplastic neurological disorders. Paraneoplastic neurological disorder development has been associated with a better control of the underlying tumour, attributed to a partly efficient anti-tumour adaptive immunity (Pignolet et al., 2013). In addition, the oncologic response to anti-CTLA4 therapy correlates with immune-related adverse events (Bertrand et al., 2015). Gynaecologic cancers and certain cancers of neuroectodermal origin are particularly associated with paraneoplastic neurological disorders. Consequently, the enhanced and diversified anti-tumour T cell response promoted by antiCTLA4 therapy could lead to paraneoplastic neurological disorder, in patients harbouring these types of cancers. As suggested, based on theoretical grounds (Pignolet et al., 2013; Steinman, 2014), our experimental model argues that immune checkpoint inhibitors may favour paraneoplastic neurological disorder development. Interestingly, two cases of autoimmune encephalomyelitis developing after the first injection of immune checkpoint inhibitors have been recently reported (Williams et al., 2016). The sero-prevalence of anti-neuronal autoantibodies can be as high as 29% in patients with small cell lung cancer (Pignolet et al., 2013; Gozzard et al., 2015) and anti-neuronal antibodies are associated with T cells with the same specificity (Albert et al., 2000). Therefore, a close monitoring of neurological symptoms is warranted, at least in the subgroup of patients with anti-neuronal autoantibodies, when immune checkpoint inhibitors are being used. Acknowledgements We acknowledge Dr Isabelle Dusart for advice regarding the study of the cerebellum, and Drs Anne Dejean, Abdelhadi Saoudi, Nicolas Blanchard, and Daniel Gonzalez-Dunia for their valuable inputs on the manuscript. The authors acknowledge the technical assistance provided by CPTP Cytometry and Imaging facilities, and histology and animal facility staff of US006. Funding This work was supported by grants from FP7-PEOPLE2012-ITN-Neurokine, ERA-NET Meltra-BBB, MidiPyrénées Region, Fondation ARC pour la recherche sur le cancer ARC, and Ligue régionale contre le cancer (R.L.). L.M.Y. was supported by grant #2013/22196-4 from São Paulo Research Foundation (FAPESP). Downloaded from http://brain.oxfordjournals.org/ by guest on September 7, 2016 melanoma-specific CD8 + T cell responses against selfantigens and, possibly, against tumour neoantigens (Kvistborg et al., 2014; Van Allen et al., 2015). Importantly, CTLA4 deficiency is associated with multitissue lymphocytic infiltration and damage of likely autoimmune origin (Waterhouse et al., 1995; Ise et al., 2010). Here again, CTLA4 maintains T cell tolerance by regulating the pathogenicity of autoantigen-specific T cells by both effector T cell autonomous effects and non-cell autonomous mechanisms, involving Treg cells (Wing et al., 2008; Ise et al., 2010). Furthermore, patients with CTLA4 haploinsufficiency present multi-tissue inflammation, including brain and cerebellar involvement (Kuehn et al., 2014; Schubert et al., 2014). Partly mirroring these data, anti-CTLA4 therapy has been associated with immune-related adverse events in 60% of treated patients (Bertrand et al., 2015). In particular, CNS autoimmunity is part of the spectrum of immune-related adverse events of anti-CTLA4 therapy. Indeed, autoimmune hypophysitis has been widely recognized as a frequent side effect of ipilimumab therapy (Iwama et al., 2014; Bertrand et al., 2015). Exacerbation of pre-existing autoimmune disease has also been reported in patients with advanced melanoma treated with ipilimumab (Johnson et al., 2016). Whether de novo development of autoimmune side effects is related to genetic susceptibility or to pre-existing serologic evidence for autoantibodies is currently unknown. In our mouse model, immune checkpoint therapy elicited migration of antigen-specific T cells into the cerebellum and subsequent killing of neurons, only when the antigen was expressed in neurons. Our data strongly suggest that CD8 + T cells play a final effector role by specifically killing MHC class I-expressing Purkinje cells in paraneoplastic cerebellar degeneration. Purkinje cells have indeed been reported to express MHC class I molecules (McConnell et al., 2009), a feature confirmed here both in vitro upon IFN- incubation and in vivo. Based on these data and on the strong production of IFN- by cerebellum-infiltrating CD8 + T cells, it is tempting to speculate that a pathogenic feed-forward loop takes place. This involves IFN- secretion by CD8 + T cells upon local recognition of onconeural antigens that further promotes MHC class I presentation by neurons, in turn rendering them vulnerable to killing by antigen-specific CD8 + T cells. A similar scenario is likely at play in human paraneoplastic neurological disorders with autoantibodies against intracellular neuronal antigens. Indeed, an IFN- signature has been reported in the CSF of such patients (Roberts et al., 2015). Additionally, CDR2-specific CD8 + T cells are present in the blood and CSF of patients with paraneoplastic cerebellar degeneration (Albert et al., 1998, 2000) and they can kill CDR2-expressing target cells in an HLA-restricted manner (Santomasso et al., 2007). Finally, in situ evidence for direct killing of neurons by cytotoxic T cells has been documented by converging studies (Bien et al., 2012). Our still images from the experimental model and from tissue of patients with paraneoplastic neurological disorder point to a possible T cell cooperation for killing, as some target neurons are contacted L. M. Yshii et al. Paraneoplastic neurological disorder Supplementary material Supplementary material is available is available at Brain online. References | 11 Kirberg J, Baron A, Jakob S, Rolink A, Karjalainen K, von Boehmer H. Thymic selection of CD8 + single positive cells with a class II major histocompatibility complex-restricted receptor. J Exp Med 1994; 180: 25–34. Kuehn HS, Ouyang W, Lo B, Deenick EK, Niemela JE, Avery DT, et al. Immune dysregulation in human subjects with heterozygous germline mutations in CTLA4. Science 2014; 345: 1623–7. Kvistborg P, Philips D, Kelderman S, Hageman L, Ottensmeier C, Joseph-Pietras D, et al. Anti-CTLA-4 therapy broadens the melanoma-reactive CD8 + T cell response. Sci Transl Med 2014; 6: 254ra128. Leach DR, Krummel MF, Allison JP. Enhancement of antitumor immunity by CTLA-4 blockade. Science 1996; 271: 1734–6. Martin-Blondel G, Pignolet B, Tietz S, Yshii L, Gebauer C, Perinat T, et al. Migration of encephalitogenic CD8 T cells into the central nervous system is dependent on the alpha4beta1-integrin. Eur J Immunol 2015; 45: 3302–12. Mason WP, Graus F, Lang B, Honnorat J, Delattre JY, Valldeoriola F, et al. Small-cell lung cancer, paraneoplastic cerebellar degeneration and the Lambert-Eaton myasthenic syndrome. Brain 1997; 120 (Pt 8): 1279–300. McConnell MJ, Huang YH, Datwani A, Shatz CJ. H2-K(b) and H2D(b) regulate cerebellar long-term depression and limit motor learning. Proc Natl Acad Sci USA 2009; 106: 6784–9. Miyata M, Miyata H, Mikoshiba K, Ohama E. Development of Purkinje cells in humans: an immunohistochemical study using a monoclonal antibody against the inositol 1,4,5-triphosphate type 1 receptor (IP3R1). Acta Neuropathol 1999; 98: 226–32. Morgan DJ, Liblau R, Scott B, Fleck S, McDevitt HO, Sarvetnick N, et al. CD8( + ) T cell-mediated spontaneous diabetes in neonatal mice. J Immunol 1996; 157: 978–83. Pellkofer H, Schubart AS, Hoftberger R, Schutze N, Pagany M, Schuller M, et al. Modelling paraneoplastic CNS disease: T-cells specific for the onconeuronal antigen PNMA1 mediate autoimmune encephalomyelitis in the rat. Brain 2004; 127 (Pt 8): 1822–30. Pignolet BS, Gebauer CM, Liblau RS. Immunopathogenesis of paraneoplastic neurological syndromes associated with anti-Hu antibodies: a beneficial antitumor immune response going awry. Oncoimmunology 2013; 2: e27384. Robert C, Thomas L, Bondarenko I, O’Day S, Weber J, Garbe C, et al. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. N Engl J Med 2011; 364: 2517–26. Roberts WK, Blachere NE, Frank MO, Dousmanis A, Ransohoff RM, Darnell RB. A destructive feedback loop mediated by CXCL10 in central nervous system inflammatory disease. Ann Neurol 2015; 78: 619–29. Ruocco MG, Pilones KA, Kawashima N, Cammer M, Huang J, Babb JS, et al. Suppressing T cell motility induced by anti-CTLA-4 monotherapy improves antitumor effects. J Clin Invest 2012; 122: 3718– 30. Saito H, Tsumura H, Otake S, Nishida A, Furukawa T, Suzuki N. L7/ Pcp-2-specific expression of Cre recombinase using knock-in approach. Biochem Biophys Res Commun 2005; 331: 1216–21. Santomasso BD, Roberts WK, Thomas A, Williams T, Blachere NE, Dudley ME, et al. A T-cell receptor associated with naturally occurring human tumor immunity. Proc Natl Acad Sci USA 2007; 104: 19073–8. Saxena A, Bauer J, Scheikl T, Zappulla J, Audebert M, Desbois S, et al. Cutting edge: multiple sclerosis-like lesions induced by effector CD8 T cells recognizing a sequestered antigen on oligodendrocytes. J Immunol 2008; 181: 1617–21. Schubert D, Bode C, Kenefeck R, Hou TZ, Wing JB, Kennedy A, et al. Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations. Nat Med 2014; 20: 1410–16. Simpson TR, Li F, Montalvo-Ortiz W, Sepulveda MA, Bergerhoff K, Arce F, et al. Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma. J Exp Med 2013; 210: 1695–710. Downloaded from http://brain.oxfordjournals.org/ by guest on September 7, 2016 Aboul-Enein F, Hoftberger R, Buxhofer-Ausch V, Drlicek M, Lassmann H, Budka H, et al. Neocortical neurones may be targeted by immune attack in anti-Yo paraneoplastic syndrome. Neuropathol Appl Neurobiol 2008; 34: 248–52. Albert ML, Austin LM, Darnell RB. Detection and treatment of activated T cells in the cerebrospinal fluid of patients with paraneoplastic cerebellar degeneration. Ann Neurol 2000; 47: 9–17. Albert ML, Darnell JC, Bender A, Francisco LM, Bhardwaj N, Darnell RB. Tumor-specific killer cells in paraneoplastic cerebellar degeneration. Nat Med 1998; 4: 1321–4. Albert ML, Darnell RB. Paraneoplastic neurological degenerations: keys to tumour immunity. Nat Rev Cancer 2004; 4: 36–44. Allison JP. Checkpoints. Cell 2015; 162: 1202–5. Aslakson CJ, Miller FR. Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Res 1992; 52: 1399–405. Bauer J, Lassmann H. Neuropathological Techniques to investigate central nervous system sections in multiple sclerosis. Methods Mol Biol 2016; 1304: 211–29. Bertrand A, Kostine M, Barnetche T, Truchetet ME, Schaeverbeke T. Immune related adverse events associated with anti-CTLA-4 antibodies: systematic review and meta-analysis. BMC Med 2015; 13: 211. Bien CG, Vincent A, Barnett MH, Becker AJ, Blumcke I, Graus F, et al. Immunopathology of autoantibody-associated encephalitides: clues for pathogenesis. Brain 2012; 135 (Pt 5): 1622–38. Dalmau J, Graus F, Villarejo A, Posner JB, Blumenthal D, Thiessen B, et al. Clinical analysis of anti-Ma2-associated encephalitis. Brain 2004; 127 (Pt 8): 1831–44. Dalmau J, Rosenfeld MR. Paraneoplastic syndromes of the CNS. Lancet Neurol 2008; 7: 327–40. Furneaux HM, Rosenblum MK, Dalmau J, Wong E, Woodruff P, Graus F, et al. Selective expression of Purkinje-cell antigens in tumor tissue from patients with paraneoplastic cerebellar degeneration. N Engl J Med 1990; 322: 1844–51. Gozzard P, Woodhall M, Chapman C, Nibber A, Waters P, Vincent A, et al. Paraneoplastic neurologic disorders in small cell lung carcinoma: a prospective study. Neurology 2015; 85: 235–9. Graus F, Saiz A, Dalmau J. Antibodies and neuronal autoimmune disorders of the CNS. J Neurol 2010; 257: 509–17. Halle S, Keyser KA, Stahl FR, Busche A, Marquardt A, Zheng X, et al. In Vivo Killing Capacity of Cytotoxic T Cells Is Limited and Involves Dynamic Interactions and T Cell Cooperativity. Immunity 2016; 44: 233–45. Hodi FS, O’Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 2010; 363: 711–23. Ise W, Kohyama M, Nutsch KM, Lee HM, Suri A, Unanue ER, et al. CTLA-4 suppresses the pathogenicity of self antigen-specific T cells by cell-intrinsic and cell-extrinsic mechanisms. Nat Immunol 2010; 11: 129–35. Iwama S, De Remigis A, Callahan MK, Slovin SF, Wolchok JD, Caturegli P. Pituitary expression of CTLA-4 mediates hypophysitis secondary to administration of CTLA-4 blocking antibody. Sci Transl Med 2014; 6: 230ra45. Johnson DB, Sullivan RJ, Ott PA, Carlino MS, Khushalani NI, Ye F, et al. Ipilimumab therapy in patients with advanced Melanoma and preexisting autoimmune disorders. JAMA Oncol 2016; 2: 234–40. BRAIN 2016: Page 11 of 12 12 | BRAIN 2016: Page 12 of 12 Steinman L. Conflicting consequences of immunity to cancer versus autoimmunity to neurons: insights from paraneoplastic disease. Eur J Immunol 2014; 44: 3201–5. Tuzun E, Zhou L, Baehring JM, Bannykh S, Rosenfeld MR, Dalmau J. Evidence for antibody-mediated pathogenesis in anti-NMDAR encephalitis associated with ovarian teratoma. Acta Neuropathol 2009; 118: 737–43. Van Allen EM, Miao D, Schilling B, Shukla SA, Blank C, Zimmer L, et al. Genomic correlates of response to CTLA-4 blockade in metastatic melanoma. Science 2015; 350: 207–11. L. M. Yshii et al. Waterhouse P, Penninger JM, Timms E, Wakeham A, Shahinian A, Lee KP, et al. Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science 1995; 270: 985–8. Williams TJ, Benavides DR, Patrice KA, Dalmau JO, de Avila AL, Le DT, et al. Association of autoimmune encephalitis with combined immune checkpoint inhibitor treatment for Metastatic cancer. JAMA Neurol 2016; 73: 928–33. doi:10.1001. Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T, Miyara M, Fehervari Z, et al. CTLA-4 control over Foxp3 + regulatory T cell function. Science 2008; 322: 271–5. Downloaded from http://brain.oxfordjournals.org/ by guest on September 7, 2016 #!!+ $" $* $ # + $* ! +*073)074"!) 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"'%'#"& " ##$%')',1 8993 8:;4867<51 7:7 #!"$ "%$ REVIEW PAPER TYPE OncoImmunology 2:12, e27384; December 2013; © 2013 Landes Bioscience Immunopathogenesis of paraneoplastic neurological syndromes associated with anti-Hu antibodies A beneficial antitumor immune response going awry Béatrice SL Pignolet1,2,3,4, Christina MT Gebauer1,2,3, and Roland S Liblau1,2,3,4,* 1 INSERM-UMR1043; Toulouse, France; 2CNRS, U5282; Toulouse, France; 3Universite de Toulouse; UPS; Centre de Physiopathologie Toulouse Purpan (CPTP); Toulouse, France; 4 CHU Toulouse Purpan; Toulouse, France Keywords: anti-Hu syndrome, autoimmunity, cancer, central nervous system, paraneoplastic neurological disorder Abbreviations: BDNF, brain-derived neurotrophic factor; CNS, central nervous system; CSF, cerebrospinal fluid; DRG, dorsal root ganglia; ELAV, embryonic lethal abnormal visual; PBMC, peripheral blood mononuclear cell; PEM, paraneoplastic encephalomyelopathy; PND, paraneoplastic neurological disorder; PSN, paraneoplastic sensory neuronopathy; SCLC, small-cell lung cancer; TCR, T cell receptor Paraneoplastic neurological disorders (PNDs) are syndromes that develop in cancer patients when an efficient antitumor immune response, directed against antigens expressed by both malignant cells and healthy neurons, damages the nervous system. Herein, we analyze existing data on the mechanisms of loss of self tolerance and nervous tissue damage that underpin one of the most frequent PNDs, the anti-Hu syndrome. In addition, we discuss future directions and propose potential strategies aimed at blocking deleterious encephalitogenic immune responses while preserving the antineoplastic potential of treatment. Introduction Paraneoplastic neurological disorders (PNDs) are neurological manifestations associated with cancer that do not originate from tumor growth or the metabolic, infectious and toxic complications of malignancy and/or its treatment. This group of syndromes is highly diverse in terms of tissue damage and clinical signs. PNDs develop in the frame of naturally occurring antitumor immune responses. The targets of such an immunological response against malignant cells are self antigens physiologically expressed by the normal nervous system and ectopically expressed by cancer cells. One of the most frequent PND is the paraneoplastic encephalomyelopathy/paraneoplastic sensory neuronopathy (PEM/PSN). PEM/PSN has also been named anti-Hu antibody-associated (or simply anti-Hu) syndrome, after *Correspondence to: Roland S Liblau; [email protected] Submitted: 10/28/2013; Revised: 11/29/2013; Accepted: 12/01/2013; Published Online: 12/10/2013 Citation: Pignolet BSL, Gebauer CMT, Liblau RS. Immunopathogenesis of paraneoplastic neurological syndromes associated with anti-Hu antibodies: A beneficial antitumor immune response going awry. OncoImmunology 2013; 2:e27384; http://dx.doi.org/10.4161/onci.27384 www.landesbioscience.com its linkage with high circulating levels of anti-Hu antibodies has been appreciated. Clinical Aspects of the Anti-Hu Syndrome In 85% of the cases, the anti-Hu syndrome is associated with lung cancer, mostly small-cell lung cancer (SCLC). More rarely, it develops in association with extrathoracic neoplasms such as neuroblastoma or intestinal, prostate, breast, bladder, and ovary carcinomas. Some patients affected by the anti-Hu syndrome present more than one primary cancer. Neurological manifestations compatible with the diagnosis of PEM/PSN have been observed in 1.4% of 432 SCLC patients and much more rarely in individuals bearing other types of cancer.1,2 A polyclonal antibody response against nuclear antigens expressed in the central nervous system (CNS), enteric neurons and dorsal root ganglion neurons, but not in other adult normal tissues was first identified in 1985 in 2 patients presenting SCLC associated with subacute sensory neuropathy.3 The expression “anti-Hu antibodies” was coined based on the first 2 letters of the surname of these patients. Anti-Hu antibodies were soon detected in multiple patients manifesting a rapid development of diverse neurological manifestations, including sensory neuropathy, cerebellar ataxia, limbic encephalitis, brainstem encephalitis, myelitis, or intestinal pseudo-obstruction. Anti-Hu antibodies are in fact frequently detected in multiple of the above-mentioned syndromes occurring in a paraneoplastic context. Interestingly, high titers of anti-Hu antibodies are absent in SCLC patients who do not exhibit neurological disorders. Of major clinical importance, these neurological manifestations precede the diagnosis of the cancer in more than 80% of the cases. Therefore, the detection of high circulating titers of antiHu antibodies in patients with a neurological condition indicates a paraneoplastic syndrome and should lead to a very thorough search of the underlying neoplasm. In some instances, the tumor OncoImmunology e27384-1 is discovered only post-mortem or never, indicating either a complete disease regression4 or the occurrence of such autoimmune neurological manifestations independent of neoplasia. Indeed, cases of limbic encephalitis associated with anti-Hu antibodies but not with cancer have been described in children.5 A spontaneously regressing or occult neuroblastoma cannot be formally excluded in these rare cases. Interestingly, tumors exhibit a significantly less severe course and a lower propensity to generate metastases in patients with PEM/PSN than in patients who do not develop PNDs.6,7 The effect on tumor spread and overall survival is however moderate, and only 30% of patients survive for more than 2 y.7,8 In as much as 60% of the cases, the prognosis of the patients is determined by the outcome of the neurological disorders rather than by tumor progression.9 Of note, SCLC patients who do not manifest PNDs but harbor low titers of anti-Hu antibodies experience tumors of low stage, frequently respond to therapy, and live longer than cancer patients not producing anti-Hu antibodies.10,11 These findings reinforce the hypothesis that effective antitumor immune responses targeting Hu antigens may be dissociated from autoimmune manifestations. As anti-Hu antibodies bind to antigens expressed both in malignant cells and healthy neurons, it was tempting to hypothesize that the anti-Hu syndrome could be an autoimmune disease triggered by cancer. This is reminiscent of vitiligo, developing in certain cases of melanoma,12 although in the case of PNDs, the cells targeted by the autoimmune process belong to a different tissue than their malignant counterparts, i.e., the immunoprivileged CNS. Hu Antigens Hu antigens in healthy neurons The search for the molecular targets of anti-Hu antibodies led to the identification of 3 proteins of approximate molecular weight of 35–38 KDa expressed in the CNS. In malignant cells, the 38 KDa band is not detected. Screening a cDNA expression library from the human cerebellum with the sera of patients affected by the anti-Hu syndrome allowed for the cloning of the highly homologous genes HuD (official name: ELAV like neuron-specific RNA binding protein 4, ELAVL4), HuC, (official name: ELAV like neuron-specific RNA binding protein 3, ELAVL3) and HuB (official name; ELAV like neuron-specific RNA binding protein 2, ELAVL2; also known as Hel-N1). The Hu family actually consists of 4 members: HuA (official name: ELAV like RNA binding protein 1, ELAVL1; also known as HuR), HuB, HuC, and HuD. These proteins share extensive homology and are highly-conserved across species.13 HuA, the most divergent, is ubiquitously expressed13,14 and is not recognized by paraneoplastic antibodies. In contrast, HuB, HuC, and HuD expression is restricted to neurons, which all express one or more of these proteins.13 The Hu proteins are highly homologous to the Drosophila melanogaster nuclear protein ELAV (embryonic lethal abnormal visual), which plays a key role in neuronal development.15,16 HuB, HuC, and HuD operate as neuron-specific RNA-binding proteins. They contain 3 distinct RNA recognition motifs that bind e27384-2 to AU-rich sequences, which often characterize the 3′ untranslated region of mRNAs and control their expression.17 Indeed, the Hu proteins have been implicated in mRNA stabilization and RNA turnover.18,19 Among various target mRNAs, HuD has recently been shown to bind, hence stabilizing, the brain-derived neurotrophic factor (BDNF)-coding mRNA.20 Moreover, neuronal Hu proteins have been shown to bind pre-mRNA species, thus regulating their alternative splicing.21 HuD is highly expressed in the nucleus, but also translocates to the cytoplasm, where it localizes within small granules.22 The cytoplasmic localization of HuD appears to be essential for neuronal differentiation.23 In addition, HuD has been shown to interact with AKT1 to trigger the outgrowth or neurites.24 Thus, Hu proteins, in particular HuD, have a major impact on multiple key neuronal processes. Hu antigens in malignant cells Whereas HuB, HuC, and HuD are all expressed in CNS neurons, HuD is Hu antigen most frequently expressed by SCLC cells. This is a consistent observation in SCLC patients, irrespective of whether they develop PEM/PSN or not.6,25 The HuDcoding mRNA has also been detected in SCLC cell lines.26 No somatic mutations or deletions/insertions affecting HuD have been observed in SCLC cell lines or tumors, including those from patients with PEM/PSN.27–29 However, a fine analysis of the post-translational modifications of HuD in cancer cells from patients developing or not PNDs has not been performed to date. Indeed, malignant cell-specific post-translational modifications of HuD may favor the development of an immune response that cross-reacts with neuronal Hu proteins. Moreover, the possible function(s) of HuD in the tumor biology, if any, has not been uncovered so far. SCLC and many cancers associated with PEM/ PSN exhibit a neuroectodermal origin. The neuroectoderm includes the neural crest (melanocytes, dorsal root ganglia, ganglia of the autonomous nervous system, adrenal glands, Schwann cells,…) and the neural tube (brain, spinal cord, retina, neurohypophysis). As a result, most SCLCs are composed of cells with a neuroendocrine phenotype, characterized by elevated expression levels of L-dopa decarboxylase, neuron-specific enolase, and neuropeptides.30 These features resemble those of neural crest-derived endocrine cells disseminated in the normal bronchial mucosa. Such a shared embryonic origin could explain the expression of HuD by SCLC cells. Autoimmune responses against Hu proteins Humoral responses The serum of patients with the anti-Hu syndrome reacts with HuB, HuC, and HuD. Since HuD is the only Hu protein consistently expressed by SCLCs,25 it is the most likely initiator of autoimmune response against Hu antigens. The presence of anti-HuD antibodies can be revealed using a large variety of techniques. The initial screening is generally performed by indirect immunohistochemistry on frozen sections of healthy human or rodent cerebral cortex, which results in a strong nuclear and weak cytoplasmic staining in the presence of anti-HuD antibodies (Fig. 1A). To firmly identify the antigenic targets, immunoblotting is performed as a complementary test OncoImmunology Volume 2 Issue 12 (Fig. 1B), using either neuronal nuclear extracts or recombinant HuD.31–33 ELISA can also be used, but its sensitivity appears to be lower than that than of immunoblotting on purified HuD.33 The titers of circulating antiHu antibodies usually ranges from 2000 to 64 000 fold dilution in patients with PEM/PSN.32,34 Elevated serum titers of anti-Hu antibodies provide a highly valuable diagnostic biomarker. Conversely, low levels of anti-Hu antibodies can be detected in the absence of PNDs in 16 to 22.5% of SCLC patients and in less than 1% of patients with other malignancies.10,33,35 The anti-HuD IgG response is polyclonal and subclass distribution analyses revealed that antiHu IgGs belong predominantly to the IgG1 and IgG3 isotypes.34,36,37 Figure 1. Detection of autoantibodies associated with the anti-Hu syndrome. (A) Patient sera containing The HuD domains encompassing Hu-specific autoantibodies specifically recognize neurons in the central nervous system (CNS) (magnifiresidues 90–101 and 171–206 are cation 200 ×). Briefly, hippocampal (left panels) and cerebellar (right panels) rat slices (EuroImmun) were recognized by all anti-Hu serum incubated with serum from either an individual not affected by paraneoplastic neurological disorders samples. Conversely, the 223–234, (PNDs) (negative control), either a patient with a confirmed anti-Hu syndrome (positive control), or a subject suspected to suffer from the anti-Hu syndrome, followed by the detection of bound IgG using biotinyl235–252, and 354–373 epitopes ated goat anti-human IgG, ABC and VIP kits (Eurobio). The test serum turned out to contain anti-neuron only react with sera of some patients antibodies exhibiting a staining pattern compatible with that of anti-Hu antibodies. (B) The molecular 38 with the anti-Hu syndrome. identification of the specificity of such neuron-specific IgGs is made by immunoblotting-based diagnostic The titers of anti-Hu antibodtests. Patient-derived serum (1/100 dilution) and cerebrospinal fluid (when available) are incubated on a ies in the cerebrospinal fluid (CSF) membrane stripped (right panel) with paraneoplastic neurological syndrome-associated antigens, including amphiphysin, CV2, PNMA2 (Ma2/Ta), Ri, Yo, and Hu proteins (EuroImmun). Upon washing, bound IgGs vary from 100 to 4000 fold diluare detected using alkaline phosphatase-conjugated anti-human IgG antibodies and their position is contion, pointing to intrathecal synfronted to a reference scheme. 32,39,40 Indeed, when adjusted thesis. to the same IgG concentration, the CSF/serum anti-Hu antibody index is > 1 in 86% of patients a study documented the expression of Hu-related antigens on the with paraneoplastic encephalomyelopathy.39 In patients exhibit- surface of SCLC and neuroblastoma cell lines.45 Whether neuing a CSF/serum antibody index > 1.5, a strong indication of rons exhibit a similar expression pattern of Hu proteins is curthe oligoclonal intrathecal synthesis of HuD-specific IgGs was rently undetermined. obtained using isoelectric focusing followed by immunoblotting Experimentally, the contribution of anti-Hu antibodies to on HuD-loaded nitrocellulose membranes.41 Collectively, these the development of PEM/PSN has been investigated in vitro, in findings indicate that anti-HuD autoantibodies are produced cell cultures, and in vivo, upon injection of human antibodies intrathecally in patients with robust CNS involvement. However, to laboratory animals. In vitro, monoclonal antibodies specific patients presenting with isolated PSNs do not exhibit intrathe- for HuD have been shown to promote the apoptotic demise of cal anti-Hu antibody synthesis.32 Interestingly, plasmapheresis neuroblastoma cell lines and primary myenteric neurons.46 The succeeds in reducing the levels of anti-Hu autoantibodies in the serum or IgG preparations from patients with the anti-Hu synserum but not in the CSF.39 drome could also kill human HuD-expressing cell lines and Anti-Hu antibodies persist over time in the serum of SCLC primary rodent neurons, a phenomenon that was accelerated patients with PEM/PSN while SCLC patients who do not in the presence of the complement system.46–48 However, this develop PND remain seronegative.42 Importantly, 8–40% of observation is not universal, and the lack of cytotoxic effects of the patients with PEM/PSN and anti-Hu antibodies also harbor anti-Hu positive sera on HuD + cancer cells or primary murine neurons has also been reported.49,50 Moreover, even when neuroautoantibodies targeting other neural antigens.35,43,44 Functional properties of anti-Hu antibodies nal cytotoxicity was observed, the specificity of the pathogenic Since anti-Hu antibodies target intracellular antigens, their autoantibodies was questioned, as the patients’ IgGs, but not an pathogenic and antitumor potential is questionable. Interestingly, anti-HuD monoclonal antibody tested alongside, mediated such www.landesbioscience.com OncoImmunology e27384-3 an effect.48 As discussed above, the serum of patients with PND often contain multiple neuron-targeting autoantibodies, some of which recognize surface receptors. Therefore, unless proper specificity assays are conducted, for instance upon pre-absorbing IgG preparations on recombinant HuD, formal conclusions regarding the impact of anti-Hu antibodies on neuronal viability are difficult to reach. The intravenous administration of IgGs from patients with PEM/PSN and high titers of anti-Hu antibody to mice did not result in any CNS lesion.51,52 In addition, all attempts to generate rodent models of the anti-Hu syndrome upon immunization with recombinant HuD failed to induce any signs of disease, CNS inflammation or neuronal degeneration, despite the development of high circulating titers of anti-HuD antibodies.51,53 Thus, although the presence of high anti-Hu titers in PND patients represents a highly valuable diagnostic tool, their role in disease pathogenesis appears at best dubious. Nevertheless, these autoantibodies reflect a strong autoimmune/antitumor immune response in which autoreactive T cells, specific for HuD or other self antigens, are likely to be involved. T cell responses The peripheral blood mononuclear cells (PBMCs) of SCLC patients with the anti-Hu syndrome exhibit an accumulation of activated HLA-DR+ CD4 + T cells and memory CD45RO + CD4 + helper T cells as compared with anti-Hu antibody-negative SCLC patients.54,55 An increase in activated CD8 + T cells in these patients has also been reported.55 CD25brightCD4 + FOXP3 + T cells of untested immunosuppressive potential are elevated in SCLC patients regardless of the development of PEM/PSN. However, a reduced expression of FOXP3-, transforming growth factor β1 (TGFβ1)- and cytotoxic T lymphocyte-associated protein 4 (CTLA4)-coding mRNAs has been detected in regulatory T cells from SCLC patients with PEM/PSN, suggesting a defect in their immunosuppressive activity.56 Consistent with the pleiocytosis observed in diagnostic CSF analyses,57 increased amounts of T cells, mostly memory CD4 + and CD8 + T cells, characterize the CSF of patients with the anti-Hu syndrome.58 The involvement of (most often circulating) autoreactive T cells in the anti-Hu syndrome has been investigated using a variety of methodological approaches. The PBMCs of SCLC patients with high anti-Hu antibody titers proliferate more vigorously in response to HuD and secrete abundant interferon γ (IFNγ), but not IL-4, as compared with PBMCs from SCLC patients without anti-Hu antibodies.54 HuD is therefore a likely antigenic target for primed autoreactive CD4 + T cells, presumably of the TH1 subtype, implicating cell-mediated immunity in the anti-Hu syndrome. However, CD4 + T-cell depletion, purification or blockade experiments using anti-HLA class II antibodies have not yet been performed, and would be needed to unambiguously identify the responding cell type. CD8 + T cells are considered to be the killer cell by excellence and are suspected to play a key pathogenic role in paraneoplastic cerebellar degeneration, another type of PND.59,60 In addition, solid tumors can express high level of MHC class I molecules and accumulating data indicate that MHC class I molecules can be upregulated on neurons, in particular during inflammatory e27384-4 conditions.61 Thus, both cancer cells and neurons could be the targets of CD8 + T cells. However, in the past decade, efforts to identify HuD-specific CD8 + T cells in SCLC patients with the anti-Hu syndrome, using a combination of cytokine release assays and flow cytometry based on HLA:HuD peptide tetramers, have yielded conflicting observations. Indeed, some investigators have reported negative results,62,63 while others have found consistent evidence for such HuD-specific CD8 + T cells.64–66 This discrepancy may relate to the methodological approaches employed by these groups. Alternatively, it could be connected to timing, as the PBMCs of patients assessed within the first 3 mo of PEM/ PSN onset exhibited more robust IFNγ ELISPOT responses to HuD-derived peptides than the PMBCs of patients tested later.65 Finally, the abovementioned discrepancy may reflect the assay used for the detection of HuD-specific CD8 + T cells. Indeed, in a study involving 4 HLA-A03:01-expressing patients with the anti-Hu syndrome, the PBMCs of only one were found to exhibit a classical cytotoxic profile with IFNγ secretion in response to the HuD140–148 peptide. Surprisingly, the PBMCs of 2 patients displayed a robust production of interleukin (IL)-5 and IL-13 in response to HuD164–172 stimulation, but almost undetectable cytotoxicity or IFNγ production.67 Of note, the PBMCs of none of the control subjects exhibited any type of response to HuDderived peptides. These data suggest that anti-HuD CD8 + T-cell responses could be skewed toward a type 2, non-cytotoxic, profile in some patients with the anti-Hu syndrome, a pattern that could be easily missed using usual functional assays. However, whether the type of CD8 + T-cell response observed in PBMCs is reflective of what occurs within neoplastic lesions and in the nervous system is unknown. If so, how the effective antitumor response and neurological damage could be mechanistically explained? Finally, it is possible that autoreactive T cells in the anti-Hu syndrome recognize additional or alternative neuronal antigens. Indeed, although antigens recognized by autoantibodies and autoreactive T cells largely overlap in most organ-specific autoimmune diseases, this is not necessarily the case.68 This aspect has not yet received adequate attention. How is immune tolerance to neuronal Hu antigens broken in patients with PEM/PSN? Studies performed in mice revealed that HuD-specific CD8 + T cells can be elicited upon immunization with recombinant adenoviruses expressing full-length HuD.69 However, the need for adjuvants and for further in vitro stimulation to detect HuDspecific CD8 + T cells point to a reduced frequency of HuDspecific T-cell precursors, their limited functional avidity, and/or to the existence of robust immunosuppressive network that operate in vivo to maintain these cells under control. The robust ex vivo CD8 + T-cell response generated in HuD-deficient, but not in wild-type, mice provided direct evidence for partial induction of tolerance to HuD under physiological conditions.69 Interestingly, HuD is expressed, although at low levels, by MHCIIhigh thymic medullary epithelial cells and thymic CD8 + dendritic cells (http://immgen.org/). Importantly, patients with the anti-Hu syndrome do not manifest autoimmune reactions in tissues other than the nervous system, underlining the selective breakdown of tolerance to HuD, but not other Hu antigens such as HuA. OncoImmunology Volume 2 Issue 12 However, up to 40% of patients with the antiHu syndrome harbor autoantibodies specific for other neuronal proteins, such as the P/Q type voltage-gated calcium channel (associated with Lambert–Eaton myasthenic syndrome), indicating that self sensitization is not exquisitely directed to Hu antigens.43 Given the constant expression of HuD by SCLC cells, it is currently unclear why some patients develop PEM/PSN whereas others do not. Parameters intrinsic to the tumor and/ or to the host may contribute to this range of variability. In patients with SCLC and the antiHu syndrome, MHC class I molecules were detected by immunohistochemistry at moderate or high levels in the neoplastic lesions of 13 out of 15 cases, whereas such levels were observed in none of 11 SCLC patients who did not develop PEM/PSN. In addition, the focal expression of HLA-DR was more frequent in Figure 2. Immunopathogenesis of the anti-Hu syndrome. An anti-Hu syndrome occurs when tumors from SLCL patients with the anti-Hu a vigorous immune response develops against the normally neuron-restricted antigen HuD syndrome than in patients without (6/15 vs ectopically expressed by an underlying tumor, most often small-cell lung carcinoma (SCLC). 42 0/11). As a cause or consequence of this HLA Tissue-resident dendritic cells capture antigens derived from malignant cells, including HuD expression pattern, tumors from PND patients (A). After the homing of these dendritic cells to tumor-draining lymph nodes, tumor-associmay be heavily infiltrated with inflammatory ated antigens, including HuD, are processed and presented to activate antigen-specific CD4+ 70 and CD8+ T cells (B). The activation of tumor-specific T cells may also occur within neoplastic cells. However, a thorough comparison of the lesions. In either case, tumor-specific T cells can reach neoplastic lesions. As cancer cells somecellular and molecular immune signature of times express MHC class I molecules and likely present HuD-derived peptides, they become neoplastic lesions from patients who developed attractive targets for HuD-specific CD8+ T cell killing. Partial (or in rare cases complete) control or not PNDs has not yet been performed. This of the tumor growth is therefore afforded (C). In parallel, the HuD-specific T cells activated is highly relevant since the type, density, and in tumor-draining lymph nodes and/or within neoplastic lesions, circulate and acquire the capacity to cross the blood-brain-barrier (D). In the central nervous system (CNS), neurons location of tumor-infiltrating immune cells as can also express MHC class I molecules, in particular under inflammatory conditions, and can well as the expression of genes related to immutherefore present peptides derived from the highly homologous HuD, HuB, HuC proteins. nological functions carry prognostic and predicThus, neurons become additional targets for HuD-specific CD8+ T cells, resulting in neuronal 71 tive value. As mentioned earlier, either cancer tissue damage and the related paraneoplastic neurological manifestations (E). cell-specific post-translational modifications of HuD and an unusual antigen processing within neoplastic lesions could favor the development of autoimmune 15% of SCLC patients harbor low circulating titers of anti-Hu responses targeting neuronal Hu. Such mechanisms have been antibodies in the absence of neurological manifestations, HuDimplicated in tissue-specific autoimmune diseases in non-para- specific immune responses might need to exceed a minimal neoplastic contexts.68,72,73 As for host factors, the HLA-DR3 and threshold to overcome the relative immune privilege of the CNS. HLA-DQ2 have been associated with the anti-Hu syndrome in Above such a threshold, the HuD-specific immune response is one study,74 but not in a second one assessing only DR alleles.75 expected to involve the CNS, as documented by the intrathecal To date, no other genetic polymorphisms have been associated synthesis of HuD-specific IgGs,41 as well as by the detection of HuD-specific CD8 + T cells in the CSF.67 This chronic autoimwith PEM/PSN. Here is a plausible scenario for the development of an auto- mune reaction ultimately leads to neuronal destruction and fixed immune reaction against Hu proteins in patients with cancer of neurological disability (Fig. 2). The stabilization of neurological neuroectodermal origin. Although all SCLCs express HuD, the signs as well as the disappearance of anti-Hu antibodies has been cross-presentation of HuD by dendritic cells and the activation of associated with complete tumor response to therapy.7,76 These data an efficient anti-Hu T-cell response is expected to occur only in suggest that malignant cells fuel the autoimmune process through a subset of patients, based on the available T-cell repertoire and the release of tumor-associated antigens. genetic polymorphisms impacting on the magnitude and quality Mechanisms of Tissue Damage of the immune response, such as those within the HLA locus. The in the Nervous System priming of a T-cell response specific for HuD (and possibly other onconeural antigens) may be sustained within the neoplastic lesion The anti-Hu paraneoplastic syndrome is a heterogeneous disitself, owing to the ability of malignant cells to express MHC molecules, possibly resulting in the inhibition of tumor growth. Since order with highly diverse clinical and pathological manifestations www.landesbioscience.com OncoImmunology e27384-5 in terms of lesion sites, inflammatory infiltrates, and neuronal damage. Despite this heterogeneity, a good correlation exists between the location and magnitude of neuronal loss or tissue damage and the type and severity of the clinical signs. Variable regions of the nervous system can be affected, with a preferential targeting of the hippocampus, lower brain stem, spinal cord, and dorsal root ganglia (DRG).9,77,78 However, the cortex, medulla, amygdala, putamen, cingulate gyrus, and pons can also be affected.77,78 Neuropathological lesions are usually more widespread than anticipated based on clinical data. In the affected areas, neuronal loss, axonal damage, reactive gliosis, and microglial nodules are conspicuous.79 In contrast, there is no evidence of astrocyte and oligodendrocyte loss or demyelination.79 Ongoing neuronal apoptosis is either lacking or only occasionally detected, possibly a result of the subacute/chronic course of the disease or the post-mortem origin of the tissue under investigation.77,79 In patients with PEM/PSN, IgG deposits are detected in the nervous tissue, predominantly in the nuclei of CNS and DRG neurons, and to a lesser degree in the cytoplasm.10,37,80 This cellular distribution is highly reminiscent of the staining pattern observed with anti-Hu antibodies. The extraction of IgGs deposited in nervous tissues revealed that at least part of these IgGs are indeed anti-Hu antibodies. In some patients, abundant IgG deposits have been detected on the neuronal surface,37 suggesting the co-existence of additional anti-neuron autoantibodies. The amount of anti-Hu IgGs in specific areas of the nervous system is higher than its serum counterpart.80 However, the major clinical signs do not correlate with the localization of anti-Hu IgGs, as assessed by histology.78,80 Furthermore, in mice transferred with IgG from patients affected by the anti-Hu syndrome, intraneuronal IgG deposits were no longer detected if the animals were perfused before brain tissue processing.51 The question of whether the anti-Hu IgGs deposited in the neuronal tissue of patients with PEM/PSN represents an artifact or a phenomenon of potential pathogenic relevance is therefore raised. Irrespective of this issue, which requires further investigation, the complement system does not seem to play a significant role in the pathogenesis of the anti-Hu syndrome. Indeed, parenchymal C3 immunoreactivity is weak or absent and no C9neo staining (a marker of complement-mediated tissue injury) is observed in affected brain areas.37,77,79,81 Inflammatory infiltrates are commonly found in DRG as well as in the CNS perivascular space and parenchyma. They are mainly composed of T cells, whereas natural killer (NK) cells are absent.37,77,79,81 B cells are occasionally detected in the perivascular cuffs and meninges but they rarely infiltrate the brain parenchyma. In inflamed tissues, macrophages are mostly restricted to the perivascular space, whereas CD68 + microglial cells are found in the parenchyma.37,77,82,83 Among T lymphocytes, CD4 + T cells are mainly located in perivascular areas, whereas CD8 + T cells represent by far the major component of the parenchymal infiltrates.37,77,79,82,83 CD8 + T cells usually form clusters and frequently localize around neurons.77,79 Immunohistochemical analyses revealed a close contact of cytotoxic granule-containing T cells with neurons in the brain tissue or DRG of patients with the anti-Hu syndrome, with, in some instances, indentation of e27384-6 the neuronal surface by the “attacking” CD8 + T cells.83 MHC class I molecules has been shown to be expressed on neurons in the inflamed nervous tissue from patients with PEM/PSN.79,84 In this context, the close apposition of CD8 + T cells with neurons and the polarization of membranous CD107a79 may reflect a cognate, antigen-dependent, in vivo interaction leading to the neuronal demise. In addition, CD8 + T cell-derived IFNγ has recently been demonstrated to promote the loss of dendrites and synapses, underlying the need to assess presence of this cytokine (or phosphorylated signal transduction and activator of transcription 1, STAT1) in brain sections from patients affected by the anti-Hu syndrome.85 In these individuals, the amount of reactive microglia is increased in all affected areas of the CNS. Microglial cells participate in the clusters of inflammatory cells in the gray matter and, together with CD8 + T cells, surround neurons.77 Collectively, these observations strongly suggest that a cytotoxic CD8 + T cell-mediated attack contributes to neuronal death and axonal damage that characterize the anti-Hu syndrome, and therefore to its neurological manifestations. However, it remains to be determined whether these CD8 + T cells are Hu-specific. Studies of the specificity of tissue-derived TCRs should help resolve this question. To date, the analysis of TCR β chain usage by immunohistochemistry has revealed an overrepresentation of certain Vβ families in 3 out of 5 patients with PEM/PSN. Lesion-infiltrating T cells expressing the expanded Vβ families were mostly CD8 + T cells. Interestingly, in some patients, similarly expanded Vβ sequences were detected in the CSF or affected areas of the nervous system as well as within neoplastic lesions.76,86 Future Directions Future investigations to obtain better insights into the mechanisms leading to antitumor immunity and nervous tissue autoimmunity in patients with PEM/PDN should integrate the in-depth molecular analysis of tissue samples (neoplastic lesions as well as neural tissue) and the development of relevant animal models. In turn, this new knowledge should assist the development of effective immunotherapies, which are dearly needed for patients suffering from the anti-Hu syndrome. The mechanisms that trigger anti-Hu antibody-associated PNDs are not yet elucidated but appear tightly linked to the development of a partly efficient antitumor immune response. The comparison of the type, density, and location of tumorinfiltrating immune cells in SCLC patients who developed or not an anti-Hu syndrome, complemented by gene-expression profiling studies, may identify signatures that are predictive of improved tumor control and/or PND development. Confronted to data emerging from a wide variety of cancers,71 this information will help investigators to assess whether the magnitude and functional orientation of intratumoral adaptive immune responses correlate with development of the anti-Hu syndrome. Similarly, the immune reaction developing in the nervous tissue of patients with PEM/PDN needs to be dissected at the molecular level using unbiased approaches. Given the dramatic and subacute course of the neurological disease in many patients with the OncoImmunology Volume 2 Issue 12 anti-Hu syndrome, post-mortem tissues are available in which neurons are actively targeted by the immune system. A thorough investigation of the antigen specificity of CNS-infiltrating CD8 + T cells using a combination of tissue laser microdissection, TCR cloning and antigen screening76,87 will inform whether HuD is the key antigenic target for pathogenic T cells or whether alternative/additional autoantigens are involved. Animal models represent valuable tools to test hypotheses regarding the initiation and the effector mechanisms of human autoimmune diseases, as well as for the development of new therapeutic strategies. A valid animal model for the anti-Hu syndrome should recapitulate the following features: (1) the existence of one or more antigens that are expressed by both malignant cells and normal neurons; (2) the development of a detectable immune response against such shared antigens; (3) the inhibition of tumor growth by this antitumor immune response; (4) the immune infiltration of the nervous system as well as neuronal death and its related clinical consequences as a result of the antitumor/autoimmune response. To date, no animal model of the anti-Hu syndrome has been successfully developed. Attempts to mimic the human disease through the transfer of patient-derived IgGs or the deliberate immunization of experimental animals with HuD have resulted in high titers of circulating anti-HuD antibodies but no neuropathological lesions.51,88 Interestingly, a mouse strain bearing a conditional deletion of both Trp53 and Rb1 in the lung develops Hu-expressing SCLCs. Although about 15% of these mice also develop high titers of anti-Hu antibodies in the serum, no neurological signs become manifest.89 Similarly, the adoptive transfer of rat TH1 cells specific for an onconeural antigen led to meningeal and perivascular CNS inflammation but failed to induce neuronal damage or overt clinical manifestations in recipient animals.90 Therefore, a robust animal model is still needed to pinpoint the immunological mechanisms leading to neurological tissue damage in patients with the anti-Hu syndrome, and to test therapeutic strategies aimed at blocking the encephalitogenic immune response without affecting productive antitumor immunity. References 1. 2. 3. 4. Darnell RB, Posner JB. Observing the invisible: successful tumor immunity in humans. Nat Immunol 2003; 4:201; http://dx.doi.org/10.1038/ni0303-201; PMID:12605223 Seute T, Leffers P, ten Velde GP, Twijnstra A. Neurologic disorders in 432 consecutive patients with small cell lung carcinoma. Cancer 2004; 100:801-6; http://dx.doi.org/10.1002/cncr.20043; PMID:14770437 Graus F, Cordon-Cardo C, Posner JB. Neuronal antinuclear antibody in sensory neuronopathy from lung cancer. Neurology 1985; 35:538-43; PMID:2984600; http://dx.doi.org/10.1212/ WNL.35.4.538 Darnell RB, DeAngelis LM. Regression of small-cell lung carcinoma in patients with paraneoplastic neuronal antibodies. Lancet 1993; 341:21-2; PMID:8093269; http://dx.doi. org/10.1016/0140-6736(93)92485-C www.landesbioscience.com 5. 6. 7. 8. An early diagnosis of PEM/PSN is possible since the antiHu antibody provides a highly specific biomarker. The effective treatment of the underlying tumor represents the main but not the sole facet of PEM/PSN management. Indeed, a wealth of data suggests that immunotherapy should be initiated before neurological symptoms have reached their plateau. To date, various approaches have been used, ranging from high-dose corticosteroids, to intravenous immunoglobulins and cyclophosphamide, but they have provided benefit to a few patients.7,44,91 A study showed no detectable adverse effects of these immunotherapies on tumor control or patient survival, although it was underpowered to detect the negative impact of a given intervention.7 Based on the information detailed above, pointing at CD8 + T cells as key immune effectors of the neurological tissue damage experienced by patients with the anti-Hu syndrome, an early and aggressive targeting of these cells should be envisioned. Approved molecules preventing the entry of T cells into the nervous system, such as anti-α4 integrin antibodies or functional antagonists of the sphingosine-1-phosphate receptor, represent very attractive possibilities in this respect.92–94 In addition, given the deleterious role of IFNγ in CD8 + T cell-mediated neurological disorders,85,95 IFNγ blockade could also constitute a valuable strategy, provided that it does not interfere with anticancer therapy. Conversely, the blockade of immune checkpoints96 should be tested with utmost caution in patients bearing tumors potentially associated with the anti-Hu syndrome, as they may unleash a dramatic neurological autoimmunity, in particular among individuals with detectable anti-Hu antibodies. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Acknowledgments We thank Jean-Yves Delattre from ICM, Pitié-Salpetrière, Paris for the Figure 1, Daniel Gonzalez-Dunia for critical reading of the manuscript and ARC, INSERM and Région MidiPyrénées for financial support. Honnorat J, Didelot A, Karantoni E, Ville D, Ducray F, Lambert L, Deiva K, Garcia M, Pichit P, Cavillon G, et al. Autoimmune limbic encephalopathy and anti-Hu antibodies in children without cancer. Neurology 2013; 80:2226-32; http:// d x.doi.org /10.1212 / W NL .0b013e318296e9c3 ; PMID:23658383 Dalmau J, Graus F, Rosenblum MK, Posner JB. Anti-Hu--associated paraneoplastic encephalomyelitis/sensory neuronopathy. A clinical study of 71 patients. Medicine (Baltimore) 1992; 71:59-72; PMID:1312211; http://dx.doi. org/10.1097/00005792-199203000-00001 Keime-Guibert F, Graus F, Broët P, Reñé R, Molinuevo JL, Ascaso C, Delattre JY. Clinical outcome of patients with anti-Hu-associated encephalomyelitis after treatment of the tumor. Neurology 1999; 53:1719-23; PMID:10563618; http://dx.doi. org/10.1212/WNL.53.8.1719 Monstad SE, Drivsholm L, Storstein A, Aarseth JH, Haugen M, Lang B, Vincent A, Vedeler CA. Hu and voltage-gated calcium channel (VGCC) antibodies related to the prognosis of small-cell lung cancer. J Clin Oncol 2004; 22:795-800; http://dx.doi. org/10.1200/JCO.2004.01.028; PMID:14990634 OncoImmunology 9. 10. 11. 12. 13. Graus F, Keime-Guibert F, Reñe R, Benyahia B, Ribalta T, Ascaso C, Escaramis G, Delattre JY. Anti-Hu-associated paraneoplastic encephalomyelitis: analysis of 200 patients. Brain 2001; 124:113848; PMID:11353730; http://dx.doi.org/10.1093/ brain/124.6.1138 Dalmau J, Furneaux HM, Gralla RJ, Kris MG, Posner JB. Detection of the anti-Hu antibody in the serum of patients with small cell lung cancer--a quantitative western blot analysis. Ann Neurol 1990; 27:54452; http://dx.doi.org/10.1002/ana.410270515; PMID:2163235 Graus F, Dalmou J, Reñé R, Tora M, Malats N, Verschuuren JJ, Cardenal F, Viñolas N, Garcia del Muro J, Vadell C, et al. Anti-Hu antibodies in patients with small-cell lung cancer: association with complete response to therapy and improved survival. J Clin Oncol 1997; 15:2866-72; PMID:9256130 Byrne KT, Turk MJ. New perspectives on the role of vitiligo in immune responses to melanoma. Oncotarget 2011; 2:684-94; PMID:21911918 Okano HJ, Darnell RB. A hierarchy of Hu RNA binding proteins in developing and adult neurons. J Neurosci 1997; 17:3024-37; PMID:9096138 e27384-7 14. Ma WJ, Cheng S, Campbell C, Wright A, Furneaux H. Cloning and characterization of HuR, a ubiquitously expressed Elav-like protein. J Biol Chem 1996; 271:8144-51; PMID:8626503; http://dx.doi. org/10.1074/jbc.271.14.8144 15. Robinow S, Campos AR, Yao KM, White K. The elav gene product of Drosophila, required in neurons, has three RNP consensus motifs. Science 1988; 242:1570-2; PMID:3144044; http://dx.doi. org/10.1126/science.3144044 16. Robinow S, White K. Characterization and spatial distribution of the ELAV protein during Drosophila melanogaster development. J Neurobiol 1991; 22:443-61; http://dx.doi.org/10.1002/ neu.480220503; PMID:1716300 17. Chung S, Jiang L, Cheng S, Furneaux H. Purification and properties of HuD, a neuronal RNA-binding protein. J Biol Chem 1996; 271:1151824; PMID:8626712; http://dx.doi.org/10.1074/ jbc.271.19.11518 18. Bolognani F, Perrone-Bizzozero NI. RNA-protein interactions and control of mRNA stability in neurons. J Neurosci Res 2008; 86:481-9; http://dx.doi. org/10.1002/jnr.21473; PMID:17853436 19. Perrone-Bizzozero N, Bolognani F. Role of HuD and other RNA-binding proteins in neural development and plasticity. J Neurosci Res 2002; 68:1216; PMID:11948657; http://dx.doi.org/10.1002/ jnr.10175 20. Allen M, Bird C, Feng W, Liu G, Li W, PerroneBizzozero NI, Feng Y. HuD promotes BDNF expression in brain neurons via selective stabilization of the BDNF long 3’UTR mRNA. PLoS One 2013; 8:e55718; http://dx.doi.org/10.1371/journal. pone.0055718; PMID:23383270 21. Ince-Dunn G, Okano HJ, Jensen KB, Park WY, Zhong R, Ule J, Mele A, Fak JJ, Yang C, Zhang C, et al. Neuronal Elav-like (Hu) proteins regulate RNA splicing and abundance to control glutamate levels and neuronal excitability. Neuron 2012; 75:106780; http://dx.doi.org/10.1016/j.neuron.2012.07.009; PMID:22998874 22. Anderson KD, Merhege MA, Morin M, Bolognani F, Perrone-Bizzozero NI. Increased expression and localization of the RNA-binding protein HuD and GAP-43 mRNA to cytoplasmic granules in DRG neurons during nerve regeneration. Exp Neurol 2003; 183:100-8; PMID:12957493; http://dx.doi. org/10.1016/S0014-4886(03)00103-1 23. Kasashima K, Terashima K, Yamamoto K, Sakashita E, Sakamoto H. Cytoplasmic localization is required for the mammalian ELAV-like protein HuD to induce neuronal differentiation. Genes Cells 1999; 4:667-83; PMID:10620013; http://dx.doi. org/10.1046/j.1365-2443.1999.00292.x 24. Fujiwara T, Fukao A, Sasano Y, Matsuzaki H, Kikkawa U, Imataka H, Inoue K, Endo S, Sonenberg N, Thoma C, et al. Functional and direct interaction between the RNA binding protein HuD and active Akt1. Nucleic Acids Res 2012; 40:1944-53; http:// dx.doi.org/10.1093/nar/gkr979; PMID:22075994 25. Manley GT, Smitt PS, Dalmau J, Posner JB. Hu antigens: reactivity with Hu antibodies, tumor expression, and major immunogenic sites. Ann Neurol 1995; 38:102-10; http://dx.doi.org/10.1002/ ana.410380117; PMID:7541976 26. Cho JH, Noguchi M. Expression of HuD (a paraneoplastic encephalomyelitis antigen) mRNA in lung cancer. J Korean Med Sci 1997; 12:305-10; PMID:9288629 27. Carpentier AF, Voltz R, DesChamps T, Posner JB, Dalmau J, Rosenfeld MR. Absence of HuD gene mutations in paraneoplastic small cell lung cancer tissue. Neurology 1998; 50:1919; PMID:9633765; http://dx.doi.org/10.1212/WNL.50.6.1919 e27384-8 28. D’Alessandro V, Muscarella LA, la Torre A, Bisceglia M, Parrella P, Scaramuzzi G, Storlazzi CT, Trombetta D, Kok K, De Cata A, et al. Molecular analysis of the HuD gene in neuroendocrine lung cancers. Lung Cancer 2010; 67:69-75; http://dx.doi.org/10.1016/j. lungcan.2009.03.022; PMID:19410329 29. Sekido Y, Bader SA, Carbone DP, Johnson BE, Minna JD. Molecular analysis of the HuD gene encoding a paraneoplastic encephalomyelitis antigen in human lung cancer cell lines. Cancer Res 1994; 54:4988-92; PMID:8069866 30. Gazdar AF, Carney DN, Becker KL, Deftos LJ, Liang V, Go W, Marangos PJ, Moody TW, Wolfsen AR, Zweig MH. Expression of peptide and other markers in lung cancer cell lines. Recent Results Cancer Res 1985; 99:167-74; PMID:2999915; http://dx.doi. org/10.1007/978-3-642-82533-0_17 31. Graus F, Elkon KB, Cordon-Cardo C, Posner JB. Sensory neuronopathy and small cell lung cancer. Antineuronal antibody that also reacts with the tumor. Am J Med 1986; 80:45-52; PMID:2417479; http://dx.doi.org/10.1016/0002-9343(86)90047-1 32. Vega F, Graus F, Chen QM, Poisson M, Schuller E, Delattre JY. Intrathecal synthesis of the anti-Hu antibody in patients with paraneoplastic encephalomyelitis or sensory neuronopathy: clinical-immunologic correlation. Neurology 1994; 44:2145-7; PMID:7969974; http://dx.doi. org/10.1212/WNL.44.11.2145 33. Verschuuren JJP, Perquin M, ten Velde G, De Baets M, Vriesman PB, Twijnstra A. Anti-Hu antibody titre and brain metastases before and after treatment for small cell lung cancer. J Neurol Neurosurg Psychiatry 1999; 67:353-7; PMID:10449558; http://dx.doi. org/10.1136/jnnp.67.3.353 34. Blaes F, Klotz M, Funke D, Strittmatter M, Kraus J, Kaps M. Disturbance in the serum IgG subclass distribution in patients with anti-Hu positive paraneoplastic neurological syndromes. Eur J Neurol 2002; 9:369-72; PMID:12099920; http://dx.doi. org/10.1046/j.1468-1331.2002.00416.x 35. Monstad SE, Knudsen A, Salvesen HB, Aarseth JH, Vedeler CA. Onconeural antibodies in sera from patients with various types of tumours. Cancer Immunol Immunother 2009; 58:1795-800; http://dx.doi.org/10.1007/s00262-009-0690-y; PMID:19294382 36. Greenlee JE, Boyden JW, Pingree M, Brashear HR, Clawson SA, Keeney PM. Antibody types and IgG subclasses in paraneoplastic neurological syndromes. J Neurol Sci 2001; 184:131-7; PMID:11239946; http://dx.doi.org/10.1016/S0022-510X(01)00442-7 37. Jean WC, Dalmau J, Ho A, Posner JB. Analysis of the IgG subclass distribution and inflammatory infiltrates in patients with anti-Hu-associated paraneoplastic encephalomyelitis. Neurology 1994; 44:140-7; PMID:8290049; http://dx.doi.org/10.1212/ WNL.44.1.140 38. Sodeyama N, Ishida K, Jaeckle KA, Zhang L, Azuma A, Yamada M, Mizusawa H, Wada Y. Pattern of epitopic reactivity of the anti-Hu antibody on HuD with and without paraneoplastic syndrome. J Neurol Neurosurg Psychiatry 1999; 66:979; PMID:9886463; http://dx.doi.org/10.1136/ jnnp.66.1.97 39. Furneaux HF, Reich L, Posner JB. Autoantibody synthesis in the central nervous system of patients with paraneoplastic syndromes. Neurology 1990; 40:1085-91; PMID:2356009; http://dx.doi. org/10.1212/WNL.40.7.1085 40. Graus F, Vega F, Delattre JY, Bonaventura I, Reñé R, Arbaiza D, Tolosa E. Plasmapheresis and antineoplastic treatment in CNS paraneoplastic syndromes with antineuronal autoantibodies. Neurology 1992; 42:536-40; PMID:1312683; http://dx.doi. org/10.1212/WNL.42.3.536 OncoImmunology 41. Rauer S, Kaiser R. Demonstration of anti-HuD specific oligoclonal bands in the cerebrospinal fluid from patients with paraneoplastic neurological syndromes. Qualitative evidence of anti-HuD specific IgG-synthesis in the central nervous system. J Neuroimmunol 2000; 111:241-4; PMID:11063845; http://dx.doi.org/10.1016/S0165-5728(00)00391-X 42. Dalmau J, Graus F, Cheung NK, Rosenblum MK, Ho A, Cañete A, Delattre JY, Thompson SJ, Posner JB. Major histocompatibility proteins, anti-Hu antibodies, and paraneoplastic encephalomyelitis in neuroblastoma and small cell lung cancer. Cancer 1995; 75:99-109; PMID:7804984; http://dx.doi. org/10.1002/1097-0142(19950101)75:1<99::AIDCNCR2820750117>3.0.CO;2-I 43. Pittock SJ, Kryzer TJ, Lennon VA. Paraneoplastic antibodies coexist and predict cancer, not neurological syndrome. Ann Neurol 2004; 56:715-9; http:// dx.doi.org/10.1002/ana.20269; PMID:15468074 44. Sillevis Smitt P, Grefkens J, de Leeuw B, van den Bent M, van Putten W, Hooijkaas H, Vecht C. Survival and outcome in 73 anti-Hu positive patients with paraneoplastic encephalomyelitis/sensory neuronopathy. J Neurol 2002; 249:745-53; http://dx.doi. org/10.1007/s00415-002-0706-4; PMID:12111309 45. Torà M, Graus F, de Bolòs C, Real FX. Cell surface expression of paraneoplastic encephalomyelitis/sensory neuronopathy-associated Hu antigens in smallcell lung cancers and neuroblastomas. Neurology 1997; 48:735-41; PMID:9065557; http://dx.doi. org/10.1212/WNL.48.3.735 46. De Giorgio R, Bovara M, Barbara G, Canossa M, Sarnelli G, De Ponti F, Stanghellini V, Tonini M, Cappello S, Pagnotta E, et al. Anti-HuD-induced neuronal apoptosis underlying paraneoplastic gut dysmotility. Gastroenterology 2003; 125:709; PMID:12851872; http://dx.doi.org/10.1016/ S0016-5085(03)00664-4 47. Greenlee JE, Parks TN, Jaeckle KA. Type IIa (‘antiHu’) antineuronal antibodies produce destruction of rat cerebellar granule neurons in vitro. Neurology 1993; 43:2049-54; PMID:8413965; http://dx.doi. org/10.1212/WNL.43.10.2049 48. Schäfer KH, Klotz M, Mergner D, Mestres P, Schimrigk K, Blaes F. IgG-mediated cytotoxicity to myenteric plexus cultures in patients with paraneoplastic neurological syndromes. J Autoimmun 2000; 15:479-84; http://dx.doi.org/10.1006/ jaut.2000.0454; PMID:11090247 49. Hormigo A, Lieberman F. Nuclear localization of anti-Hu antibody is not associated with in vitro cytotoxicity. J Neuroimmunol 1994; 55:205-12; PMID:7829670; http://dx.doi. org/10.1016/0165-5728(94)90011-6 50. Tanaka K, Ding X, Tanaka M. Effects of antineuronal antibodies from patients with paraneoplastic neurological syndrome on primary-cultured neurons. J Neurol Sci 2004; 217:25-30; PMID:14675605; http://dx.doi.org/10.1016/j.jns.2003.08.006 51. Sillevis Smitt PA, Manley GT, Posner JB. Immunization with the paraneoplastic encephalomyelitis antigen HuD does not cause neurologic disease in mice. Neurology 1995; 45:1873-8; PMID:7477985; http://dx.doi.org/10.1212/WNL.45.10.1873 52. Uchuya M, Graus F, Vega F, Reñé R, Delattre JY. Intravenous immunoglobulin treatment in paraneoplastic neurological syndromes with antineuronal autoantibodies. J Neurol Neurosurg Psychiatry 1996; 60:388-92; PMID:8774401; http://dx.doi. org/10.1136/jnnp.60.4.388 53. Sakai K, Gofuku M, Kitagawa Y, Ogasawara T, Hirose G. Induction of anti-Purkinje cell antibodies in vivo by immunizing with a recombinant 52-kDa paraneoplastic cerebellar degeneration-associated protein. J Neuroimmunol 1995; 60:135-41; PMID:7642741; http://dx.doi.org/10.1016/0165-5728(95)00063-8 Volume 2 Issue 12 54. Benyahia B, Liblau R, Merle-Béral H, Tourani JM, Dalmau J, Delattre JY. Cell-mediated autoimmunity in paraneoplastic neurological syndromes with anti-Hu antibodies. Ann Neurol 1999; 45:162-7; PMID:9989617; http://dx.doi.org/10.1002/15318249(199902)45:2<162::AID-ANA5>3.0.CO;2-R 55. de Beukelaar JW, Sillevis Smitt PA, Hop WC, Kraan J, Hooijkaas H, Verjans GM, Gratama JW. Imbalances in circulating lymphocyte subsets in Hu antibody associated paraneoplastic neurological syndromes. Eur J Neurol 2007; 14:1383-91; http:// d x.doi.org /10.1111/j.1468 -1331.20 07.01986.x ; PMID:18028190 56. Tani T, Tanaka K, Idezuka J, Nishizawa M. Regulatory T cells in paraneoplastic neurological syndromes. J Neuroimmunol 2008; 196:166-9; http://dx.doi.org/10.1016/j.jneuroim.2008.03.002; PMID:18455243 57. Psimaras D, Carpentier AF, Rossi C; PNS Euronetwork. Cerebrospinal fluid study in paraneoplastic syndromes. J Neurol Neurosurg Psychiatry 2010; 81:42-5; http://dx.doi.org/10.1136/ jnnp.2008.159483; PMID:19324868 58. de Jongste AH, de Graaf MT, van den Broek PD, Kraan J, Smitt PA, Gratama JW. Elevated numbers of regulatory T cells, central memory T cells and classswitched B cells in cerebrospinal fluid of patients with anti-Hu antibody associated paraneoplastic neurological syndromes. J Neuroimmunol 2013; 258:85-90; http://dx.doi.org/10.1016/j.jneuroim.2013.02.006; PMID:23566401 59. Albert ML, Darnell JC, Bender A, Francisco LM, Bhardwaj N, Darnell RB. Tumor-specific killer cells in paraneoplastic cerebellar degeneration. Nat Med 1998; 4:1321-4; http://dx.doi.org/10.1038/3315; PMID:9809559 60. Santomasso BD, Roberts WK, Thomas A, Williams T, Blachère NE, Dudley ME, Houghton AN, Posner JB, Darnell RB. A T-cell receptor associated with naturally occurring human tumor immunity. Proc Natl Acad Sci U S A 2007; 104:19073-8; PMID:18045792; http://dx.doi.org/10.1073/pnas.0704336104 61. Liblau RS, Gonzalez-Dunia D, Wiendl H, Zipp F. Neurons as targets for T cells in the nervous system. Trends Neurosci 2013; 36:315-24; http://dx.doi. org/10.1016/j.tins.2013.01.008; PMID:23478065 62. de Beukelaar JW, Verjans GM, van Norden Y, Milikan JC, Kraan J, Hooijkaas H, Sintnicolaas K, Gratama JW, Sillevis Smitt PA. No evidence for circulating HuD-specific CD8+ T cells in patients with paraneoplastic neurological syndromes and Hu antibodies. Cancer Immunol Immunother 2007; 56:1501-6; http://dx.doi.org/10.1007/s00262-0070295-2; PMID:17597332 63. de Jongste AH, de Graaf MT, Martinuzzi E, van den Broek PD, Kraan J, Lamers CH, Mallone R, Gratama JW, Sillevis Smitt PA. Three sensitive assays do not provide evidence for circulating HuD-specific T cells in the blood of patients with paraneoplastic neurological syndromes with anti-Hu antibodies. Neuro Oncol 2012; 14:841-8; http://dx.doi.org/10.1093/ neuonc/nos118; PMID:22591661 64. Plonquet A, Garcia-Pons F, Fernandez E, Philippe C, Marquet J, Rouard H, Delfau-Larue MH, Kosmatopoulos K, Lemonnier F, Farcet JP, et al. Peptides derived from the onconeural HuD protein can elicit cytotoxic responses in HHD mouse and human. J Neuroimmunol 2003; 142:93-100; PMID:14512168; http://dx.doi.org/10.1016/ S0165-5728(03)00269-8 65. Rousseau A, Benyahia B, Dalmau J, Connan F, Guillet JG, Delattre JY, Choppin J. T cell response to Hu-D peptides in patients with anti-Hu syndrome. J Neurooncol 2005; 71:231-6; http://dx.doi. org/10.1007/s11060-004-1723-1; PMID:15735910 66. Tanaka M, Tanaka K. Pathogenesis and treatment of paraneoplastic neurologic syndrome. Expert Rev Neurother 2002; 2:901-9; http://dx.doi. org/10.1586/14737175.2.6.901; PMID:19810923 www.landesbioscience.com 67. Roberts WK, Deluca IJ, Thomas A, Fak J, Williams T, Buckley N, Dousmanis AG, Posner JB, Darnell RB. Patients with lung cancer and paraneoplastic Hu syndrome harbor HuD-specific type 2 CD8+ T cells. J Clin Invest 2009; 119:2042-51; http://dx.doi. org/10.1172/JCI36131; PMID:19509467 68. Meresse B, Malamut G, Cerf-Bensussan N. Celiac disease: an immunological jigsaw. Immunity 2012; 36:907-19; http://dx.doi.org/10.1016/j. immuni.2012.06.006; PMID:22749351 69. DeLuca I, Blachère NE, Santomasso B, Darnell RB. Tolerance to the neuron-specific paraneoplastic HuD antigen. PLoS One 2009; 4:e5739; http://dx.doi.org/10.1371/journal.pone.0005739; PMID:19492067 70. Rosenblum MK. Paraneoplasia and autoimmunologic injury of the nervous system: the anti-Hu syndrome. Brain Pathol 1993; 3:199-212; PMID:8293180; ht t p : //d x .doi.or g /10.1111/j.1750 -3 639.19 93. tb00747.x 71. Galon J, Angell HK, Bedognetti D, Marincola FM. The continuum of cancer immunosurveillance: prognostic, predictive, and mechanistic signatures. Immunity 2013; 39:11-26; http:// d x . d o i . o r g /10 .1016 / j . i m m u n i . 2 013 . 0 7. 0 0 8 ; PMID:23890060 72. Marrack P, Kappler JW. Do MHCII-presented neoantigens drive type 1 diabetes and other autoimmune diseases? Cold Spring Harb Perspect Med 2012; 2:a007765; http://dx.doi.org/10.1101/cshperspect. a007765; PMID:22951444 73. Sollid LM, Jabri B. Celiac disease and transglutaminase 2: a model for posttranslational modification of antigens and HLA association in the pathogenesis of autoimmune disorders. Curr Opin Immunol 2011; 23:732-8; http://dx.doi.org/10.1016/j. coi.2011.08.006; PMID:21917438 74. de Graaf MT, de Beukelaar JW, Haasnoot GW, Levering WH, Rogemond V, Didelot A, Honnorat J, Gratama JW, Smitt PA. HLA-DQ2+ individuals are susceptible to Hu-Ab associated paraneoplastic neurological syndromes. J Neuroimmunol 2010; 226:147-9; http://dx.doi.org/10.1016/j.jneuroim.2010.05.035; PMID:20547426 75. Uchuya M, Fleury A, Graus F, Costagliola D, Liblau R, Merle-Beral H, Théodorou I, Delattre JY. Lack of association between human leukocyte antigens and the anti-Hu syndrome in patients with small-cell lung cancer. Neurology 1998; 50:565-6; PMID:9484403; http://dx.doi.org/10.1212/WNL.50.2.565 76. Pellkofer HL, Voltz R, Goebels N, Hohlfeld R, Dornmair K. Cross-reactive T-cell receptors in tumor and paraneoplastic target tissue. Arch Neurol 2009; 66:655-8; http://dx.doi.org/10.1001/archneurol.2009.56; PMID:19433667 77. Bernal F, Graus F, Pifarré A, Saiz A, Benyahia B, Ribalta T. Immunohistochemical analysis of antiHu-associated paraneoplastic encephalomyelitis. Acta Neuropathol 2002; 103:509-15; http://dx.doi. org/10.1007/s00401-001-0498-0; PMID:11935268 78. Dalmau J, Posner JB. Neurologic paraneoplastic antibodies (anti-Yo; anti-Hu; anti-Ri): the case for a nomenclature based on antibody and antigen specificity. Neurology 1994; 44:2241-6; PMID:7991105; http://dx.doi.org/10.1212/WNL.44.12.2241 79. Bien CG, Vincent A, Barnett MH, Becker AJ, Blümcke I, Graus F, Jellinger KA, Reuss DE, Ribalta T, Schlegel J, et al. Immunopathology of autoantibody-associated encephalitides: clues for pathogenesis. Brain 2012; 135:1622-38; http://dx.doi. org/10.1093/brain/aws082; PMID:22539258 80. Dalmau J, Furneaux HM, Rosenblum MK, Graus F, Posner JB. Detection of the anti-Hu antibody in specific regions of the nervous system and tumor from patients with paraneoplastic encephalomyelitis/ sensory neuronopathy. Neurology 1991; 41:175764; PMID:1944905; http://dx.doi.org/10.1212/ WNL.41.11.1757 OncoImmunology 81. Graus F, Campo E, Cruz-Sanchez F, Ribalta T, Palacin A. Expression of lymphocyte, macrophage and class I and II major histocompatibility complex antigens in normal human dorsal root ganglia. J Neurol Sci 1990; 98:203-11; PMID:1700807; http:// dx.doi.org/10.1016/0022-510X(90)90261-K 82. Antoine JC, Mosnier JF, Honnorat J, Convers P, Absi L, Lapras J, Michel D. Paraneoplastic demyelinating neuropathy, subacute sensory neuropathy, and anti-Hu antibodies: clinicopathological study of an autopsy case. Muscle Nerve 1998; 21:8507; PMID:9626244; http://dx.doi.org/10.1002/ ( S I C I ) 10 9 7- 459 8 ( 19 9 8 0 7 ) 21 : 7< 85 0 : : A I D MUS2>3.0.CO;2-5 83. Wanschitz J, Hainfellner JA, Kristoferitsch W, Drlicek M, Budka H. Ganglionitis in paraneoplastic subacute sensory neuronopathy: a morphologic study. Neurology 1997; 49:1156-9; PMID:9339709; http:// dx.doi.org/10.1212/WNL.49.4.1156 84. Plonquet A, Gherardi RK, Créange A, Antoine JC, Benyahia B, Grisold W, Drlicek M, Dreyfus P, Honnorat J, Khouatra C, et al. Oligoclonal T-cells in blood and target tissues of patients with antiHu syndrome. J Neuroimmunol 2002; 122:1005; PMID:11777548; http://dx.doi.org/10.1016/ S0165-5728(01)00452-0 85. Kreutzfeldt M, Bergthaler A, Fernandez M, Brück W, Steinbach K, Vorm M, Coras R, Blümcke I, Bonilla WV, Fleige A, et al. Neuroprotective intervention by interferon-γ blockade prevents CD8+ T cell-mediated dendrite and synapse loss. J Exp Med 2013; 210:2087-103; http://dx.doi.org/10.1084/ jem.20122143; PMID:23999498 86. Voltz R, Dalmau J, Posner JB, Rosenfeld MR. T-cell receptor analysis in anti-Hu associated paraneoplastic encephalomyelitis. Neurology 1998; 51:114650; PMID:9781545; http://dx.doi.org/10.1212/ WNL.51.4.1146 87. Siewert K, Malotka J, Kawakami N, Wekerle H, Hohlfeld R, Dornmair K. Unbiased identification of target antigens of CD8+ T cells with combinatorial libraries coding for short peptides. Nat Med 2012; 18:824-8; http://dx.doi.org/10.1038/nm.2720; PMID:22484809 88. Carpentier AF, Rosenfeld MR, Delattre JY, Whalen RG, Posner JB, Dalmau J. DNA vaccination with HuD inhibits growth of a neuroblastoma in mice. Clin Cancer Res 1998; 4:2819-24; PMID:9829748 89. Kazarian M, Calbo J, Proost N, Carpenter CL, Berns A, Laird-Offringa IA. Immune response in lung cancer mouse model mimics human antiHu reactivity. J Neuroimmunol 2009; 217:38-45; http://dx.doi.org/10.1016/j.jneuroim.2009.08.014; PMID:19765830 90. Pellkofer H, Schubart AS, Höftberger R, Schutze N, Pagany M, Schüller M, Lassmann H, Hohlfeld R, Voltz R, Linington C. Modelling paraneoplastic CNS disease: T-cells specific for the onconeuronal antigen PNMA1 mediate autoimmune encephalomyelitis in the rat. Brain 2004; 127:1822-30; http://dx.doi. org/10.1093/brain/awh205; PMID:15201193 91. Oh SJ, Dropcho EJ, Claussen GC. Anti-Hu-associated paraneoplastic sensory neuropathy responding to early aggressive immunotherapy: report of two cases and review of literature. Muscle Nerve 1997; 20:157682; PMID:9390671; http://dx.doi.org/10.1002/ ( SIC I )1097- 4598 (199712 ) 2 0 :12 <1576 : : A I D MUS13>3.0.CO;2-Z 92. Brinkmann V, Billich A, Baumruker T, Heining P, Schmouder R, Francis G, Aradhye S, Burtin P. Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis. Nat Rev Drug Discov 2010; 9:883-97; PMID:21031003; http:// dx.doi.org/10.1038/nrd3248 e27384-9 93. Ifergan I, Kebir H, Alvarez JI, Marceau G, Bernard M, Bourbonnière L, Poirier J, Duquette P, Talbot PJ, Arbour N, et al. Central nervous system recruitment of effector memory CD8+ T lymphocytes during neuroinflammation is dependent on α4 integrin. Brain 2011; 134:3560-77; http://dx.doi.org/10.1093/ brain/awr268; PMID:22058139 94. Planas R, Jelčić I, Schippling S, Martin R, Sospedra M. Natalizumab treatment perturbs memory- and marginal zone-like B-cell homing in secondary lymphoid organs in multiple sclerosis. Eur J Immunol 2012; 42:790-8; http://dx.doi.org/10.1002/ eji.201142108; PMID:22144343 e27384-10 95. Scheikl T, Pignolet B, Dalard C, Desbois S, Raison D, Yamazaki M, Saoudi A, Bauer J, Lassmann H, Hardin-Pouzet H, et al. Cutting edge: neuronal recognition by CD8 T cells elicits central diabetes insipidus. J Immunol 2012; 188:4731-5; http://dx.doi. org/10.4049/jimmunol.1102998; PMID:22504649 96. Riley JL. Combination checkpoint blockade--taking melanoma immunotherapy to the next level. N Engl J Med 2013; 369:187-9; http://dx.doi.org/10.1056/ NEJMe1305484; PMID:23724866 OncoImmunology Volume 2 Issue 12 ! "" !)( &! !( # !%( ( ! "( !( ("!!(!!! " " """$(,*+- +-- 3302 Guillaume Martin-Blondel et al. DOI: 10.1002/eji.201545632 Eur. J. Immunol. 2015. 45: 3302–3312 Migration of encephalitogenic CD8 T cells into the central nervous system is dependent on the α4β1-integrin Guillaume Martin-Blondel ∗1,2,3 , Béatrice Pignolet ∗2,3,5 , Silvia Tietz4 , Lidia Yshii2,3 , Christina Gebauer2,3 , Therese Perinat4 , Isabelle Van Weddingen2 , Claudia Blatti4 , Britta Engelhardt ∗∗4 and Roland Liblau ∗∗2,3 1 Department of Infectious and Tropical Diseases, Toulouse University Hospital, Toulouse, France 2 INSERM U1043 - CNRS UMR 5282, Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse, France 3 Université Toulouse III, Toulouse, France 4 Theodor Kocher Institute, University of Bern, Bern, Switzerland 5 Department of Clinical Neurosciences, Toulouse University Hospital, Toulouse, France Although CD8 T cells are key players in neuroinflammation, little is known about their trafficking cues into the central nervous system (CNS). We used a murine model of CNS autoimmunity to define the molecules involved in cytotoxic CD8 T-cell migration into the CNS. Using a panel of mAbs, we here show that the α4β1-integrin is essential for CD8 T-cell interaction with CNS endothelium. We also investigated which α4β1-integrin ligands expressed by endothelial cells are implicated. The blockade of VCAM-1 did not protect against autoimmune encephalomyelitis, and only partly decreased the CD8+ T-cell infiltration into the CNS. In addition, inhibition of junctional adhesion molecule-B expressed by CNS endothelial cells also decreases CD8 T-cell infiltration. CD8 T cells may use additional and possibly unidentified adhesion molecules to gain access to the CNS. Keywords: α4β1-Integrin r CD8 T cell r Junctional adhesion molecule-B r Migration r Neuroinflammation Additional supporting information may be found in the online version of this article at the publisher’s web-site Introduction The central nervous system (CNS) is considered as a unique immune-privileged environment allowing a basal immune surveillance under physiological conditions, and restraining potentially deleterious inflammatory reactions in disease states [1, 2]. A tight control of the trafficking of immune cells toward the CNS is a Correspondence: Dr. Roland Liblau e-mail: [email protected] C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim major parameter contributing to this immune-privileged status [3]. Indeed, circulating immune cells have to cross protective barriers using a complex multistep cascade that involves distinct trafficking cues both at the surface of the CNS endothelial cells and of immune cells [4]. The current knowledge regarding T-cell migration into the CNS derives mainly from CD4 T cells whereas little is known for CD8 ∗ These authors contributed equally to this work. These authors are co-principal investigators of this work. ∗∗ www.eji-journal.eu Eur. J. Immunol. 2015. 45: 3302–3312 T cells. Under inflammatory conditions, either due to autoimmune responses or infection, the inflamed blood-brain barrier (BBB) endothelium upregulates the expression of adhesion molecules (selectins and cell adhesion molecules of the immunoglobulin superfamily). Several surface molecules expressed by CD4 T cells, such as P-selectin glycoprotein ligand-1, αLβ2, α4β1, CD6, or α6β1, regulate sequential steps for transmigration that include tethering, rolling, capture, firm adhesion, and finally diapedesis of bloodborne circulating T cells across the endothelial layer [4]. Antigen recognition in the subarachnoidal spaces then allow T cells to migrate through the glia limitans superficialis into the CNS parenchyma, leading to clinical disease [5]. In this scenario, a pivotal role for the interaction between the α4β1-integrin heterodimer expressed by activated T helper 1 CD4 cells, and vascular cell adhesion protein 1 (VCAM-1), its main ligand on BBB endothelial cells, has been demonstrated [6–10]. However, as Th17 CD4 cells were shown to infiltrate the brain in the absence of α4integrin in an αLβ2-dependent manner [10, 11], α4-independent routes to the CNS exist for other crucial T-cell subsets. Although cumulative evidence points to a key role for CD8 T cells in several inflammatory CNS disorders such as MS, autoimmune encephalitis, or immune reconstitution inflammatory syndrome (IRIS) affecting the CNS [12–14], little is known about trafficking of CD8 T cells into the CNS. Interaction between P-selectin glycoprotein ligand-1 and P-selectin contributes to the recruitment of human CD8 T cells in brain vessels [15]. CD8 T-cell migration is not affected by blocking interactions between αLβ2/ICAM-1, ALCAM/CD6, PECAM-1/PECAM-1, or CCL2/CCR2, while all of them were previously shown to partake in the recruitment of other immune cell populations to the CNS [16, 17]. The conflicting results about neutralization of α4-integrin in EAE and in coronavirus-induced encephalitis, restricting or not CD8 T-cell infiltration of the CNS, suggest that these cells may use alternative and possibly unidentified adhesion molecules to gain access to the CNS, raising the potential for selective control of their trafficking into the brain [10, 17, 18]. Using a murine model of CNS autoimmunity, the aim of our study was to define the role of α4-integrins in the CD8 T-cell migration across the BBB during neuroinflammation, and to specify which of the heterodimers containing the α4-integrin subunit, namely α4β1 and α4β7, is implicated. Results α4β1-Integrin blockade protects CamK-HA mice from developing CD8 T-cell-mediated encephalomyelitis CamK-HA mice selectively express the Haemagglutinin (HA) of the Influenza virus in neurons under the control of the calmodulin kinase promoter. These mice, but not control littermates, exhibited severe encephalomyelitis characterized by weight loss, neurological signs, and death in 40–50% of recipients after the adoptive transfer of 5 × 106 in vitro generated cytotoxic HA-specific CD8 T cells. Diabetes insipidus caused by the CD8-mediated C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Cellular immune response destruction of arginine-vasopressin hypothalamic neurons was a prominent clinical manifestation in the surviving CamK-HA recipients [19]. Endogenous CD4 and CD8 T cells also infiltrated the CNS of CamK-HA mice developing encephalomyelitis (Supporting Information Fig. 1A–B). However, because CD4 T cell-depleted recipients underwent weight loss and diabetes insipidus, endogenous CD4 T cells are not required for disease development (Supporting Information Fig. 1C–D). To delineate in this model the role of α4-integrin in CD8 T-cell migration to the CNS, we first showed that in vitro differentiated cytotoxic HA-specific CD8 T cells express both α4- and β7-integrin subunits, and the α4β7-integrin heterodimer (Fig. 1A). Because there is no mAb against the α4β1-integrin heterodimer, we used an in vitro binding assay to indirectly show that in vitro differentiated cytotoxic HA-specific CD8 T cells express the α4β1-integrin heterodimer. While HA-specific CD8 T cells untreated or treated with a control IgG bind to recombinant ICAM-1 and VCAM-1, an anti-α4-integrin mAb completely inhibited binding to VCAM-1 (one-way ANOVA, p < 0.00001), but did not affect binding to ICAM-1 (Fig. 1B). As VCAM-1 is the main ligand of the α4β1integrin heterodimer, this suggests that HA-specific CD8 T cells express the functional α4β1-integrin heterodimer. We then assessed whether treatment of CamK-HA mice with anti-α4-integrin mAb could protect against CD8 T-cell-mediated encephalomyelitis. Therefore, we treated CamK-HA recipients with an anti-α4-integrin or a control IgG at day 0, 4, and 8 after the adoptive transfer of 5 × 106 cytotoxic HA-specific CD8 T cells. Compared to recipients receiving the control IgG, CamK-HA recipients treated by anti-α4-integrin mAb lost less weight (one-way ANOVA, p < 0.0001) and showed no death (0/10 versus 5/10, Log-rank test, p = 0.006). In addition, survivors experienced less diabetes insipidus (2/10 versus 4/5) (Fig. 1C–E). Since α4-integrin is a subunit shared by both α4β1- and α4β7-integrin heterodimers, we then investigated which one is involved in CD8 T-cell migration into the CNS. Currently no mAb is available that specifically recognizes the α4β1-integrin heterodimer, while the DATK32 mAb recognizes and blocks specifically the α4β7-integrin heterodimer [7]. We therefore treated CamK-HA recipients with this anti-α4β7integrin mAb. Recipients underwent the same clinical phenotype as mice receiving the control mAb. Indirectly, this strongly suggests that the α4β1-integrin is the essential heterodimer for activated CD8 T-cell migration into the CNS in our model. However, α4-integrin blockade did not fully abrogate the neurological signs induced by HA-specific CD8 T-cell transfer in CamK-HA mice as significant weight loss developed when compared to control littermates (Fig. 1C), and central diabetes insipidus was detected in 20% of treated recipients. Blockade of the α4β1-integrin reduces CD8 T-cell infiltration and microglial activation in the CNS We next investigated the impact of α4β1-integrin blockade on CNS inflammatory infiltrate composition and tissue damage. Eight days after the adoptive transfer of 5 × 106 cytotoxic HA-specific CD8 www.eji-journal.eu 3303 3304 Guillaume Martin-Blondel et al. Eur. J. Immunol. 2015. 45: 3302–3312 Figure 1. α4β1-integrin blockade protects CamK-HA mice from developing CD8 T-cellmediated encephalomyelitis. (A) FACS analysis of α4-integrin subunit, β7-integrin subunit, and α4β7-integrin heterodimer expression on in vitro differentiated cytotoxic HA-specific CD8 T cells (one experiment representative of three experiments of in vitro differentiation of HAspecific cytotoxic CD8 T cells).(B) In vitro binding assay of cytotoxic HA-specific CD8 T cells to recombinant ICAM-1 or VCAM-1 IgG and a control protein (DNER-IgG), after pretreatment with isotype control rat IgG2b or antiα4-integrin-IgG. Pooled data from three independent experiments, each point representing the mean of triplicates. One-way ANOVA ****p < 0.00001. (C and D) Adoptive transfer at day 0 of 5 × 106 cytotoxic HA-specific CD8 T cells in nontransgenic mice or in CamK-HA mice treated at day 0, 4, and 8 by an anti-α4, an anti-α4β7, or a control IgG mAb. Impact on the weight (C) and on survival (D) of the recipient mice is shown. (C) One-way ANOVA ***p < 0.0001. (D) Log-rank (Mantel–Cox) test *p < 0.01. (E) Incidence of diabetes insipidus among survivors at day 24 after the adoptive transfer of 5 × 106 cytotoxic HAspecific CD8 T cells in nontransgenic mice or in CamK-HA mice treated at day 0, 4, and 8 by an anti-α4, an anti-α4β7, or a control IgG mAb. Diabetes insipidus is defined using dipsticks by a urine-specific gravity ࣘ 1010 on three consecutive days. (C), (D), and (E) are pooled data from three independent experiments, each involving three to five mice per group. Data represent mean ± SEM. T cells, CamK-HA recipients treated with an anti-α4-integrin mAb or a control IgG were sacrificed and their CNS-infiltrating cells were analyzed by flow cytometry. A drastic reduction in the number of CD45high Thy1.2+ T cells infiltrating the CNS was observed in recipients treated with the anti-α4-integrin mAb (1333 ± 263 versus 35 452 ± 5748 cells/CNS, respectively, Mann–Whitney test, p < 0.0001; Fig. 2A–B). Ex vivo analysis of splenocytes showed that the anti-α4-integrin mAb did not deplete CD8 T cells in the periphery, and even promoted the accumulation of transferred cells in the secondary lymphoid organs (Supporting Information Fig. 2A–B). Moreover, ex vivo analyses and restimulation with the cognate HA peptide showed that the anti-α4-integrin mAb also did not affect activation of transferred CD8 T cells collected from spleen and lymph nodes (Supporting Information Fig. 2C–F). In the CNS, we observed a decrease in microglial activation, evaluated by MHC class II expression on CD45int CD11b+ cells, in C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim recipients treated by the anti-α4-integrin mAb (MFI for MHC class II 589 ± 127 versus 6825 ± 1172, Mann–Whitney test, p = 0.0002; Fig. 2C–D). Histological analysis also corroborated the marked reduction in the parenchymal CD8 T-cell infiltration in recipients treated by the anti-α4-integrin mAb-treated animals (Mann– Whitney test, p = 0.0002; Fig. 2E–G), while some meningeal CD8 T cells could still be detected. Antagonization of α4β1-integrin, therefore inhibits CD8 T-cell infiltration and microglial activation in the CNS parenchyma of recipient CamK-HA mice. VCAM-1 blockade retains the clinical phenotype and only partly decreases CD8 T cell CNS infiltration To further investigate how α4β1-integrin contributes to CD8 T cell trafficking to the CNS, we investigated which ligand expressed www.eji-journal.eu Eur. J. Immunol. 2015. 45: 3302–3312 Cellular immune response Figure 2. Blockade of the α4β1-integrin reduces T-cell infiltration and microglial activation in the CNS. (A–D) FACS analysis of CNS-infiltrating cells at day 8 after adoptive transfer of 5 × 106 cytotoxic HA-specific CD8 T cells in CamK-HA mice treated at day 0 and 4 by an anti-α4 or a control IgG mAb. (A) Illustration of the gating strategy. (B) Absolute number of CD45high Thy1.2+ T cells infiltrating the CNS. Each dot shows an individual animal. Mann–Whitney test *** p < 0.0001. (C) Overlay of MHC class II expression on CD45int CD11b+ cells in mice treated by a control IgG (black line), or anti-α4 mAb (red line). One representative out of three independent experiments is shown. (D) The MFI of MHC class II expression on CD45int CD11b+ microglial cells is shown. Mann–Whitney test *** p = 0.0002. Each dot shows an individual animal. (B and D) are pooled data from three independent experiments, each involving three to five mice per group. Data represent mean ± SEM. (E–G) Brain CD8 T-cell infiltration at day 8 after adoptive transfer of 5 × 106 cytotoxic HA-specific CD8 T cells in CamK-HA mice treated at day 0 and 4 by a control IgG (E) or an anti-α4-integrin mAb (F; bar = 50 μm). (G) Histological scoring for parenchymal CD8 T-cell infiltration. Pool of three independent experiments, each involving three to five mice per group. Mann–Whitney test *** p = 0.0002. Data represent mean ± SEM. on brain microvascular endothelial cells may interact with α4β1integrin. First, we blocked in vivo VCAM-1, the major ligand for α4β1-integrin [20], using two distinct mAbs. As both mAbs provided identical results (Fig. 3A), the two groups were pooled in Figure 3B and C. CamK-HA recipients treated by anti-VCAM-1 mAb lost more weight (one-way ANOVA, p < 0.0001) and developed diabetes insipidus more frequently (9/11 versus 1/6, chi-square test with Yates’ correction, p = 0.036) compared to mice treated by the anti-α4-integrin mAb (Fig. 3A–C). Clinical outcomes (weight loss, diabetes insipidus, and death) were not significantly different between recipient mice treated with the anti-VCAM-1 mAbs or with the control IgG. To assess the impact of VCAM-1 inhibition on CD8 T-cell infiltration of the CNS, and to unambiguously identify and enumerate the transferred cells, CD45.1+ HA-specific CD8 T cells were C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim injected in CD45.2+ CamK-HA mice (Fig. 3D). Flow cytometry analysis of CNS-infiltrating cells from CamK-HA recipients 8 days after the adoptive transfer revealed a significant reduction in the number of transferred CD45.1+ CD8 T cells in mice treated with anti-VCAM-1 mAbs compared to mice receiving the control IgG (7990 ± 1862 versus 14 331 ± 1582 cells, Mann–Whitney test, p = 0.01; Fig. 3E). However, VCAM-1 blockade reduced the number of CNS-infiltrating transferred CD8 T cells to a much lesser extent than the use of anti-α4-integrin mAb (161 ± 48 cells, Mann– Whitney test, p < 0.0001). Collectively, these data indicate that blockade of VCAM-1 partially inhibits the α4β1-dependent migration of CD8 T cells across the BBB, but does not protect mice from developing CNS tissue damage, suggesting that additional vascular ligands of α4β1-integrin might be implicated in CD8 T cell trafficking into the CNS. www.eji-journal.eu 3305 3306 Guillaume Martin-Blondel et al. Eur. J. Immunol. 2015. 45: 3302–3312 Figure 3. VCAM-1 blockade retains the clinical phenotype and only partly decreases CD8 T cell CNS infiltration. (A–C) Adoptive transfer of 5 × 106 cytotoxic HA-specific CD8 T cells in CamK-HA mice treated at day 0, 4, and 8 by an anti-α4-integrin, a control IgG, anti-VCAM-1 (clone 429 or 6C7.1), or an anti-JAM-B mAb. Impact on the weight (A), survival (B), and on development of diabetes insipidus (C). Pool of two independent experiments, each involving three to six mice per group. (A) One-way ANOVA ***p < 0.0001. (B) Log-rank (Mantel–Cox) test, ns. Data represent mean ± SEM. (D–F) FACS analysis of CNS-infiltrating cells at day 8 after adoptive transfer of 5 × 106 CD45.1+ cytotoxic HA-specific CD8 T cells in CD45.2+ CamK-HA mice treated at day 0 and 4 by a control IgG, an anti-α4-integrin, an anti-VCAM-1 (clone 6C7.1), an anti-JAM-B, or both an anti-VCAM-1 and anti-JAM-B mAbs. (D) Illustration of the general gating strategy. (E) Absolute number of CD45.1+ CD8+ T cells infiltrating the CNS. Pool of two to three independent experiments using three to five mice per group. Mann–Whitney test, *p < 0.05, **p < 0.001, ***p < 0.0001. Data represent mean ± SEM. (F) Percentage of CD45. 1+ CD8+ T cells that are CD44+ CD62L− and that produce IFN-γ and TNF-α. Pool of two independent experiments using two to three mice per group. One-way ANOVA, ns. Data represent mean ± SEM. JAM-B is a ligand for α4β1-integrin implicated in CD8 T-cell migration to the CNS Because junctional adhesion molecule-B (JAM-B) had been reported to be a ligand for α4β1-integrin on endothelial cells [21], we assessed the contribution of JAM-B as another putative BBB endothelial ligand for α4β1-integrin in CD8 T-cell migration into the CNS. Indeed, CNS microvascular endothelial cells express JAM-B in addition to PECAM-1 (Fig. 4). We investigated whether JAM-B could be potential ligand for α4β1-integrin expressed on CD8 T cells. We treated CamK-HA recipients with an anti-JAM-B mAb at day 0, 4, and 8 after the adoptive transfer of 5 × 106 cytotoxic HA-specific CD8 T cells. While CamK-HA recipients treated by the anti-JAM-B mAb displayed significantly less weight loss compared to recipients treated by control IgG or anti-VCAM-1 C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim mAbs, they displayed the same incidence of diabetes insipidus and death (Fig. 3A–C). Flow cytometry analysis of the CNS of CamKHA recipients treated by anti-JAM-B mAb showed a significant decrease in the number of infiltrating CD45.1+ CD8 transferred cells compared to mice receiving the control IgG (7016 ± 2029 versus 14 331 ± 1582 cells/CNS, Mann–Whitney test, p = 0.017; Fig. 3E). This decrease was similar to that achieved when blocking VCAM-1 (Mann–Whitney test, ns), but clearly inferior to the inhibition achieved by the anti-α4-integrin mAb (Mann–Whitney test, p = 0.0017; Fig. 3E). Increased dose of anti-JAM-B mAb did not improve the reduction of CNS-infiltrating CD45.1+ CD8+ transferred cells, suggesting that saturating concentrations of the antibody were obtained (Supporting Information Fig. 3). Therefore, JAM-B expressed by BBB endothelial cells might represent a ligand for α4β1-integrin-mediated migration of CD8 T cells into www.eji-journal.eu Eur. J. Immunol. 2015. 45: 3302–3312 Cellular immune response Figure 4. CNS microvascular endothelial cells express JAM-B in addition to PECAM-1. (A–C) Immunofluorescence staining for JAM-B in the mouse CNS was evaluated on cryosections (6 μm) of brains of CamK-HA mice day 8 after adoptive transfer of 5 × 106 cytotoxic HA-specific CD8 T cells. Double immunofluorescence staining of JAM-B (A) and PECAM-1 (B) show colocalization of JAM-B and PECAM-1 staining (C) on all vessels in the mouse cortex. Arrowheads point to representable JAM-B positive (A) and JAM-B/PECAM-1 double positive vessels (C). (D–F) Double immunofluorescence staining of JAM-B (D, arrowheads) and VCAM-1 (E) show a colocalization of JAM-B and VCAM-1 (F) on blood vessels (arrowhead), however JAM-B immune staining can additionally be detected on endothelial cells staining negative for VCAM-1. (F) Arrowheads with stars point to JAM-B positive vessels that do not stain positive for VCAM-1. (G–I) Isotype control staining with rabbit IgG control antibody (G) and PECAM-1 (H) shows no staining for the rabbit IgG (G and I). The variability in background with the anti-JAM-B antibody observed in (A), (D), and (J) is related to the area in the CNS and the inflammatory status of the brain. All sections were counter-stained with DAPI to show cell nuclei. Bars are 50 μm. Panel shown above constitutes one representative staining; in total three individual tissues were processed. the CNS. In order to test the hypothesis that VCAM-1 and JAM-B are complementary ligands for α4β1-integrin, we treated recipients with both anti-JAM-B and anti-VCAM-1 mAbs. Coadministration of both mAbs did not demonstrate a synergistic reduction in the number of CNS-infiltrated CD45.1+ CD8 transferred T cells (Fig. 3E). We finally investigated the phenotype of CD8 T cells that entered the CNS of CamK-HA recipients after blockade of α4integrin, α4β7-integrin, VCAM-1, or JAM-B. Whereas their absolute numbers differed importantly between groups, the proportion of transferred CD45.1+ CD8 T cells that are CD44+ CD62L− , and that produce both IFN-γ and TNF-α did not significantly differ between groups (Fig. 3F). C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Discussion Molecular cues used by CD8 T cells to migrate into the CNS are poorly defined. Using a murine model of CNS autoimmune neuroinflammation, we showed that the migration of cytotoxic CD8 T cells to the CNS relies on the α4β1-integrin heterodimer. We also showed that VCAM-1 and JAM-B expressed by BBB endothelial cells are likely implicated in this process, but that their inhibition is insufficient to completely block CD8 T-cell infiltration into the CNS or to mitigate the clinical encephalomyelitis signs. Two studies using animal models of neuroinflammation and in vitro transmigration assays demonstrated that blocking the www.eji-journal.eu 3307 3308 Guillaume Martin-Blondel et al. α4-integrin decreases migration of CD8 T cells across CNS vascular structures [17, 18]. However, because the α4-integrin is common to α4β1- and α4β7-integrin heterodimers [22], it is unknown which one is implicated. Our model showed that blockade of α4-integrin, but not of the α4β7-integrin heterodimer, prevents encephalomyelitis and tissue damage by interfering with cytotoxic CD8 T-cell entry into the CNS. These results strongly suggest that the α4β1-integrin is the essential heterodimer for the interaction of activated CD8 T cells with the BBB. Similarly, blocking α4-integrin can abrogate the development of EAE mediated by encephalitogenic CD4 Th1 cells [7–10]. Those cells critically rely on β1integrin to accumulate in the CNS during EAE [6]. Moreover, blocking the α4β7-integrin heterodimer fails to inhibit EAE development in mice and Rhesus monkeys [7, 23], and ectopic expression of the α4β7-integrin ligand MAdCAM-1 in CNS endothelial cells fails to trigger CNS recruitment of α4β7-expressing T cells [24]. Thus, α4β1-integrin rather than α4β7-integrin mediates both CD4 and CD8 T-cell interaction with CNS endothelium. However, as α4-integrin blockade did not totally abrogate clinical signs and CNS infiltration of transferred CD8 T cells in our model, an α4β1-independent route, although accessory, remains operative. Its molecular bases are still to be determined. The most studied brain endothelial ligand for α4β1-integrin is VCAM-1 [20]. VCAM-1 is constitutively expressed at low level on the BBB endothelium, and mediates both the initial capture and the subsequent G-protein-dependent arrest of CD4 Th1 cells upon interaction with the α4β1-integrin [8]. Expression of VCAM-1 is upregulated in inflammatory conditions on endothelial cells of the BBB and of the blood-leptomeningeal barrier, as well as on choroid plexus epithelium of the blood-cerebrospinal fluid barrier, further increasing α4β1/VCAM-1-mediated T-cell adhesion to inflamed CNS endothelium [25, 26]. It was previously shown that VCAM-1 blockade inhibits clinical or histopathological signs of EAE mediated by encephalitogenic CD4 Th1 cells [7]. In a model of OVA-expressing Listeria monocytogenes infection, access of OVA-specific CD8 T cells to the CNS was inhibited by the antibody-mediated blockade of VCAM-1 to the same extent as did the α4-integrin blockade [18]. Unexpectedly, blockade of VCAM1 in our model only had a partial effect on CD8 T-cell migration to the CNS, as the number of CNS-infiltrated CD8 T cells was reduced by 46% compared to mice treated by the control IgG (Fig. 3D). This decrease was not clinically relevant as recipients treated with different clones of anti-VCAM-1 mAb experienced weight loss, death, and diabetes insipidus to the same extent as mice treated by the control IgG. Therefore, VCAM-1 blockade was not sufficient in our model to inhibit α4β1-expressing CD8 T-cells migration into the CNS. Several differences between our study and that of Young et al. [18] might explain those discordant results, including the genetic background of the mice, BALB/c or (BALB/c × C57BL6) F1 versus C57BL6/J, the localization of the HA and OVA antigens in different cells, neurons and oligodendrocytes, respectively, and the state of differentiation of the encephalitogenic CD8 T cells (terminally differentiated highly cytotoxic versus shortly activated CD8 T cells). Moreover, the L. monocytogenes infection within the CNS in this model might induce additional C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Immunol. 2015. 45: 3302–3312 triggers including increased expression of VCAM-1 at the BBB, allowing for increased VCAM-1-dependence of CD8 T-cell migration into the CNS. It was suggested that natalizumab, a humanized mAb against the human α4 subunit of α4β1-integrin and α4β7, might also affect CD4 and CD8 T-cell expression of α4-integrinunrelated molecules such as CD62L, CXCR3, and αLβ2 in longterm treated natalizumab patients [27, 28]. Here, we have used a very short treatment. In addition, the high specificity of PS/2 for the murine α4-integrin subunit makes this antibody unlikely to cross-react with other integrin subunit or unrelated molecules such as selectins. Indeed, PS/2 recognizes the functional epitope B2 on the α4-integrin subunit and was shown to specifically block α4β1-mediated lymphocyte binding to VCAM-1 and fibronectin as well as α4β7-dependent binding to VCAM-1 and MAdCAM-1 in vitro [29, 30]. Another explanation could be that, in our model, VCAM-1 is not the sole, or even not the main, ligand for α4β1integrin. In that respect, we investigated if other known α4β1-integrin ligands might be implicated. At least two additional vascular ligands have been described, the CS1 domain of a spliced variant of fibronectin [31, 32], and JAM-B [21, 33]. Since no reliable blocking antibody against CS1 domain is currently available, the role of fibronectin in the migration process of CD8 T cells could not be explored yet. Because α4β1–JAM-B interaction has only been investigated in vitro or in vivo in skin microvasculature [21], and because JAM-B is expressed by the BBB in our model, we focused on this putative ligand for CD8 T-cell migration to the CNS. Members of the classical JAM family are transmembrane type 1 proteins with two immunoglobulin domains highly expressed in cells that present well organized tight junctions, such as the endothelium of the BBB [34]. JAM-A, interacting with αLβ2-integrin, is implicated in cell migration to the CNS. A blocking mAb directed to JAM-A significantly inhibited leukocyte infiltration in the CSF and the brain parenchyma in a model of cytokine-induced meningitis [35]. However, those results were not confirmed in infectious meningitis [36]. JAM-B plays a central role in the regulation of paracellular permeability [34], but also impacts T-cell rolling and firm adhesion [21]. Although a lack of JAM-B expression at steady state on brain endothelial cells was suggested [37], we show that JAM-B is expressed on brain endothelial cells in our murine model of CNS autoimmunity. Because blockade of JAM-B was associated to a partial protection against encephalomyelitis and a significant reduction in the number of infiltrated CD8 T cells, however to a lesser extent than α4β1-integrin blockade, our data suggest that JAM-B participates to the CD8 T-cell migration process to the CNS. The discovery of the role of α4β1–VCAM-1 interaction in T-cell migration to the CNS led to the development of natalizumab, which has proven to be highly beneficial in relapsingremitting MS [38]. Unfortunately, natalizumab is associated with an increased risk of developing progressive multifocal leukoencephalopathy (PML), an opportunistic disease of the CNS caused by John Cunningham (JC) virus infection of oligodendrocytes [39]. Development of PML following natalizumab therapy likely relies on a multifactorial scenario including permissiveness for active JC virus replication and impaired immune surveillance www.eji-journal.eu Eur. J. Immunol. 2015. 45: 3302–3312 [40–43]. Compared with untreated MS patients, natalizumab treatment induces a decrease in cerebrospinal fluid CD4 and CD8 T cells [44, 45]. Because CD4 and CD8 T cells are crucial in controlling JC virus reactivation and dissemination [46, 47], our results suggest that development of PML is, at least in part, due to natalizumab-mediated inhibition of CNS immunosurveillance by JC virus-specific CD8 T cells. Furthermore, natalizumab-associated PML in MS patients is often complicated after natalizumab withdrawal by IRIS, darkening again the prognosis [48]. Although CD4 T cells might participate [49], a clear dominance of CD8 T cells in CNS-infiltrates has been observed in natalizumab-associated PML-IRIS occurring in patients with MS [50], reminiscent of the PML-IRIS pathology described in HIV-infected patients [51]. This indirectly supports the idea that the discontinuation of natalizumab allows migration of CD8 T cell to the CNS. Migration of encephalitogenic CD8 T cells to the CNS is dependent on the α4β1-integrin. α4β1-Integrin blockade in MS is beneficial but exposes to opportunistic viral infections due to the disruption of CNS immune surveillance. Because CD8 T cells requirements for CNS migration are distinct from those of other immune cells, future work should help identify novel molecular targets to block specifically CNS recruitment of destructive immune cells while leaving the migration of protective immune cell subsets into the CNS unaffected. Materials and methods Mice The CL4-TCR transgenic mouse line expresses an H-2Kd -restricted TCR (Vα10; Vβ8.2) against the Influenza virus HA transmembrane peptide amino acids 512–520 (IYSTVASSL) [52]. The CamK-iCre [53] and the Rosa26tm(HA)1Lib [54] mice have been described previously. CamK-HA mice were obtained by crossing the CamK-iCre transgenic mice with the Rosa26tm(HA)1Lib mice [19]. Cre-mediated genomic DNA recombination and the resulting transcription of HA occurred only in CamK-HA mice and were confined to the brain and spinal cord [19]. Mice were backcrossed at least six times on the BALB/c background. In some experiments, to benefit from a congenic marker allowing the distinction between transferred HA-specific CD8 T cells (CD45.1) and recipient T cells (CD45.2), donor and recipient mice on a (BALB/c × C57BL6) F1 background were used. Of note, the number of T cells infiltrating the CNS of CamK-HA mice did not differ between the BALB/c and the (BALB/c × C57BL6) F1 backgrounds (Supporting Information Fig. 4). All mice were 6–9-week-old at the onset of the experiments. Mice were kept under specific pathogen-free conditions. This study was carried out in strict accordance with EU regulations and with the recommendations of the French national chart for ethics of animal experiments (articles R 214-87–90 of the “Code rural”). The protocol was approved by the committee on the ethics of animal experiments of the région Midi-Pyrénées (permit numbers: 04-U563-DG-06 and MP/18/26/04/04). All procedures were per C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Cellular immune response formed under deep anesthesia as described below and all efforts were made to minimize animal suffering. In vitro differentiation and adoptive transfer of HA-specific cytotoxic CD8 T cells HA-specific cytotoxic CD8 T cells were generated as previously described [55]. Briefly, 5.105 purified naive CD8 T cells from CL4TCR mice were stimulated with 5.106 irradiated syngeneic splenocytes in DMEM supplemented with 10% FCS (Life Technologies, Paisley, Scotland) containing 1 mM HA peptide, 1 ng/mL IL-2, and 20 ng/mL IL-12. On day 5, cells were collected by Ficoll density separation and 5 × 106 living cytotoxic CD8 T cells were injected intravenously in recipient mice. Cells routinely contained >98% CD8+ CD3+ Vβ8.2+ T cells and >95% of them produce high levels of granzyme B and IFN-γ. Antibodies used for in vivo experiments mAbs were injected intravenously. Entotoxin-free rat mAb against mouse α4-integrin (clone PS/2, IgG2b, 250 μg/injection), α4β7-integrin (clone DATK32, IgG2a, 500 μg/injection), VCAM-1 (clone 6C7.1, IgG1, 250 μg/injection), and antiendoglin (control IgG, clone MJ7/18, IgG2a, 250 μg/ injection) were produced in B.E.’s lab. The rat anti-CD4-depleting mAb (clone GK1.5, IgG2b, 500 μg/injection) was produced in R.L.’s lab. The anti-VCAM-1 clone 429 (IgG2a, 100 μg/injection) and the anti-JAM-B clone 150005 (IgG2a, 100 μg/injection, or 200 μg/injection where indicated) were purchased from eBioscience and R&D Systems, respectively. Purification of mononuclear cells Mice were deeply anesthetized and perfused with 20 mL of PBS. For purification of mononuclear cells, brains were collected in PBS and dissociated using a glass Potter. Brain suspensions were enzymatically digested for 1 h at 37°C in RPMI 1640 medium (Invitrogen) containing collagenase D (1 mg/mL), and DNase I (10 mg/mL). The digested suspensions were filtered (70 mm cell strainer, Falcon), CNS-infiltrating mononuclear cells were collected after Percoll density separation and directly used for FACS staining. For purification of mononuclear cells from spleen and cervical draining lymph nodes, organs were collected in PBS and dissociated using a glass Potter. After red blood cell lysis, mononuclear cells were used for FACS staining. Ex vivo restimulation of HA-specific CD8 T cells Mononuclear cells from spleen and cervical lymph nodes (4 × 106 ) were plated in a 24-well plate for 6 h and restimulated with 1 μM of HA peptide or of H-2Kd -binding noncognate antigen www.eji-journal.eu 3309 3310 Guillaume Martin-Blondel et al. (Cw3 peptide, RYLKNGKETL). GolgiPlugTM (BD Pharmingen) was added for the last 2 h before mononuclear cells were used for FACS staining. FACS analysis In vitro differentiated cytotoxic HA-specific CD8 T cells were stained using a viability dye and anti-CD8α (53-6.7, BD Pharmingen), anti-α4-integrin (hybridoma supernatant, clone PS/2), antiβ7-integrin (hybridoma supernatant, clone FIB504), or anti-α4β7integrin heterodimer (hybridoma supernatant, clone DATK32). Mononuclear cells were stained using a viability dye and antiCD45 (30-F11, BD Pharmingen), anti-CD45.1 (A20, Biolegend), anti-CD45.2 (104, BD Pharmingen), anti-CD11b (M1/70, eBioscience), anti-Thy1.2 (53-2.1, eBioscience), anti-CD4 (RM4-5, BD Pharmingen), anti-CD8 (53-6.7, BD Pharmingen), anti-MHC class II (M5/114.15.2, eBioscience), anti-CD44 (IM7, Biolegend), anti-CD62L (MEL-14, BD Pharmingen), and anti-CD25 (PC61, BD Pharmingen) mAbs. After fixation and permeabilization, cells were intracellularly stained for IFN-γ (XMG1.2, BD Biosciences) and TNF-α (MP6-XT22, BD Pharmingen). Data were collected on an LSRII or FACSCalibur (Becton Dickinson) and analyzed with FlowJo software (Tree Star). In vitro binding assay system Wells of diagnostic microscope slides (ER-202W-CE24, Thermo Scientific) were coated with protein A (20 μg/mL in PBS pH 9) for 1 h at 37°C. After protein A incubation, wells were washed three times with PBS (pH 7.4) and blocked with 1.5% BSA in PBS for 30 min at 37°C. Subsequently, wells were washed once with PBS (pH 7.4) and recombinant proteins (100 nM) were incubated for 2 h at 37°C (recombinant DNER-Fc chimera, R&D 2254-DN; recombinant mouse ICAM-1-Fc chimera R&D 796-IC; recombinant VCAM-1-Fc chimera, R&D 643-VM). After recombinant proteincoating, wells were washed twice with PBS (pH 7.4) and blocked with 1.5% BSA in PBS for 30 min at room temperature. To perform the in vitro binding assay, HA-specific cytotoxic CD8 T cells were collected at 5 × 106 cells/mL in assay medium (DMEM, 25 mM HEPES, 5% calf serum, 2% L-glutamine) and incubated with either the blocking antibody (PS/2, 10 μg/mL) or the isotype control antibody (rat IgG2b, 10 μg/mL) for 20 min at room temperature. Antibodies were washed away by centrifugation for 3 min at 1400 rpm. Diagnostic microscope slides were placed on a rotating platform and 20 μL cell suspension (1 × 105 cells/well) was added and incubated on the recombinant proteins for 30 min. Slides were washed twice with PBS and fixed in 2.5% v/v glutaraldehyde in PBS. The number of adherent cells was evaluated by counting bound cells per field of view under the microscope using a grid ocular. Immunofluorescence staining Preparation of mouse tissue and staining procedures were performed as published previously [56]. Briefly, mice were perfused C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Immunol. 2015. 45: 3302–3312 with PBS or 1% formaldehyde in PBS through the left ventricle of the heart. Dissected tissues were embedded in Tissue-Tek (Sakura, the Netherlands), frozen, and stored at −80°C. For immunofluorescence staining of JAM-B, 6 μm cryostat sections were fixed with ice-cold methanol for 20 s, followed by blocking with blocking buffer (2% BSA, 1% FCS, 1% donkey serum in PBS). Subsequently, primary rabbit anti-JAM-B antibody (αJB829 1:250; kindly provided by Beat Imhof, Geneva, Switzerland) and either rat antimouse PECAM-1 (Mec 13.3, 20 μg/mL) or rat antimouse VCAM-1 (6C7.1, 10 μg/mL) were incubated with the sections for 1 h. In between and after antibody incubation, sections were washed with PBS. Sections were then incubated with secondary antibodies goat antirat Alexa 488 (1:200, Invitrogen MP A11006) and donkey antirabbit Cy3 (1:200, JIR 111-165-144) and DAPI (0.5 μg/mL, Invitrogen D3571) for 1 h and coverslipped with Mowiol (Sigma–Aldrich, USA). For immunofluorescence staining of CD8, cryostat sections were fixed in ethanol for 10 min at 4°C, followed by acetone for 1 min at room temperature. Subsequently, sections were dried for 30 min at room temperature. Blocking solution, skimmed milk (5% in TBS) was incubated for 20 min. Primary rat-antimouse CD8 antibody (Lyt 2, eBiosciences) and secondary goat-antirat IgG-Cy3 antibody (eBiosciences) were diluted in blocking solution. Antibody incubation was performed for 1 h at room temperature. To visualize the cell nuclei, DAPI (0.5 μg/mL, Invitrogen D3571) was incubated for 5 min at room temperature. Sections were washed with TBS between incubation steps and finally mounted with Mowiol (Sigma–Aldrich) and analyzed using a Nikon Eclipse 600 fluorescence microscope. Statistical analysis Differences between two sets of data were evaluated by a twotailed Mann–Whitney U test. For analysis of scatter plots comparing ࣙ3 groups of mice, one-way ANOVA with Bonferroni’s posttest was used. Survival curves were plotted by the Kaplan– Meier method and compared with the log-rank test. Data represent mean ± SEM. Probability values of p ࣘ 0.05 were considered statistically significant. All tests were carried out using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). Acknowledgments: This work was supported by an international ARSEP grant (to B.E. and R.L.), the Toulouse University Hospital, the French National Institute for Medical Research (INSERM), the French National Research Agency (ANR-13-BSV3-0003-01), and European Union, FP7-PEOPLE-2012-ITN NeuroKine to R.L., and the Swiss National Science Foundation (Grant No. 149420) and the Swiss Multiple Sclerosis Society to B.E. S.T. has been supported by a fellowship from the German Research Foundation. We thank Dr. Beat Imhof (Geneva, Switzerland) for kindly providing the anti-JAM-B antibody. www.eji-journal.eu Eur. J. Immunol. 2015. 45: 3302–3312 Conflict of interest: The authors declare no financial or commercial conflict of interest. Cellular immune response promotes leukocyte trafficking into the central nervous system. Nat. Immunol. 2008. 9: 137–145. 17 Ifergan, I., Kebir, H., Alvarez, J. I., Marceau, G., Bernard, M., Bourbonniere, L., Poirier, J. et al., Central nervous system recruitment of effector References 1 Galea, I., Bechmann, I. and Perry, V. H., What is immune privilege (not)? Trends Immunol. 2007. 28: 12–18. 2 Liblau, R. S., Gonzalez-Dunia, D., Wiendl, H. and Zipp, F., Neurons as targets for T cells in the nervous system. Trends Neurosci. 2013. 36: 315– 324. 3 Wilson, E. H., Weninger, W. and Hunter, C. A., Trafficking of immune cells in the central nervous system. J. Clin. Invest. 2010. 120: 1368–1379. 4 Engelhardt, B. and Ransohoff, R. M., Capture, crawl, cross: the T cell code to breach the blood-brain barriers. Trends Immunol. 2012. 33: 579–589. 5 Bartholomaus, I., Kawakami, N., Odoardi, F., Schlager, C., Miljkovic, D., Ellwart, J. W., Klinkert, W. E. et al., Effector T cell interactions with meningeal vascular structures in nascent autoimmune CNS lesions. Nature 2009. 462: 94–98. 6 Bauer, M., Brakebusch, C., Coisne, C., Sixt, M., Wekerle, H., Engelhardt, B. and Fassler, R., Beta1 integrins differentially control extravasation of inflammatory cell subsets into the CNS during autoimmunity. Proc. Natl. Acad. Sci. USA 2009. 106: 1920–1925. 7 Engelhardt, B., Laschinger, M., Schulz, M., Samulowitz, U., Vestweber, D. and Hoch, G., The development of experimental autoimmune encephalomyelitis in the mouse requires alpha4-integrin but not alpha4beta7-integrin. J. Clin. Invest. 1998. 102: 2096–2105. 8 Vajkoczy, P., Laschinger, M. and Engelhardt, B., Alpha4-integrin-VCAM1 binding mediates G protein-independent capture of encephalitogenic T cell blasts to CNS white matter microvessels. J. Clin. Invest. 2001. 108: 557–565. 9 Yednock, T. A., Cannon, C., Fritz, L. C., Sanchez-Madrid, F., Steinman, L. and Karin, N., Prevention of experimental autoimmune encephalomyelitis by antibodies against alpha 4 beta 1 integrin. Nature 1992. 356: 63–66. 10 Rothhammer, V., Muschaweckh, A., Gasteiger, G., Petermann, F., Heink, S., Busch, D. H., Heikenwalder, M. et al., Alpha4-integrins control viral meningoencephalitis through differential recruitment of T helper cell subsets. Acta Neuropathol. Commun. 2014. 2: 27. 11 Rothhammer, V., Heink, S., Petermann, F., Srivastava, R., Claussen, M. C., Hemmer, B. and Korn, T., Th17 lymphocytes traffic to the central nervous system independently of alpha4 integrin expression during EAE. J. Exp. Med. 2011. 208: 2465–2476. 12 Martin-Blondel, G., Delobel, P., Blancher, A., Massip, P., Marchou, B., Liblau, R. S. and Mars, L. T., Pathogenesis of the immune reconstitution inflammatory syndrome affecting the central nervous system in patients infected with HIV. Brain 2011. 134: 928–946. 13 Saxena, A., Martin-Blondel, G., Mars, L. T. and Liblau, R. S., Role of CD8 T cell subsets in the pathogenesis of multiple sclerosis. FEBS Lett. 2011. 585: 3758–3763. 14 Bien, C. G., Vincent, A., Barnett, M. H., Becker, A. J., Blumcke, I., Graus, F., Jellinger, K. A. et al., Immunopathology of autoantibody-associated encephalitides: clues for pathogenesis. Brain 2012. 135: 1622–1638. 15 Battistini, L., Piccio, L., Rossi, B., Bach, S., Galgani, S., Gasperini, C., Ottoboni, L. et al., CD8+ T cells from patients with acute multiple sclerosis display selective increase of adhesiveness in brain venules: a critical role for P-selectin glycoprotein ligand-1. Blood 2003. 101: 4775–4782. 16 Cayrol, R., Wosik, K., Berard, J. L., Dodelet-Devillers, A., Ifergan, I., Kebir, H., Haqqani, A. S. et al., Activated leukocyte cell adhesion molecule C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim memory CD8+ T lymphocytes during neuroinflammation is dependent on alpha4 integrin. Brain 2011. 134: 3560–3577. 18 Young, K. G., Maclean, S., Dudani, R., Krishnan, L. and Sad, S., CD8+ T cells primed in the periphery provide time-bound immune-surveillance to the central nervous system. J. Immunol. 2011. 187: 1192–1200. 19 Scheikl, T., Pignolet, B., Dalard, C., Desbois, S., Raison, D., Yamazaki, M., Saoudi, A. et al., Cutting edge: neuronal recognition by CD8 T cells elicits central diabetes insipidus. J. Immunol. 2012. 188: 4731–4735. 20 Elices, M. J., Osborn, L., Takada, Y., Crouse, C., Luhowskyj, S., Hemler, M. E. and Lobb, R. R., VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site. Cell 1990. 60: 577–584. 21 Ludwig, R. J., Hardt, K., Hatting, M., Bistrian, R., Diehl, S., Radeke, H. H., Podda, M. et al., Junctional adhesion molecule (JAM)-B supports lymphocyte rolling and adhesion through interaction with alpha4beta1 integrin. Immunology 2009. 128: 196–205. 22 Luo, B. H., Carman, C. V. and Springer, T. A., Structural basis of integrin regulation and signaling. Annu. Rev. Immunol. 2007. 25: 619–647. 23 Haanstra, K. G., Hofman, S. O., Lopes Estevao, D. M., Blezer, E. L., Bauer, J., Yang, L. L., Wyant, T. et al., Antagonizing the alpha4beta1 integrin, but not alpha4beta7, inhibits leukocytic infiltration of the central nervous system in rhesus monkey experimental autoimmune encephalomyelitis. J. Immunol. 2013. 190: 1961–1973. 24 Doring, A., Pfeiffer, F., Meier, M., Dehouck, B., Tauber, S., Deutsch, U. and Engelhardt, B., TET inducible expression of the alpha4beta7-integrin ligand MAdCAM-1 on the blood-brain barrier does not influence the immunopathogenesis of experimental autoimmune encephalomyelitis. Eur. J. Immunol. 2011. 41: 813–821. 25 Laschinger, M. and Engelhardt, B., Interaction of alpha4-integrin with VCAM-1 is involved in adhesion of encephalitogenic T cell blasts to brain endothelium but not in their transendothelial migration in vitro. J. Neuroimmunol. 2000. 102: 32–43. 26 Steiner, O., Coisne, C., Cecchelli, R., Boscacci, R., Deutsch, U., Engelhardt, B. and Lyck, R., Differential roles for endothelial ICAM-1, ICAM-2, and VCAM-1 in shear-resistant T cell arrest, polarization, and directed crawling on blood-brain barrier endothelium. J. Immunol. 2010. 185: 4846–4855. 27 Jilek, S., Mathias, A., Canales, M., Lysandropoulos, A., Pantaleo, G., Schluep, M. and Du Pasquier, R. A., Natalizumab treatment alters the expression of T-cell trafficking marker LFA-1 alpha-chain (CD11a) in MS patients. Mult. Scler. 2013 [Epub ahead of print]. 28 Schwab, N., Schneider-Hohendorf, T., Posevitz, V., Breuer, J., Gobel, K., Windhagen, S., Brochet, B. et al., L-selectin is a possible biomarker for individual PML risk in natalizumab-treated MS patients. Neurology 2013. 81: 865–871. 29 Berlin, C., Berg, E. L., Briskin, M. J., Andrew, D. P., Kilshaw, P. J., Holzmann, B., Weissman, I. L. et al., Alpha 4 beta 7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1. Cell 1993. 74: 185–195. 30 Kamata, T., Puzon, W. and Takada, Y., Identification of putative ligandbinding sites of the integrin alpha 4 beta 1 (VLA-4, CD49d/CD29). Biochem. J. 1995. 305: 945–951. 31 Man, S., Tucky, B., Bagheri, N., Li, X., Kochar, R. and Ransohoff, R. M., Alpha4 Integrin/FN-CS1 mediated leukocyte adhesion to brain microvascular endothelial cells under flow conditions. J. Neuroimmunol. 2009. 210: 92–99. www.eji-journal.eu 3311 3312 Guillaume Martin-Blondel et al. Eur. J. Immunol. 2015. 45: 3302–3312 32 van der Laan, L. J., van der Goes, A., Wauben, M. H., Ruuls, S. R., 46 Gasnault, J., Kahraman, M., de Goer de Herve, M. G., Durali, D., Del- Dopp, E. A., De Groot, C. J., Kuijpers, T. W. et al., Beneficial effect of fraissy, J. F. and Taoufik, Y., Critical role of JC virus-specific CD4 T-cell modified peptide inhibitor of alpha4 integrins on experimental allergic responses in preventing progressive multifocal leukoencephalopathy. encephalomyelitis in Lewis rats. J. Neurosci. Res. 2002. 67: 191–199. AIDS 2003. 17: 1443–1449. 33 Cunningham, S. A., Rodriguez, J. M., Arrate, M. P., Tran, T. M. and Brock, 47 Du Pasquier, R. A., Schmitz, J. E., Jean-Jacques, J., Zheng, Y., Gordon, T. A., JAM2 interacts with alpha4beta1. Facilitation by JAM3. J. Biol. Chem. J., Khalili, K., Letvin, N. L. et al., Detection of JC virus-specific cytotoxic 2002. 277: 27589–27592. T lymphocytes in healthy individuals. J. Virol. 2004. 78: 10206–10210. 34 Martin-Padura, I., Lostaglio, S., Schneemann, M., Williams, L., Romano, 48 Tan, I. L., McArthur, J. C., Clifford, D. B., Major, E. O. and Nath, M., Fruscella, P., Panzeri, C. et al., Junctional adhesion molecule, a novel A., Immune reconstitution inflammatory syndrome in natalizumab- member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration. J. Cell Biol. 1998. 142: 117–127. 35 Del Maschio, A., De Luigi, A., Martin-Padura, I., Brockhaus, M., Bartfai, T., Fruscella, P., Adorini, L. et al., Leukocyte recruitment in the cerebrospinal fluid of mice with experimental meningitis is inhibited by an antibody to junctional adhesion molecule (JAM). J. Exp. Med. 1999. 190: 1351–1356. 36 Lechner, F., Sahrbacher, U., Suter, T., Frei, K., Brockhaus, M., Koedel, U. and Fontana, A., Antibodies to the junctional adhesion molecule cause disruption of endothelial cells and do not prevent leukocyte influx into the meninges after viral or bacterial infection. J. Infect. Dis. 2000. 182: 978–982. 37 Aurrand-Lions, M., Johnson-Leger, C., Wong, C., Du Pasquier, L. and Imhof, B. A., Heterogeneity of endothelial junctions is reflected by differential expression and specific subcellular localization of the three JAM family members. Blood 2001. 98: 3699–3707. 38 Polman, C. H., O’Connor, P. W., Havrdova, E., Hutchinson, M., Kappos, L., Miller, D. H., Phillips, J. T. et al., A randomized, placebo-controlled trial of natalizumab for relapsing multiple sclerosis. N. Engl. J. Med. 2006. 354: 899–910. 39 Bloomgren, G., Richman, S., Hotermans, C., Subramanyam, M., Goelz, S., Natarajan, A., Lee, S. et al., Risk of natalizumab-associated progressive multifocal leukoencephalopathy. N. Engl. J. Med. 2012. 366: 1870–1880. 40 Bonig, H., Wundes, A., Chang, K. H., Lucas, S. and Papayannopoulou, T., Increased numbers of circulating hematopoietic stem/progenitor cells are chronically maintained in patients treated with the CD49d blocking antibody natalizumab. Blood 2008. 111: 3439–3441. 41 del Pilar Martin, M., Cravens, P. D., Winger, R., Frohman, E. M., Racke, M. K., Eagar, T. N., Zamvil, S. S. et al., Decrease in the numbers of dendritic cells and CD4+ T cells in cerebral perivascular spaces due to natalizumab. Arch. Neurol. 2008. 65: 1596–1603. 42 Perkins, M. R., Ryschkewitsch, C., Liebner, J. C., Monaco, M. C., Himelfarb, D., Ireland, S., Roque, A. et al., Changes in JC virus-specific associated PML. Neurology 2011. 77: 1061–1067. 49 Aly, L., Yousef, S., Schippling, S., Jelcic, I., Breiden, P., Matschke, J., Schulz, R. et al., Central role of JC virus-specific CD4+ lymphocytes in progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome. Brain 2011. 134: 2687–2702. 50 Metz, I., Radue, E. W., Oterino, A., Kumpfel, T., Wiendl, H., Schippling, S., Kuhle, J. et al., Pathology of immune reconstitution inflammatory syndrome in multiple sclerosis with natalizumab-associated progressive multifocal leukoencephalopathy. Acta Neuropathol. 2012. 123: 235–245. 51 Martin-Blondel, G., Bauer, J., Cuvinciuc, V., Uro-Coste, E., Debard, A., Massip, P., Delisle, M. B. et al., In situ evidence of JC virus control by CD8+ T cells in PML-IRIS during HIV infection. Neurology 2013. 81: 964– 970. 52 Morgan, D. J., Liblau, R., Scott, B., Fleck, S., McDevitt, H. O., Sarvetnick, N., Lo, D. et al., CD8(+) T cell-mediated spontaneous diabetes in neonatal mice. J. Immunol. 1996. 157: 978–983. 53 Casanova, E., Fehsenfeld, S., Mantamadiotis, T., Lemberger, T., Greiner, E., Stewart, A. F. and Schutz, G., A CamKIIalpha iCre BAC allows brainspecific gene inactivation. Genesis 2001. 31: 37–42. 54 Saxena, A., Bauer, J., Scheikl, T., Zappulla, J., Audebert, M., Desbois, S., Waisman, A. et al., Cutting edge: multiple sclerosis-like lesions induced by effector CD8 T cells recognizing a sequestered antigen on oligodendrocytes. J. Immunol. 2008. 181: 1617–1621. 55 Vizler, C., Bercovici, N., Heurtier, A., Pardigon, N., Goude, K., Bailly, K., Combadiere, C. et al., Relative diabetogenic properties of islet-specific Tc1 and Tc2 cells in immunocompetent hosts. J. Immunol. 2000. 165: 6314– 6321. 56 Wyss, L., Schafer, J., Liebner, S., Mittelbronn, M., Deutsch, U., Enzmann, G., Adams, R. H. et al., Junctional adhesion molecule (JAM)-C deficient C57BL/6 mice develop a severe hydrocephalus. PLoS One 2012. 7: e45619. Abbreviations: BBB: Blood-brain barrier · IRIS: Immune reconstitution T cell responses during natalizumab treatment and in natalizumab- inflammatory syndrome · JAM-B: Junctional adhesion molecule-B · PML: associated progressive multifocal leukoencephalopathy. PLoS Pathog. Progressive multifocal leukoencephalopathy 2012. 8: e1003014. 43 Warnke, C., Mausberg, A. K., Stettner, M., Dehmel, T., Nekrich, L., Meyer zu Horste, G., Hartung, H. P. et al., Natalizumab affects the T-cell receptor repertoire in patients with multiple sclerosis. Neurology 2013. 81: 1400– 1408. 44 Schneider-Hohendorf, T., Rossaint, J., Mohan, H., Boning, D., Breuer, J., Full correspondence: Dr. Roland Liblau, INSERM U1043 - CNRS UMR 5282, Centre de Physiopathologie de Toulouse Purpan (CPTP), Purpan Hospital, Place du Docteur Baylac TSA 40031, 31059 Toulouse Cedex 9, France Fax: +33-561-77-21-38 e-mail: [email protected] Kuhlmann, T., Gross, C. C. et al., VLA-4 blockade promotes differential routes into human CNS involving PSGL-1 rolling of T cells and MCAMadhesion of TH17 cells. J. Exp. Med. 2014. 211: 1833–1846. 45 Stuve, O., Marra, C. M., Jerome, K. R., Cook, L., Cravens, P. D., Cepok, S., Frohman, E. M. et al., Immune surveillance in multiple sclerosis patients treated with natalizumab. Ann. Neurol. 2006. 59: 743–747. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Received: 5/3/2015 Revised: 21/8/2015 Accepted: 7/9/2015 Accepted article online: 31/8/2015 www.eji-journal.eu #!"$ "%& 2712 Lidia Yshii et al. DOI: 10.1002/eji.201545759 Eur. J. Immunol. 2015. 45: 2712–2720 ECI 2015 Review Series Neurons and T cells: Understanding this interaction for inflammatory neurological diseases Lidia Yshii1,2,3 , Christina Gebauer1,2 , Raphaël Bernard-Valnet1,2 and Roland Liblau1,2 1 INSERM U1043 - CNRS UMR 5282, Centre de Physiopathologie Toulouse-Purpan, Toulouse, France 2 Université Toulouse III, Toulouse, France 3 Institute of Biomedical Sciences I, University of Sao Paulo, Sao Paulo, Brazil Central nervous system (CNS) inflammation occurs in a large number of neurological diseases. The type and magnitude of CNS inflammation, as well as the T-cell contribution, vary depending on the disease. Different animal models of neurological diseases have shown that T cells play an important role in CNS inflammation. Furthermore, recent studies of human neurological disorders have indicated a significant role for T cells in disease pathology. Nevertheless, how individual T-cell subsets affect neuronal survival, damage and/or loss remains largely unclear. In this review we discuss the processes by which T cells mediate either beneficial or deleterious effects within the CNS, with emphasis on the direct interaction between T cells and neurons, as occurs in multiple sclerosis, paraneoplastic cerebellar degeneration, and viral encephalitis. The therapeutic approaches targeting T cells and their mediators as treatment for neurological diseases are also described here. Keywords: Autoimmunity r Central nervous system Neurons r T cells r Viral infection r Inflammation Introduction Due to its limited regenerative capacity, the central nervous system (CNS) needs to protect itself against potentially damaging exuberant immune responses. This is achieved through a singular state of reduced exposure to the immune system, known as “immune privilege” (reviewed in [1, 2]). This “immune privilege” occurs thanks to the blood–brain barrier (BBB), limited major histocompatibility complex (MHC) molecule expression in the brain, and neuronal expression of molecules with immunosuppressive properties, such as TGF-β and CD200 [3]. All these contribute to protect neurons and axons from microgliaand/or T-cell-induced damage although it is not as drastic as previously thought. Activated immune cells can enter the CNS Correspondence: Prof. Roland Liblau e-mail: [email protected] C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim r Neurodegeneration r through various routes under specific conditions through selective receptor–ligand interactions (reviewed in [4]). Recent studies have identified an unconventional lymphatic drainage system from the CNS interstitial fluid and cerebrospinal fluid (CSF) to the deep cervical lymph nodes [5, 6]. Whether immune cells exit the CNS using these lymphatic vessels is currently under investigation. Inflammation occurs in a large number of neurological diseases with etiologies as diverse as ischemia, trauma, infection, autoimmunity, or neurodegeneration [7, 8]. The type and magnitude of CNS inflammation, and the possible contribution of T cells, vary largely depending on the considered disease. A pivotal role has been attributed to T cells in animal models of CNS inflammation, and similar observations are emerging from the study of human neurological disorders (reviewed in [7, 9]). Moreover, CNS-resident cells, including some neurons, constitutively express MHC class I molecules [10, 11], challenging the classical view that neurons might be excluded from cytotoxic T-cell (CTL) www.eji-journal.eu Eur. J. Immunol. 2015. 45: 2712–2720 recognition [12]. In addition, MHC class I expression on neurons and glial cells increases strikingly under pathological conditions [13]. Importantly, recent data indicate that neurons can process and present viral or exogenous antigens to CTLs, resulting in neuronal cell death [14, 15]. In this review, we discuss the processes by which T cells can have either beneficial or deleterious effects within the CNS, with a special emphasis on the direct interaction between T cells and neurons. We also review the different therapeutical approaches targeting T cells and their mediators for the treatment of neurological diseases. T-cell migration into the CNS T cells enter into the CNS through migration across the BBB, the blood–leptomeningeal barrier, or the choroid plexus (reviewed in [16]). Integrins, selectins, and chemokine receptors expressed on T cells regulate this transmigration, and T-cell ligands are upregulated on CNS endothelial cells during neuroinflammation [16]. In particular, the interaction between α4β1-integrin, expressed by activated T cells, and vascular cell adhesion protein 1 (VCAM1), expressed on blood vessels, has been shown to have a pivotal role for CD4+ and CD8+ T-cell migration into the CNS [17–19]. Th17 cells, however, have been shown to adhere to endothelial cells and extravasate into the CNS independently of α4β1 [20], using either a melanoma cell adhesion molecule (MCAM)- [21], or lymphocyte function-associated antigen 1 (LFA1)-dependent pathway [22]. Moreover, the chemokine receptor CCR6 has been shown to control the migration of Th17 cells into the CNS through the choroid plexus in a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) [23]. Additionally, CD4+ FoxP3+ T regulatory (Treg) cells depend on LFA-1 to migrate into the CNS in the absence of α4-integrin during EAE [24]. In humans, blocking CCR5 appears to limit the traffic of CD8+ T cells into the CNS of patients suffering from progressive multifocal leukoencephalopathy-associated immune reconstitution inflammatory syndrome (PML-IRIS), which is an unbalanced immune reaction to the JC virus responsible for PML [25, 26]. Moreover, CCR5 deficiency in humans is a strong risk factor for CNS infection by West Nile virus (WNV) [27], most likely because CCR5-deficient cells limit the local migration of effector T cells. Collectively, these data suggest that different immune cell subsets use various molecular cues to penetrate the CNS. These specific pathways offer the possibility of blocking only the immune cell type involved in a particular disease. Neuronal protection against T-cell-mediated inflammation While T cells are considered destructive in many neurological disorders (see “Inflammatory and neurodegenerative diseases mediated by T cells”), they can also promote neuroprotection in the C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim HIGHLIGHTS same experimental situations [28]. For instance, Th1 and Th2 CD4+ T cells have been shown to produce neurotrophins, which are neurotrophic growth factors that contribute to the development, function, and survival of neurons [29]. The production of the neurotrophin BDNF (brain-derived neurotrophic factor), in particular, is enhanced upon antigen stimulation of T cells, which in turn has been shown to support neuronal survival by inducing axonal outgrowth and regeneration [30]. Additionally, both BDNF and neurotrophin-3 were detected in activated T cells infiltrating the CNS during EAE, suggesting that the damaging consequences of T cells can be counteracted by neurotrophic factors produced by T cells [28]. Importantly, the interactions between T cells and neurons are bidirectional. Neurons have evolved mechanisms to directly protect the CNS from T-cell-mediated inflammation. For example, neurons have been shown to promote T-cell apoptosis through Fas/FasL interactions [31] and produce antiinflammatory cytokines, such as TGF-β, which is associated with resistance to EAE [32, 33]. Furthermore, a pro-inflammatory environment results in increased production of TGF-β and enhanced surface expression of CD80/CD86 by neurons [32]. In turn, TGF-β and CD80/CD86 maintain the antigen-independent CD4+ T-cell proliferation and promote the conversion of pathogenic CD4+ T cells into TGFβ+ CTLA-4+ Treg cells, which then suppress encephalitogenic T cells and inhibit EAE [32]. In addition, Treg cells counteract effector immune mechanisms within tissues, preserving neuronal function. In particular, Treg cells can control CNS inflammation in EAE [34, 35]. For instance, Treg cells expressing the transcription factor FoxA1 (FoxA1+ Treg cells) have been shown to enhance their own expression of programmed cell death ligand 1 (PD-L1) and kill pathogenic CD4+ T cells, thereby suppressing EAE in a FoxA1- and PD-L1-dependent way [36]. There is also evidence that CD8+ CD122+ T cells produce IL-10, which inhibits proliferation of CTLs as well as their IFN-γ production, resulting in suppression of EAE, especially in the late phase [37]. In addition, after CNS injury, CD4+ T-cell infiltration occurs independently of MHC class II expression. The CNS-invading CD4+ T cells exhibit increased IL-4 output as compared with that of their peripheral counterparts, as a result of MyD88 signaling triggered by damage-associated molecular pattern molecules (DAMPs) [38]. The T-cell-derived IL-4 indirectly triggers neuroprotection and axon growth through activation of IL-4 receptors on neurons, enhancing the neurotrophin signaling pathway [38]. In a model of acute spinal cord injury, both Th1 cells and Treg cells have been shown to be required for recovery [39]. Likewise, in amyotrophic lateral sclerosis (ALS) mouse model, Treg cells are recruited to attenuate the cytotoxic activity of microglia and, consequently, promote neuroprotection [40, 41]. Nevertheless, within the CNS, effector T cells mostly contribute to inflammation and neurodegeneration in both sterile and nonsterile situations [7]. We therefore concentrate below on pathological conditions in which T cells promote neuronal/axonal damage and discuss the mechanisms involved and www.eji-journal.eu 2713 2714 Lidia Yshii et al. how to therapeutically interfere with disease-promoting T-cell populations. T-cell activities in the CNS during viral infections The interaction between CD8+ T cells and neurons during viral infections differs according to the virus involved. Exposure to the neurotropic herpes simplex virus type 1 (HSV1) results in chronic infection of neurons in the trigeminal ganglia. During latency, no infectious viral particles are produced and HSV-1 proteins are not detectable [42]. HSV-1-specific CTLs have been shown to localize in close proximity to HSV-1-infected neuronal cell body in human trigeminal ganglia [43]. HSV-1-specific CTL activation or retention involves CD4+ T cells, as well as local APCs [44]. CD8+ T cells have been shown to use at least two noncytolytic mechanisms to maintain HSV-1 latency [45]. First, granzyme B delivered by HSV-1-specific CD8+ T cells to infected neurons can directly cleave ICP4, a viral protein required for efficient transcription of early and late viral genes, without impairing neuronal survival in vitro [45]. Second, IFN-γ secretion by HSV1-specific CTLs has been implicated in inhibiting the expression of HSV-1 gene products required for viral reactivation [45]. Thus, Granzyme B and IFN-γ are essential to control the reactivation of HSV-1 from latency. In contrast to the noncytolytic mechanisms employed by CTLs in response to HSV-1 infection, mouse models for WNV infection have revealed that CD8+ T cells are required to clear the virus from infected neurons in an MHC class I-dependent manner [46]. Mice lacking MHC I molecules have an increased and sustained WNV infection in both peripheral and CNS tissues [46]. Indeed, CD8+ T cells have been shown to directly kill infected neurons through both the perforin and Fas/FasL ligand pathways to eliminate WNV from the CNS [47]. Additionally, CD8+ T cells produce TNF-related apoptosis-inducing ligand (TRAIL) to restrict WNV infection of CNS neurons [48]. Furthermore, upon WNV infection, production of CXCL10 by neurons is induced by IFN-γ, thereby promoting the recruitment of CXCR3-expressing CD8+ T cells into the CNS [49]. However, CXCL10 expression can vary between neuronal subsets: cerebellar granule neurons present higher expression of CXCL10 in comparison to cortical neurons, indicating a differential regulation of inflammatory responses according to the neuron subtypes [49]. The neurotropic Borna disease virus (BDV) is another example in which CD8+ T cells clear the virus through direct killing of infected neurons. Interestingly, BDV triggers up-regulation of MHC class I on primary murine neurons, thereby allowing cognate interaction between these neurons and virus-specific CTLs [15]. This leads to morphological and functional alterations of the infected neurons, preceding their subsequent apoptosis [15]. In the viral déjà vu model (antiviral CTLs targeting an epitope shared by the initial virus persisting in neurons and the secondary virus) in mice infected with a lymphocytic choriomeningitis virus (LCMV) variant, an original non-neurotoxic phenomenon results from the CTL attack on neurons in vivo [50]. Indeed, infection C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Immunol. 2015. 45: 2712–2720 with this variant results in a rapid loss of dendrites and synapses, induced by the release of IFN-γ by CTLs in close contact with neurons, and subsequent IFN-γ receptor signaling in neurons, independent of Fas/FasL or perforin involvement [50]. Of note, in CNS lesions of Rasmussen encephalitis patients, a similar phosphorylation of STAT1 observed in the murine déjà vu model is often found within neurons in close contact with CD8+ T cells [50]. IFN-γ produced by CD8+ T cells may promote neurotoxicity through additional mechanisms. For example, the IFN-γ receptor can form a complex with the AMPA receptor subunit GluR1 in neurons, increasing the excitability and vulnerability to glutamate excitotoxicity [51]. In addition, IFN-γ signaling in neurons has been shown to reduce the synaptic conduction below a critical threshold and enhances the expression of MHC class I molecules on neurons [51]. These effects may render neurons more susceptible to CD8+ T cell-mediated cytolysis during the course of inflammatory diseases. Thus, the collective effect of TRAIL, FasL, and perforin, and the increased expression of MHC class I molecules, underlies some of the molecular tools by which effector CD8+ T cells directly interact with and kill infected neurons to contain viral infection [47, 48]. Moreover, production of CXCL10 by neurons further attracts effector CD8+ T cells at sites of viral infection. However, the molecular tools used by T cells to control the viral infection of CNS neurons seems to be dependent on the virus involved and may even differ according to the infected neuronal subset. Inflammatory and neurodegenerative diseases mediated by T cells The potential implication of T cells, and particularly CD8+ T cells, in CNS autoimmune diseases is receiving increasing attention [7, 8, 52, 53]. Indeed, the list of human neurological diseases in which T cells have been suggested to target neurons or axons, directly or indirectly, is continuously growing (see Table 1). We describe below three selected diseases in which T cells target neurons through different mechanisms. In multiple sclerosis (MS) and paraneoplastic cerebellar degeneration (PCD) the adaptive immune system is a key mediator of CNS tissue damage whereas Parkinson’s disease (PD), a primary neurodegenerative disease with secondary inflammation, is mostly mediated by innate immune cells [8]. In MS, axonal transection and neuronal loss have been shown to develop in the context of a complex T-cell-mediated chronic inflammation [54]. Contrary to this, in PCD, a rare autoimmune disease associated with gynecologic cancers, there is evidence suggesting that the selective loss of Purkinje neurons likely results from direct interaction between CTLs and Purkinje neurons [55]. Finally, recent data highlight the possible contribution of T cells in PDs [14]. Multiple sclerosis and EAE, its popular animal model MS is very likely an autoimmune disease of the CNS which leads to multifocal demyelination and neurodegeneration [9]. MS www.eji-journal.eu HIGHLIGHTS Eur. J. Immunol. 2015. 45: 2712–2720 Table 1. Human neurological diseases involving targeting of CNS neurons by infiltrating T cells Disease Infectious diseases HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP)a) JC virus PML-IRISb) Viral encephalitisc) Acute/subacute postviral encephalitisd) Toxoplasmic encephalitise) (Plausible) Autoimmune diseases Narcolepsyf) Anti-Hu syndromeg) Encephalitis associated with anti-Ma1/2 antibody Stiff-person syndromeh) Paraneoplastic cerebellar degenerationi) Idiopathic central diabetes insipidus j) Multiple sclerosisk, l) Febrile infection-related epilepsy syndrome (FIRES)m) Other diseases Parkinson’s diseasen) Rasmussen’s diseaseo) CLIPPERSp) Hemophagocytic lymphohistiocytosisq) CTLA-4 haploinsufficiencyr) Targeted neurons Pathogenic T-cell population(s) Axonal loss in spinal cord Cytotoxic CD8+ T cells Cerebellar granule cells; cortical neurons Infected neurons Neurons Infected neurons CD8+ T cells/CD4+ T cells Mostly CD8+ T cells CD8+ and CD4+ T cells CD8+ T cells Hypothalamic Orexin neurons Most neurons Diencephalic neurons CD8+ T cells/CD4+ T cells CD8+ T cells CD8+ T cells GABAergic neurons Purkinje neurons Neurohypophysis AVP neurons Most axons/neurons Temporal neurons and basal ganglia CD8+ T cells CD8+ T cells CD8+ T cells Th17; CD8+ T cells T cells Substantia nigra dopaminergic neurons Cortical neurons Brainstem and cerebellum Gray and white matter Gray and white matter CD4+ T cells CD8+ T cells CD4+ > CD8+ T cells CD8+ T cells (and NK cells) Unknown a) Matsuura et al., J. Neuropathol. Exp. Neurol. (2015) 74:2 Schippling et al., Ann. Neurol. (2013) 74:622 c) Phares et al., J Virol (2012) 86:2416 d) Gogate et al., J Neuropathol Exp Neurol (1996) 55:435 e) Blanchard et al., Parasite Immunol (2015) 37:150 f) Liblau et al., Lancet Neurol (2015) 14:318 g) Pignolet et al., Oncoimmunology (2013) 2:e27384 h) Poh et al., J Neurol Sci (2014) 337:235 i) Albert et al., Nat Med (1998) 4:1321 j) Scheikl et al., J Immunol (2012) 188:4731 k) Aktas et al., Neuron (2005) 46:421 l) Huseby et al., J Exp Med (2001) 194:669 m) Rebillard et al., Lancet Neurology Autoimmune Disorders Conference (2015) personal communication n) Brochard et al., J Clin Invest (2009) 119:182 o) Varadkar et al., Lancet Neurol (2014) 13:195 p) Simon et al., J Neurol Neurosurg Psychiatry (2012) 83:15 q) Deiva et al., Neurology (2012) 78:1150 r) Kuehn et al., Science (2014) 345:1623. b) development involves both genetic [56] and environmental factors [57]. Extensive axonal/neuronal damage is observed in MS pathology, with transected axons and reduced neuronal density resulting in brain and spinal cord atrophy [54, 58]. These findings are associated with focal infiltration by macrophages and T cells, which form demyelinated plaques within the white matter and in the cortex. These infiltrating immune cells secrete proinflammatory cytokines, chemokines and reactive oxygen species that affect the neurons, due to chronic inflammation and oxidative stress, as well as mitochondrial and ion channel dysfunction, culminating in neuronal death [9]. Extensive studies in EAE mice and MS patients have suggested a key role for several T-cell subsets in MS pathogenesis, but their C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim contribution to the disease is still under investigation. While the initial animal models of EAE emphasized the role of CD4+ T cells [59], Lucchinetti et al. [60] demonstrated that CD8+ T-cell infiltration is present in more than 70% of human MS cortical plaque biopsies. Moreover, CD8+ T cells have been shown to be far more numerous than CD4+ T cells in active parenchymal MS lesions [61]. Notably, higher levels of IL-17A have been detected in the cerebrospinal fluid (CSF) of MS patients compared with that from healthy subjects, and a large proportion (approx. 80%) of CNSinfiltrating T cells express IL-17 in active MS lesions [62, 63]. In the classical EAE model, MOG-specific Th17 cells have been shown to form direct physical contacts with neurons and their processes within demyelinating lesions [64]. These contacts were shown to www.eji-journal.eu 2715 2716 Lidia Yshii et al. be LFA-1-dependent, but antigen- and MHC class II-independent [64]. Nevertheless, Th17 cells induced a marked, localized and partially reversible raise in Ca2+ concentration within axons that could lead to axon transection and neuronal soma degeneration [64], while Th1 cells were much less efficient at killing neurons through this mechanism [65]. In contrast, encephalitogenic CD4+ T cells have been shown to contribute to neuronal damage by releasing TRAIL and activate caspase 3 in neurons [65]. Increasing evidence indicates that CD8+ T cells contribute to MS pathogenesis, and animal models in which CD8+ T cells are the main effectors of disease have been developed [66]. For instance, myelin basic protein (MBP)-specific CD8+ T cells have been shown to cause severe CNS autoimmune disease in mice [67]. The MBPspecific CTLs induce demyelination and neuronal apoptosis, in an IFN-γ-dependent fashion, leading to motor function impairment [67]. In a humanized mouse model of EAE, it was demonstrated that MHC class I alleles and CD8+ T cells are directly implicated. In fact, MHC class I-restricted CD8+ T cells efficiently initiated the first attack [68]. Furthermore, T-cell infiltrates were located predominantly in the spinal cord and cerebellum, with signs of demyelination and neuronal degeneration [68]. Collectively, these mouse models highlight the potential for auto reactive CD4+ and CD8+ T cells to contribute directly or indirectly to axonal or neuronal damage. The advances in two-photon in vivo imaging techniques, which allow real-time intravital monitoring of T-cell dynamics and function, will undoubtedly further our understanding of the underlying mechanisms [69]. Paraneoplastic cerebellar degeneration PCD is a rare and severe neurological disease, caused by the selective loss of Purkinje neurons in the cerebellum. PCD develops mainly in patients with breast or ovarian carcinomas [53, 70]. An autoimmune response targeting a cytoplasmic protein (named cdr2) expressed in both tumor cells and Purkinje neurons [55] is thought to ultimately lead to neuroinflammation [71]. However, the presence of cdr2-expressing tumor cells is not sufficient to induce PCD. Actually, 60% of ovarian tumors and 25% of breast tumors express cdr2 in the absence of PCD [70]. The immunopathogenesis of PCD is not fully clarified. Although patients are diagnosed based on high titers of anti-cdr2 autoantibodies, the direct pathogenic contribution of anti-cdr2 autoantibodies is very unlikely, given the intracellular location of the target auto-antigen [72]. However, a recent report of a study using cerebellar organotypic slice culture demonstrated that anti-cdr2 antibody internalization caused deregulation of Ca2+ homeostasis, leading to morphological changes of Purkinje cells and alterations in the cell signaling pathways, resulting in neurodegeneration [73]. According to one possible scenario, dendritic cells that have phagocytosed apoptotic tumor cells would activate tumorspecific CD4+ and CD8+ T cells in local lymph nodes [72]. As a possible result, most tumors detected in PCD patients have limited disease spread [74]. However, cdr2-specific CTLs would migrate to the CNS and target Purkinje neurons [55]. Indeed, CD8+ T cells C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Immunol. 2015. 45: 2712–2720 are found in the cerebellum of patients with PCD [75]. Moreover, granzyme B-containing CD8+ T cells are observed close to the Purkinje neurons [75], which can express MHC class-I molecules [10]. Although cdr2-specific CTLs were found in the blood and CSF of PCD patients [55, 76], there is no evidence that they cause cerebellar pathology. This is in part due to the lack of appropriate animal models of PCD. Parkinson’s disease PD, the most prevalent movement disorder, is caused by the selective degeneration of dopaminergic neurons in the substantia nigra (SN), which are located in the midbrain. CD4+ and CD8+ T cells have been detected within the SN in postmortem specimens from PD patients [77]. The same observation was made in the mouse model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)induced dopaminergic neuronal death [77]. Further investigation indicated that CD4+ T cells contribute to the neuronal loss induced by MPTP, through activation of the Fas/FasL pathway [77]. Moreover, MHC class I was detected in dopaminergic neurons of the SN in both healthy control and PD postmortem tissues [14]. The same study showed that human stem cell-derived dopaminergic neurons can display MHC class I upon IFN-γ treatment [14]. High exposure to the dopamine precursor, L-DOPA, has been shown to induce MHC class I expression on murine dopaminergic neurons, prior to their subsequent destruction by CTLs [14]. Both studies concur to suggest that T cells contribute to the neuronal loss observed in PD [14, 77]. However, the precise T-cell subset at play remains to be firmly determined. Furthermore, other immune cells, such as activated microglia and astrocytes are believed to cause neuronal death in a nonantigen-specific way [78]. This neuroinflammation in PD could well participate to dopaminergic neuronal demise. However, although actively studied, therapeutic immunomodulation has not yet been proved successful in PD. Therapeutic implications of understanding the consequences of neuron/T-cell interaction In experimental models such as EAE, the range of possibilities to therapeutically target pathogenic T cells is remarkable. Translation to humans may take 15 years or more and, unfortunately, it has not been consistently correlated with success. The use of T-cell depletion strategies has been revived thanks to the approval for, and strong beneficial effect of, Alemtuzumab in active MS [79, 80]. Alemtuzumab, a humanized anti-CD52 mAb, induces long-lasting (2–3 years) CD8+ and CD4+ T-cell depletion [81]. In view of the data described above, it is noteworthy that the progressive T-cell reconstitution after cycles of Alemtuzumab injection favors cells with a regulatory phenotype (CD25+ FOXP3+ CD127low ), as well as CD4+ and CD8+ T cells producing IL-10 and TGF-β [82]. A transient increase in circulating IL-4+ T cells has also been noted [82]. During the reconstitution www.eji-journal.eu HIGHLIGHTS Eur. J. Immunol. 2015. 45: 2712–2720 phase, T cells also produce high levels of neurotrophic factors, such as BDNF, CNTF, and PDGF, which favor survival and differentiation of neurons and oligodendrocytes in vitro [83]. Whether these reconstituted T cells can penetrate the CNS and exert neuroprotective functions, however, remains uncertain. Due to the lack of appropriate techniques, the selective elimination of functional T-cell subsets such as Th1, Th17, effector or memory cytotoxic CD8+ T cells has not been clinically tested. Daclizumab, a humanized anti-CD25 mAb inhibiting signaling through the high-affinity IL-2R, was initially investigated in MS to blunt activated pathogenic CD25+ T cells [84, 85]. Unexpectedly, Daclizumab treatment led to only a marginal reduction in conventional CD4+ and CD8+ T-cell numbers and function, but elicited regulatory mechanisms involving CD56bright NK cells [84, 86]. Blocking the migration of T cells (as well as other leukocytes) from the blood to the CNS using anti-integrin monoclonal antibodies or a structural analog of sphingosine has proven highly successful [87, 88]. Indeed, the use of anti-α4 integrin mAb, which inhibits VLA/VCAM1 interaction, or functional antagonists of sphingosine1-phosphate (S1P) receptors, which prevent lymphocyte exit from secondary lymphoid organs into the blood, have produced remarkable therapeutic advances for people with MS [87, 88]. It is reasonable to predict that other CNS inflammatory diseases could benefit from this therapeutic approach. However, the weak selectivity of this approach remains a major downside, putting, inter alia, the CNS at risk of opportunistic infections. More selective strategies, which interfere with given T-cell subsets, such as Th17 or cytotoxic CD8+ T cells, will rely on the growing knowledge on the molecular cues governing their transmigration into the CNS. This idea is nevertheless dependent upon two key parameters: (i) the identification of selective/preferential pathways for given T-cell subsets; and (ii) the absence of molecular redundancy for their penetration into the CNS. The inhibition of effector T-cell entry to the CNS by the CCR5 antagonist, maraviroc, in patients with PML-IRIS may be a first encouraging example of a strategy that addresses both parameters [25, 26]. Selective inhibition of given immune molecules has been a major therapeutic revolution. The use of monoclonal antibodies targeting soluble T-cell mediators, in particular cytokines, or their receptors is a novel therapeutical approach for patients with CNS inflammatory diseases. mAbs neutralizing IL-17A or GM-CSF have been tested in small trials with MS patients with suggestive efficacy [89, 90]. Blocking IFN-γ in MS, as well as in other neurological diseases (Table 1) could be another therapeutical approach. Indeed, IFN-γ is strongly expressed in active MS lesions and intravenous injection of recombinant IFN-γ has been shown to increase MS exacerbation rate [91]. Moreover, the administration of neutralizing anti-IFN-γ mAb could be used to treat patients with hemophagocytic lymphohistiocytosis [92]. However, the severe opportunistic infections that have been shown to develop mostly in Asian patients who had been treated with autoantibodies neutralizing IFN-γ raise serious concerns for the prolonged use of this strategy [93]. The inhibition of key effector molecules within T cells, such as Granzyme B and IL-17, represents exciting novel strategies for future development. Pharmacologic inhibitors of C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim master transcription factors involved in T-cell differentiation are being developed. For instance, siRNAs target precise molecules within cells, but their efficient delivery to the desired cell population in vivo remains a challenge [94]. The use of ‘smart ligands’, such as aptamers targeting a specific surface receptor linked to siRNA, may help solve this challenge [95]. The ideal goal in the treatment of CNS autoimmune diseases would be to precisely neutralize the pathogenic autoreactive T cells, instead of deleting large proportions of T cells or targeting molecules needed for a protective immune response. Spectacular results in preclinical animal models of MS with antigen-specific tolerance protocols have been presented [96], supporting this therapeutic approach. However, this has not yet met clinical expectations, possibly due to the ill-defined nature of both the target autoantigens and the pathogenic lymphocyte subsets. Nevertheless, innovative antigen-specific strategies, such as myelin peptide coupled to cells or nanobeads are being followed in MS [97, 98]. Conclusion In this review, we have focused on the interactions between T cells and neurons and their consequences in CNS disease. In recent years, the role of T cells in tissue damage (and repair) has been increasingly investigated, given the possible therapeutic implications. Nevertheless, the molecular mechanisms through which each T-cell subset mediates neuronal loss or survival remain largely unclear. Until now, T-cell-associated CNS diseases, such as MS, are gradually responsive to drugs blocking T-cell entry into the CNS. However, more precise therapeutic approaches blocking specific T-cell functions in the CNS are still needed. The emerging knowledge on the genetic bases of CNS inflammatory diseases will, in all likelihood, help design more specific therapies. Acknowledegment: This work was supported by FAPESP 2013/22196-4 (LY), by INSERM, CNRS, Toulouse III University (RL), and grants from ARSEP, ANR T cell-CNS and the European Union, FP7-PEOPLE-2012-ITN NeuroKine (RL). Conflict of interest: The authors declare no financial or commercial conflict of interest. References 1 Bechmann, I., Galea, I. and Perry, V. H., What is the blood-brain barrier (not)? Trends Immunol. 2007. 28: 5–11. 2 Billingham, R. E. and Boswell, T., Studies on the problem of corneal homografts. Proc. R Soc. Lond. B Biol. Sci. 1953. 141: 392–406. www.eji-journal.eu 2717 2718 Lidia Yshii et al. 3 Chitnis, T., Imitola, J., Wang, Y., Elyaman, W., Chawla, P., Sharuk, M., Raddassi, K. et al., Elevated neuronal expression of CD200 protects Wlds mice from inflammation-mediated neurodegeneration. Am. J. Pathol. 2007. 170: 1695–1712. 4 Ransohoff, R. M. and Engelhardt, B., The anatomical and cellular basis of immune surveillance in the central nervous system. Nat. Rev. Immunol. 2012. 12: 623–635. Eur. J. Immunol. 2015. 45: 2712–2720 routes into human CNS involving PSGL-1 rolling of T cells and MCAMadhesion of TH17 cells. J. Exp. Med. 2014. 211: 1833–1846. 22 Rothhammer, V., Heink, S., Petermann, F., Srivastava, R., Claussen, M. C., Hemmer, B. and Korn, T., Th17 lymphocytes traffic to the central nervous system independently of alpha4 integrin expression during EAE. J. Exp. Med. 2011. 208: 2465–2476. 23 Reboldi, A., Coisne, C., Baumjohann, D., Benvenuto, F., Bottinelli, D., 5 Louveau, A., Smirnov, I., Keyes, T. J., Eccles, J. D., Rouhani, S. J., Peske, Lira, S., Uccelli, A. et al., C-C chemokine receptor 6-regulated entry of J. D., Derecki, N. C. et al., Structural and functional features of central TH-17 cells into the CNS through the choroid plexus is required for the nervous system lymphatic vessels. Nature 2015. 523: 337–341. initiation of EAE. Nat. Immunol. 2009. 10: 514–523. 6 Aspelund, A., Antila, S., Proulx, S. T., Karlsen, T. V., Karaman, S., Det- 24 Glatigny, S., Duhen, R., Arbelaez, C., Kumari, S. and Bettelli, E., Integrin mar, M., Wiig, H. et al., A dural lymphatic vascular system that drains alpha L controls the homing of regulatory T cells during CNS autoimmu- brain interstitial fluid and macromolecules. J. Exp. Med. 2015. 212: 991– nity in the absence of integrin alpha 4. Sci. Rep. 2015. 5: 7834. 999. 25 Martin-Blondel, G., Bauer, J., Uro-Coste, E., Biotti, D., Averseng- 7 Liblau, R. S., Gonzalez-Dunia, D., Wiendl, H. and Zipp, F., Neurons as Peaureaux, D., Fabre, N., Dumas, H. et al., Therapeutic use of CCR5 antag- targets for T cells in the nervous system. Trends Neurosci. 2013. 36: 315– onists is supported by strong expression of CCR5 on CD8(+) T cells in 324. progressive multifocal leukoencephalopathy-associated immune recon- 8 Waisman, A., Liblau, R. S. and Becher, B., Innate and adaptive immune responses in the CNS. Lancet Neurol. 2015. 14: 945–955. 9 Mahad, D. H., Trapp, B. D. and Lassmann, H., Pathological mechanisms in progressive multiple sclerosis. Lancet Neurol. 2015. 14: 183–193. 10 McConnell, M. J., Huang, Y. H., Datwani, A. and Shatz, C. J., H2-K(b) and H2-D(b) regulate cerebellar long-term depression and limit motor learning. Proc. Natl. Acad. Sci. USA 2009. 106: 6784–6789. 11 Needleman, L. A., Liu, X. B., El-Sabeawy, F., Jones, E. G. and McAllister, A. K., MHC class I molecules are present both pre- and postsynaptically in the visual cortex during postnatal development and in adulthood. Proc. Natl. Acad. Sci. USA 2010. 107: 16999–17004. 12 Joly, E. and Oldstone, M. B., Neuronal cells are deficient in loading peptides onto MHC class I molecules. Neuron 1992. 8: 1185–1190. stitution inflammatory syndrome. Acta Neuropathol. 2015. 129: 463–465. 26 Giacomini, P. S., Rozenberg, A., Metz, I., Araujo, D., Arbour, N., Bar-Or, A. and Maraviroc in Multiple Sclerosis-Associated, P. M. L. I. G., Maraviroc and JC virus-associated immune reconstitution inflammatory syndrome. N. Engl. J. Med. 2014. 370: 486–488. 27 Glass, W. G., McDermott, D. H., Lim, J. K., Lekhong, S., Yu, S. F., Frank, W. A., Pape, J. et al., CCR5 deficiency increases risk of symptomatic West Nile virus infection. J. Exp. Med. 2006. 203: 35–40. 28 Hammarberg, H., Lidman, O., Lundberg, C., Eltayeb, S. Y., Gielen, A. W., Muhallab, S., Svenningsson, A. et al., Neuroprotection by encephalomyelitis: rescue of mechanically injured neurons and neurotrophin production by CNS-infiltrating T and natural killer cells. J. Neurosci. 2000. 20: 5283–5291. 29 Fargali, S., Sadahiro, M., Jiang, C., Frick, A. L., Indall, T., Cogliani, V., 13 Hoftberger, R., Aboul-Enein, F., Brueck, W., Lucchinetti, C., Rodriguez, Welagen, J. et al., Role of neurotrophins in the development and function M., Schmidbauer, M., Jellinger, K. et al., Expression of major histocom- of neural circuits that regulate energy homeostasis. J. Mol. Neurosci. 2012. patibility complex class I molecules on the different cell types in multiple sclerosis lesions. Brain Pathol. 2004. 14: 43–50. 48: 654–659. 30 Kerschensteiner, M., Gallmeier, E., Behrens, L., Leal, V. V., Misgeld, T., 14 Cebrian, C., Zucca, F. A., Mauri, P., Steinbeck, J. A., Studer, L., Scherzer, C. Klinkert, W. E., Kolbeck, R. et al., Activated human T cells, B cells, and R., Kanter, E. et al., MHC-I expression renders catecholaminergic neurons monocytes produce brain-derived neurotrophic factor in vitro and in susceptible to T-cell-mediated degeneration. Nat. Commun. 2014. 5: 3633. inflammatory brain lesions: a neuroprotective role of inflammation? J. 15 Chevalier, G., Suberbielle, E., Monnet, C., Duplan, V., Martin-Blondel, G., Exp. Med. 1999. 189: 865–870. Farrugia, F., Le Masson, G., Liblau, R. et al., Neurons are MHC class I- 31 Flugel, A., Schwaiger, F. W., Neumann, H., Medana, I., Willem, M., Wek- dependent targets for CD8 T cells upon neurotropic viral infection. PLoS erle, H., Kreutzberg, G. W. et al., Neuronal FasL induces cell death of Pathog. 2011. 7: e1002393. 16 Engelhardt, B. and Ransohoff, R. M., Capture, crawl, cross: the T cell code to breach the blood-brain barriers. Trends Immunol. 2012. 33: 579–589. 17 Yednock, T. A., Cannon, C., Fritz, L. C., Sanchez-Madrid, F., Steinman, L. and Karin, N., Prevention of experimental autoimmune encephalomyeli- encephalitogenic T lymphocytes. Brain Pathol. 2000. 10: 353–364. 32 Liu, Y., Teige, I., Birnir, B. and Issazadeh-Navikas, S., Neuron-mediated generation of regulatory T cells from encephalitogenic T cells suppresses EAE. Nat. Med. 2006. 12: 518–525. 33 Cautain, B., Damoiseaux, J., Bernard, I., van Straaten, H., van Breda tis by antibodies against alpha 4 beta 1 integrin. Nature 1992. 356: 63–66. Vriesman, P., Boneu, B., Druet, P. et al., Essential role of TGF-beta in 18 Young, K. G., Maclean, S., Dudani, R., Krishnan, L. and Sad, S., CD8+ the natural resistance to experimental allergic encephalomyelitis in rats. T cells primed in the periphery provide time-bound immune-surveillance to the central nervous system. J. Immunol. 2011. 187: 1192–1200. 19 Ifergan, I., Kebir, H., Alvarez, J. I., Marceau, G., Bernard, M., Bourbon- Eur. J. Immunol. 2001. 31: 1132–1140. 34 McGeachy, M. J., Stephens, L. A. and Anderton, S. M., Natural recovery and protection from autoimmune encephalomyelitis: contribution niere, L., Poirier, J. et al., Central nervous system recruitment of effector of CD4+CD25+ regulatory cells within the central nervous system. J. memory CD8+ T lymphocytes during neuroinflammation is dependent Immunol. 2005. 175: 3025–3032. on alpha4 integrin. Brain 2011. 134: 3560–3577. 35 Korn, T., Reddy, J., Gao, W., Bettelli, E., Awasthi, A., Petersen, T. R., 20 Glatigny, S., Duhen, R., Oukka, M. and Bettelli, E., Cutting edge: loss of Backstrom, B. T. et al., Myelin-specific regulatory T cells accumulate in alpha4 integrin expression differentially affects the homing of Th1 and the CNS but fail to control autoimmune inflammation. Nat. Med. 2007. Th17 cells. J. Immunol. 2011. 187: 6176–6179. 13: 423–431. 21 Schneider-Hohendorf, T., Rossaint, J., Mohan, H., Boning, D., Breuer, J., 36 Liu, Y., Carlsson, R., Comabella, M., Wang, J., Kosicki, M., Carrion, B., Kuhlmann, T., Gross, C. C. et al., VLA-4 blockade promotes differential Hasan, M. et al., FoxA1 directs the lineage and immunosuppressive C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu HIGHLIGHTS Eur. J. Immunol. 2015. 45: 2712–2720 properties of a novel regulatory T cell population in EAE and MS. Nat. Med. 2014. 20: 272–282. 37 Lee, Y. H., Ishida, Y., Rifa’i, M., Shi, Z., Isobe, K. and Suzuki, H., Essential role of CD8+CD122+ regulatory T cells in the recovery from experimental autoimmune encephalomyelitis. J. Immunol. 2008. 180: 825–832. 38 Walsh, J. T., Hendrix, S., Boato, F., Smirnov, I., Zheng, J., Lukens, J. R., Gadani, S. et al., MHCII-independent CD4+ T cells protect injured CNS neurons via IL-4. J. Clin. Invest. 2015. 125: 699–714. 39 Raposo, C., Graubardt, N., Cohen, M., Eitan, C., London, A., Berkutzki, T. and Schwartz, M., CNS repair requires both effector and regulatory T cells with distinct temporal and spatial profiles. J. Neurosci. 2014. 34: 10141–10155. 40 Kunis, G., Baruch, K., Miller, O. and Schwartz, M., Immunization with a myelin-derived antigen activates the brain’s choroid plexus for recruitment of immunoregulatory cells to the CNS and attenuates disease progression in a mouse model of ALS. J. Neurosci. 2015. 35: 6381–6393. 53 Blachere, N. E., Orange, D. E., Santomasso, B. D., Doerner, J., Foo, P. K., Herre, M., Fak, J. et al., T cells targeting a neuronal paraneoplastic antigen mediate tumor rejection and trigger CNS autoimmunity with humoral activation. Eur. J. Immunol. 2014. 44: 3240–3251. 54 Peterson, J. W., Bo, L., Mork, S., Chang, A. and Trapp, B. D., Transected neurites, apoptotic neurons, and reduced inflammation in cortical multiple sclerosis lesions. Ann. Neurol. 2001. 50: 389–400. 55 Albert, M. L., Darnell, J. C., Bender, A., Francisco, L. M., Bhardwaj, N. and Darnell, R. B., Tumor-specific killer cells in paraneoplastic cerebellar degeneration. Nat. Med. 1998. 4: 1321–1324. 56 International Multiple Sclerosis Genetics, C., Beecham, A. H., Patsopoulos, N. A., Xifara, D. K., Davis, M. F., Kemppinen, A., Cotsapas, C. et al., Analysis of immune-related loci identifies 48 new susceptibility variants for multiple sclerosis. Nat. Genet. 2013. 45: 1353–1360. 57 Ascherio, A., Munger, K. L. and Lunemann, J. D., The initiation and prevention of multiple sclerosis. Nat. Rev. Neurol. 2012. 8: 602–612. 41 Endo, F., Komine, O., Fujimori-Tonou, N., Katsuno, M., Jin, S., Watanabe, 58 Schirmer, L., Antel, J. P., Bruck, W. and Stadelmann, C., Axonal loss and S., Sobue, G. et al., Astrocyte-derived TGF-beta1 accelerates disease pro- neurofilament phosphorylation changes accompany lesion development gression in ALS mice by interfering with the neuroprotective functions and clinical progression in multiple sclerosis. Brain Pathol. 2011. 21: 428– of microglia and T cells. Cell Rep. 2015. 11: 592–604. 440. 42 Stevens, J. G., Wagner, E. K., Devi-Rao, G. B., Cook, M. L. and Feldman, L. 59 Ben-Nun, A., Kaushansky, N., Kawakami, N., Krishnamoorthy, G., Berer, T., RNA complementary to a herpesvirus alpha gene mRNA is prominent K., Liblau, R., Hohlfeld, R. et al., From classic to spontaneous and human- in latently infected neurons. Science 1987. 235: 1056–1059. ized models of multiple sclerosis: impact on understanding pathogenesis 43 Theil, D., Derfuss, T., Paripovic, I., Herberger, S., Meinl, E., Schueler, and drug development. J. Autoimmun. 2014. 54: 33–50. O., Strupp, M. et al., Latent herpesvirus infection in human trigeminal 60 Lucchinetti, C. F., Popescu, B. F., Bunyan, R. F., Moll, N. M., Roemer, ganglia causes chronic immune response. Am. J. Pathol. 2003. 163: 2179– S. F., Lassmann, H., Bruck, W. et al., Inflammatory cortical demyeli- 2184. 44 van Velzen, M., Jing, L., Osterhaus, A. D., Sette, A., Koelle, D. M. and Verjans, G. M., Local CD4 and CD8 T-cell reactivity to HSV-1 anti- nation in early multiple sclerosis. N. Engl. J. Med. 2011. 365: 2188– 2197. 61 Babbe, H., Roers, A., Waisman, A., Lassmann, H., Goebels, N., Hohlfeld, gens documents broad viral protein expression and immune compe- R., Friese, M. et al., Clonal expansions of CD8(+) T cells dominate the tence in latently infected human trigeminal ganglia. PLoS Pathog. 2013. 9: T cell infiltrate in active multiple sclerosis lesions as shown by microma- e1003547. nipulation and single cell polymerase chain reaction. J. Exp. Med. 2000. 45 Knickelbein, J. E., Khanna, K. M., Yee, M. B., Baty, C. J., Kinchington, P. 192: 393–404. R. and Hendricks, R. L., Noncytotoxic lytic granule-mediated CD8+ T cell 62 Kostic, M., Dzopalic, T., Zivanovic, S., Zivkovic, N., Cvetanovic, A., Sto- inhibition of HSV-1 reactivation from neuronal latency. Science 2008. 322: janovic, I., Vojinovic, S. et al., IL-17 and glutamate excitotoxicity in 268–271. 46 Shrestha, B. and Diamond, M. S., Role of CD8+ T cells in control of West Nile virus infection. J. Virol. 2004. 78: 8312–8321. 47 Shrestha, B. and Diamond, M. S., Fas ligand interactions contribute to CD8+ T-cell-mediated control of West Nile virus infection in the central nervous system. J. Virol. 2007. 81: 11749–11757. 48 Shrestha, B., Pinto, A. K., Green, S., Bosch, I. and Diamond, M. S., CD8+ T cells use TRAIL to restrict West Nile virus pathogenesis by controlling infection in neurons. J. Virol. 2012. 86: 8937–8948. 49 Zhang, B., Chan, Y. K., Lu, B., Diamond, M. S. and Klein, R. S., CXCR3 mediates region-specific antiviral T cell trafficking within the central nervous system during West Nile virus encephalitis. J. Immunol. 2008. 180: 2641–2649. 50 Kreutzfeldt, M., Bergthaler, A., Fernandez, M., Bruck, W., Steinbach, K., Vorm, M., Coras, R. et al., Neuroprotective intervention by interferongamma blockade prevents CD8+ T cell-mediated dendrite and synapse loss. J. Exp. Med. 2013. 210: 2087–2103. 51 Mizuno, T., Zhang, G., Takeuchi, H., Kawanokuchi, J., Wang, J., Sonobe, Y., Jin, S. et al., Interferon-gamma directly induces neurotoxicity through a neuron specific, calcium-permeable complex of IFN-gamma receptor and AMPA GluR1 receptor. FASEB J. 2008. 22: 1797–1806. 52 Steinman, L., Conflicting consequences of immunity to cancer versus autoimmunity to neurons: insights from paraneoplastic disease. Eur. J. Immunol. 2014. 44: 3201–3205. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim the pathogenesis of multiple sclerosis. Scand. J. Immunol. 2014. 79: 181– 186. 63 Tzartos, J. S., Friese, M. A., Craner, M. J., Palace, J., Newcombe, J., Esiri, M. M. and Fugger, L., Interleukin-17 production in central nervous systeminfiltrating T cells and glial cells is associated with active disease in multiple sclerosis. Am. J. Pathol. 2008. 172: 146–155. 64 Siffrin, V., Radbruch, H., Glumm, R., Niesner, R., Paterka, M., Herz, J., Leuenberger, T. et al., In vivo imaging of partially reversible th17 cellinduced neuronal dysfunction in the course of encephalomyelitis. Immunity 2010. 33: 424–436. 65 Aktas, O., Smorodchenko, A., Brocke, S., Infante-Duarte, C., Schulze Topphoff, U., Vogt, J., Prozorovski, T. et al., Neuronal damage in autoimmune neuroinflammation mediated by the death ligand TRAIL. Neuron 2005. 46: 421–432. 66 Saxena, A., Martin-Blondel, G., Mars, L. T. and Liblau, R. S., Role of CD8 T cell subsets in the pathogenesis of multiple sclerosis. FEBS Lett. 2011. 585: 3758–3763. 67 Huseby, E. S., Liggitt, D., Brabb, T., Schnabel, B., Ohlen, C. and Goverman, J., A pathogenic role for myelin-specific CD8(+) T cells in a model for multiple sclerosis. J. Exp. Med. 2001. 194: 669–676. 68 Friese, M. A., Jakobsen, K. B., Friis, L., Etzensperger, R., Craner, M. J., McMahon, R. M., Jensen, L. T. et al., Opposing effects of HLA class I molecules in tuning autoreactive CD8+ T cells in multiple sclerosis. Nat. Med. 2008. 14: 1227–1235. www.eji-journal.eu 2719 2720 Lidia Yshii et al. Eur. J. Immunol. 2015. 45: 2712–2720 69 Sorbara, C. D., Wagner, N. E., Ladwig, A., Nikic, I., Merkler, D., Kleele, 86 Bielekova, B., Catalfamo, M., Reichert-Scrivner, S., Packer, A., Cerna, M., T., Marinkovic, P. et al., Pervasive axonal transport deficits in multiple Waldmann, T. A., McFarland, H. et al., Regulatory CD56(bright) natural sclerosis models. Neuron 2014. 84: 1183–1190. killer cells mediate immunomodulatory effects of IL-2Ralpha-targeted 70 Darnell, J. C., Albert, M. L. and Darnell, R. B., Cdr2, a target antigen of naturally occuring human tumor immunity, is widely expressed in gynecological tumors. Cancer Res. 2000. 60: 2136–2139. 71 Verschuuren, J., Chuang, L., Rosenblum, M. K., Lieberman, F., Pryor, A., Posner, J. B. and Dalmau, J., Inflammatory infiltrates and complete absence of Purkinje cells in anti-Yo-associated paraneoplastic cerebellar degeneration. Acta Neuropathol. 1996. 91: 519–525. 72 Dalmau, J. and Rosenfeld, M. R., Paraneoplastic syndromes of the CNS. Lancet Neurol. 2008. 7: 327–340. 73 Schubert, M., Panja, D., Haugen, M., Bramham, C. R. and Vedeler, C. A., Paraneoplastic CDR2 and CDR2L antibodies affect Purkinje cell calcium homeostasis. Acta Neuropathol. 2014. 128: 835–852. 74 Giometto, B., Grisold, W., Vitaliani, R., Graus, F., Honnorat, J., Bertolini, G. and Euronetwork, P. N. S., Paraneoplastic neurologic syndrome in the PNS Euronetwork database: a European study from 20 centers. Arch. Neurol. 2010. 67: 330–335. 75 Aye, M. M., Kasai, T., Tashiro, Y., Xing, H. Q., Shirahama, H., Mitsuda, M., Suetsugu, T. et al., CD8 positive T-cell infiltration in the dentate nucleus of paraneoplastic cerebellar degeneration. J. Neuroimmunol. 2009. 208: 136–140. 76 Albert, M. L., Austin, L. M. and Darnell, R. B., Detection and treatment of activated T cells in the cerebrospinal fluid of patients with paraneoplastic cerebellar degeneration. Ann. Neurol. 2000. 47: 9–17. 77 Brochard, V., Combadiere, B., Prigent, A., Laouar, Y., Perrin, A., BerayBerthat, V., Bonduelle, O. et al., Infiltration of CD4+ lymphocytes into the brain contributes to neurodegeneration in a mouse model of Parkinson disease. J. Clin. Invest. 2009. 119: 182–192. 78 Biber, K., Owens, T. and Boddeke, E., What is microglia neurotoxicity (Not)? Glia 2014. 62: 841–854. 79 Cohen, J. A., Coles, A. J., Arnold, D. L., Confavreux, C., Fox, E. J., Hartung, H. P., Havrdova, E. et al., Alemtuzumab versus interferon beta 1a as firstline treatment for patients with relapsing-remitting multiple sclerosis: a randomised controlled phase 3 trial. Lancet 2012. 380: 1819–1828. 80 Coles, A. J., Twyman, C. L., Arnold, D. L., Cohen, J. A., Confavreux, C., Fox, E. J., Hartung, H. P. et al., Alemtuzumab for patients with relapsing multiple sclerosis after disease-modifying therapy: a randomised controlled phase 3 trial. Lancet 2012. 380: 1829–1839. 81 Hill-Cawthorne, G. A., Button, T., Tuohy, O., Jones, J. L., May, K., Somerfield, J., Green, A. et al., Long term lymphocyte reconstitution after alemtuzumab treatment of multiple sclerosis. J. Neurol. Neurosurg. Psychiatry 2012. 83: 298–304. 82 Zhang, X., Tao, Y., Chopra, M., Ahn, M., Marcus, K. L., Choudhary, N., Zhu, H. et al., Differential reconstitution of T cell subsets following immunodepleting treatment with alemtuzumab (anti-CD52 monoclonal antibody) in patients with relapsing-remitting multiple sclerosis. J. Immunol. 2013. 191: 5867–5874. 83 Jones, J. L., Anderson, J. M., Phuah, C. L., Fox, E. J., Selmaj, K., Margolin, D., Lake, S. L. et al., Improvement in disability after alemtuzumab treat- therapy (daclizumab) in multiple sclerosis. Proc. Natl. Acad. Sci. USA 2006. 103: 5941–5946. 87 Polman, C. H., O’Connor, P. W., Havrdova, E., Hutchinson, M., Kappos, L., Miller, D. H., Phillips, J. T. et al., A randomized, placebo-controlled trial of natalizumab for relapsing multiple sclerosis. N. Engl. J. Med. 2006. 354: 899–910. 88 Kappos, L., Radue, E. W., O’Connor, P., Polman, C., Hohlfeld, R., Calabresi, P., Selmaj, K. et al., A placebo-controlled trial of oral fingolimod in relapsing multiple sclerosis. N. Engl. J. Med. 2010. 362: 387–401. 89 Constantinescu CS, A. A., Fryze, W. Safety and pharmacokinetics of MOR103, a human antibody to granulocyte-macrophage colonystimulating factor, in patients with multiple sclerosis ACTRIMS-ECTRIMS 2014, pp Abstract FC 2.4. 90 Havrdova, E., Belova, A., Goloborodko, A., Tisserant, A., Jones, I., Garren, H. and Johns, D., Positive proof of concept of AIN457, an antibody against interleukin-17A, in relapsing-remitting multiple sclerosis Actrims. Multiple Scler. J. 2012, 18: 513–513. 91 Panitch, H. S., Hirsch, R. L., Haley, A. S. and Johnson, K. P., Exacerbations of multiple sclerosis in patients treated with gamma interferon. Lancet 1987. 1: 893–895. 92 Agency, E. M., Public summary of opinion on orphan designation: Recombinant human anti-interferon gamma monoclonal antibody for the treatment of haemophagocytic lymphohistiocytosis European Medicines Agency. European Medicines Agency, Committee for Orphan Medicinal Products, London, United Kingdom 2010. 93 Browne, S. K., Burbelo, P. D., Chetchotisakd, P., Suputtamongkol, Y., Kiertiburanakul, S., Shaw, P. A., Kirk, J. L. et al., Adult-onset immunodeficiency in Thailand and Taiwan. N. Engl. J. Med. 2012. 367: 725–734. 94 Keefe, A. D., Pai, S. and Ellington, A., Aptamers as therapeutics. Nat. Rev. Drug Discov. 2010. 9: 537–550. 95 Wang, C. W., Chung, W. H., Cheng, Y. F., Ying, N. W., Peck, K., Chen, Y. T. and Hung, S. I., A new nucleic acid-based agent inhibits cytotoxic T lymphocyte-mediated immune disorders. J. Allergy Clin. Immunol. 2013. 132: 713–722 e711. 96 Anderton, S., Burkhart, C., Metzler, B. and Wraith, D., Mechanisms of central and peripheral T-cell tolerance: lessons from experimental models of multiple sclerosis. Immunol. Rev. 1999. 169: 123–137. 97 Lutterotti, A., Yousef, S., Sputtek, A., Sturner, K. H., Stellmann, J. P., Breiden, P., Reinhardt, S. et al., Antigen-specific tolerance by autologous myelin peptide-coupled cells: a phase 1 trial in multiple sclerosis. Sci. Transl. Med. 2013. 5: 188ra175. 98 Getts, D. R., Shea, L. D., Miller, S. D. and King, N. J., Harnessing nanoparticles for immune modulation. Trends Immunol. 2015. 36: 419–427. Full correspondence: Proof. Roland Liblau, INSERM U1043 - CNRS UMR 5282, Centre de Physiopathologie Toulouse-Purpan, Purpan Hospital, Place du Docteur Baylac TSA 40031, 31059 Toulouse Cedex 9, France Fax: +33 561 77 21 38 e-mail: [email protected] ment of multiple sclerosis is associated with neuroprotective autoimmunity. Brain 2010. 133: 2232–2247. 84 Pfender, N. and Martin, R., Daclizumab (anti-CD25) in multiple sclerosis. See all the ECI 2015 Reviews at http://onlinelibrary.wiley.com/journal/ 10.1002/(ISSN)1521-4141/homepage/eci2015.htm Exp. Neurol. 2014. 262 Pt A: 44–51. 85 Gold, R., Giovannoni, G., Selmaj, K., Havrdova, E., Montalban, X., Radue, E. W., Stefoski, D. et al., Daclizumab high-yield process in relapsing-remitting multiple sclerosis (SELECT): a randomised, doubleblind, placebo-controlled trial. 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