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DATI 1° AUTORE PER LA CORRISPONDENZA:
Nome: WALTER
Cognome: ARANCIO
Data di nascita 19 maggio 1977
Ente di appartenenza: Università degli studi di Palermo
CF:RNCWTR77E19G273D
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Anaplastic Large T Cell Lymphoma (ALCL) is characterized by high expression of P-Selectin
Glycoprotein Ligand 1 (PSGL-1) that positively correlates with CD30 expression and TCR signaling
pathway.
Walter Arancio1, Alessandro Gulino1, Valeria Cancila1, Pier Paolo Piccaluga2, Paolo Macor3, Arianna di
Napoli4, Sabina Sangaletti5, Ada Maria florena6.
1
Laboratorio di Immunologia dei Tumori, ProSAMI, Università degli Studi di Palermo.
Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, ALMA MATER STUDIORUM - Università di Bologna.
3
Dipartimento di Scienze della Vita, Università degli Studi di Trieste.
4
Dipartimento di Medicina Clinica E Molecolare, Università degli Studi di Roma "La Sapienza".
5
Struttura Complessa Immunologia Molecolare, Fondazione IRCSS Istituto Nazionale dei Tumori, Milano.
6
Scienze per la Promozione della Salute e Materno Infantile "G.D'Alessandro” Università degli Studi di Palermo.
2
INTRODUCTION: _________________________________________________________________________
P-selectin glycoprotein ligand-1 (PSGL-1), coded by the SELPLG gene, is one of the major ligands of selectins,
it is normally expressed in microvilli of polymorphonucleated leukocytes, monocytes, activated T-lymphocytes,
plasma cells and activated platelets, where it has a major role in regulating the tethering, rolling and
extravasation(1). It may bind also von Willerbrand factor (vWF) and chemokines such as CCL21 and CCL19 to
facilitate T-cell lymph node entry.
PSGL-1 is a homodimeric disulfide-linked glycoprotein, which is highly post-translationally modified. It may
bind with variable affinity to its different ligands depending on the status of its modofications.
PSGL-1 cytoplasmic domain transduces signals upon activation via spleen tyrosine kinase (Syk) after clustering
within lipid rafts; the signaling leads to the secretion of cytokines or the activation of membrane integrins to
promote extravasation. Noteworthy, PSGL-1 is involved in core molecular programs such as Syk, PLC2,
PI3K or MAPK pathways that let envisage a role for this molecule beyond cell adhesion and extravasation.
The cross-linking of PSGL-1 in activated T cells is able to induce apoptosis, and PSGL-1 acts as an immune
checkpoint regulator that promoting T cell exhaustion also through Programmed-cell death-protein-1 (PD-1)
upregulation, and IL-2 signaling quencing (2,3).
PSGL-1 expression in pathological conditions has been reported in lymphoproliferative disorders showing
plasmacytic differentiation and in multiple myeloma, (MM) (4,5), where it has been suggested as a potential
target for humoral immunotherapy (4).
In T-cell lymphomas, the expression of PSGL-1 and its molecular correlates have been not investigated so far.
Most T-cell lymphomas are classified as peripheral T-cell lymphomas (PTCL) a heterogeneous group of
aggressive non-Hodgkin lymphomas (NHLs). The most common subtypes of PTCL, are PTCL not otherwise
specified (PTCL-NOS), anaplastic large-cell lymphoma (ALCL), and angioimmunoblastic T-cell lymphoma
(AITL). Up to date, no effective therapeutic targets have been identified, except for the TNF receptor CD30,
targeted by the anti-CD30 drug conjugated antibody Brentuximab-Vedotin (6).
In this study we investigated the expression of PSGL-1 and its molecular co-relates in T-cell lymphomas and
speculated on its suitability as functional target.
METHODS:_______________________________________________________________________________
Gene Expression Profiles (GEPs) have been examined in a panel of 180 T-cell lymphomas and control samples
and in a set of 498 B-cell lymphomas and control samples in order to evaluate the expression of selected genes
and their correlations. Gene set enrichment and cluster analyses have been performed. PSGL-1 expression has
been evaluated by Immuno-Histochemistry (IHC) on Tissue Macro Arrays (TMAs) comprising 110 ALCLs and
50 PTCLs-NOS. Immunolocalization has been performed as previously reported (4,5). CIBERSORT software
has been applied on GEP data in order to infer the enrichment of specific bystander immune elements. In vitro
experiments have been performed on Karpas229, SUDHL1, L82 and TS cell lines. Cellular viability was
assessed by MTT assay. Inhibition of adhesion was evaluated as the ability of cells to remain adherent to
endothelial Ea.hy926 cells. Capping of PSGL-1 was manually scored on confocal microscopy.
RESULTS:________________________________________________________________________________
We evaluated the expression of SELPLG in 678 samples, comprising both T and B cells, and lymphomatous
samples. SELPLG was first investigated in B-cell setting, being plasma cells neoplasms and naïve B-cells
positive and negative controls respectively. Among B-cell lymphomas, Classical Hodgking Lymphoma (CHL)
and T-cell/histiocyte-rich diffuse large B-cell Lymphoma(TCRBL) showed the highest expression of SELPLG.
Notably, these histotypes are characterized by a very rich environment comprising T cells and myeloid cells. In
the T cell setting consistent expression of SELPLG was detected, with ALCL showing the highest expression
(Fig.1). ALCL encompasses different clinical entities that histologically share the presence of large
pleomorphic T-cells expressing CD30. ALCLs can be classified according to the expression of Anaplastic
Lymphoma Kinase (ALK). Surprisingly, no statistically significant difference was detected in SELPLG
expression between ALK+ and ALK- ALCL specimens. Based on data from GEP analysis, we evaluated the
expression of PSGL-1 in TMAs of 160 PTCL, confirming that PSGL-1 was consistently highly expressed in
ALCL (Fig.2). Prompted by GEP and IHC analyses, where CD30+ ALCL and CHL resulted highly expressing
SELPLG/PSGL-1, we hypothesized that a positive correlation between SELPLG and TNFRSF8 (CD30 coding
gene) might occur. Our data confirmed this hypothesis. The correlation was observed both in T and B
lymphoma settings, suggesting that it is not cell-type intrinsic. Network analysis by Genemania pointed to six
genes potentially correlated with both SELPLG and TNFRSF8, namely MSC, SNX20, TNFRSF4, TNFRSF25,
TNIP1 and TRAF1. We next evaluated the genes that showed a positive Pearson’s product-moment correlation
(coefficient 0.75 to 1) with SELPLG in different settings. Strikingly, ALCL showed the highest number of
genes significantly correlated with SELPLG, which do not overlap with the other settings analyzed, suggesting
that SELPLG activity might be peculiar in ALCL. GSEA of such genes pointed to specific pathways and
cellular functions, many of which previously associated with cancer, including RNA splicing and RNA
processing functions. Subsequently, we tried to classify ALCLs according to SELPLG expression in order to
identify relevant differences in the transcriptional program related with SELPLG. Almost 4000 genes
differentially clustered together with high or low cases (Fig.3), TNIP1 (from Genemania) and T cell receptor
(TCR) signaling genes were among them. Among TCR signaling genes, LCK, LAT, ZAP70, SYK proved to be
correlated with SELPLG. These data suggest that PSGL-1 signaling correlates with TCR signaling in ALCL. In
order to functionally characterize PSGL-1 in ALCL cells, we first investigated its expression on cells with two
different antibodies: KPL1 (a known blocking antibody against PSGL-1) and TB5 (with non-blocking
functions). Coherently, KPL1 was able to interfere with ALCL adhesion in vitro, while TB5 was not effective.
As PSGL-1 required raft formation during signaling, we evaluated the capability of two antibodies to induce
capping and found that only TB5 was able to do so. Consistently, since ALCL has an activated phenotype, TB5
was effective in induce apoptosis though variably in the different ALCL cell lines. Finally, we evaluated via
CIBERSORT method if SELPLG expression might influence the microenvironment characterizing the
enrichment of specific bystander immune elements. ALCLs result enriched in Mast cells and neutrophils in
comparison with other PTCLs and normal controls, and this enrichment was particularly relevant in highlyexpressing SELPLG ALCLs.
Fig. 3
Fig. 1
Fig. 2
CONCLUSIONS:___________________________________________________________________________
SELPLG expression characterize T-cell lymphoma, in particular ALCL and it correlates with CD30 expression.
SELPLG expression correlates with relevant pathways such as TCR signaling.
In ALCL cell lines, PSGL-1 has a functionally expression that can be used to modulate adhesion, signaling and
death.
1.
Platelet/polymorphonuclear leukocyte interaction: P-selectin triggers protein-tyrosine phosphorylation-dependent
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2.
PSGL-1 Is an Immune Checkpoint Regulator that Promotes T Cell Exhaustion. Tinoco R, et Al. Immunity. 2016 May
17;44(5):1190-203
3.
In vivo regulation of Bcl6 and T follicular helper cell development. Poholek AC, et Al. J Immunol. 2010 Jul 1;185(1):313-26.
4.
P-selectin glycoprotein ligand-1 as a potential target for humoral immunotherapy of multiple myeloma. Tripodo C, et Al.
Curr Cancer Drug Targets. 2009 Aug;9(5):617-25.
5.
Identification of CD162 in plasma-cell dyscrasia.. Florena AM, et Al. Lancet Oncol. 2005 Aug;6(8):632..
6.
Antibody-drug conjugates for cancer therapy. Thomas A, et Al. Lancet Oncol. 2016 Jun;17(6):e254-62.