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UvA-DARE (Digital Academic Repository) Homeostasis and function of T cells in healthy individuals and renal transplant recipients Havenith, S.H.C. Link to publication Citation for published version (APA): Havenith, S. H. C. (2012). Homeostasis and function of T cells in healthy individuals and renal transplant recipients General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl) Download date: 19 Jun 2017 3 CXCR5+CD4+ follicular helper T cells accumulate in resting human lymph nodes and have superior B cell helper activity Simone H.C. Havenith*,†, Ester B.M. Remmerswaal*,†, Mirza M. Idu‡, Karlijn A.M.I. van Donselaar - van der Pant †, Nelly van der Bom*, Fréderike J. Bemelman†, Ester M.M. van Leeuwen*, Ineke J. M. ten Berge†, and René A. W. van Lier§ Department of Experimental Immunology, Academic Medical Center, Amsterdam, the Netherlands † Renal Transplant Unit, Department of Internal Medicine and ‡ Department of Surgery, Academic Medical Center, Amsterdam, the Netherlands, § Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, Amsterdam, the Netherlands. * 45 CD4 + T cells in human lymph nodes Abstract Although many relevant immune reactions are initiated in the lymph nodes, this compartment has not been systematically studied in humans. Analyses have been performed on immune cells derived from tonsils, but as this tissue is most often inflamed, generalization of these data is difficult. Here, we analyzed the phenotype and function of the human CD4+ T cell subsets and lineages in paired resting lymph node and peripheral blood samples. Naïve, central memory and effector memory cells as well as Th1, Th2, Th17 and Tregs were equally represented in both compartments. On the other hand, cytotoxic CD4+ T cells were strikingly absent in the lymph nodes. CXCR5+CD4+ T cells, representing putative follicular T helper (Tfh) cells were overrepresented in lymph nodes and expressed higher levels of Tfh markers than their peripheral blood counterparts. Compared to the circulating pool, lymph node derived CXCR5+CD4+ T cells were superior in providing help to B cells. Thus, functionally competent Tfh cells accumulate in resting human lymph nodes providing a swift induction of naïve and memory antibody responses upon antigenic challenge. 3 46 CD4 + T cells in human lymph nodes Introduction 3 CD4+ T cells play a critical role in forming adaptive immune responses against various pathogens 1-3. Furthermore, they are known to be involved in the pathogenesis of autoimmune diseases, asthma and allergic responses 4;5. CD4+ T cells consist of several different T helper (Th) lineages, including Th1, Th2, Th17 and regulatory T cells (Tregs) 6;7. Additionally, in CMV-infected individuals cytotoxic CD4+ T cells are found in the circulation 8-10. In recent years, follicular T helper cells (Tfh) have been identified as the CD4+ T cell subset specialized in supporting activation, expansion and differentiation of B cells and they are required for the formation of germinal centers (GCs) 11;12. Expression of CXCR5 and downregulation of CCR7 allows these cells to migrate from the T cell zone to the primary and secondary follicles, where they encounter antigen primed B cells 13;14. Human tonsillar Tfh cells express high initiate levels of ICOS, CD40L, and PD-1 15;16 and the transcription factor BCL-6 17;18 and they produce the B-helper cytokine IL-21 19. In the human peripheral blood (PB) CXCR5+CD4+ T cells have a CCR7+ central memory phenotype. However, these circulating CXCR5+CD4+ T cells lack the expression of ICOS, PD-1 and BCL-6 20. Although adaptive immune responses are initiated in the lymph nodes (LN), human CD4+ T cells and their different lineages have only been thoroughly studied in the peripheral blood (PB), also in relation to disease 21-23. The fact that the human secondary lymphoid compartment is rarely studied can be attributed to the difficulty in attaining lymphoid tissue. Tonsils, which are surgically removed because of recurrent or chronic inflammation, have been utilized in the past to study the secondary lymphoid compartment. However, because of the chronic inflammatory conditions, these studies likely reflect a skewed image. Data on the different Th lineages and in particular on Tfh cells in resting human LNs are still lacking. In the present study, we had the unique opportunity to study CD4+ T cell subsets in paired human LN and PB samples. We studied the presence of the different CD4+ T cell subsets and lineages in the LNs and compared this to their presence in the PB compartment. Furthermore, we analysed phenotypical, but also functional characteristics of CXCR5+ cells from human LN in comparison to the PB. Materials and methods Subjects We studied paired heparinized peripheral blood mononuclear cells (PBMC) and lymph node mononuclear cells (LNMC), which were isolated prior to, respectively during living donor kidney transplantation. EBV- and CMV-serostatus of the patients and their ages are set out in supplemental table 1. All patients were treated with quadruple immunosuppression, consisting of CD25mAb induction therapy, prednisolone, calcineurin-inhibitor and mycophenolic acid. Except for CD25mAb, immunosuppressive treatment was started immediately after transplantation. At the time the lymph nodes were gathered, the first dose of CD25mAb was already administered. However, we 47 CD4 + T cells in human lymph nodes have demonstrated that CD25mAb (basiliximab, Novartis Pharma, Basel, Switzerland) was not detectable in LNMC and that CD25 expression on LNMC could ex vivo be blocked with CD25mAb 24. The Medical Ethics Committee of the Academic Medical Center, Amsterdam, approved the study and all subjects gave written informed consent. Isolation of mononuclear cells from peripheral blood and lymph nodes PBMCs were isolated using standard density gradient centrifugation as described previously 25. Lymph nodes were collected from kidney transplant recipients during living donor kidney transplantation. Briefly, LNMCs were isolated from surgical residual material of the recipient, gathered during the implantation of the transplanted kidney. Before anastomising the arteria and vena renalis, the iliac artery and vein are dissected free. The residual tissue removed in this procedure to obtain adequate vascular exposure, often contains lymph nodes. Directly after extraction, the gathered lymph nodes were chopped in small pieces. A cell suspension was obtained by grinding the material through a flow-through chamber. PBMCs and LNMCs were subsequently cryopreserved until the day of analysis. Virological analysis To determine CMV serostatus, anti-CMV immunoglobulin G (IgG) was measured in serum using the AxSYM microparticle enzyme immunoassay (Abbott Laboratories, Abbott Park, IL, USA) according to the manufacturer’s instructions. Measurements were calibrated relative to a standard serum. EBV serostatus was determined by qualitative measurement of specific immunoglobulin G (IgG) against the viral capsid antigen (VCA) and against nuclear antigen of EBV using respectively the anti-EBV VCA IgG enzyme-linked immunosorbent assay and the anti-EBV nuclear antigen of EBV IgG enzyme-linked immunosorbent assay (Biotest, Dreieich, Germany). All tests were performed following the instructions of the manufacturers. Immunofluorescence staining; flow cytometry PBMCs and LNMCs were washed in phosphate-buffered saline (PBS) containing 0.01% NaN3 and 0.5% bovine serum albumin. Half a million PBMCs and LNMCs were incubated with fluorescence-labeled monoclonal antibodies (mAbs) for 30 minutes at 4°C, protected from light, at concentrations according to the manufacturer’s instructions. The following surface antibodies were used: CD8 V450, CD3 V500, CCR7 PE-Cy7, CD25 PE, CXCR5 Alexa Fluor 488, CXCR5 Alexa Fluor 647, ICOS PE (BD Biosciences, San Jose, CA, USA), CD27 APC-Alexa Fluor 780, CD4 PerCP-eFluor 710, CD45RA eFluor 605NC, PD1 PE (eBioscience Inc, San Diego, CA, USA), CX3CR1 FITC, CCR6 Alexa Fluor 647, CD69 Alexa Fluor 700 (BioLegend, San Diego, CA, USA), CXCR3 PE (R&D Systems, Abingdon, UK). For intracellular stainings, cells were fixed after surface staining with FACS Lysing Solution (BD Biosciences). Following permeabilization (FACS Permeabilizing Solution 2 (BD Biosciences)) cells were stained with Ki-67 FITC (BD Biosciences) or granzyme B PE (Sanquin) or granzyme K FITC (Immunotools, Friesoythe, Germany). For intracellular FOX-P3 stainings, cells were 3 48 CD4 + T cells in human lymph nodes 3 fixed and permeabilized using the Foxp3 Staining Buffer Set and subsequently stained with Foxp3 APC (both eBioscience). Live/Dead fixable red cell stain kit (Invitrogen, Paisley, UK) was used in every staining to exclude dead cells from the analysis. Cells were measured on a LSR-Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software (FlowJo, Ashland, OR, USA) Detection of cytokines Cytokine release after peptide or PMA/Ionomycin stimulation was performed as described by Lamoreaux et al 26, PBMCs and LNMCs were thawed and rested overnight in suspension flasks (Greiner) in RPMI supplemented with 10% FCS, penicilline and streptomycine (culture medium). Half a million cells were stimulated for 4 hours at 37ºC 5% CO2 with or without PMA (10ng/ml) and Ionomycin(1μg/ml) in culture medium, in the presence of αCD28 (15E8) (2μg/ml), αCD29 (TS 2/16) (1 μg/ml) (Sanquin, Amsterdam, The Netherlands), brefeldin A (Invitrogen) (10μg/ml) and Golgistop (BD Biosciences) in a final volume of 200μl. Stimulations were performed in non-treated round bottom 96-well plates (Corning). Subsequently, cells were washed twice and incubated with CD3 V500, CD8 V450, CD4 PE-Cy5.5 and Live/Dead fixable red cell stain for 30 min at 4ºC. Then, they were washed twice, fixed and permeabilized (Cytofix/Cytoperm reagent, BD) and subsequently incubated with a combination of the following intracellular mAbs: anti-IFNγ APC-Alexa Fluor 750 (Invitrogen), -IL-4 PE-Cy7, -IL-17A APC-Alexa Fluor 780, -IL-21 Alexa Fluor 647 (eBioscience), -IL-2 PE, -IL-5 PE (BioLegend) for 30 min at 4ºC. Cells were washed twice and measured on a LSR Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software (FlowJo). Cell sorting For isolation of CXCR5+ memory T cells, cells were labeled with CD3 PE (eBioscience), CD4 APC, CD45RA PE Cy7, CXCR5 AF488 (BD Biosciences) and CCR7 PerCPCy5.5 (BioLegend). For isolation of B cells, cells were labeled with CD19 PE and CD20 APC (BD Biosciences). Cells were sorted on a FACSARIA (BD Biosciences). Purity of the obtained sorted cells was verified by flowcytometry and was at least 95%. B cell co-culture and stimulation Sorted LN and PB derived CXCR5+CD4+ T cells were co-cultured with LN and PB derived B cells for 7 days. Cells were cultured in RPMI (Invitrogen) containing 10% fetal calf serum, 100U/ml sodium penicillin G (Brocades Pharma BV, Leiderdorp, The Netherlands) and 100ug/ml streptomycin sulphate (Invitrogen). For stimulation we added soluble 1ug/ml anti-CD3 (clone 1XE) and soluble 5ug/ml anti-CD28 (clone 15E8) to the cell culture. ELISA IgG IgM Supernatants were tested for secreted IgM and IgG with an in-house ELISA using polyclonal rabbit anti-human IgG and IgM reagents and a serum protein calibrator all from Dako (Heverlee, Belgium), as described previously in more detail 27. 49 CD4 + T cells in human lymph nodes Statistical analysis Statistical analysis of paired samples was done by two tailed Wilcoxon signed rank test with a 95% confidence interval. Results LN and PB contain equal percentages of naïve, central memory and effector memory CD4+ T cells We collected paired LN and PB samples in which we compared the presence of total CD4+ T cells and of naïve, central-memory and effector-memory CD4+ T cells. The percentage of CD4+ T cells within the CD3+ subset was similar in LNs and PB (figure 1A). We phenotypically distinguished naïve, central memory and effector memory CD4+ T cells based upon their expression of CCR7 and CD45RA 28. We demonstrated that CCR7+CD45RA+ naïve (Tn), CCR7+CD45RA- central memory (Tcm) and CCR7- effector memory (Tem) CD4+ T cells were equally represented in LN and PB (figure 1B). Th1, Th2, Th17 and Tregs are evenly divided over the LN and PB compartment To learn more about the distribution of the different T helper cell lineages over LN and PB, we analysed the chemokine-receptor expression pattern and cytokine producing capacity of the CD4+ T cells in both compartments. The chemokine-receptors CXCR3 and CCR6 can help distinguish between Th1, Th2 and Th17 cells 29-33. We found that the percentages of CXCR3+CCR6- Th1, CXCR3-CCR6- Th2 and CXCR3-CCR6+ Th17 memory CD4+ T cells were equal in PB and LN (figure 2A and B). Markedly, the Figure 1) CD4+ T cells are equally represented in LN and PB. (A) CD4+ T cells as a percentage of total T cells in LN compared to PB. (B) Percentage of CCR7+CD45RA+ naïve (Tn), CCR7+CD45RA- central memory (Tcm) and CCR7-CD45RA- effector memory (Tem) CD4+ T cells within total CD4+ T cells in LN compared to PB. PB and LN are paired samples, derived from the same patient. Statistical analysis was done using Wilcoxon signed rank test. Data are representative for 17 individuals. 3 50 CD4 + T cells in human lymph nodes 3 percentage of CXCR3-CCR6- Th2 cells in the PB showed a strong correlation with the percentage of Th2 cells present in the LN (figure 2C), whereas the percentage of Th1 and Th17 cells in PB and LN did not correlate significantly (figure 2D and E). Next, we functionally discriminated Th cells according to their cytokine producing capacity. PB CD4+ T cells produced significantly more IFNγ (p=0.003), while IL-2 was equally expressed by both LN and PB CD4+ T cells (figure 2F and G). The percentage of CD4+ T cells producing both IL-4 and IL-5, although very low, was significantly higher in PB (p=0.008; figure 2H and I). The percentage of IL-5 single positive cells was also significantly higher in the PB (p=0.006), while IL-4 single positive cells were equally present in LN and PB (data not shown). IL-17 was produced in similar percentages by PB and LN CD4+ T cells (figure 2J and K). Finally, we questioned how the percentage of LN regulatory CD4+ T cells relates to their percentage in the PB. Therefore, we analysed FoxP3, CD25 and IL-7Rα (CD127) expression on both LN and PB derived CD4+ T cells. We found that FoxP3+CD25brightCD127low Tregs were equally distributed between LN and PB (figure 3A and B). The amount of Tregs in the PB did not correlate with the amount in the LNs (figure 3C). Overall, these data imply that the different Th lineages, consisting of Th1, Th2, Th17 and Tregs are equally distributed over PB and LN. However, IFNγ and IL-4/IL-5 double producing CD4+ T cells were somewhat over-represented in the PB compartment. Cytotoxic, granzyme B+ CD4+ T cells are not detectable in the LN Latent infection with cytomegalovirus (CMV) has a great impact on the composition of the PB CD8+ T cell compartment but not on the LN CD8+ T cell compartment 24. We studied cytotoxic, granzyme B+, CD4+ T cells which have been shown to appear as a consequence of CMV infection 9;10;34. In the PB, cytotoxic CD4+ T cells are characterized by the lack of expression of the costimulatory molecules CD27 and CD28 9. Furthermore, they express CX3CR1 35, which provides them with the capability to migrate to inflamed vascular endothelium. We found that CD27-CD28- (figure 4A and B), CX3CR1+CCR7- (figure 4C and D) and granzyme B+ (figure 4E and F) CD4+ T cells were not at all detectable in LNs. CXCR5+CD4+ T cells accumulate in LNs and express higher levels of Tfh-markers as compared to their PB counterparts Tfh cells express CXCR5 and are specialized in providing help to B cells in the LN. In humans, but not in mice, CXCR5 expressing CD4+ T cells are also found in the PB. The percentage of CXCR5 expressing CD4+ T cells in resting LN (mean 27%, SD ±13%) was significantly higher than in the circulation (mean 12%, SD ±5%) (figure 5A). Interestingly, similar to Th2 cells, the percentage of CXCR5+CD4+ T cells showed a strong correlation between PB and LN (figure 5B). LN CXCR5+CD4+ T cells expressed significantly more ICOS (figure 5C and H) and PD-1 (figure 5D and I) than their PB counterparts. Furthermore, LN CXCR5+CD4+ T cells expressed significantly less CCR7 (figure 5E and J) and more CD69 as compared to PB derived CXCR5+CD4+ T cells (figure 5F and K). IL-21 is a cytokine known to play an important role in class switch 51 CD4 + T cells in human lymph nodes 3 Figure 2) LN and PB contain equal percentages of CD4+ T cell lineages Th1, Th2, Th17. (A) Comparison of CXCR3 (x-axis) and CCR6 (y-axis) expression on LN and PB memory CD4+ T cells. Dotplots are gated on CD3+CD4+CD45RA- lymphocytes. The left panel represents the LN and the right panel the PB derived memory CD4+ T cells. (B) Paired analysis of the percentage of CXCR3+CCR6- (Th1), CXCR3-CCR6- (Th2) and CXCR3-CCR6+ (Th17) memory CD4+ T cells in LN as compared to PB. Correlation between the percentage of (C) Th1 CXCR3+CCR6- (R2 = 0.3), (D) Th2 CXCR3-CCR6- (R2=0.56, p= 0.02) and (E) Th17 CXCR3-CCR6+ (R2=0.006) memory CD4+ T cells in LN on the x-axis and PB on the y-axis. (F) Comparison of IFN-γ (x-axis) and IL-2 (y-axis) expression by LN (left panel) and PB (right panel) CD4+ T cells. (G) Paired analysis of the percentage of IFNγ (left panel) and IL-2 (right panel) producing CD4+ T cells in LN compared to PB. (H) Comparison of IL-5 (x-axis) and IL-4 (y-axis) producing CD4+ T cells in LN (left panel) and PB (right panel). (I) Paired analysis of the percentage of IL-4/IL-5 double producing CD4+ T cells in LN and PB. (J) IL-17 (x-axis) producing CD4+ T cells in LN (left panel) as compared to PB (right panel). (K) Paired analysis of the percentage of IL-17 producing CD4+ T cells. In figures D, F, H and J the dotplots were gated on CD3+CD4+ lymphocytes. Statistical analysis was done using Wilcoxon signed rank test. The data are representative for 9 individuals. * = p<0.05, ** = p<0.01 52 CD4 + T cells in human lymph nodes 3 Figure 3) Tregs are equally distributed over LN and PB. (A) Comparison of FoxP3 (x-axis) and CD25 (y-axis) expression on LN (left panel) and PB (right panel) CD4+ T cells. (B) Paired analysis of FoxP3+CD25brightCD127dim CD4+ T cells in LN compared to PB. Dotplots were gated on CD3+CD4+ lymphocytes. Statistical analysis was done using Wilcoxon signed rank test. The data are representative for 9 individuals. * = p<0.05, ** = p<0.01 recombination, plasma cell differentiation and proliferation of B cells. Indeed, also IL-21 secreting CD4+ T cells reside more in the LNs (figure 5G and L). In conclusion, we demonstrated that the percentage of CXCR5+CD4+ T cells was higher in LNs than in the circulating pool and possessed more Tfh characteristics. Compared to PB, LN CXCR5+CD4+ T cells provide superior B cell help for Ig production Previous data have demonstrated that tonsil derived CXCR5+CD4+ T cells exhibit higher B helper cell activity in vitro as compared to CXCR5-CD4+ T cells 36. Furthermore, PB CXCR5+ cells have also been shown to induce more Ig production by B-cells as compared to PB CXCR5-CD4+ T cells 37. However, a paired functional comparison, of PB and LN derived CXCR5+CD4+ T cells, has not been performed previously. To determine B cell helper capacity, we sorted CXCR5+CD4+ T cells from both LN and PB and added each of them in a 1:1 concentration to either sorted LN or PB derived B cells. Subsequently, the cells were stimulated for 7 days with a TCR stimulus and costimulation. LN CXCR5+CD4+ T cells induced more IgG and IgM production as compared to PB CXCR5+CD4+ T cells when co-cultured with either LN or PB derived B cells (Figure 6A and B). Furthermore, LN derived B cells were more potent producers of IgG as compared to PB derived B cells (Figure 6A and B). With regard to the production of IgM, the difference between LN and PB B cells was less obvious (Figure 6A and B). As shown in figure 6C and D, LNs contained more switched memory phenotype CD27+IgD- B cells, which may explain the difference found in IgG production by LN and PB B cells. Together, these data show that LN derived CXCR5+CD4+ T cells have superior capacity to provide help to B cells required for IgG and IgM production. 53 CD4 + T cells in human lymph nodes 3 Figure 4) Cytotoxic CD4+ T cells are excluded from the LNs. Comparison of (A) CD27 (x-axis) and CD28 (y-axis), (C) CX3CR1 (x-axis) and CCR7 (y-axis) and (E) Granzyme K (x-axis) and Granzyme B (y-axis) expression on LN (left panels) and PB (right panels) CD4+ T cells. Dotplots are gated on CD3+CD4+ lymphocytes. Paired analysis of the percentage of (B) CD27-CD28- CD4+ T cells, (D) CCR7-CX3CR1+ CD4+ T cells and (F) Granzyme B+ CD4+ T cells in LN as compared to PB. Statistical analysis was done using Wilcoxon signed rank test. Data are representative for 11 individuals. * = p<0.05, ** = p<0.01 54 CD4 + T cells in human lymph nodes 3 Figure 5) CXCR5+CD4+ T cells expressing ICOS, PD-1 and CD69 are over-represented in LN as compared to the PB compartment. (A) Paired comparative analysis of the percentage of CXCR5+ cells within LN and PB CD4+ T cells. (B) Correlation of the percentage CXCR5+ cells within LN (x-axis) and PB (y-axis) CD4+ T cells. Comparison of (C) CXCR5 (x-axis) and ICOS (y-axis), (D) CXCR5 (x-axis) and PD1 (y-axis), (E) CXCR5 (x-axis) and CCR7 (y-axis), (F) CXCR5 (x-axis) and CD69 (y-axis) expression and (G) IL-21 (x-axis) production in LN (left) and PB (right). Dotplots are gated on CD3+CD4+ lymphocytes. Paired comparative analysis of the percentage of (H) ICOS, (I) PD1, (J) CCR7 and(K) CD69 expression on LN and PB CXCR5+ CD4+ T cells. (L) Paired analysis of IL-21 production by LN and PB derived CD4+ T cells. Statistical analysis was done using Wilcoxon signed rank test. Data are representative for 9 individuals. * = p<0.05, ** = p<0.01, *** = p< 0.001, **** = p<0.0001 55 CD4 + T cells in human lymph nodes 3 Figure 6) LN CXCR5+CD4+ T cells provide superior help to B cells. LN CXCR5+CD4+ T cells (black bars) and PB CXCR5+CD4+ T cells (gray bars) were co-cultured with LN B cells (A) or PB B cells (B) and subsequently stimulated with αCD3αCD28. We measured IgG and IgM in the culture supernatants by means of ELISA. IgG and IgM are expressed in percentages, where 100% is the highest amount of IgG or IgM produced within each separate individual. Figure 1A and 1B represent 4 separate experiments. Absolute values ranged from 65 to 3172 ng/ml of IgG and from 82 to 21622 ng/ml of IgM. (C) IgD and CD27 expression on LN (right panel) and PB (left panel) B cells. Dotplots are gated on CD19+CD20+ lymphocytes (D) Paired analysis of CD27+IgD- memory B cells in LN compared to PB (n=6). Statistical paired analysis was done using Wilcoxon signed rank test. Data are representative for 9 individuals. ** = p<0.01 Discussion Although most adaptive immune responses are initiated in the LNs, data on the distribution of differentiated Th cells within resting LNs and PB were still lacking. We are the first to compare the circulating CD4+ T-cell compartment to CD4+ T cells residing in resting LNs. Overall Th1, Th2, Th17 cells and Tregs were found to be equally spread over the two compartments, whereas cytotoxic CD4+ T cells were absent from the LNs. Furthermore, the resting human LN compartment comprised significantly more CXCR5+CD4+ T cells than the PB compartment. Apart from significantly higher expression levels of ICOS, PD-1 and CD69 and lower expression of CCR7, these cells also provide stronger helper activity for Immunoglobulin (Ig) production in vitro than their PB counterparts. The chemokine-receptor CCR7 and its ligands play an important role in directing the migration of T cells into the LNs 28. Surprisingly, we found that Tn and Tcm cells, which both express CCR7, were not enriched in the LNs and that Tem, which lack CCR7 expression, were also equally present in PB and LN. This might be explained 56 CD4 + T cells in human lymph nodes 3 by the fact that the Tcm cells, once they have reached the LN, down-regulate CCR7 upon contact with its ligand CCL19 (25,26). Moreover, Tem cells might reach the LN by means of inflammatory chemokines and their receptors, such as CXCL10 and its receptor CXCR3 38. Based on the low expression of cell activation markers such as Ki67 and HLA-DR (data not shown), the LNs analyzed in our study are presumably in a resting state. Still, they originate from the para-iliac region draining the pelvic area and parts of the intestine, areas which are constantly exposed to pathogens. Thus, low levels of CXCL10 might continuously be produced in these LNs, attracting CXCR3 expressing T cells from the peripheral blood compartment. The percentages of both CXCR5+CD4+ T cells and CXCR3-CCR6- Th2 cells show a strong correlation between PB and LN. Apparently, the frequencies of circulating CXCR5+CD4+ T cells and Th2 cells are a reflection of the frequencies present in the LNs. This is in agreement with the fact that LNs are the most important effector site for both Tfh and Th2 cells. On the other hand the percentages of Th1, Th17 cells and Tregs do not correlate between LN and PB. The fact that we could not find a correlation here can perhaps be explained by the fact that Th1, Th17 cells and Tregs are also distributed over other compartments, such as the spleen, peripheral tissues and/or sites of infection 39;40. Moreover, they do not have LNs as their primary effector site. LN and PB samples analysed in this study are derived from patients undergoing kidney transplantation. We can’t exclude that end stage renal disease (ESRD) might have influenced our results. Loss of renal function has been associated with decreased responses to vaccination such as HBV 41. Indeed, patients with ESRD have an increased susceptibility to infectious diseases and increased mortality caused by sepsis 42. Furthermore, ESRD and uraemia have been associated with a depletion of naïve and central memory T cells and B cells 43. Although these findings may point to a defect in T helper function, our in vitro studies demonstrated proper helper activity of the isolated CD4 populations. Judging from their chemokine-receptor expression pattern, Th1 and Th2 cells are equally distributed over the two compartments. However, the Th1-cytokine IFN-γ and the Th2-cytokines IL-4/IL-5 are produced slightly more by the PB CD4+ T cells. The higher percentage of IFN-γ producing CD4+ T cells in the PB is possibly due to the fact that the PB contains more cytotoxic CD28-CD4+ T cells, whereas these cells are absent from the LNs. We have previously shown that a larger proportion of CD28-CD4+ T cells produce IFN-γ as compared to CD28+CD4+ T cells 10. The discrepancy between Th1/Th2 chemokinereceptor expression and cytokine production can also be caused by a disproportionate up- or down-regulation of chemokine-receptors in the different compartments. Remarkably, we did not detect any cytotoxic GranzymeB+ CD4+ T cells in LNs, whereas they were present in the PB compartment of CMV seropositive individuals. Previously, we have established that circulating cytotoxic CD4+ T cells appear as a consequence of CMV infection 9;10. Like CMV-specific CD8+ T cells, which were also shown to be mainly present in the PB compartment 24, the cytotoxic CD4+ T cells express CX3CR1 and lack the expression of CCR7, favoring their migration to CMVinfected vascular endothelium 44-47. 57 CD4 + T cells in human lymph nodes In humans, several studies have been performed on the presence and function of CXCR5+CD4+ so-called follicular T helper (Tfh) cells in tonsillar lymphoid tissue. Functional studies demonstrate that tonsil CXCR5+CD4+ T cells are superior in supporting Ig production as compared to tonsil CXCR5-CD4+ T cells 36. The same holds true for PB CXCR5+ as compared to CXCR5-CD4+ T cells 37;48. Because of the scarce accessibility of LNs for human research, until now, no studies on CXCR5+CD4+ T cells in resting LN have been performed. We are the first to demonstrate that CXCR5+CD4+ T cells expressing Tfh cell markers are present in significantly higher numbers in the LNs than in the PB. Subsequently, we show that LN derived CXCR5+CD4+ T cells are better at providing B-cell help required for Ig production than their PB counterparts. When comparing our data to previous studies on tonsil derived CXCR5+CD4+ T cells, we noticed that the percentage of CXCR5+CD4+ T cells we found in LNs is much lower than the percentage previously found in human tonsils 15;36;49. This difference might be explained by the fact that most tonsillectomies are performed because of recurrent or chronic inflammation, whereas the LNs we studied, are not inflamed. Furthermore, in human tonsillar tissue, different subsets of Tfh cells have been described: CXCR5hiICOShi and CXCR5loICOSloCD4+ T cells 49;50. The CXCR5hiICOShiCD4+ T cells express high amounts of PD-1 and are found in the germinal centers (GCs), where they provide help to GC-B cells. In contrast, CXCR5loICOSloCD4+ T cells express lower amounts of PD-1 and have been shown to be Tfh-committed and to help B-cell responses outside the GCs 49. Likely, CXCR5loICOSloCD4+ T cells function at the T cell – B cell border during the initial phase of B cell activation and they contain precursors from which CXCR5hiICOShiCD4+ T cells, GC Tfh cells, emerge in a later stage 12. We did not detect CXCR5hiICOShiCD4+ T cells in the LNs. Again this might be explained by the fact that in tonsils an immune response is ongoing, while the LNs studied here represent lymphoid tissue during homeostatic condition. In a recent study, that primarily focused on HIV infected individuals, a high percentage of CXCR5+PD-1hi cells in peripheral LNs is described 51, which may be explained by an ongoing immune response because of viral persistence in these HIV patients. Increased differentiation toward Tfh cells has also been observed in LCMV mouse model for persisting viral infection 52. We did find CXCR5+CD4+ T cells expressing low amounts of ICOS and PD-1 in the LNs while PB CXCR5+CD4+ T cells do not express ICOS or PD-1. As mentioned earlier, the resting LNs analyzed in the present study are most likely continuously exposed to a low burden of pathogens, which may explain the presence of the CXCR5loICOSloCD4+ T cells. Apparently a limited exposure to pathogens does not induce CXCR5hiICOShiCD4+ T cells which are necessary for GC reactions. In conclusion, we show that Th1, Th2, Th17 and Tregs are equally distributed over LN and PB, while cytotoxic granzymeB+ CD4+ effector T cells are only detectable at their site of action, namely in the PB compartment. Furthermore, as could be expected from their capacity to provide help in antibody production, indeed CXCR5+CD4+ T cells are dominantly present in resting LNs. Apart from that, these cells possess both functional and phenotypic characteristics that resemble the Tfh cells as characterized 3 58 CD4 + T cells in human lymph nodes 3 in previous studies. 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J.Exp.Med. 2011;208:987-999. 61 CD4 + T cells in human lymph nodes supplementary material Supplemental Table 1 patient Immunosuppressive regimen CMV EBV age figure Pt682 P/MMF/FK/CD25mAb - + 62 1 Pt701 P/MMF/FK/CD25mAb - + 68 1 Pt466 P/MMF/FK/CD25mAb + + 47 1,3 Pt498 P/MMF/FK/CD25mAb + + 52 1,3 Pt516 P/MMF/FK/CD25mAb + + 58 1,3 Pt532 P/MMF/FK/CD25mAb + + 41 1,3 Pt560 P/MMF/FK/CD25mAb + + 65 1,3 Pt567 P/MMF/FK/CD25mAb + + 63 1,3 Pt647 P/MMF/FK/CD25mAb + + 26 1,3 Pt651 P/MMF/FK/CD25mAb + + 55 1,3 Pt670 P/MMF/FK/CD25mAb + + 49 1,3 Pt692 P/MMF/FK/CD25mAb + + 65 1,3 Pt524 P/MMF/FK/CD25mAb - + 53 1,2,4 Pt719 P/MMF/FK/CD25mAb + + 26 1,2,4 Pt753 P/MMF/FK/CD25mAb - + 73 1,2,4 Pt797 P/MMF/FK/CD25mAb + + 25 1,2,4 Pt813 P/MMF/FK/CD25mAb - - 33 1,2,4 Pt827 P/MMF/FK/CD25mAb - + 22 1,2,4,5 Pt868 P/MMF/FK/CD25mAb - + 58 1,2,4,5 Pt778 P/MMF/FK/CD25mAb + - 57 1,2,4,5 PtSi P/MMF/FK/CD25mAb + + 41 1,2,4,5 3