Vγ9/Vδ2 T cells are a minor subset of T cells in human blood and differ from all other lymphocytes by their specific responsiveness to (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), a metabolite produced by a large range of microbial pathogens. Vγ9/Vδ2 T cells can be skewed towards distinct effector functions, in analogy to, and beyond, the emerging plasticity of CD4+ T cells. As such, depending on the microenvironment, Vγ9/Vδ2 T cells can assume features reminiscent of Th1, Th2, Th17 and Treg cells as well as professional APCs. We here demonstrate that Vγ9/Vδ2 T cells express markers associated with follicular B helper T (TFH) cells when stimulated with HMB-PP in the presence of IL-21. HMB-PP induces upregulation of IL-21R on Vγ9/Vδ2 T cells. In return, IL-21 plays a co-stimulatory role in the expression of the B-cell-attracting chemokine CXCL13, the CXCL13 receptor CXCR5 and the inducible co-stimulator by activated Vγ9/Vδ2 T cells, and enhances their potential to support antibody production by B cells. The interaction between HMB-PP-responsive Vγ9/Vδ2 T cells, IL-21-producing TFH cells and B cells in secondary lymphoid tissues is likely to impact on the generation of high affinity, class-switched antibodies in microbial infections.
The establishment of long-term humoral immunity depends on the production of high-affinity antibodies capable of neutralising and opsonising invading pathogens. These antibodies are generated through somatic hypermutation, class switch recombination and affinity maturation of activated B cells, processes that take place in the germinal centres (GCs) of secondary lymphoid organs and depend on cognate help provided by CD4+ follicular B helper T (TFH) cells 1, 2. Human TFH cells are defined by the expression of characteristic markers 3–7: the transcriptional repressor Bcl-6 that directs TFH lineage commitment; the chemokine receptor CXCR5 that enables TFH cells to migrate into the B-cell follicles; the CXCR5 ligand CXCL13 that attracts further CXCR5+ cells such as naïve B cells and early activated CD4+ T cells; the co-stimulatory molecules, inducible co-stimulator (ICOS) and programmed cell death 1 (PD-1), which interact with their corresponding ligands on B cells; and the cytokine IL-21 that steers B-cell differentiation and antibody production. Murine TFH cells express the same panel of markers with the exception of CXCL13.
While the crucial role of TFH cells in providing B-cell help is undisputed, other T-cell subsets including CD8+ T cells, NKT cells and γδ T cells contribute to the outcome of humoral immune responses 8–10. γδ T cells support antibody production in immunised and infected mice 11–13. Of note, GCs are present in TCR-α−/− and TCR-β−/− mice and develop in SCID mice upon adoptive transfer of γδ T cells and B cells, demonstrating that γδ T cells are sufficient to orchestrate follicular responses 14–16. In humans, γδ T cells can be found in secondary lymphoid tissues 17, 18, where they are scattered throughout the T zone and clustered within GCs 8. Early studies in lupus patients led to the isolation of human γδ T-cell lines capable of inducing autoantibody production by autologous B cells 19. Subsequent investigations demonstrated that in vitro activated human γδ T cells can readily provide help to B cells via contact-dependent mechanisms involving CD40L and ICOS and soluble factors including IL-4 and IL-10 8, 20. Activated human γδ T cells may also express additional markers of relevance for a possible interaction with B cells including OX40, PD-1, CD27 and CD70 8, 21, 22.
Vγ9/Vδ2+ γδ T cells comprise an enigmatic human γδ T-cell subset that normally constitutes 1–5% of circulating T cells 23 but expands rapidly in response to infections with microbes producing the metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) 24. For reasons that are not yet understood, Vγ9/Vδ2 T cells and the responsiveness to HMB-PP are only found in higher primates including humans despite the ubiquitous distribution of potentially harmful, HMB-PP producing micro-organisms 25. Freshly isolated human Vγ9/Vδ2 T cells are characterised by a largely pro-inflammatory cytokine profile comprising IFN-γ, TNF-α and GM-CSF although co-expression of IL-4 is frequently observed 26–28. Importantly, however, they can be skewed towards distinct effector functions depending on the microenvironment, in analogy to, and even beyond, the emerging plasticity of CD4+ αβ cells. As such, under appropriate culture conditions, Vγ9/Vδ2 T cells divert from the ‘default’ Th1/CTL-like phenotype and assume features reminiscent of Th2 cells 27, 29, Th17 cells 30, 31, Treg cells 32 and even professional APCs 33. Our earlier microarray studies revealed that Vγ9/Vδ2 T cells express a range of TFH-associated molecules when stimulated with HMB-PP in the presence of IL-21 suggesting a potential role in the GC reaction 27. We here sought to provide a functional validation of these findings and investigated the effect of IL-21 on the acquisition of TFH-associated features by Vγ9/Vδ2 T cells. Our present data demonstrate that HMB-PP induces upregulation of IL-21R on Vγ9/Vδ2 T cells, thereby enhancing their responsiveness to IL-21. In return, IL-21 plays a co-stimulatory role in the expression of CXCL13, CXCR5 and ICOS by activated Vγ9/Vδ2 T cells and their potential to provide B-cell help as evidenced by supporting antibody production.
HMB-PP induces upregulation of the IL-21 receptor on human γδ T cells
We recently reported the expression of IL-21R mRNA by Vγ9/Vδ2 T cells 27, yet no data are available to date on the protein level nor on the activation-dependent regulation of IL-21R expression by human γδ T cells. Here, a monoclonal antibody against human IL-21R 34, 35 specifically stained B cells, CD4+ T cells and Vγ9/Vδ2 T cells but not monocytes in freshly isolated PBMCs (Fig. 1A). Similarly, IL-21R was expressed by both CD4+ T cells and Vγ9/Vδ2 T cells in tonsils.
The IL-2Rα chain CD25 confers high-affinity responsiveness to IL-2, a cytokine closely related to IL-21, and is an early activation marker on T cells, including Vγ9/Vδ2 T cells. We therefore examined whether the expression of IL-21R is similarly activation-dependent. Indeed, incubation of PBMCs in the presence of HMB-PP led to a significant upregulation not only of CD25 but also of IL-21R on Vγ9/Vδ2 T cells. In the same PBMC cultures, no effect was seen on CD25 or IL-21R expression by CD4+ T cells, B cells and monocytes (not shown), demonstrating the specificity of HMB-PP for Vγ9/Vδ2 T cells. IL-21R was predominantly found on activated Vγ9/Vδ2 T cells that co-expressed CD25 and CD69 (Fig. 1B). Time-course experiments showed that stimulation with HMB-PP led to a transient upregulation of IL-21R on Vγ9/Vδ2 T cells, peaking at 48 h and returning to baseline levels within 4–5 days (Fig. 1C). Taken together, these data suggest that HMB-PP-activated Vγ9/Vδ2 T cells are able to respond in vitro to exogenously added IL-21 and in vivo to IL-21 released from bystander cells in the microenvironment, such as TFH cells in secondary lymphoid tissues.
IL-21 co-stimulates the expression of CXCL13 by activated γδ T cells
Migration and positioning of T cells and B cells in lymph node follicles are tightly controlled by the chemokine CXCL13 and its receptor CXCR5. We previously demonstrated on the mRNA level that CXCL13 is expressed by Vγ9/Vδ2 T cells stimulated with HMB-PP in the presence of IL-21 but not of IL-2 or IL-4, and that the secretion of CXCL13 protein into the supernatants of PBMCs stimulated with HMB-PP and IL-21 depends on the presence of Vγ9/Vδ2 T cells 27. To directly confirm γδ T cells as the source of CXCL13, we established a flow cytometric method to detect intracellular CXCL13. As a control in these experiments, a proportion of tonsillar CD4+ T cells stained positive for CXCL13 when stimulated with PMA and ionomycin (Fig. 2A). Our data unambiguously show that activated, CD25+ Vγ9/Vδ2 T cells expressed CXCL13 when co-cultured with B cells in the presence of HMB-PP and IL-21 (Fig. 2A). These findings were further validated by immunofluorescence microscopy, where intracellular CXCL13 was clearly detectable in a proportion of Vγ9/Vδ2 T cells co-cultured with B cells in the presence of HMB-PP and IL-21 (Fig. 2B). Despite the low percentage of CXCL13+ Vγ9/Vδ2 T cells under these co-culture conditions, the levels of CXCL13 secreted into the supernatant were substantial (Fig. 2C). Of note, substituting IL-21 with IL-2 almost completely abrogated the CXCL13 production (not shown), confirming IL-21 as the main co-stimulatory factor in inducing CXCL13 expression by Vγ9/Vδ2 T cells.
IL-21 co-stimulates the expression of CXCR5 on tonsillar but not on peripheral γδ T cells
The expression of the chemokine receptor CXCR5 allows activated T cells to migrate into the CXCL13-rich B-cell follicles of secondary lymphoid tissues, where they instruct B cells to undergo somatic hypermutation and differentiate into plasma cells. CXCR5 was clearly expressed on B cells and on a subset of CD4+ T cells in freshly isolated PBMCs and in tonsils, in line with previous reports 36, yet we failed to detect CXCR5 on Vγ9/Vδ2 T cells (Fig. 2D). Of note, stimulation with neither PHA nor HMB-PP induced upregulation of CXCR5 on peripheral Vγ9/Vδ2 T cells (Fig. 2E), whereas peripheral CD4+ T cells readily upregulated CXCR5 expression in response to PHA (not shown). However, and in contrast to peripheral Vγ9/Vδ2 T cells, tonsillar Vγ9/Vδ2 T cells did express CXCR5 after stimulation with HMB-PP, and even more so (p<0.05) after stimulation with HMB-PP and IL-21 (Fig. 2E). CXCR5 expression on activated tonsillar Vγ9/Vδ2 T cells reached up to 30% within 24 h and declined after 3 days (Fig. 2F). Taken together, these data suggest that Vγ9/Vδ2 T cells activated in the periphery may reach the draining lymph nodes and start expressing CXCR5 and CXCL13 upon exposure to TFH cell-derived IL-21, thereby contributing to the formation of the GC.
IL-21 co-stimulates the expression of ICOS by peripheral and tonsillar γδ T cells
Co-expression of high levels of CXCR5 and ICOS is a defining feature of TFH cells 37, and the ability of TFH cells 38 as well as Vγ9/Vδ2 T cells 20 to provide follicular B-cell help is in part mediated through ICOS. Here, resting peripheral Vγ9/Vδ2 T cells did not express ICOS but readily upregulated its expression in response to HMB-PP (Fig. 3A). Of note, IL-21 showed a strong co-stimulatory effect on ICOS, with expression levels reaching 50% of all Vγ9/Vδ2 T cells over a period of 6 days, at a time point of considerable proliferative expansion (data not shown and 27). In parallel with ICOS, activated Vγ9/Vδ2 T cells also transiently expressed the co-stimulatory molecule OX40, although the effect of IL-21 on OX40 levels was less pronounced (Fig. 3A).
When comparing the responsiveness of peripheral and tonsillar Vγ9/Vδ2 T cells, tonsillar cells showed an increased sensitivity to IL-21 with regard to ICOS expression, while OX40 was preferentially induced on peripheral Vγ9/Vδ2 T cells (Fig. 3B). Other markers that were upregulated similarly on both peripheral and tonsillar Vγ9/Vδ2 T cells exposed to a combination of HMB-PP and IL-21 included CD25, CD70, CD86 and HLA-DR (Fig. 3B). Taken together, our data demonstrate a role for IL-21 in the acquisition by Vγ9/Vδ2 T cells of markers associated with B-cell help. In particular, the activation of tonsillar Vγ9/Vδ2 T cells in the presence of IL-21 leads to the expansion of ICOS+ Vγ9/Vδ2 T cells with a TFH cell-like phenotype.
IL-21 enhances the potential of human γδ T cells to provide B-cell help
We next tested the effect of IL-21 on the potential of peripheral Vγ9/Vδ2 T cells to stimulate autologous B cells. In the presence of HMB-PP, Vγ9/Vδ2 T cells underwent potent crosstalk with B cells as evidenced by the upregulation of CD25, CD69, CD40 and CD86 on co-cultured B cells (Fig. 4). This effect was comparable to the effect of treatment of B cells with the mitogen Staphylococcus aureus Cowan 1 (SAC), indicating that HMB-PP-activated Vγ9/Vδ2 T cells provided a strong stimulus for B cells. This activation of B cells occurred irrespective of the presence of IL-21 in the medium, suggesting that under the conditions tested IL-21 on its own did not have a direct effect on B cells. However, addition of IL-21 to the medium significantly enhanced the resulting levels of IgM, IgG and IgA antibodies secreted by γδ T-cell/B-cell co-cultures, indicating that HMB-PP and IL-21 acted synergistically (Fig. 5A). IgE antibodies were not detectable under these conditions (data not shown). Of note, the stimulatory effect of γδ T cells on autologous B cells depended on the γδ T-cell/B-cell ratio, confirming the exquisite potential of HMB-PP-activated γδ T cells to provide the B-cell help, especially when co-stimulated in the presence of IL-21 (Fig. 5B). Finally, we confirmed the role of IL-21 in enhancing B-cell helper function of γδ T cells by using co-cultures of tonsillar Vγ9/Vδ2 T cells and autologous tonsillar CXCR5+ B cells. While IL-21 or HMB-PP alone induced IgM production by tonsillar B cells, the combination of both had a synergistic effect on the overall levels produced and resulted in IgM levels comparable to those obtained with CXCR5+ B cells stimulated polyclonally with SAC+IL-2 or co-cultured with autologous tonsillar CXCR5+ TFH cells (Fig. 5C).
We here provide evidence that IL-21 contributes to the acquisition of B-cell helper functions by human Vγ9/Vδ2 T cells. We used an in vitro system of autologous Vγ9/Vδ2 T cells and B cells from tonsils or blood, the microbial metabolite HMB-PP and the TFH-derived cytokine IL-21 to try to mimic the physiological conditions in the GC. Our report is the first to show that resting Vγ9/Vδ2 T cells express IL-21R on their surface, and that the expression of IL-21R is greatly enhanced upon stimulation. Binding of IL-21 to γδ T cells had so far only been described in mice 39. Our findings imply that resting Vγ9/Vδ2 T cells are directly responsive to IL-21 in the microenvironment, and even more so shortly after activation with HMB-PP. Vγ9/Vδ2 T cells are rapidly drawn to sites of infection where they will encounter invading pathogens and interact with other immune cells including neutrophils, monocytes and DCs, thus defining an innate-like ‘first line of defence’ or sentinel function 24, 25, 28. However, activation of both human and mouse γδ T cells triggers changes in their migratory pattern and induces upregulation of CCR7 8, 40. Once recruited to secondary lymphoid tissues, pre-activated Vγ9/Vδ2 T cells may thus encounter IL-21 produced by TFH cells and as a consequence express a distinct set of molecules associated with providing B-cell help. Lymph node-resident Vγ9/Vδ2 T cells may also become stimulated by HMB-PP draining from the site of infection.
The acquisition of TFH-associated markers by Vγ9/Vδ2 T cells and their dependence on IL-21 was first suggested by our recent microarray studies 27. IL-21 turned out to have a similar capacity as the related cytokine IL-2 to support Vγ9/Vδ2 T-cell proliferation yet without promoting the supposedly ‘signatory’ molecules IFN-γ and TNF-α 41, thus highlighting a much greater plasticity of Vγ9/Vδ2 T-cell responses than previously appreciated. The demonstration that IL-21 drives the expression of the B-cell-attracting chemokine CXCL13 suggests a role for IL-21-stimulated Vγ9/Vδ2 T cells in orchestrating immune cell trafficking to the GC. Our present data indicate that IL-21 also plays a role in supporting the expression of the CXCL13 receptor, CXCR5, by tonsillar Vγ9/Vδ2 T cells. Of note, we were unable to induce significant levels of CXCR5 on peripheral Vγ9/Vδ2 T cells, in line with previous reports 8 but at odds with others 20. Control experiments confirmed the expression of CXCR5 on peripheral CD4+ T cells and B cells, and further upregulation of CXCR5 on CD4+ T cells stimulated with PHA, thus validating the reagents and methodology used. We also observed lower CXCR5 expression values on tonsillar Vγ9/Vδ2 T cells than those reported by Caccamo et al. 20. However, stimulation of tonsillar Vγ9/Vδ2 T cells with HMB-PP and IL-21 resulted in our hands in the rapid induction of CXCR5 expression within 24 h. Thus, our data confirm that activated tonsillar Vγ9/Vδ2 T cells can clearly express CXCR5 20, providing a molecular explanation for their clustering in GCs 8, 27. An increased and more rapid responsiveness to IL-21 of tonsillar Vγ9/Vδ2 T cells, compared with that of peripheral Vγ9/Vδ2 T cells, was also apparent with respect to the induction of ICOS. These data indicate that IL-21 drives Vγ9/Vδ2 T cells to assume a TFH-like phenotype, thus evoking the crucial effect of IL-21 in the generation of CD4+ TFH cells 35.
The phenotypic characterisation was further supported by the functional demonstration that co-stimulation with IL-21 of co-cultures of peripheral Vγ9/Vδ2 T cells and B cells enhanced the production of IgM, IgG and IgA in the presence of HMB-PP, and that a similar co-stimulatory effect of IL-21 was seen on IgM levels produced by co-cultures of tonsillar Vγ9/Vδ2 T cells and B cells. We are aware that the interpretation of these co-culture assays is hampered by a possible direct effect of IL-21 on B cells 42, 43. Although IL-21 on its own had no significant effect on antibody production by peripheral B cells, partial activation of B cells by IL-21 may have played a role in these experiments. In co-cultures of tonsillar cells, there was a clear effect of IL-21 alone, suggesting that under those conditions HMB-PP and IL-21 acted synergistically. To rule out any direct effect on B cells, we pre-stimulated purified Vγ9/Vδ2 T cells with HMB-PP and IL-21 before adding them to autologous B cells. However, no conclusive data were obtained (data not shown), most likely because any soluble factors of importance for supporting antibody production released during this pre-stimulation period would have been washed away before setting up Vγ9/Vδ2 T-cell/B-cell co-cultures.
Finally, the aspect of antigen specificity deserves attention. Earlier studies in mice demonstrated that GC reactions that solely depend on γδ T cells are somewhat inefficient and result in relatively high titres of class-switched, self-reactive antibodies 12, 44. γδ T cells may thus provide general B-cell help and enhance humoral immune responses, while MHC-restricted αβ T cells ensure antigen specificity by regulating affinity maturation and clonal selection. This view is supported by the fact that HMB-PP is present in the majority of bacterial pathogens, in malaria parasites and in Toxoplasma, and hence activation of human Vγ9/Vδ2 T cells is likely to occur in a broad range of infections 25. A ‘bystander’ role for Vγ9/Vδ2 T cells in driving humoral responses may result in different antibody levels, isotypes or affinities upon infection with HMB-PP-producing or HMB-PP-deficient micro-organisms. While such a role can only be postulated in humans and at best supported by indirect epidemiological evidence, infection and/or vaccination experiments in non-human primates might address the contribution of Vγ9/Vδ2 T cells to the generation of high-affinity, class-switched antibodies. Such studies could exploit the availability of convenient pathogen models such as specially engineered HMB-PP-deficient strains of Listeria monocytogenes45 or HMB-PP overproducing strains of Mycobacterium tuberculosis46.
Recent findings also point towards a more antigen-restricted role of Vγ9/Vδ2 T cells in providing B-cell help, given their potential to take up exogenous antigens and present them to CD4+ and CD8+ T cells 33, 47. Antigen-presenting γδ T cells may thus be able to interact directly with TFH cells. In mice, epidermal γδ T cells leave the skin upon activation and reach the draining lymph node where they may help the production of antibodies specific for non-self-antigen applied onto, or artificially expressed within, the skin 40, 48. It remains to be investigated whether HMB-PP or related compounds with activity on Vγ9/Vδ2 T cells, such as the aminobisphosphonate zoledronate, might be useful in boosting humoral responses. A recent vaccine trial in cynomolgus monkeys did address a possible adjuvant effect of the HMB-PP analogue Picostim on the immune response against mycobacterial antigens yet did not measure the levels of anti-mycobacterial antibodies 49. Similarly, the effect of aminobisphosphonate treatment on anti-tumour antibody responses in cancer patients has so far been overlooked.
Taken together, our present findings complement and extend previous observations on the potential of γδ T cells to provide B-cell help and ascribe an important role to IL-21 in contributing to this potential. The interaction between HMB-PP-responsive Vγ9/Vδ2 T cells with IL-21-producing TFH cells and with B cells in secondary lymphoid tissues is likely to impact on the generation of high affinity, class-switched antibodies in microbial infections.
Materials and methods
PBMCs were isolated from peripheral blood of healthy volunteers using Lymphoprep (Axis-Shield). To study cells from secondary lymphoid tissues, mononuclear cells were also isolated from fresh tonsils of patients undergoing tonsillectomy. Ethical approval was obtained from the South East Wales Local Ethics Committee (08/WSE04/17) and conducted according to the principles expressed in the Declaration of Helsinki. All patients and volunteers provided written informed consent.
For functional assays, cell subpopulations were purified by MACS (Miltenyi): peripheral B cells (>98%) using anti-CD19 microbeads (for feeder cells) or the untouched B-cell isolation kit (for antibody production); tonsillar CXCR5+ B cells (>95%) using the untouched B-cell isolation kit followed by CXCR5-PE mAbs (51505.111; R&D Systems) and anti-PE microbeads; tonsillar CXCR5+ TFH cells (>85%) using CD4-FITC mAbs and the anti-FITC multisort kit followed by CXCR5-PE mAbs and anti-PE microbeads; and peripheral Vγ9/Vδ2 T cells (>95%) using Vδ2-PE (B6.1; BD Biosciences) or Vγ9-PE-Cy5 (Immu360; Beckman-Coulter) mAbs and anti-PE microbeads. Tonsillar Vγ9/Vδ2 T cells (>99%) were enriched from CD4-depleted tonsillar cells using Vδ2-PE mAbs and anti-PE microbeads, followed by further purification on a MoFlo cell sorter (Dako Cytomation) as Vγ9+ Vδ2+ CD3+ CD4− CD19− lymphocytes. Purities of all cell populations obtained were determined by flow cytometry.
Cells were acquired on a nine-colour CyAn ADP (Beckman Coulter) and analysed with FloJo 7.5 (TreeStar), using mAbs directed against TCR-Vδ2 (B6.1), CD3 (SK7, UCHT1, HIT3a), CD4 (RPA-T4), CD14 (MOP9), CD25 (M-A251), CD27 (1A4CD27), CD45RA (HI100), CD45RO (UCHL-1), CD69 (FN50), CD70 (Ki-24), CD86 (2331), CD134/OX40 (ACT35), CD278/ICOS (DX29), HLA-DR (L243) and IgD (IA6-2) (all from BD Biosciences) and TCR-Vγ9 (Immu360), CD4 (SFCI12T4D11) and CD40 (mAB89) from Beckman Coulter; CD19 (SJ25C1) from eBioscience; CD19 (HD37) from Dako; IL-21R (9N1; 34, 35); and rat anti-CCR7 (3D12; 50); together with appropriate isotype controls and secondary reagents. Cells of interest were gated based on their appearance in side scatter and forward scatter area/height and exclusion of live/dead staining (fixable Aqua; Invitrogen). For intracellular detection of CXCL13, brefeldin A (Sigma) was added to cultures at 10 μg/mL 4 h prior to harvesting; cells were then fixed and permeabilised using the Fix&Perm kit (eBioscience) and stained with mouse anti-CXCL13 (53610; R&D Systems) followed by an goat anti-mouse secondary antibody (Dako) and surface staining as above.
The medium used was RPMI-1640 with 2 mM L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 50 μg/mL penicillin/streptomycin, 50 μM β-mercaptoethanol and 10% foetal calf serum (Invitrogen). PBMCs or tonsillar cells were cultured for up to 7 days in the presence of 10 nM synthetic, i.e. LPS-free HMB-PP 51 with or without 10–100 U/mL IL-2 (Proleukin; Chiron) or 50–100 ng/mL IL-21 (Zymogenetics). As controls, cells were stimulated with 1 μg/mL PHA (Sigma) for up to 3 days or with 10 ng/mL PMA (Sigma) and 1 μg/mL ionomycin (Sigma) for 6 h. For functional assays, purified B cells were co-cultured with purified γδ T cells or TFH cells at a ratio of 1–5 B cells per T cell, unless otherwise indicated. In some experiments, B cells alone were stimulated with 0.01% SAC (Pansorbin; Merck) in the presence of 100 U/mL IL-2.
Immunofluorescence microscopy was performed using a Nikon microscope (Eclipse 80i, Nikon). After 3 days of culture, cells were fixed, permeabilised and stained with mouse anti-CXCL13, followed by Alexa Fluor488-conjugated goat anti-mouse IgG1 (green), anti-Vδ2-PE (red) and counterstaining with DAPI (blue). Images were captured with a Nikon digital camera (DXM1200F) controlled by ACT-1 2.70 imaging software and processed with Adobe Photoshop 6.0.
Cell culture supernatants were analysed for CXCL13 by ELISA (R&D Systems). Antibody levels were determined using the human IgM, IgG, IgA and IgE quantitation kits (Universal Biologicals). All samples were measured in duplicate on a Dynex MRX II reader.
Data were analysed using two-tailed Student's t-tests or two-way ANOVA (GraphPad Prism 4.0), with differences considered significant as indicated in the figures: *p<0.05; **p<0.01; ***p<0.001.
The authors are grateful to Gareth Williams, Marco-Domenico Caversaccio and Bernd Werle for providing human tissue samples. We also thank Hassan Jomaa for providing HMB-PP; Donald Foster for IL-21; Martin Lipp for CCR7 antibodies; Janet Fisher and Chris Pepper for cell sorting; Simone Meuter for help with immunofluorescence microscopy; and Francesco Dieli, Derek Doherty and David Vermijlen for the stimulating discussion. This research was supported by an MRC PhD Studentship to R.R.B. and a RCUK Fellowship in Translational Research and a Wellcome Trust VIP Award to M. E. B. M. is in receipt of a Royal Society Wolfson Research Merit Award.
Conflict of interest: The authors declare no financial or commercial conflict of interest.