SAP-controlled T-B cell interactions underlie germinal centre formation

doi:10.1038/nature07345

Nature. Author manuscript; available in PMC 2009 April 9.

Published in final edited form as:

Nature. 2008 October 9; 455(7214): 764–769.

doi: 10.1038/nature07345

PMCID: PMC2652134

NIHMSID: NIHMS67294

SAP-controlled T-B cell interactions underlie germinal centre formation

Hai Qi,¹^* Jennifer L. Cannons,²^* Frederick Klauschen,¹ Pamela L. Schwartzberg,²^# and Ronald N. Germain¹^#

¹ Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases National Institutes of Health, Bethesda, MD 20892, USA

² Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA

Correspondence and requests for materials should be addressed to R.N.G (Email: rgermain/at/niaid.nih.gov) or P.L.S. (Email: pams/at/nhgri.nih.gov)

^*These authors contributed equally to this work.

^#These authors contributed equally to this work.

The publisher's final edited version of this article is available at Nature

See other articles in PMC that cite the published article.

Abstract

Generation of long-term antibody-mediated immunity depends on the germinal centre (GC) reaction, which requires cooperation between antigen-specific T and B lymphocytes. In the human X-linked lymphoproliferative disease and its gene-targeted mouse model, loss-of-function mutations in signalling lymphocyte activation molecule-associated protein (SAP, encoded by SH2D1a) cause a profound defect in GC formation by an as yet unknown mechanism. Using two-photon intravital imaging, here we show that SAP deficiency selectively impairs the ability of CD4⁺ T cells to stably interact with cognate B cells but not antigen-presenting dendritic cells. This selective defect results in a failure of antigen-specific B cells to receive adequate levels of contact-dependent T cell help to expand normally, despite sap^−/− T cells exhibiting the known characteristics of otherwise competent helper T cells. Furthermore, lack of stable interactions with B cells renders sap^−/− T cells unable to be efficiently recruited to and retained in a nascent GC to sustain the GC reaction. These results offer a compelling explanation for the GC defect due to SAP deficiency and provide novel insights into the bi-directional communication between cognate T and B cells in vivo.

The germinal centre (GC) reaction, which supports antibody affinity maturation and the generation of B cell memory¹^–³, requires activation of CD4⁺ T cells by antigen-bearing dendritic cells (DCs), followed by differentiation of these T cells and their physical interactions with antigen-activated B cells⁴. The activated T cells promote the survival, proliferation, and differentiation of the B cells by delivering contact-dependent helper signals in an antigen-specific fashion⁵^–⁸. Follicular helper T (T_FH) cells, a subset of activated CD4⁺ T cells that highly express CXCR5, CD40L, ICOS, and signalling lymphocyte activation molecule-associated protein (SAP), are particularly important for a sustained GC response⁹.

In human X-linked lymphoproliferative disease (XLP) and its gene-targeted mouse model, lack of functional SAP protein in T cells causes a profound defect in GC formation and humoral immunity¹⁰^–¹⁷, but how SAP regulates the GC-promoting T helper cell response is unknown. An intracellular adaptor protein, SAP binds to signalling lymphocyte activation molecule (SLAM) and other transmembrane molecules of the SLAM family¹⁸^,¹⁹. It modulates T cell antigen receptor signalling and promotes type 2 T helper (T_H2) cell differentiation¹³^,¹⁴^,²⁰. However, impaired T_H2 differentiation alone is not an adequate explanation for the profound GC defect seen in SAP-deficient animals¹⁷^,²¹^,²². To better understand how SAP deficiency results in impaired GC formation, we took a combined approach of classical cellular immunology and intravital 2-photon imaging, which allowed us to examine not only the activation phenotype of T cells but also the dynamics of their critical interactions with DCs and B cells in vivo²³.

Sap^−/− T cells undergo normal activation by DCs

Because optimal T cell activation and differentiation in vivo require long-lasting T-DC conjugation²⁴^–²⁷, we first examined the impact of SAP deficiency on T-DC interactions using OT-2 TCR transgenic T cells specific to I-A^b complexed with an ovalbumin-derived peptide (OVA₃₂₃). Behaviours of sap^−/− and sap^+/+ T cells in the same draining lymph node (LN) were compared following co-transfer into mice previously injected s.c. with OVA₃₂₃-pulsed DCs. Sap^−/− and sap^+/+ OT-2 T cells showed equivalent contact times with DCs presenting OVA₃₂₃ (Figs. 1a and 1b, Supplementary Movie 1). Consistent with this result, sap^−/− and sap^+/+ OT-2 T cells proliferated and accumulated to a similar extent (Fig. 1c). Within the expanded pool of activated T cells of either genotype, similar frequencies of CXCR5⁺ICOS⁺ T_FH precursors were observed and both groups of T cells migrated into the B cell follicular areas of the LN (Fig. 1d and 1e). These data indicate SAP deficiency does not grossly alter initial T cell interaction with or activation by antigen-presenting DCs in vivo.

Figure 1

Sap^−/− T cells normally interact with and are activated by DCs in vivo

Sap^−/− T cells exhibit a selective defect in stable interactions with B cells

During a T-dependent B cell response, helper T cells activated by DCs must subsequently engage antigen-triggered B cells based on class II major histocompatibility complex (MHC)-restricted antigen recognition⁷^,²⁸. This process is characterized by long-lasting, mobile conjugate pairs formed between cognate T and B cells²⁹. Based on these prior observations, we used intravital imaging to assess whether SAP deficiency affects antigen-dependent T-B interactions in vivo. Sap^−/− and sap^+/+ OT-2 T cells were co-transferred into mice together with wild-type B cells expressing the MD4 transgenic B cell receptor, which recognizes hen egg lysozyme (HEL). As antigen, cross-linked conjugates of HEL and OVA (HEL-OVA) were used to allow activation of both T and B transgenic cells and to permit MHC class II-dependent antigen presentation to the OT-2 T cells by the MD4 B cells. Following immunization, sap^+/+ OT-2 T cells engaged migrating MD4 B cells and formed long-lasting mobile conjugate pairs, while sap^−/− OT-2 T cells showed predominantly brief contacts with the same cohort of MD4 B cells (Figs. 2a and 2b; Supplementary Movies 2 and 3; mean±SEM of the median T-B contact time by sap^+/+ vs. sap^−/− T cells: 19.2±2.6 vs. 8.2±1.9 minutes, 5 experiments). Thus, are in striking contrast to the lack of significant effects on the duration of T-DC interactions (Fig. 1a and 1b), the absence of SAP expression in T cells severely reduces the longevity of T-B interactions in vivo.

Figure 2

Sap^−/− T cells are defective in adhesion to cognate B cells

To examine more quantitatively the differential effect of SAP deficiency on T-APC interactions, a flow cytometry-based in vitro assay was utilized in which shear stress was imposed on unfixed conjugates between activated OT-2 T cell blasts and OVA₃₂₃-pulsed DCs or activated B cells. While a dose-dependent increase in T-B conjugate efficiency was observed with sap^+/+ OT-2 T cells, conjugate formation by sap^−/− T cells was low at all peptide concentrations tested (Fig. 2c). In contrast, within the same non-saturating peptide dose range, sap^−/− T cells were as efficient as wild-type T cells in forming conjugates with DCs (Fig. 2d). To determine whether the defective adhesion to B cells by activated sap^−/− T cells might have been programmed during their priming interactions with DCs and whether SAP expression is specifically required during T-B interactions, DNA constructs encoding either green fluorescent protein (GFP) or a GFP-SAP fusion protein were independently transfected into sap^−/− T cell blasts. Complementation with GFP-SAP but not GFP alone fully rescued T-B conjugate formation by transfected sap^−/−T cell blasts (Fig. 2e; also see Supplementary Fig. 1). Thus, the requirement for SAP expression in T cells for optimal adhesion to B cells is contemporaneous with T-B interaction. SAP facilitates the recruitment of Fyn kinase to cytoplasmic tails of SLAM-related proteins, which is important for SAP regulation of Th2 cytokine production²⁰^,³⁰ but less so for SAP-controlled GC development¹⁷^,³¹. Consistent with these latter results, we found that SAP^R78A, a mutant SAP capable of binding SLAM-related molecules but severely impaired in binding Fyn kinase²⁰^,³⁰, rescued T-B conjugate formation in vitro (Supplementary Fig. 2). Taken together, our analyses of T cell-APC conjugation establish that SAP critically regulates the contact duration and the adhesive strength of cognate T-B but not antigen-driven T-DC interactions.

B cells fail to acquire sufficient help from sap^−/− T cells

The cell numbers used in the preceding experiments can result in competition among T cells for antigen on presenting cells in vivo³², raising the question of whether sap^−/− T cells would, if present alone, form more stable contacts with antigen-specific B cells or provide B cells with a summed contact time from sequential brief interactions comparable to the total interaction time with wild-type T cells. To examine these issues, a small number (6×10⁴) of sap^+/+ or sap^−/− OT-2 T cells were separately transferred into recipient B6 mice that concomitantly received 3×10⁵ MD4 B cells. Cumulative interactions between B and T cells were assessed by continuous intravital imaging over a 3-hour period of time between 60 to 72 hours post- immunization, when substantial clonal expansion has begun and physical co-localization of activated T and B cells can be observed ⁸^,²⁹^,³³^,³⁴. Under these modified conditions, sap^−/− OT-2 cells still demonstrated abbreviated contacts with activated MD4 B cells following immunization with HEL-OVA (2.1±0.2 minutes [mean±SEM] vs. 8.9±0.8 minutes for sap^+/+ T cells, p<0.0001; Fig. 3a, Supplementary Movie 4). While many contacts involving sap^−/− T cells were brief and similar to non-cognate T-B contacts in duration, on the population level they lasted for a statistically longer period of time (2.1±0.2 vs. 1.3±0.1 minutes for non-cognate interactions, p=0.0014), indicating that antigen-specific interactions occurred between sap^−/− OT-2 cells and MD4 B cells but were not sustained. Individual B cells were also tracked for their interactions with different T cells. MD4 cells often accumulated >1 hour of interactions with sap^+/+ OT-2 T cells, while they were rarely able to accumulate more than 20 minutes of contact with sap^−/− OT-2 T cells over the same period (Figs. 3b–d; Supplementary Movies 4 and 5). B cells remained equally trackable under the two conditions and interacted with similar numbers of sap^−/− or sap^+/+ T cells (Supplementary Fig. 3). The severe reduction in cumulative time of contact with sap^−/− OT-2 T cells correlated with a pronounced impairment of MD4 B cell clonal expansion (Fig. 3e). Nonetheless, sap^−/− OT-2 T cells expanded comparably to their sap^+/+ counterparts and expressed similar levels of CD40L, ICOS, OX40, and CXCR5 (Fig. 3f; Supplementary Fig. 4) at this time. The latter is a phenotype considered characteristic of activated T cells capable of effectively promoting humoral responses and with features of T_FH precursor cells as commonly defined⁹^,³⁵. In concert with the previous data, these findings suggest that it is not an intrinsic inability of SAP-deficient T cells to express key molecular signals, but rather a failure to exchange these signals during sufficiently long periods of contact with B cells that accounts for their markedly reduced capacity to promote B cell expansion and subsequent GC development.

Figure 3

B cells fail to receive contact-dependent help from SAP-deficient T cells

Sap^−/− T cells are not efficiently recruited into and retained in forming GCs

Recruitment of helper T cells to a forming GC is necessary for maintaining the GC reaction³⁶, and GC-localized T_FH cells are required for effective immunoglobulin class-switching and antibody affinity maturation⁹. To explore whether SAP deficiency also impairs T cell recruitment to and retention within nascent GCs, we transferred equal numbers (3×10⁴) of CFP-expressing sap^+/+ OT-2 cells and GFP-expressing sap^−/− OT-2 cells into B6 mice together with 3×10⁵ non-fluorescent MD4 cells. Six to 8 days after HEL-OVA immunization, GCs developed within the draining LN, seen as GL7⁺ areas that largely excluded IgD⁺ naïve B cells (Supplementary Fig. 5). While the IgD⁺ follicular mantle zone was populated by both sap^+/+ and sap^−/− OT-2 cells, the GC area predominantly contained sap^+/+ OT-2 cells, consistent with a failure of GC recruitment and/or retention of sap^−/− T cells.

To examine this issue dynamically, recipient mice were also given dye-labelled naïve B cells 24 to 48 hours before intravital imaging to differentiate follicular mantle and GC areas. Although individual naïve B cells migrate in and out of GCs³⁷, as a population they remain substantially excluded from GCs (Supplementary Fig. 5), allowing simultaneous identification of the follicular area and approximation of the GC-mantle border using time-averaged images (see Supplementary Fig. 6 for details). Sap^+/+ and sap^−/− OT-2 T cells exhibited strikingly different dynamic patterns within the follicle (Supplementary Movie 6 and Fig. 4a). While sap^+/+ OT-2 T cells migrated freely into and accumulated within the GCs, sap^−/− OT-2 T cells mainly swarmed in the follicular mantle. To quantify migratory behaviours of the two types of T cells around GCs, a tessellation algorithm was used to retrieve the GC surface (Supplementary Fig. 6) and define a virtual mantle-GC interface zone was then defined as encompassing spatial coordinates within 10 μm of the GC outer surface. T cell tracks were subsequently classified according to their interactions with this interface zone (Fig. 4b and 4c). When reaching the interface from the follicular mantle, sap^+/+ OT-2 T cells were more likely to continue into the GC than to return to the mantle. Sap^−/− T cells exhibited the opposite behaviour, being more likely to turn back than to cross into the GC. Conversely, when reaching the interface zone from within the GC, sap^+/+ T cells were more likely to return to the GC than to escape into the mantle, whereas sap^−/− T cells were much more likely to escape than to return. For those tracks that started from the interface zone, sap^−/− T cells exhibited a preference for migrating into the mantle as compared to moving into the GC, while for sap^+/+ T cells the movement in these two directions was comparable. Consistent with these distinct migration patterns, sap^−/− T cells also exhibited significantly shorter GC retention times (Supplementary Fig. 7). Therefore, in the absence of SAP, T cells are not efficiently recruited into or retained within a nascent GC. These data suggest SAP-deficient T cells cannot act as effective GC-associated T_FH cells to sustain the GC reaction, a defect that would contribute to the profound impairment of GC responses in SAP-deficient hosts.

Figure 4

Defective GC recruitment and retention of sap^−/− T cells due to lack of efficient cognate interactions with B cells

To address whether the inefficient GC recruitment and retention of sap^−/− T cells resulted from their reduced antigen-specific interactions with B cells, radiation bone-marrow chimeras were constructed so that endogenous B cells were deficient in class II MHC expression and thus unable to engage in antigen-specific interactions with T cells, while T cells could still interact with and be activated by non-B antigen presenting cells including DCs (Supplementary Fig. 8; see a methodological diagram in Fig. 4d). Following co-transfer of both sap^+/+ and sap^−/− OT-2 T cells together with MD4 B cells into these chimeric animals, they were immunized with a mixture of HEL-BSA and intact OVA proteins. In this setting, MD4 B cells cannot engage transferred OT-2 T cells as cognate partners but can still form GCs (Supplementary Fig. 9a) by utilizing cognate help from endogenous CD4⁺ T cells. OT-2 cells in these mice can be normally activated by OVA-presenting DCs, but are deprived of cognate interactions with B cells, which are either transferred MD4 cells that do not present OVA or endogenous B cells that are derived from class II MHC-deficient bone marrow progenitors. While both sap^+/+ and sap^−/− OT-2 T cells upregulated CXCR5 and ICOS expression in this experimental system (Supplementary Fig. 9b), now they both failed to be efficiently recruited into and retained within GCs (Figs. 4e and 4f, Supplementary Fig. 7, Supplementary Movies 7 and 8). When the same chimeric mice were immunized with HEL-OVA, a condition in which OT-2 T cells could engage MD4 B cells as cognate partners, sap^+/+ OT-2 T cells were recruited to the GC, while sap^−/− OT-2 T cells were not (data not shown). These findings indicate that T cells require cognate interactions with B cells to be recruited into and retained within GCs and suggest that, by failing to engage productively in such cell-cell interactions, SAP-deficient T cells are unable to physically localize to the GC to effectively sustain the GC reaction.

Conclusions

This study demonstrates that SAP expression in T cells is critical for stable antigen-dependent T-B adhesion but dispensable for T-DC interactions, revealing that distinct molecular rules govern the stable interactions of CD4⁺ T cells with two major antigen presenting cell types in vivo. Stabilization of T-B interactions may rely on a T cell-autonomous mechanism orchestrated by SAP and its associated SLAM family molecules at the cell-cell interface, while stable T-DC association might be actively promoted via DC-mediated mechanisms, such as those involving cytoskeletal and membrane mechanics³⁸^,³⁹. In accord with this notion, B cells but not DCs express high levels of SLAM, CD84, Ly9, and Ly108 molecules (Supplementary Fig. 10), which are also highly expressed by T cells activated in vitro and by T_FH cells in vivo (ref. ⁴⁰ and data not shown). The differential SAP regulation of T-DC and T-B interactions correlates with our observations that CD4⁺ T cell activation in vivo is grossly normal irrespective of SAP expression, while otherwise competent effector T cells fail to efficiently deliver contact-dependent help to cognate B cells in the absence of SAP. At the cellular level, these results provide a likely explanation for why SAP deficiency specifically impairs T-dependent humoral immunity but leaves intact or even exaggerates cell-mediated immune responses that also require CD4⁺ T cell activity¹³^–¹⁵^,⁴¹^,⁴². While additional factors may also contribute to the overall defects of humoral immunity seen in SAP-deficient hosts⁴³, our results strongly suggest the predominant mechanism is the disruption of antigen-specific T-B adhesion. Future studies are necessary to determine the molecular basis for how SAP selectively controls cognate T-B association, to identify SAP-associated SLAM family proteins involved in this process, and to elucidate the cascade of intercellular signal exchange that transpires across a stable T-B synapse.

In conclusion, our findings offer a compelling explanation for the profound impairment of the GC response in SAP-deficient mice and suggest a mechanism contributing to the B cell-centric pathologies associated with the XLP syndrome. They provide novel insights into the operating principles of intercellular communication between T cells and diverse antigen presenting cells and serve as a striking example of the importance of a direct visualization approach in understanding immune system function in vivo.

METHODS SUMMARY

Cell conjugation assay in vitro

OT-2 T cells (5×10⁵/well) were incubated for 30 min at 37°C in 96-well U-bottom plates with DCs (10⁶/well) or LPS-activated B cells (2×10⁶ per well) that had been pulsed with antigen. Conjugates were enumerated by flow cytometry after the cell mixture was stained at 4°C for CD4, CD11c, and CD19 and repeatedly washed.

Adoptive transfer, cell phenotyping, and intravital imaging

To visualize T-DC interactions (Fig. 1), OVA₃₂₃-pulsed DCs were injected subcutaneously at 2×10⁶ per mouse 24 hours prior to intravenous transfer of sap^+/+ and sap^−/− T cells (3×10⁶ each). Imaging was conducted 12 to 24 hours later. To examine activation phenotypes, 10⁶ DCs and 2×10⁵ GFP-expressing T cells per mouse were used. To visualize interactions between B cells and sap^+/+ and sap^−/− T cells in the same LN (Fig. 2), 3×10⁶ OT-2 T cells of each genotype were co-transferred into mice together with 5×10⁶ wild-type B cells. Immunization was done 12 hours prior to cell transfer, and imaging was conducted 24 to 36 hours thereafter. To visualize T-B interactions under non-competitive conditions and to assay T and B cell expansion (Fig. 3), 6×10⁴ GFP-expressing OT-2 T cells were co-transferred together with 3×10⁵ CFP-expressing B cells 24 hours prior to immunization. Imaging and cytometric analyses were conducted 60–72 and 96 hours later, respectively. To visualize GC recruitment and retention of T cells (Fig. 4), 3×10⁴ CFP-expressing sap^+/+ and 3×10⁴ GFP-expressing sap^−/− OT-2 T cells were co-transferred with 3×10⁵ non-fluorescent MD4 B cells. Imaging was conducted 6 to 8 days post immunization. Dye-labelled naïve B cells (2–4×10⁷) were given 1 day before imaging to provide follicle/GC landmarks. The imaging set-up was essentially as previously described⁴⁴. For imaging sessions longer than 2 hours, the animal’s hydration was maintained by lactated Ringer’s solution given via a catheter. The typical X-Y-Z dimension was 0.5–1.1×0.5–1.1×3 μm, and the time resolution was 30–45s. For experiments involving co-transfer of two types of dye-labelled T cells, the cells were always reciprocally labelled to control for potential dye-induced behavioural differences.

Statistical analysis

The Mann-Whitney rank sum test was used to calculate p values for highly skewed distributions. For Gaussian-like distributions, two-tailed t tests were used.

Supplementary data

Supplementary Figures and Movie Legends

This file contains Supplementary Figures 1-10 and Supplementary movie legends.

Click here to view.^{(17M, pdf)}

Supplemental Methods

Click here to view.^{(161K, pdf)}

Supplementary Movie 1

Click here to view.^{(2.6M, mov)}

Supplementary Movie 2

Click here to view.^{(4.3M, mov)}

Supplementary Movie 3

Click here to view.^{(2.7M, mov)}

Supplementary Movie 4

Click here to view.^{(9.8M, mov)}

Supplementary Movie 5

Click here to view.^{(11M, mov)}

Supplementary Movie 6

Click here to view.^{(9.8M, mov)}

Supplementary Movie 7

Click here to view.^{(9.0M, mov)}

Supplementary Movie 8

Click here to view.^{(9.3M, mov)}

Acknowledgments

H.Q. is in debt to Y. Hong for support, encouragement, and inspiration. This work was funded by the intramural research programs of the NIAID and NHGRI, NIH, U.S.A.

Footnotes

Reprints and permission information is available online at www.nature.com/reprints. The authors declare no competing financial interests.

References

1. Coico RF, Bhogal BS, Thorbecke GJ. Relationship of germinal centers in lymphoid tissue to immunologic memory. VI Transfer of B cell memory with lymph node cells fractionated according to their receptors for peanut agglutinin. J Immunol. 1983;131:2254–2257. [PubMed]

2. Berek C, Berger A, Apel M. Maturation of the immune response in germinal centers. Cell. 1991;67:1121–1129. [PubMed]

3. Jacob J, Kelsoe G, Rajewsky K, Weiss U. Intraclonal generation of antibody mutants in germinal centres. Nature. 1991;354:389–392. [PubMed]

4. MacLennan IC, et al. The changing preference of T and B cells for partners as T-dependent antibody responses develop. Immunol Rev. 1997;156:53–66. [PubMed]

5. Raff MC. Role of thymus-derived lymphocytes in the secondary humoral immune response in mice. Nature. 1970;226:1257–1258. [PubMed]

6. Mitchison NA. The carrier effect in the secondary response to hapten-protein conjugates. II Cellular cooperation. Eur J Immunol. 1971;1:18–27. [PubMed]

7. Lanzavecchia A. Antigen-specific interaction between T and B cells. Nature. 1985;314:537–539. [PubMed]

8. Garside P, et al. Visualization of specific B and T lymphocyte interactions in the lymph node. Science. 1998;281:96–99. [PubMed]

9. Vinuesa CG, Tangye SG, Moser B, Mackay CR. Follicular B helper T cells in antibody responses and autoimmunity. Nat Rev Immunol. 2005;5:853–865. [PubMed]

10. Purtilo DT, Cassel CK, Yang JP, Harper R. X-linked recessive progressive combined variable immunodeficiency (Duncan’s disease) Lancet. 1975;1:935–940. [PubMed]

11. Grierson HL, et al. Immunoglobulin class and subclass deficiencies prior to Epstein-Barr virus infection in males with X-linked lymphoproliferative disease. Am J Med Genet. 1991;40:294–297. [PubMed]

12. Ma CS, Nichols KE, Tangye SG. Regulation of cellular and humoral immune responses by the SLAM and SAP families of molecules. Annu Rev Immunol. 2007;25:337–379. [PubMed]

13. Wu C, et al. SAP controls T cell responses to virus and terminal differentiation of TH2 cells. Nat Immunol. 2001;2:410–414. [PubMed]

14. Czar MJ, et al. Altered lymphocyte responses and cytokine production in mice deficient in the X-linked lymphoproliferative disease gene SH2D1A/DSHP/SAP. Proc Natl Acad Sci U S A. 2001;98:7449–7454. [PubMed]

15. Crotty S, et al. SAP is required for generating long-term humoral immunity. Nature. 2003;421:282–287. [PubMed]

16. Morra M, et al. Defective B cell responses in the absence of SH2D1A. Proc Natl Acad Sci U S A. 2005;102:4819–4823. [PubMed]

17. Cannons JL, et al. SAP regulates T cell-mediated help for humoral immunity by a mechanism distinct from cytokine regulation. J Exp Med. 2006;203:1551–1565. [PMC free article] [PubMed]

18. Coffey AJ, et al. Host response to EBV infection in X-linked lymphoproliferative disease results from mutations in an SH2-domain encoding gene. Nat Genet. 1998;20:129–135. [PubMed]

19. Sayos J, et al. The X-linked lymphoproliferative-disease gene product SAP regulates signals induced through the co-receptor SLAM. Nature. 1998;395:462–469. [PubMed]

20. Cannons JL, et al. SAP regulates T_H2 differentiation and PKC-θ-mediated activation of NF-κB1. Immunity. 2004;21:693–706. [PubMed]

21. Dent AL, Hu-Li J, Paul WE, Staudt LM. T helper type 2 inflammatory disease in the absence of interleukin 4 and transcription factor STAT6. Proc Natl Acad Sci U S A. 1998;95:13823–13828. [PubMed]

22. Andoh A, et al. Absence of interleukin-4 enhances germinal center reaction in secondary immune response. Immunol Lett. 2000;73:35–41. [PubMed]

23. Huang AY, Qi H, Germain RN. Illuminating the landscape of in vivo immunity: insights from dynamic in situ imaging of secondary lymphoid tissues. Immunity. 2004;21:331–339. [PubMed]

24. Stoll S, Delon J, Brotz TM, Germain RN. Dynamic imaging of T cell-dendritic cell interactions in lymph nodes. Science. 2002;296:1873–1876. [PubMed]

25. Mempel TR, Henrickson SE, Von Andrian UH. T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature. 2004;427:154–159. [PubMed]

26. Miller MJ, Safrina O, Parker I, Cahalan MD. Imaging the single cell dynamics of CD4⁺ T cell activation by dendritic cells in lymph nodes. J Exp Med. 2004;200:847–856. [PMC free article] [PubMed]

27. Celli S, Lemaitre F, Bousso P. Real-time manipulation of T cell-dendritic cell interactions in vivo reveals the importance of prolonged contacts for CD4⁺ T cell activation. Immunity. 2007;27:625–634. [PubMed]

28. Abbas AK, Haber S, Rock KL. Antigen presentation by hapten-specific B lymphocytes. II Specificity and properties of antigen-presenting B lymphocytes, and function of immunoglobulin receptors. J Immunol. 1985;135:1661–1667. [PubMed]

29. Okada T, et al. Antigen-engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells. PLoS Biol. 2005;3:e150. [PMC free article] [PubMed]

30. Davidson D, et al. Genetic evidence linking SAP, the X-linked lymphoproliferative gene product, to Src-related kinase FynT in T_H2 cytokine regulation. Immunity. 2004;21:707–717. [PubMed]

31. McCausland MM, et al. SAP regulation of follicular helper CD4 T cell development and humoral immunity is independent of SLAM and Fyn kinase. J Immunol. 2007;178:817–828. [PubMed]

32. Garcia Z, et al. Competition for antigen determines the stability of T cell-dendritic cell interactions during clonal expansion. Proc Natl Acad Sci U S A. 2007;104:4553–4558. [PubMed]

33. Kelsoe G, Zheng B. Sites of B-cell activation in vivo. Curr Opin Immunol. 1993;5:418–422. [PubMed]

34. Liu YJ, et al. Sites of specific B cell activation in primary and secondary responses to T cell-dependent and T cell-independent antigens. Eur J Immunol. 1991;21:2951–2962. [PubMed]

35. Moser B, Schaerli P, Loetscher P. CXCR5⁺ T cells: follicular homing takes center stage in T-helper-cell responses. Trends Immunol. 2002;23:250–254. [PubMed]

36. de Vinuesa CG, et al. Germinal centers without T cells. J Exp Med. 2000;191:485–494. [PMC free article] [PubMed]

37. Schwickert TA, et al. In vivo imaging of germinal centres reveals a dynamic open structure. Nature. 2007;446:83–87. [PubMed]

38. Al-Alwan MM, Rowden G, Lee TD, West KA. The dendritic cell cytoskeleton is critical for the formation of the immunological synapse. J Immunol. 2001;166:1452–1456. [PubMed]

39. Benvenuti F, et al. Requirement of Rac1 and Rac2 expression by mature dendritic cells for T cell priming. Science. 2004;305:1150–1153. [PubMed]

40. Chtanova T, et al. T follicular helper cells express a distinctive transcriptional profile, reflecting their role as non-Th1/Th2 effector cells that provide help for B cells. J Immunol. 2004;173:68–78. [PubMed]

41. Hron JD, et al. SH2D1A regulates T-dependent humoral autoimmunity. J Exp Med. 2004;200:261–266. [PMC free article] [PubMed]

42. Crotty S, et al. Hypogammaglobulinemia and exacerbated CD8 T-cell-mediated immunopathology in SAP-deficient mice with chronic LCMV infection mimics human XLP disease. Blood. 2006;108:3085–3093. [PubMed]

43. Ma CS, et al. Impaired humoral immunity in X-linked lymphoproliferative disease is associated with defective IL-10 production by CD4⁺ T cells. J Clin Invest. 2005;115:1049–1059. [PMC free article] [PubMed]

44. Qi H, Egen JG, Huang AY, Germain RN. Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells. Science. 2006;312:1672–1676. [PubMed]