(CD150) Lymphocytic Activation Molecule Receptor IL-4 Production Is Dependent on Signaling Germinal Center T Follicular Helper C

Germinal Center T Follicular Helper Cell IL-4 Production Is Dependent on Signaling Lymphocytic Activation Molecule Receptor (CD150) This information is current as of September 25, 2021. Isharat Yusuf, Robin Kageyama, Laurel Monticelli, Robert J. Johnston, Daniel DiToro, Kyle Hansen, Burton Barnett and Shane Crotty J Immunol 2010; 185:190-202; Prepublished online 4 June 2010; Downloaded from doi: 10.4049/jimmunol.0903505 http://www.jimmunol.org/content/185/1/190 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2010/06/02/jimmunol.090350 Material 5.DC1 References This article cites 96 articles, 48 of which you can access for free at: http://www.jimmunol.org/content/185/1/190.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Germinal Center T Follicular Helper Cell IL-4 Production Is Dependent on Signaling Lymphocytic Activation Molecule Receptor (CD150)

Isharat Yusuf,*,1 Robin Kageyama,* Laurel Monticelli,* Robert J. Johnston,*,† Daniel DiToro,* Kyle Hansen,* Burton Barnett,* and Shane Crotty*,†

CD4 T cell help is critical for the generation and maintenance of germinal centers (GCs), and T follicular helper (TFH) cells are the CD4 T cell subset required for this process. Signaling lymphocytic activation molecule (SLAM)-associated protein (SAP [SH2D1A]) expression in CD4 T cells is essential for GC development. However, SAP-deficient mice have only a moderate defect + in TFH differentiation, as defined by common TFH surface markers. CXCR5 TFH cells are found within the GC, as well as along the boundary regions of T/B cell zones. In this study, we show that GC-associated T follicular helper (GC TFH) cells can be Downloaded from identified by their coexpression of CXCR5 and the GL7 epitope, allowing for phenotypic and functional analysis of TFH and GC TFH populations. GC TFH cells are a functionally discrete subset of further polarized TFH cells, with enhanced B cell help capacity and a specialized ability to produce IL-4 in a TH2-independent manner. Strikingly, SAP-deficient mice have an absence of the GC 2 TFH cell subset and SAP TFH cells are defective in IL-4 and IL-21 production. We further demonstrate that SLAM (Slamf1, CD150), a surface receptor that uses SAP signaling, is specifically required for IL-4 production by GC TFH cells. GC TFH cells require IL-4 and -21 production for optimal help to B cells. These data illustrate complexities of SAP-dependent SLAM family http://www.jimmunol.org/ receptor signaling, revealing a prominent role for SLAM receptor ligation in IL-4 production by GC CD4 T cells but not in TFH cell and GC TFH cell differentiation. The Journal of Immunology, 2010, 185: 190–202.

ong-term humoral immunity is provided by long-lived TFH cells to home to the B cell follicle (8–10). We and other plasma cells and memory B cells, both of which arise from investigators recently demonstrated that Bcl6 is the master regu- L the germinal center (GC) reaction and are critical compo- lator of TFH cell differentiation (11–13). In the absence of TFH nents of protective immunity to many pathogens (1–5). GCs re- cells, as a result of the absence of Bcl6 (11–13) or the presence of quire CD4 T cells, and T follicular helper (TFH) cells are the CD4 B lymphocyte-induced maturation protein (Blimp-1) (11), GC T cell lineage specialized in B cell help (6, 7). A central property responses are lost, as is most T-dependent Ab production. by guest on September 25, 2021 of TFH cells is their ability to migrate to the B cell follicle, allow- SAP (SLAM-associated protein), a small Src homology domain ing them to provide help to B cells to initiate and maintain the GC 2-domain adaptor protein encoded by the gene SH2D1A, is integral response. Expression of the chemokine receptor CXCR5 allows to T cell-dependent humoral immunity. Like TFH cells, SAP is required for GC formation (14). GCs are absent in SAP2 mice postinfection with lymphocytic choriomeningitis virus (LCMV) *Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology; and (14), influenza (15), Schistosoma mansoni eggs (16), or vaccinia †Department of Medicine, University of California, San Diego, School of Medicine, La Jolla, CA 92037 virus (17), as well as after protein immunizations (16, 18). SAP 1Current address: Department of Respiratory, Inflammation, and Autoimmunity, expression in CD4 T cells is required for GC responses (14–16, MedImmune, Gaithersburg, MD. 19). SAP is likely not needed in B cells (19), although this is Received for publication October 28, 2009. Accepted for publication April 12, 2010. disputed (20). Studies also showed important roles for SAP This work was supported by a Pew Scholar award, La Jolla Institute for Allergy and in NKT cell development (21, 22), CD4 T cell TH1/TH2 differen- Immunology Institutional Funds, a Cancer Research Institute award, and National tiation (23–25), and regulation of NK cell killing (26). Institutes of Health National Institute of Allergy and Infectious Diseases Grants R01 The essential role of SAP in T cell-dependent humoral immunity 072543 and R01 063107. I.Y. and R.J. were supported by fellowships from the University of California, San Diego/La Jolla Institute for Allergy and Immunology is conserved in humans. SH2D1A mutations result in the lethal National Institutes of Health Training Grant. immunodeficiency X-linked lymphoproliferative disease (27). Raw microarray signal data have been deposited at the National Center for Biotech- Patients with this disease have severe GC and memory B cell nology Information GEO database (www.ncbi.nlm.nih.gov/geo/) under accession deficiencies (28–30). numbers GSE21379–GSE21381. The SLAM family of receptors, which bind SAP through their Address correspondence and reprint requests to Dr. Shane Crotty, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037. E-mail immunotyrosine switch motif, are expressed on T cells, B cells, and address: [email protected] other hematopoietic cells (27, 31, 32). The SLAM family consists of The online version of this article contains supplemental material. nine receptors closely grouped on chromosome 1 in mice and Abbreviations used in this paper: Blimp-1, B lymphocyte-induced maturation pro- humans, most of which (with the exception of 2B4) form homophilic- tein; BTLA, B and T lymphocyte attenuator; GC, germinal center; GC TFH cell, binding interactions (27). SLAM (CD150) is the prototypic member germinal center-associated T follicular helper cell; LCMV, lymphocytic choriomeningitis virus; MFI, mean fluorescence intensity; PD-1, programmed death 1; qPCR, of the family. SLAM family receptors are an important genetic sus- quantitative PCR; SAP, signaling lymphocytic activation molecule-associated pro- ceptibility locus for autoantibody production and systemic lupus tein; SLAM, signaling lymphocytic activation molecule; SMtg, SMARTATCR trans- erythematosus in mice and humans (33, 34). genic; T , T follicular helper; WT, wild-type. FH SAP has a major role in T cell–B cell adhesion (35). In the Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 absence of SAP expression in CD4 T cells, conjugates between www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903505 The Journal of Immunology 191 cognate CD4 T cells and B cells were short-lived in vitro and Andre Veillette (Clinical Research Institute of Montreal, Montreal, QC, in vivo (35), although the development of CXCR5hiICOShi putative Canada) and was used as previously described (19) after direct conjugation to Alexa Fluor 647, according to the kit instructions (Invitrogen). TFH CD4 T cells was not significantly diminished (35). It has not been known which of the SAP-binding SLAM family receptors is Histology required for T cell help to B cells (27, 35) or whether adhesion is Standard immunofluorescence histology was performed, as described the only role for SAP and the SLAM family receptors in the GC. previously (39), using an upright microscope (DM6000B) with an SP5 To better understand the generation of potent GC and Ab confocal head (Leica Microsystems, Deerfield, IL). The following Abs responses, we took the complementary approach of studying the were used: GL7 FITC rat anti-mouse (BD Pharmingen), B220 Alexa Fluor roles of SAP and SLAM in the development and function of T 647 (RA3-6B2; eBioscience), and CD45.1 biotin (A20; eBioscience) with FH Streptavidin Alexa Fluor 555 (Invitrogen). cells. We found a phenotypically and functionally distinct subpopulation of TFH cells that resides within GCs. These GC- GC CD4 T cell calculations resident TFH cells (GC TFH cells) produce IL-4 and are absent in Based on histology 5–10% of GC cells are CD4 T cells. Mice have ∼7.5 3 5 5 SAP-deficient mice. GC TFH cell production of IL-4 is dependent 10 virus-specific GC B cells and 2.9 3 10 virus-specific TFH cells at day on SLAM. Furthermore, IL-4 and IL-21 production by GC TFH 8 after LCMV infection, as measured by flow cytometry. Therefore, the cells is required for optimal Ab production. quantity of virus-specific TFH cells was estimated to exceed the number of CD4 T cells inside of GCs by a factor of 3:1. Materials and Methods RNA, gene expression microarrays, and quantitative PCR Mice Splenocytes were isolated, and Ag-specific CD4 T cells were enriched using C57BL/6J (B6) mice were purchased from The Jackson Laboratory (Bar anti-CD45.1–FITC and magnetic bead purification (Miltenyi Biotec). WT Downloaded from 2 2 2 2 2 + + + 2 2 Harbor, ME). SAP (36), OTII CD45.1, SLAM / SAP , and SMARTA and SAP TFH cells and non-TFH CD4 CD45.1 TCRb CD19 7AAD b + cells were sorted on the basis of CXCR5 expression (CXCR5hi versus TCR transgenic (SMtg; LCMV gp66-77 I-A specific) (37) CD45.1 mice lo CXCR5 ), using a FACSAria (BD Biosciences, San Jose, CA). TFH cell were all on a fully B6 background and were bred at the La Jolla Institute for hi lo Allergy and Immunology. B6.Sle1b mice were a gift of Edward Wakeland gating was confirmed by CXCR5 and SLAM costaining on an aliquot of 3 6 (UT Southwestern Medical Center, Dallas, TX) (38). SLAM mice were cells. Approximately 1 10 cells from each condition (in duplicate) were generated by Y. Yunagi on the 129 background (24) and backcrossed for sorted directly into RNAlater (Ambion, Austin, TX). Naive SMtg CD4 T cells were obtained from intact SMtg mice, sorting for CD4+CD45.1+ 11 generations to the B6 background at Kyushu University and the La Jolla lo hi 2 http://www.jimmunol.org/ Institute for Allergy and Immunology. Whole-genome microsatellite anal- CD44 CD62L 7AAD . RNA was isolated using RNeasy Mini spin ysis, through the University of California, Los Angeles, Southern California columns (Qiagen, Valencia, CA), including Qiashredder and on-column Genotyping Consortium, verified that the SLAM2/2 mice were 99% B6. digestion of genomic DNA. Then, some RNA samples were concentrated The remaining 1% was of the Sv129 background around the SLAM locus, using MinElute spin columns (Qiagen). incorporating the region between the single nucleotide polymorphism RNA quality of all samples was confirmed by BioAnalyzer Nano gel markers mCv22849619 and rs13476259. SAP2 mice were .99% B6 by (Agilent, Palo Alto, CA), and then probes were generated by single round single nucleotide polymorphism analysis, with a small region of the X linear amplification using the Ovation Pico system (Nugen) and used on chromosome remaining Sv129. All animal experiments were conducted Affymetrix 430 2.0 chips. Data were analyzed using Genespring (Agilent), in accordance with approved animal protocols. Microsoft Excel (Microsoft, Redmond, WA), and Prism 5.0 (GraphPad, San Diego, CA). Raw microarray signal data have been submitted to

Adoptive transfers, infections, and immunizations National Center for Biotechnology Information GEO database (www.ncbi. by guest on September 25, 2021 nlm.nih.gov/geo/) under accession numbers GSE21379 and GSE21381. Cell transfers into host mice were performed by i.v. injection via the retro- cDNA synthesis was performed using SuperScript II Reverse Tran- orbital sinus. SMtg CD4 T cells were transferred so that each mouse received scriptase (Invitrogen) with oligonucleotide dT and random hexamer primed 5,000 SMtg CD4+ T cells (∼12,000 total splenocytes, as determined for each 3 3 reactions, which were then pooled. Quantitative PCR (qPCR) reactions were experiment by FACS). For mixed-cell experiments, 25 10 wild-type performed in triplicate using iTaq SYBR Green Supermix with ROX (Bio- (WT) SMtg cells and 25 3 103 SAP2 SMtg cells were transferred into 3 3 Rad, Hercules, CA) on a Roche LightCycler 480 (Roche, Mannheim, a common CD4-depleted host (50 10 SMtg cells/mouse); CD4 deple- Germany). Primers were as described (11). For GATA3, two independent tions were done as previously described (39). LCMV stocks were prepared primer sets were used for confirmation: one primer set within the GATA3 and quantified as described (39). All infections were done by i.p. injection of 3 5 + coding region and one primer set within the GATA3 39 untranslated region. 1–2 10 PFU LCMV Armstrong per mouse. For OTII CD4 T cell trans- b-actin was used as the reference for normalization, and expression levels fers, naive CD4+ T cells were purified from spleen by negative selection 3 3 were then normalized to the naive CD4 T cell control. using magnetic beads (Miltenyi Biotec, Auburn, CA); 250 10 OTII cells For Figs. 1, 2, and 4, CD4 T cells were purified by negative selection were transferred per mouse. NP-OVA/alum was prepared by mixing NP(19)- using magnetic beads (Miltenyi Biotec). Enriched cells were sorted as OVA (Vector Laboratories, Burlingame, CA) in PBS with alum (Pierce, CD4+CD44hi7AAD2 and on the basis of CXCR5 and GL7 expression: Rockford, IL) at a 3:1 ratio for 60 min at 4˚C. NP-OVA/alum immunizations lo 2 + 2 non-TFH cells (CXCR5 GL7 ), TFH cells (CXCR5 GL7 ), and GC TFH consisted of 100 mg given i.p. cells (CXCR5+GL7+). RNA isolation, cDNA synthesis, and qPCR analysis Flow cytometry were performed as described above. Raw microarray signal data have been submitted to National Center for Biotechnology Information GEO Single-cell suspensions of spleen were prepared by standard gentle mechan- database under accession numbers GSE21380 and GSE21381. ical disruption. Surface staining for flow cytometry used mAbs to B220 For Fig. 7, purified SLAM2/2 and SLAM+/+ CD4 T cells were sorted in (RA3-6B2), GL7 (FITC [BD Pharmingen, San Diego, CA] or purified triplicate (each replicate consisting of four pooled SLAM2/2 or SLAM+/+ [eBioscience]) and Fas (BD Pharmingen). Also used were mAbs to CD4 spleens) into non-TFH,TFH, and GC TFH populations for qPCR analysis. (LT34 and GK1.5), B and T lymphocyte attenuator (BTLA) (6F7), ICOS (7E.17G9), programmed death 1 (PD-1) (J43), CD45.1 (A20), CD45.2 ELISA (104), CD62L (MEL-14), IFN-g (XMG1.2), and CD44 (IM7) (all from Serum was used from B6 mice 30 d after LCMVinfection. Anti-LCMV IgG1 eBioscience). Abs against SLAM (TC15-12F12.2) were from BioLegend. and IgG2c were quantified by ELISA using LCMV-infected cell lysate as the FITC-labeled peanut agglutinin was from Vector Laboratories. CXCR5 capture Ag. Ninety-six–well polysorp microtiter plates (Nunc, Rochester, staining was done using purified anti-CXCR5 (BD Pharmingen), followed NY) were coated overnight with LCMV-infected cell lysate in PBS. Follow- by biotinylated anti-rat IgG (Jackson ImmunoResearch Laboratories, West ing incubation of sample serum, HRP-conjugated goat anti-mouse IgG1 and Grove, PA), and then PE- or allophycocyanin-labeled streptavidin (Caltag IgG2c (Jackson ImmunoResearch Laboratories) were used for detection. Laboratories, Burlingame, CA), with each staining step done in PBS + 0.5% BSA + 2% FCS + 2% normal mouse serum on ice; samples were In vitro help assay acquired without fixation. Doublets were gated out during FlowJo analysis to exclude possible B–T cell conjugates. For intracellular cytokine stain- Purified CD4+ T cells from B6 mice 8 d post LCMV infection were sorted ing, splenocytes were stimulated with PMA and ionomycin for 4 h, fol- into GL7hiCXCR5hi, GL7loCXCR5hi, and GL7loCXCR5lo populations. lowed by surface staining. Intracellular staining was performed with Abs T cells (5 3 104) were cultured for 7 d with purified B cells (5 3 105)in against IFN-g (BD Pharmingen). Anti-SAP mAb(12C4) was a gift of the presence of IL-2 and b-ME. We used the blocking Abs aIL-4 and 192 SLAM-DEPENDENT GC TFH IL-4 PRODUCTION

aIL-21 at 20 and 40 ng/ml, respectively. Supernatants were collected, and We identified Ag-specific TFH cells after an acute LCMV infection total IgG was quantified by ELISA. Goat anti-mouse IgG+IgA+IgM (Cal- + as CXCR5 CD4 T cells that coexpress a set of TFH surface markers tag Laboratories) was the capture Ab, biotinylated goat anti-mouse IgG (ICOShiPD-1hiSLAMloBTLAhiCD200hi) validated by our labora- (Caltag Laboratories) was the secondary Ab, and HRP Avidin D was added thereafter (Vector Laboratories). For Fig. 8C, purified CD4+ T cells from tory for transgenic (Fig. 1A,1B) (11) and nontransgenic CD4 SLAM2/2 and SLAM+/+ mice 8 d post-LCMV infection were sorted for T cells (11) in the context of a viral infection and validated by GL7hiCXCR5hi populations. T cells (15 3 104) were cultured for 7 d with other laboratories in the context of other infections or immuniza- 3 5 purified B cells (5 10 ) in the presence of IL-2 and b-ME. Quantification tions (42–46). T cells are required for GC formation in vivo after was done by ELISA, as described above. FH viral infection (11). However, only ∼20% of virus-specific TFH Statistical analysis cells were inside GCs (see Materials and Methods for calcula- Statistical tests were performed using Prism 5.0, and p values were cal- tions). Therefore, we examined whether the CD4 T cells residing culated using two-tailed unpaired Student t tests, with a 95% confidence within the GC could be phenotypically identified as a specific sub- 2 interval. Error bars represent the SEM. The x test (95% confidence) was population of TFH cells, similar to the model proposed by used to analyze GC CD45.1 CD4 T cell counts by histology versus GL7+ neg McHeyzer-Williams et al. (41). and GL7 CD45.1 CD4 T cell counts by flow cytometry. A two-tailed We performed a search for markers of GC T cells and identified Mann–Whitney U test (95% confidence) was used for Fig. 8A. FH GL7 as a specific surface marker (Fig. 1C). A subpopulation of TFH cells in LCMV-infected mice stained with the GL7 mAb, whereas Results + hi the majority of TFH cells did not (Fig. 1C). A similar GL7 CXCR5 GC T cells are a distinct subpopulation of TFH cells PD-1hi staining pattern was seen within CD44hi OTII CD4 T cells

Murine TFH cells are most frequently deﬁned as a singular pop- after NP-Ova immunization (Fig. 5D, left panel). CD4 T cells within Downloaded from ulation (7), but subpopulations have also been proposed (40, 41). GCs stained positive with GL7 by histology (Fig. 1E), and GL7+ http://www.jimmunol.org/ by guest on September 25, 2021

+ FIGURE 1. GC T cells (GC TFH) are distinct from TFH cells in their ability to produce IL-4. A and B, CD45.1 SMtg CD4 T cells were adoptively transferred into C57BL/6 (B6)recipient micesubsequentlyinfectedwith LCMV. A, SMtgCD4 T cells inspleen, day8 postinfection. B, ICOS,PD-1, BTLA, and CXCR5 expression hi 2/ onSMtgcells; TFH cells are boxed. C, GL7 Ag and CXCR5 expression on nontransgenicCD44 CD4 T cells, day8 after LCMVinfection. Non-TFH cells (CXCR5 lo 2 hi 2 hi + 2 ), GL7 TFH cells (CXCR5 GL7 ),and GC TFH cells (CXCR5 GL7 )areboxed.D, PD-1 expression on GC TFH,GL7 TFH,non-TFH, and naive CD4 cells. Data are representative of at least three independent experiments. E, Immunofluorescence histology of spleen, 8 d after CD45.1+ OTII CD4 T cell transfer and NP-OVA immunization. GL7 (green), CD45.1 (red), and B220 (blue). CD45.1+ CD4 T cells inside the GC are marked by white boxes, and representative CD45.1+ CD4 T cells outside the GC are marked by yellow boxes. Enlarged views of the boxed CD4 T cells are shown to the right. Original magnification 3200. F, Quantitation of + 2 + hi GL7 and GL7 CD45.1 CD4 T cells outside GCs. G–P, mRNA expression by polyclonal LCMV-specific CD44 CD4 T cells, day 8 postinfection. Non-TFH lo hi 2 hi + + lo (CXCR5 ), TFH (CXCR5 GL7 ), and GC TFH (CXCR5 GL7 ) cells. Naive cells (CD4 CD44 ). Expression levels were quantitated in reference to b-actin mRNA and then normalized to naive CD4 T cells, except CXCR5, which was quantified by gene-expression microarray. G,Cmah.H,Bcl6.I,PD-1.J,ICOS. K,BTLA.L, CD200. M,IL-21.N,CXCR5.O, Prdm1/Blimp-1. P,IL-4.n = 2/group, with 10 spleens pooled per sample. ppp , 0.01; pppp , 0.001. The Journal of Immunology 193

+ Ag-specific (CD45.1 ) CD4 T cells were not present outside of GCs The strong TH1 bias of the antiviral immune response was (0 versus 2372; p , 0.0001 by x2 test) (Fig. 1F). confirmed by several experimental approaches. The majority (95%) 2/lo GL7 is a rat mAb that recognizes a2,6-linked N- of CXCR5 (non-TFH) virus-specific CD4 T cells produced hi acetylneuraminic acid (Neu5Ac) on glycan chains (47, 48). In- IFN-g (Fig. 2A,2B). Most (57%) CXCR5 TFH cells also triguingly, Neu5Ac is highly expressed on GC B cells, and GL7 produced IFN-g (Fig. 2A,2B). Both populations also expressed has long been used as a marker for GC B cells. Theoretically, high levels of T-bet (non-TFH cells versus naive CD4 T cells, + + Neu5Ac (GL7 ) CD4 T cells could acquire the Neu5Ac sugar 400-fold, p , 0.001; TFH cells versus naive, 100-fold, p , 0.001) + from the surface of adjacent Neu5Ac GC B cells. We examined (Fig. 2C). Consistent with the expression of IFN-g by the non-TFH hi + the capacity of GC TFH cells to synthesize Neu5Ac. Cmah is the and TFH populations, GC TFH cells (CXCR5 GL7 ) also expressed enzyme that converts Neu5Ac to the more abundant sugar substantial levels of IFN-g (Fig. 2D). The Ab response to LCMV is Neu5Gc, which is not bound by GL7 (48). A decreased expression highly dominated by IgG2c (Fig. 2E), consistent with the heavily of Cmah results in higher levels of Neu5Ac (48). Gene-expression TH1-biased IFN-g–producing antiviral CD4 T cell response. analysis of naive, non-TFH,TFH, and GC TFH cells revealed To directly demonstrate IL-4 production by GC TFH cells, we that GC TFH cells had lower Cmah expression than the other used IL-4 GFP reporter mice. After LCMV infection, IL-4 produc- CD4 T cell populations (p , 0.0001) (Fig. 1G). Therefore, the tion was observed by CXCR5+ but not CXCR52/lo virus-specific + + increased Neu5Ac GL7 epitope on GC TFH cells is the result of CD4 T cells (Fig. 3A). GL7 CXCR5 CD4 T cells were the primary metabolic changes within the CD4 T cells. Neu5Gc is a potent IL-4 producers (p , 0.0001) (Fig. 3B). No IL-4 was made by CD22 ligand that strongly inhibits B cell activation (48–50). The non-TFH CD4 T cells, again confirming that the IL-4 production absence of Neu5Gc on GC B cells (48) and GC TFH cells (Fig. 1) was TFH specific and not TH2 specific (Fig. 3A,3C). The majority of Downloaded from eliminates the tonic negative signaling through CD22 during GC the IL-4 was produced by the highest CXCR5-expressing cells, T–B interactions, thereby likely providing an elegant mechanism even within the TFH population (p = 0.0002) (Fig. 3C). This cor- to enhance GC B cell proliferation. roborates the data that GC TFH cells coordinately express the high- After protein immunizations, Cyster and colleagues (10) iden- est levels of GL7, Bcl6, PD-1, CXCR5, and IL-4 (Fig. 1) and + int tified a CXCR5 PD-1 TFH cell population and a higher PD-1– correlates with human data showing that GC TFH cells have the

expressing GC-associated T cell population. In studying the CD4 highest level of CXCR5 (12, 58). http://www.jimmunol.org/ T cell response to LCMV, we were unable to distinguish these two SAP-deficient mice have a severe block in GC TFH cell TFH-related populations based on PD-1 and CXCR5 expression alone (Fig. 1B), likely as a result of the broad upregulation of differentiation PD-1 on T cells after a viral infection (Fig. 1D) (51). Backgating SAP mRNA expression was increased in TFH and GC TFH cells analysis revealed that PD-1 expression was highest within the (p , 0.0001) (Fig. 4A). These data correlated well with data + GL7 GC TFH population (Fig. 1D). showing increased expression of SAP in human tonsillar TFH cells compared with other T cell subsets (59, 60). We also examined GC TFH cells produce IL-4 SAP proteins levels in CD4 T cells and found graded increases by guest on September 25, 2021 Having identified GC TFH cells and non-GC TFH cells as distin- from naive cells to non-TFH cells to TFH cells to GC TFH cells, guishable populations of Ag-specific CD4 T cells, distinct from with SAP expression elevated 4-fold in GC TFH cells over naive non-TFH cells, we assessed the phenotypic properties of these CD4 T cells (p , 0.0001) (Fig. 4B,4C). cells. Assessment of the expression of the TFH master regulator Given that SAP expression by CD4 T cells is a requirement for transcription factor Bcl6 and its antagonist Blimp-1 (11) GC development (14), we examined whether TFH cell differenti- 2 indicated that GC TFH cells are a further polarized state of TFH ation was dependent on SAP. Up to a 50% reduction in SAP TFH differentiation (Fig. 1H,1O). Expression of IL-21, PD-1, ICOS, cell differentiation was observed in vivo for SMtg CD4 T cells CXCR5, BTLA, and CD200 were all increased in TFH cells versus responding to an LCMV infection (p = 0.0013) (Fig. 4G). Several non-TFH cells, and expression of these TFH-associated molecules independent TFH cell markers were used, including ICOS, PD-1, was further increased in GC TFH cells (Fig. 1I–N), corroborating and BTLA (Fig. 4D–F). Overall, CD4 T cell activation and pro- 2 that GC TFH cells are a further polarized state of TFH cell differen- liferation were normal, because total numbers of SAP and WT tiation. An exception to this pattern was IL-4, which was uniquely SMtg CD4 T cells were equivalent (data not shown, Supplemental 2 expressed by GC TFH cells but not GL7 TFH cells (Fig. 1P). Fig. 1). A partial reduction in TFH cell frequency was also seen in hi Although IL-4 was reported to be expressed by TFH or GC- the polyclonal (CD44 ) LCMV-specific CD4 T cell response in 2 associated CD4 T cells, those findings were in the context of SAP mice (28%; p , 0.05) (Fig. 4H). TFH cell differentiation is TH2-biased infections or immunizations (46, 52, 53). The obser- unusual in that it is heavily dependent on the presence of cognate vation that GC TFH cells produced IL-4 after an LCMV infection B cells (6, 10, 11). Because B cell defects can cause TFH cell was surprising, because CD4 T cell responses to viral infections, deficiencies, we used WT:SAP mixed adoptive transfers to con- 2 including LCMV, exhibit a strong TH1 bias (11, 54–57). It was firm that the SAP TFH cell differentiation defect was CD4 T cell also intriguing that the expression was exquisitely selective to the intrinsic (p , 0.0001) (Supplemental Fig. 1). 2 GC TFH cells. This indicated that GC TFH cells are a specific dif- Although TFH cell differentiation was reduced in SAP mice, ferentiation state of TFH cells, and the ability to produce IL-4 is this #2-fold reduction in TFH cell frequency failed to account for a delineating feature of GC TFH cells, independent of the type of the profound impairment of GC B cell development (Fig. 4I). + immunization or infection. Furthermore, no loss of TFH cell (CXCR5 ) differentiation was Interestingly, the expression of IL-4 by the GC TFH population observed in a second model, protein-immunization experiments 2 was TH2 independent, because GATA3 expression was not in- with SAP OTII (Fig. 4J,4K), indicating that there is not a uni- creased above basal levels in any virus-specific CD4 T cells, in- versal requirement for SAP in TFH cell differentiation. This is cluding the GC TFH subpopulation (Fig. 2). C-Maf expression was consistent with the study of Qi et al. (35), which was published also unchanged among non-TFH,TFH, and GC TFH cells (Fig. 2G). while this work was ongoing. + Consistent with the absence of GATA3, TH2 cytokine IL-5 mRNA In light of the identification of the GL7 GC-associated GC TFH was undetectable (data not shown). subset of TFH cells, as well as the high levels of SAP observed in 194 SLAM-DEPENDENT GC TFH IL-4 PRODUCTION Downloaded from

FIGURE 2. LCMV-speciﬁc TFH cells and GC TFH cells exhibit TH1, but not TH2, attributes. A and B, Day 8 after LCMV infection of B6 mice that were 2 + recipients of SMtg CD4 T cells. IFN-g expression by CXCR5 non-TFH cells and CXCR5 TFH SMtg CD4 T cells after a 4-h in vitro restimulation with

PMA/ionomycin. A, Gated SMtg CD4 T cells are shown. Boxes show non-TFH and TFH cell gates. B, IFN-g expression by cells gated in A (n = 5/group). http://www.jimmunol.org/ Data are representative of three independent experiments. T-bet (C) and IFN-g (D) mRNA expression by polyclonal LCMV-speciﬁc CD44hi CD4+ T cells, 8 d after LCMV infection, directly ex vivo. Normalized as in Fig. 1. pppp , 0.001. E, Serum Ab levels (mg/ml) of IgG2c and IgG1 in LCMV-infected B6 mice. GATA3 (F) and c-Maf (G) mRNA expression, quantitated as above.

2 GC TFH cells (Fig. 4B,4C), we hypothesized that GC TFH cell after LCMV infection (Fig. 6A) revealed that SAP TFH cells had 2 differentiation may be impaired in SAP-deficient mice. We found predominantly normal TFH cell gene expression (WT versus SAP , 2 a striking 4–7-fold reduction in the GC TFH cell population in R = 0.9905) (Fig. 6B), including normal expression levels of SAP2 mice after LCMV infection (p , 0.0001) (Fig. 5A,5B). the T master regulator transcription factor Bcl6 (Fig. 6C), FH by guest on September 25, 2021 2 A comparable reduction in SAP GC TFH cell differentiation was downregulation of the TFH differentiation antagonist Blimp-1 observed after protein immunization (p , 0.0001) (Fig. 5C–E). (Fig. 6D), and upregulation of the TFH-related genes ICOS and + hi An almost complete absence of GC TFH cells (GL7 CXCR5 ) CD200 (Fig. 6E,6F). A 2-fold reduction in IL-21 was seen (Fig. 2 also correlated with the absence of the very highest PD-1– 6G). Strikingly, IL-4 expression was completely lost in SAP TFH expressing CXCR5hi CD4 T cells (Fig. 5F). cells (Fig. 6H). qPCR confirmed that IL-4 mRNA levels in SAP2 TFH cells were reduced to ,5% of that produced in WT TFH cells TFH cells in SAP-deficient mice exhibit IL-4 and IL-21 defects (p , 0.003). These data matched our earlier observation that GC We examined the effects of SAP deficiency on TFH cell gene TFH cells, but not TFH cells, produce IL-4 (Fig. 1P). Because 2 expression. Global gene-expression analysis comparing purified SAP CD4 T cells fail to differentiate into GC TFH cells, 2 hi hi SAP TFH cells (CXCR5 ) versus WT TFH cells (CXCR5 )8d the absence of IL-4 and a reduction in IL-21 production by

FIGURE 3. IL-4 production by GC TFH cells but not TFH or non-TFH cells. IL-4 production was tracked in 4get IL-4/GFP reporter mice after LCMV infection. A, IL-4 production by CXCR5+ CD4 T cells. Gated CD44hi CD4 T cells are shown. B, CXCR5+ CD4 T cells were then gated on the basis of GL7 + 2 + hi expression (GL7 GC TFH, GL7 TFH, as per Fig. 1C), and GFP IL-4 producing cells were quantiﬁed. C, Antiviral CD44 CD4 T cells were gated on the basis of CXCR5 expression, and GFP+ IL-4–producing cells in each population were quantiﬁed. ppp , 0.01; pppp , 0.001. The Journal of Immunology 195 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

2 hi FIGURE 4. SAP mice have a reduced TFH population after LCMV infection. A, SAP/SH2D1A mRNA expression in polyclonal LCMV-specific CD44 + 2/lo hi 2 hi + CD4 non-TFH cells (CXCR5 ), TFH cells (CXCR5 GL7 ), and GC TFH cells (CXCR5 GL7 ), day 8 after LCMV infection. Expression levels 2 lo lo hi hi + hi hi normalized as in Fig. 1. B, SAP protein expression in SAP naive (CD44 ), WT naive (CD44 ), GC TFH (CD44 CXCR5 GL7 ), TFH (CD44 CXCR5 2 hi 2/lo GL7 ), and non-TFH (CD44 CXCR5 ) CD4 T cells by flow cytometry. C, Quantitation of SAP protein mean fluorescence intensity (MFI) from B. Data are representative of two independent experiments (n = 6). D–G, WT or SAP2 SMtg CD4 T cells were adoptively transferred into B6 recipient mice subsequently infected with LCMV. Flow cytometric analysis of ICOS (D), PD-1 (E), and BTLA (F), gated on SMtg CD4 T cells at day 8 postinfection. G, hi + SMtg TFH cell quantitation. Data are representative of three experiments. H,TFH differentiation of polyclonal LCMV-specific (CD44 ) CD4 T cells in WT and SAP2 mice, 8 d postinfection. Naive (CD44lo) CD4 T cells are also shown. I, GC B cell frequency in WT and SAP- mice, 8 d after LCMV infection. Data are representative of .10 independent experiments. J, WT or SAP2 OTII CD4 T cells were adoptively transferred into B6 recipient mice subsequently lo 2 + immunized with NP-OVA. CXCR5 expression on naive CD44 B6 CD4 T cells, WT OTII cells, and SAP OTII CD4 T cells. K, Quantitation of TFH data in J. Data are representative of four independent experiments. ppp , 0.01; pppp , 0.001.

2 SAP TFH cells are interpreted as a direct result of the lack of the LCMV infection, SLAM is upregulated on non-TFH CD4 T cells GC TFH subpopulation. but not TFH CD4 T cells (11) (Fig. 7B). When virus-speciﬁc GC TFH cells were examined, an increase in SLAM expression from SLAM signaling drives the majority of IL-4 production by GC 2 GL7 TFH cells was noted (Fig. 7D,7E). SLAM can be involved TFH cells in IL-4 production in a SAP-dependent manner (23, 24). SLAM is SLAM is expressed on all CD4 T cells (39) and is upregulated on a self-associating receptor and is expressed on B cells. SLAM activated CD4 T cells (24, 39, 61–63), and we observed SLAM expression is substantially upregulated on GC B cells (Fig. 7F). expression on TH1 and TH2 CD4 T cells in vitro (Fig. 7A). After Therefore, SLAM–SLAM interaction likely occurs between GC 196 SLAM-DEPENDENT GC TFH IL-4 PRODUCTION Downloaded from

2 FIGURE 5. SAP is required for GC TFH cell differentiation. A, B,GCTFH cell differentiation in WT or SAP mice after LCMV infection. A, Flow hi + hi cytometry of GC TFH cell frequencies (CXCR5 GL7 ). Gated CD44 CD4 T cells are shown. B,GCTFH cell quantitation from data in A (n = 5/group). 2

Data are representative of three independent experiments. C–E, Congenically marked WT or SAP OTII CD4 T cells were transferred into B6 mice http://www.jimmunol.org/ subsequently immunized with NP-OVA. Eight days later, OTII CD4 T cells were analyzed for GC TFH cell differentiation. C, Total CD4 T cells are shown. 2 hi lo + Box shows OTII cell gate. D,GCTFH cell differentiation by WT and SAP OTII cells. CD44 CD62L CD4 T cells are shown. GC TFH cells are boxed. E, hi lo GC TFH cell percentage of OTII CD44 CD62L CD4 T cells (n = 4/group). Data are representative of three independent experiments. F, PD-1 expression 2 by WT and SAP TFH cells. pppp , 0.001.

+ B cells and GC TFH cells, allowing for adhesion and bidirectional GL7 GC TFH subpopulation provided quantitatively different 2 signaling. We hypothesized that interaction may induce GC TFH amounts of B cell help. Purified GL7 TFH cells provided cell differentiation or IL-4 production. efficient help to cocultured B cells in vitro, inducing sig- by guest on September 25, 2021 We tested whether SLAM–SAP signaling regulates GC TFH nificantly greater levels of IgG production than non-TFH cells cell differentiation or function, using SLAM-deficient mice (p , 0.01) or no T cells (p , 0.001) (Fig. 8A). GC TFH cells 2/2 (slamf1 ). Given that all SLAM family receptors are closely were even more potent than TFH cells at stimulating IgG located on chromosome 1 and have one or more roles in autoim- production (p , 0.01) (Fig. 8A). Blocking IL-21 eliminated IgG munity (27, 33, 34, 64), it was necessary to use 129Sv SLAM production (p , 0.001), confirming the critical role of IL-21 in family locus (sle1b) B6 congenic mice as the appropriate plasma cell differentiation (Fig. 8B). Importantly, blocking +/+ 2/2 Slamf1 control (34, 65). We analyzed Slamf1 versus IL-4 in the GC TFH–B cell cocultures decreased the levels of +/+ Slamf1 CD4 T cells for TFH cell and GC TFH cell differentiation secreted IgG by 75% (p , 0.04) (Fig. 8B), a level comparable 2/2 2 in vivo and found normal frequencies of Slamf1 TFH cells and to that of GL7 TFH–B cell cocultures. These results illustrate that GC TFH cells (Fig. 7G–I). Therefore, although SAP was necessary GC TFH cells produce physiologically meaningful levels of for the differentiation of GC TFH cells SLAM was not. This was IL-4, and TFH and GC TFH cells are functionally distinct. confirmed using SLAM2/2SAP2 double-deficient mice, which Based on those findings, we hypothesized that if SLAM en- showed that SAP is epistatic to SLAM for GC TFH cell differen- gagement is a primary regulator of IL-4 production by GC TFH 2/2 tiation and GC development (Supplemental Fig. 2). Therefore, cells, Slamf1 GC TFH cells should exhibit defective B cell 2/2 SAP is required for positive signaling and not inhibition of an help. We tested this using sorted Slamf1 GC TFH cells and +/+ alternative fate-determination signal through SLAM. congenic Slamf1 GC TFH cell controls cocultured with B cells. 2/2 Although Slamf1 GC TFH cells were present, IL-4 produc- B cells incubated with SLAM-deficient GC TFH cells produced 2/2 tion by Slamf1 GC TFH cells was markedly reduced (77% loss 74% less IgG than B cells incubated with WT GC TFH cells (p , 2/2 compared with WT; p = 0.0014) (Fig. 7J). As was observed for 0.0001) (Fig. 8C). Slamf1 GC TFH cells produced levels of WT GC TFH cells, no GATA3 mRNA induction was observed in IL-21 comparable to WT cells (Fig. 7K), and the defective 2/2 2/2 Slamf1 GC TFH cells (p .. 0.05 versus naive CD4 T cells). Slamf1 GC TFH B cell help can be fully accounted for by IL-21 production was normal (Fig. 7K). These Slamf12/2 CD4 the decrease in IL-4 production, given that SLAM deficiency or T cell data demonstrate that SLAM signaling is required blockade of IL-4 resulted in a comparable loss of GC TFH B cell for IL-4 production by GC TFH cell differentiation in a TH2- help activity (Fig. 8B,8C, Fig. 9). independent manner.

IL-4 production by GC TFH cells is required for optimal B cell Discussion help In this study we report four ﬁndings of note: GC TFH cells are The production of IL-4 by GC TFH cells led us to hypothesize that a phenotypically and functionally distinct further differentiated IL-4 is required for optimal GC TFH cell function and B cell help. subpopulation of TFH cells; the GC TFH cells are specialized 2 We examined whether GL7 TFH cells and the IL-4–producing producers of IL-4; SAP is not required for TFH cell differentiation, The Journal of Immunology 197 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

2 FIGURE 6. SAP-deﬁcient TFH cells do not express IL-4. WT or SAP SMtg CD4 T cells were adoptively transferred into B6 recipient mice subsequently 2 infected with LCMV. A, WT and SAP TFH SMtg CD4 T cells were sorted by FACS on the basis of CXCR5 expression at day 8 postinfection. TFH cell phenotype was conﬁrmed by staining for CXCR5hiSLAMlo expression on reserved aliquots of cells. Isolated mRNA from sorted cells was used for gene- 2 expression microarray. B, Scatter plot of WT TFH cell versus SAP TFH cell microarray gene-expression data, log2 scale. Black lines indicate 3-fold expression changes. R2 = 0.9905; 45,101 gene probes shown. Average signal of biological replicates is plotted. Data are shown from one of two independent lo 2/lo 2 2/lo experiments (n = 2/group). C–H, Gene expression in naive (CD44 , from uninfected mice), WT non-TFH (CXCR5 ), SAP non-TFH (CXCR5 ), WT hi 2 hi TFH (CXCR5 ), and SAP TFH (CXCR5 ) SMtg CD4 T cells, by Affymetrix microarray (n = 2/group). Each sample consisted of cells from 10 pooled spleens. mRNA expression of each gene was normalized to naive CD4 T cell controls. C, Bcl6. D, Prdm1/Blimp-1. E, ICOS. F, CD200. G, IL-21. H, IL-4.

but is required for GC TFH cell differentiation and the resultant TFH cells can outnumber GC resident CD4 T cells. Migration + IL-4 production; and IL-4 production by GC TFH cells is depen- studies demonstrated that CXCR5 CD4 T cells home to B cell dent on SLAM. follicles and the T/B border zones (8, 10) (Supplemental Fig. 3). 198 SLAM-DEPENDENT GC TFH IL-4 PRODUCTION Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 7. SLAM-deficient GC TFH cells exhibit impaired IL-4 production. A, Flow cytometric analysis of SLAM expression on naive CD4 T cells and + in vitro polarized TH1 and TH2 CD4 T cells. B, Flow cytometric analysis of SLAM and CXCR5 expression on CD45.1 SMtg CD4 T cells, day 8 after lo lo hi LCMV infection. C, Microarray analysis of SLAM mRNA in naive (CD44 , from uninfected mice), non-TFH (CXCR5 ), and TFH (CXCR5 ) SMtg CD4 lo hi T cells. D and E, Flow cytometry on splenocytes, day 8 after LCMV infection of B6 mice. D, SLAM expression on naive (CD44 ), non-TFH (CD44 lo hi lo 2 hi + + CXCR5 ), TFH (CD44 CXCR5 GL7 ), and GC TFH (CD44 CXCR5 GL7 ) endogenous CD4 T cells. E, SLAM mean fluorescence intensity (MFI), quantified from experiments in D. F, SLAM MFI on IgD+ (naive) and GC B cells, day 8 after LCMV infection of B6 mice. Data are representative of three hi + hi 2 2/lo 2/2 to five independent experiments. G,GCTFH (CXCR5 GL7 ), TFH (CXCR5 GL7 ), and non-TFH (CXCR5 ) CD4 T cell populations in Slamf1 2/2 +/+ +/+ hi + (SLAM ) and Slamf1 (SLAM ) mice, 8 d after LCMV infection. Gated CD44 CD4 T cells are shown. Quantitated TFH cell (H) and GC TFH cell (I) frequencies in SLAM+/+ and SLAM2/2 mice, from experiment shown in G (n = 6/group). Data are representative of three independent experiments. 2/2 +/+ J and K,GCTFH,TFH, and non-TFH CD4 T cells, as gated in G, were sorted from SLAM and SLAM mice, 8 d after LCMV infection. Expression of +/+ 2/2 IL-4 (J) and IL-21 (K) mRNA in SLAM and SLAM GC TFH cells. Expression levels by qPCR were quantitated in reference to a control gene and then normalized to naive CD4 T cells. Data are representative of two experiments (n = 3/group). ppp , 0.01; pppp , 0.001. The Journal of Immunology 199

FIGURE 9. Two-stage model of TFH cell differentiation, showing the regulation and impact of SLAM and SAP on TFH and GC TFH cells. Downloaded from tally tested. This model highlights the likely interrelatedness of TFH and GC TFH cells. The use of TFH and GC TFH nomenclature (41) seems most appropriate to describe these cell types, given their apparent close relationship by gene expression (Fig. 1) and their putative interconversion (Fig. 9). FIGURE 8. IL-4 is needed for optimal GC TFH B cell help. A, Non-TFH

hi lo 2 hi hi 2 http://www.jimmunol.org/ (CD44 CXCR5 GL7 ), TFH (CD44 CXCR5 GL7 ), and GC TFH Roles of SAP and SLAM in TFH and GC TFH cells (CD44hiCXCR5+GL7+) CD4 T cells were sorted from spleens of LCMV-infected mice and cultured with puriﬁed B cells. IgG was quanti- The expression of SLAM on GC TFH cells and GC B cells suggests tated in day 7 culture supernatants by ELISA. Data are representative of that GC TFH cells initiate SAP signaling though SLAM–SLAM hi hi 2 four experiments (n = 3/group). B,TFH (CD44 CXCR5 GL7 ) and GC engagement upon T–B conjugate formation, which, in turn, drives hi + + TFH (CD44 CXCR5 GL7 ) CD4 T cells were sorted from spleens of IL-4 production. Importantly, the presence of GC TFH cells in LCMV-infected mice and cultured with puriﬁed B cells in the presence 2/2 2 Slamf1 mice, but not SAP mice, indicates that GC TFH cell or absence of aIL4 or aIL21 mAb. IgG was quantitated in day 7 culture differentiation, although dependent on SAP, is controlled by supernatants by ELISA. Data are representative of two experiments (n =3/ hi + + a SLAM family receptor other than SLAM. Alternatively, SLAM

group). C,GCTFH (CD44 CXCR5 GL7 ) CD4 T cells were sorted from by guest on September 25, 2021 2 2 may contribute to GC T cell differentiation, with other SLAM SLAM / and SLAM+/+ LCMV-infected mice and then cocultured with FH purified B cells. IgG was quantitated in day 7 culture supernatants by family receptors sharing overlapping functions. Qi et al. (35) ELISA. Data are representative of two experiments (n = 3/group). pp , showed that SAP controls T cell–B cell adhesion. SLAM may 0.05; ppp , 0.01; pppp , 0.001. participate in that adhesion function, but its role in IL-4 production seems to be distinct and nonoverlapping. Other SAP- binding members of the SLAM receptor family, such as CD84 We propose that CD4 T cells differentiate to TFH cells after and Ly108, form considerably higher-affinity homophilic interac- interacting with cognate B cells at the T/B border zones, via B cell tions and are likely to be stronger contributors to SAP-mediated induction of Bcl6 expression in the CD4 T cells (6, 11). These TFH T cell–B cell adhesion (66–69). Indeed, it was recently shown cells can provide B cell help at the T/B border zones, or they can that CD84 contributes to optimal SAP-dependent GC development migrate into GCs, becoming GC TFH cells and providing survival (70). Nonetheless, our data show that SLAM’s role in IL-4 and differentiation signals to GC B cells. Signals from SLAM production by GC TFH cells is not redundant with other SLAM family receptor engagement may directly drive GC TFH cell dif- family receptors and that SLAM is the primary receptor respon- ferentiation in a SAP-dependent manner, or adhesion may only be sible for driving IL-4 production by GC TFH cells during a viral required for prolonged T–B conjugate formation, with GC TFH infection (Figs. 7J,8C). cell differentiation induced by other signals provided by the cog- The severe GC defect phenotype seen in the absence of SAP has nate B cells. In most cases, with the exception of IL-4 production, not been recapitulated in any single SLAM family receptor 2/2 GC TFH cells seem to be highly activated TFH cells [enhanced knockout mouse (24, 27, 31, 32, 70–73), including SLAM Bcl6, ICOS, and IL-21 (Fig. 1)], consistent with ongoing Ag (39). At least five SLAM family receptors are expressed on CD4 recognition, which would be explained by their localization in T cells. Functional redundancies in signaling among the SLAM GCs with cognate GC B cells presenting Ag. In addition, intravital family receptor members can occur, as seen in NKT cell de- microscopy studies indicated that CD4 T cells in GCs are not velopment (22). Overlapping SAP signaling through SLAM sessile (9) and regularly exit GCs (35). Incorporating those obser- family receptors likely occurs in TFH and GC TFH cells during vations, we infer that GC TFH cells are most likely not terminally CD4 T cell help to B cells, where normally different receptors differentiated and GC TFH cells exchange with the bulk TFH cell have different functions, or are expressed at different stages of population (Fig. 9). The gene expression and intravital microscopy the immune response; however, sufficient overlap exists that the data suggest that GC TFH cells are in a transient differentiation loss of any single SLAM family receptor is compensated for by state induced by interaction with GC B cells. GC TFH cells exit the the presence of other SLAM family receptors. Because they GC with some frequency and, thereby, presumably return to are encoded by adjacent genes, double or triple SLAM family 2 int 2 a GL7 Bcl6 IL-4 TFH state until they re-enter a GC and re- mutants cannot be generated by breeding, and redundancies will engage cognate B cells. This possibility remains to be experimen- have to be addressed with alternative approaches. 200 SLAM-DEPENDENT GC TFH IL-4 PRODUCTION

Non-TFH CD4 T cell expression of SAP is substantially lower GC TFH cell IL-4 regulation than that of GC TFH cells. All CD4 T cells express SLAM (39). How is IL-4 produced in a TH2-independent manner? In this ar- This suggests that the coordinated expression of SAP in GC TFH ticle, we demonstrated that IL-4 production by GC TFH cells is cells (Fig. 4B) is critical for SLAM-dependent IL-4 production dependent on SLAM and SAP signaling. Previous work showed (Fig. 9). Collectively, these data define a novel role for SLAM that a SLAM-SAP-Fyn-PKCu signaling axis can induce and SAP in GC TFH cell function and development while also IL-4 production by CD4 T cells in vitro (23, 24, 31, 96). A providing new insights into the complexity of SAP-dependent SAP-Fyn-PKCu signaling axis also controls the development of SLAM family receptor signaling. unconventional thymocyte-selected CD4 T cells (97). SLAM recruits SAP, which recruits Fyn kinase via R78, and Fyn kinase TFH and GC TFH cell-specific cytokines activity can result in PKCu recruitment (23, 24). Further work What is the key cytokine produced by TFH? IL-4 was originally will help to determine whether Fyn and PKCu are downstream identified as a B cell growth and differentiation factor, and it has components of IL-4 induction in GC TFH cells. Consistent long been known to be a potent cytokine for murine or human with previous studies (24, 96), IFN-g levels were moderately B cells (74). Those observations led to a long-standing conclusion increased in the TH1 (non-TFH), TFH, and GC TFH populations that TH2 cells were the primary CD4 T cells responsible for 2/2 2/2 2/2 in the absence of SLAM (data not shown). Elegant work by B cell help in vivo. However, IL-4 , IL-4R ,orSTAT6 Schwartzberg and colleagues (23) demonstrated that the SAP2 mice exhibit grossly normal GC development and Ag-specific IL-4 production defect is independent of the presence of elevated IgG responses (43, 75–77), although with significant isotype sub- IFN-g, because SAP2IFN-g2/2 CD4 T cells still displayed class changes (75, 78). TH2 cells are not required for B cell help a severe IL-4 defect in vitro. Downloaded from in vivo. A newly identified subset of CD4 T cells (TFH cells) is Notably, GATA3 is not induced in the GC TFH cells, and the TH2 specialized in B cell help (7) and required for GC development cytokine IL-5 is not produced. There are multiple precedents for in vivo (6, 11–13). IL-4 production in the absence of GATA3 induction. NKT cells IL-21 has become the leading candidate to be the primary B cell express IL-4 but have only low levels of GATA3, and NKT cell help cytokine produced by TFH cells (7, 79). However, like IL-4, the IL-4 transcription is NFATc1 dependent (98). In conventional absence of IL-21 or IL-21R had minimal impact on GC develop- CD4 T cells, STAT5 can induce IL-4 production, but not IL-5 http://www.jimmunol.org/ ment or TFH cell differentiation in a majority of in vivo models (56, transcription, in the absence of GATA3 induction (99). 80–84), although GC and TFH cell defects were observed in some The relationships among different CD4 T cells lineages have systems (43, 85). Furthermore, IL-21 production is not unique to become more and more complex. We recently proposed that al- TFH cells; it is also produced by TH17, TH2, and TH1 cells (86). though TFH cells are a distinct CD4 T cell lineage with a master In this article, we showed that IL-21 and IL-4 are abundantly regulator transcription factor, specialized gene-expression profile, produced by Ag-specific GC TFH CD4 T cells, and production and unique biological function, differentiation of a cell to a TFH of those cytokines is defective in the absence of SAP. Several cell does not outright preclude TH1, TH2, or TH17 characteristics, recent studies showed selective IL-4 production by GC TFH cells including cytokine and master regulator transcription factor ex- by guest on September 25, 2021 during TH2-biased parasitic infections (46, 52, 53). Our data in- + pression (6, 11). Similar ideas of overlapping differentiation pro- dicated that the IL-4 GC TFH subset of TFH cells that we grams between TFH cells and other canonical CD4 lineages were identified in the context of a TH1-biased viral infection is analo- proposed by other investigators (41, 46, 100). In this article, we gous cells, and the production of IL-4 is controlled by the TFH further defined the complexity of TFH cell-differentiation stages transcriptional program. Recent human data indicated that human and TFH cell cytokine regulation. Our findings highlight that TFH TFH cells exhibit similar phenotypic properties: tonsillar int cell differentiation is a multistage process requiring Bcl6 induc- CXCR5 CD4 T cells exhibited many TFH cell characteristics hi tion (11–13), cognate B cells (11), and then SAP-dependent dif- but did not express IL-4, whereas CXCR5 CD4 T cells expressed ferentiation and functions (14, 35), including SLAM-dependent the highest levels of TFH cell markers and expressed IL-4 (58). IL-4 induction. Additional studies to understand the integration A reasonable model of TFH cytokines is that IL-21 and IL-4 are of these signals and the TFH cell to GC TFH cell transition will be the two main TFH B cell help cytokines in vivo, and the absence of important for developing improved vaccine strategies for long- one can largely be compensated for by the other. This model stems term Ab responses. from the observations that IL-21 and IL-4 are produced by GC T cells, whereas IL-21, but not IL-4, is made in large quantities FH Acknowledgments by the whole TFH population during a viral infection, and both We thank Lindsay Crickard, Elisabeth Krow-Lucal, Danelle Eto, Cheryl cytokines provide B cell help in vitro when produced by GC TFH cells (Fig. 8B). The absence of either cytokine alone had a modest Kim, Kurt Van Gunst, and Sacha Garcia for technical assistance. We also thank Andre Veillette for the gift of anti-SAP mAb and Edward Wakeland impact on GCs in several in vivo mouse systems. However, the for B6.sle1b mice. combined absence of IL-4 and IL-21R resulted in a severe loss of GCs and Ab responses (80). The antiapoptotic impact of IL-4 on B cells is well documented (87–89), and recent work by Locksley Disclosures and colleagues (46) elegantly showed a role for IL-4 in somatic The authors have no financial conflicts of interest. hypermutation and affinity maturation. IL-4 can also contribute to sustaining GCs (52). IL-21 was shown to be a potent inducer of References plasma cell differentiation by human B cells (90–93) and murine 1. Schoenberger, S. P., and S. Crotty. 2008. Immunologic memory. In Funda- B cells (94, 95). In our TFH cell studies, blocking IL-21 in GC TFH mental Immunology, 6th Ed., W. E. Paul, ed. Lippincott Williams & Wilkins, cell–B cell cultures reduced IgG production to near background Philadelphia. p. 862–898. 2. McHeyzer-Williams, M. 2008. B Lymphocyte biology. In Fundamental levels (Fig. 8B). This is consistent with studies of human TFH cells (92). Therefore, although it seems that the functions of these two Immunology, 6th Ed., W. E. Paul, ed. 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