Human tonsil B-cell lymphoma 6 (BCL6)-expressing PNAS PLUS CD4+ T-cell subset specialized for B-cell help outside germinal centers

Salah-Eddine Bentebibela,b, Nathalie Schmitta, Jacques Banchereaua,c,1, and Hideki Uenoa,c,2 aBaylor Institute for Immunology Research, Baylor Research Institute, Dallas, TX 75204; bInstitute of Biomedical Studies, Baylor University, Waco, TX 76798; and cInstitut National de la Santé et de la Recherche Médicale U899, Center for Human Vaccines, Dallas, TX 75204 AUTHOR SUMMARY

− CD4+ helper T cells derived (IgD+CD38 CD19+), memory − − from the thymus play a funda- (IgD CD38 CD19+), and ger- − mental role in the generation of minal center B (IgD CD38+ antibodies against specific anti- CD19+) cells. T- and B-cell gens, an important function cocultures revealed that in immune response. Among CXCR5loICOSlo CD4+ T cells CD4+ helper T cells, a subset and Tfh cells (i) differentially called T follicular helper (Tfh) secrete cytokines and chemo- cells provides help to antibody- kines and (ii) differentially help producing B cells in germinal B-cell subsets (Fig. P1). Thus, centers, structures found in sec- Tfh cells secrete large amounts ondary lymphoid organs such of the chemokine CXCL13 as lymph nodes and spleen. In among tonsillar CD4+ T-cell germinal centers, B cells are se- populations, whereas CXCR5lo lected through their interaction ICOSlo CD4+ T cells secrete with Tfh cells and follicular large amounts of cytokines IL-10 dendritic cells. Selected B cells and IL-21. Tfh cells are efficient then differentiate into either at helping germinal center B long-lived, antibody-producing cells, and they promote their plasma cells or memory B cells, survival and Ig production. In lo lo + which produce large amounts of contrast, CXCR5 ICOS CD4 IMMUNOLOGY antibodies upon a secondary T cells are far more efficient than antigen challenge (1). There- Fig. P1. Two distinct Tfh-committed cells in human tonsils Tfh cells at inducing naïve B cells differentially help B-cell subsets. CXCR5loICOSlo CD4+ T cells localize fore, germinal center response exclusively outside germinal centers (GC) and induce naïve and to proliferate and differentiate is central to the generation of memory B cells to become antibody-secreting cells. CXCR5hiICOShi GC- into antibody secreting cells. + high-affinity antibodies. CD4 T Tfh cells are specialized to help B cells in GCs. Whereas CXCR5loICOSlo Blocking experiments showed lo lo + cells also provide help to B cells CD4+ T cells produce higher levels of IL-21 upon interaction with B cells, that CXCR5 ICOS CD4 outside germinal centers and GC-Tfh cells produce higher levels of CXCL13. CXCR5loICOSlo CD4+ T T cells help naïve B cells in induce the differentiation of B cells and GC-Tfh cells show similar expression profiles of BCL6 and a manner dependent on multiple PRDM1 transcripts. Our study suggests that tonsillar CXCR5loICOSlo cells into either short-lived + cytokines and surface molecules, plasma cells (extrafollicular CD4 T cells represent Tfh-committed extrafollicular helper cells and/or including IL-21, IL-10, ICOS, precursors of GC-Tfh cells. plasma cells) or B cells that and CD40L. The difference be- participate in germinal center tween the CXCR5loICOSlo responses. However, the identity of such CD4+ T cells involved CD4+ T cells and Tfh cells in the capacity to help naïve B cells was, in the assistance of B cells outside germinal centers remains at least partly, explained by the differences in IL-21 secretion. unclear, particularly in humans. In this study, we identified Notably, CXCR5loICOSloCD4+ T cells failed to help germinal a CD4+ T-cell subset in human tonsils. This CD4+ T-cell sub- center B cells and did not maintain their survival. We hypothe- set shares properties with Tfh cells but exclusively localizes size that CXCR5loICOSloCD4+ T cells might induce the death outside germinal centers. of germinal center B cells, which express high amounts of We analyzed the phenotype and functions of distinct CD4+ surface molecules delivering a death signal called FAS. Indeed, T-cell subsets in human tonsils. Human tonsillar Tfh cells soluble FAS ligand was detected in cocultures of germinal coexpress the chemokine receptor 5 (CXCR5) and the inducible costimulator (ICOS) at high density (2) but not a receptor pro- + tein called IL-7 receptor. The tonsillar CD4 T-cell subset spe- Author contributions: S.-E.B., N.S., J.B., and H.U. designed research; S.-E.B. and N.S. per- cialized for the help of B cells outside germinal centers expressed formed research; S.-E.B., N.S., and H.U. analyzed data; and S.-E.B., J.B., and H.U. wrote the CXCR5 and ICOS only at low levels (here called CXCR5lo paper. ICOSlo CD4+ T cells) but expressed the marker IL-7 receptor The authors declare no conflict of interest. abundantly. Furthermore, although a majority of Tfh cells ex- This article is a PNAS Direct Submission. press the protein marker programmed death-1 (PD-1), only 1Present address: Inflammation and Virology Discovery and Translational Areas, Hoffmann- a minor fraction of CXCR5loICOSlo CD4+ T cells expressed La Roche, Inc., Nutley, NJ 07110. PD-1 at very low density. 2To who correspondence should be addressed. E-mail: [email protected]. To determine their functions, the isolated tonsillar CD4+ T-cell See full research article on page E488 of www.pnas.org. subsets were cocultured with B-cell subsets, including naïve Cite this Author Summary as: PNAS 10.1073/pnas.1100898108.

www.pnas.org/cgi/doi/10.1073/pnas.1100898108 PNAS | August 16, 2011 | vol. 108 | no. 33 | 13371–13372 Downloaded by guest on September 26, 2021 center B cells with CXCR5loICOSlo CD4+ T cells but not with ICOSloCD4+ T cells were exclusively localized outside Tfh cells. Consistently, FASLG transcript (encoding FAS-L) germinal centers. Therefore, CXCR5loICOSloCD4+ T cells lo lo + was expressed by CXCR5 ICOS CD4 T cells but was might represent extrafollicular helper cells engaged in inducing expressed very little by Tfh cells. Upon blocking the FAS/FAS– the differentiation of B cells into extrafollicular plasma cells. ligand interaction with a neutralizing anti-FAS antibody, lo lo + This hypothesis is supported by the demonstration in lupus- CXCR5 ICOS CD4 T cells were able to maintain the prone mice models of the presence of extrafollicular helper survival of germinal center B cells and induce them to produce cells that share the property with Tfh cells, including Bcl-6 antibodies. These observations indicate that CXCR5loICOSlo + expression (4). We cannot exclude, however, the possibility CD4 T cells induce apoptosis of germinal center B cells lo lo + through FAS/FAS–ligand interaction. that CXCR5 ICOS CD4 T cells represent precursors of Tfh cells. We also analyzed the expression of two transcriptional re- lo lo + pressors, B-cell lymphoma 6 (Bcl6) and B lymphocyte-induced Our study shows that CXCR5 ICOS CD4 T cells represent maturation protein 1 (encoded by PRDM1 gene), by tonsillar Tfh-committed cells helping B-cell responses outside germinal lo lo + CD4+ T-cell populations. Bcl6 is expressed at high levels by centers. Additional characterization of CXCR5 ICOS CD4 tonsillar Tfh cells. Mouse studies indicate that Tfh cell genera- T cells together with determination of their developmental tion in vivo is positively regulated by Bcl6 and negatively re- mechanisms will bring significant insights to the pathogenesis gulated by B lymphocyte-induced maturation protein 1 (3). of human autoimmune diseases, where extrafollicular B-cell lo lo + We found that the CXCR5 ICOS CD4 T cells express BCL6 responses are involved (4). Such studies might also benefit the and PRDM1 transcripts at equivalent levels with Tfh cells design of vaccines against infectious diseases. (thus, abundant BCL6 and few PRDM1 transcripts). lo lo + Collectively, our study suggests that CXCR5 ICOS CD4 1. King C, Tangye SG, Mackay CR (2008) T follicular helper (TFH) cells in normal and T cells are committed to the Tfh lineage; they showed similar dysregulated immune responses. Annu Rev Immunol 26:741–766. profiles of BCL6 and PRDM1 expression with Tfh cells, and 2. Rasheed AU, Rahn HP, Sallusto F, Lipp M, Müller G (2006) Follicular B helper T cell activity they helped B cells in a manner dependent on IL-21, IL-10, is confined to CXCR5(hi)ICOS(hi) CD4 T cells and is independent of CD57 expression. Eur J ICOS, and CD40L, thus sharing functions with Tfh cells. Immunol 36:1892–1903. lo lo + 3. Johnston RJ, et al. (2009) Bcl6 and Blimp-1 are reciprocal and antagonistic regulators of Furthermore, CXCR5 ICOS CD4 T cells robustly up-regu- T follicular helper cell differentiation. Science 325:1006–1010. lated ICOS and PD-1 expression upon activation, thus sharing a 4. Poholek AC, et al. (2010) In vivo regulation of Bcl6 and T follicular helper cell lo phenotype with Tfh cells. At variance with Tfh cells, CXCR5 development. J Immunol 185:313–326.

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