That Provide Help for B Cells Their Role As Non-Th1/Th2 Effector Cells
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T Follicular Helper Cells Express a Distinctive Transcriptional Profile, Reflecting Their Role as Non-Th1/Th2 Effector Cells That Provide Help for B Cells This information is current as of September 30, 2021. Tatyana Chtanova, Stuart G. Tangye, Rebecca Newton, Nita Frank, Martin R. Hodge, Michael S. Rolph and Charles R. Mackay J Immunol 2004; 173:68-78; ; doi: 10.4049/jimmunol.173.1.68 Downloaded from http://www.jimmunol.org/content/173/1/68 Supplementary http://www.jimmunol.org/content/suppl/2004/06/17/173.1.68.DC1 http://www.jimmunol.org/ Material References This article cites 63 articles, 32 of which you can access for free at: http://www.jimmunol.org/content/173/1/68.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 30, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology T Follicular Helper Cells Express a Distinctive Transcriptional Profile, Reflecting Their Role as Non-Th1/Th2 Effector Cells That Provide Help for B Cells1 Tatyana Chtanova,*‡ Stuart G. Tangye,† Rebecca Newton,* Nita Frank,§ Martin R. Hodge,§ Michael S. Rolph,*‡ and Charles R. Mackay2*‡ Effector T cell responses have long been viewed in the context of the Th1/Th2 paradigm. Recently, a third major subset of nonpolarized effector T cells that provides help to B cells has been identified. These T cells, termed T follicular helper (TFH) cells, home to the B cell areas of secondary lymphoid tissue, through interactions mediated via the chemokine receptor CXCR5 and its ligand CXCL13. Affymetrix microarrays were used to identify transcription factors, cytokines, and cell surface molecules that ؉ underlie the differentiation pathways and functional properties of the TFH subset. The transcriptional profile of human CXCR5 Downloaded from TFH cells was compared with that of Th1 and Th2 cells, which enabled the identification of numerous genes expressed preferen- tially by TFH cells, over the other effector subsets. Certain TFH genes were also expressed by B cells and thus appear to be particularly relevant for humoral immunity. Abs were used to confirm the expression of several factors. In particular, CD84 and CD200, the cytokine IL-21, and the transcription factor BCL6 were all strongly associated with TFH cells. Gene microarrays reveal a highly distinctive transcriptional profile for a third subset of effector T cells that differs markedly from Th1 and Th2 cells. The Journal of Immunology, 2004, 173: 68–78. http://www.jimmunol.org/ cell help for Ab production is an important component of CXCR5 by TFH and B cells allows the colocalization of these cells the immune response. It occurs first in T cell areas and to sites of CXCL13 production (i.e., follicles), thus enabling pro- later in B cell follicles, within secondary lymphoid organs ductive T-B cell interactions. T ϩ (1, 2). T cell help to B cells was long thought to be solely attrib- Several features of CXCR5 TFH cells have been described. utable to Th2 cells (3–5), because IL-4, a Th2 cytokine, was found Like other effector T cells, TFH express activation markers such as to stimulate B cell proliferation and class switching and induce CD69, and low levels of CCR7 and CD62L (similar to Th1 and up-regulation of costimulatory molecules such as CD40. However, Th2 cells), and exhibit effector function, namely, help for Ab pro- B cell help still occurs in the absence of IL-4, because IL-4- and ϩ by guest on September 30, 2021 duction (8–10). CXCR5 TFH cells are nonpolarized with respect Stat6-deficient mice still make Abs (6, 7). to Th1/Th2 cytokine production. Interestingly, CXCR5 is lost on Recently, a non-Th1, non-Th2 effector T cell capable of provid- fully differentiated Th1 and Th2 cells (13), suggesting that these ing help for B cells was identified in lymphoid tissues through effector subsets may be excluded from B cell follicles. A cytokine expression of the chemokine receptor CXCR5 (8–10). The expres- profile that distinguishes TFH from Th1 and Th2 cells has yet to be sion of CXCR5 by these T cells, termed follicular B-helper T 3 described. In addition, transcription factors such as T-bet and (TFH) cells (8–10), allows them to localize to B cell follicles, GATA3, which determine Th1 and Th2 differentiation (14), have where they provide help to B cells. CXCR5 is also expressed by B yet to be identified for T cells. cells, and is required for the development of B cell follicles in FH Expression of CXCR5 by T cells does not necessarily define T secondary lymphoid tissues. Mice lacking either CXCR5 or its cells capable of providing B cell help. In one study, only a small ligand CXCL13 (B lymphocyte chemoattractant) display major ab- ϩ subset of human CXCR5 T cells that coexpressed CD57 was errations in splenic follicular architecture and reduced numbers of capable of effector function (10). These T cells were reported as lymph nodes and Peyer’s patches (11, 12). The expression of CD57ϩCD45ROϩCXCR5ϩCD4ϩ and CCR7Ϫ, made up 15–25% of total tonsillar CXCR5ϩ T cells, and specifically localized to ϩ ϩ *Garvan Institute of Medical Research, Darlinghurst, †Centenary Institute of Cancer germinal centers (GC) (10). CD57 CXCR5 T cells supported Medicine and Cell Biology, Camperdown, and ‡Cooperative Research Center for Asthma, University of Sydney, Camperdown, New South Wales, Australia; and §Mil- production of IgG, IgA, and IgM by human tonsil B cells; pro- lenium Pharmaceuticals, Cambridge, MA 02139 duced IL-10, IL-2, IL-4, IFN-␥, and TNF-␣ following activation; Received for publication February 11, 2004. Accepted for publication April 22, 2004. and expressed the costimulatory molecules OX40 and CD40L ϩ The costs of publication of this article were defrayed in part by the payment of page (10). However, others have reported that CD57 GC T cells dis- charges. This article must therefore be hereby marked advertisement in accordance play a phenotype consistent with anergy (15). In addition, a small with 18 U.S.C. Section 1734 solely to indicate this fact. proportion of circulating CD4ϩCD45ROϩ T cells expresses 1 This work was supported by the Cooperative Research Center for Asthma, and National Health and Medical Research Council, Australia, and in part by grants from CXCR5. These cells are incapable of providing help to B cells, the New South Wales Cancer Council (awarded to S.G.T.). have poor cytokine production capabilities, coexpress CD62L and 2 Address correspondence and reprint requests to Dr. Charles R. Mackay, Garvan CCR7, and probably represent a subpopulation of circulating, cen- Institute of Medical Research, 384 Victoria Street, Darlinghurst, New South Wales tral memory T cells (8). 2010, Australia. E-mail address: [email protected] We sought to understand the function, identity, and molecular 3 Abbreviations used in this paper: TFH, T follicular helper; TCM, T central memory; interactions of this third subset of human effector T cells, TFH, TEM, T effector memory; GC, germinal center; SLAM, signaling lymphocytic acti- vation molecule; SAP, SLAM-associated protein. particularly because the discovery of transcription factors, cytokines, Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 69 and homing molecules on Th1 and Th2 cells has provided consider- using a CD4 negative-selection kit (Miltenyi Biotec). Polarized Th1 and able insight into the biological role of these effector subsets. We iso- Th2 cells were generated as previously described (17) by stimulation with lated CD57ϩCXCR5ϩCD4ϩ and CD57ϪCXCR5ϩCD4ϩ T cells immobilized anti-CD3 at 1–5 g/ml (clone TR66), soluble anti-CD28 (1 g/ml; BD Pharmingen, San Diego, CA), and IL-2 (100 U/ml). Th1 cul- from tonsils and assessed gene expression using Affymetrix oligonu- tures also received IL-12 (5 ng/ml) and neutralizing anti-IL-4 (1 g/ml), cleotide microarrays. We also compared TFH cells to the other major and Th2 cultures received IL-4 (10 ng/ml) and neutralizing anti-IFN-␥ (1 effector subsets, Th1 and Th2, and to effector memory (TEM) and g/ml; BD Pharmingen). The cells were cultured for 3–4 days, harvested, central memory (T ) T cells isolated from peripheral blood. This and restimulated under the same conditions for another 3–4 days. At this CM stage, cells were harvested for RNA isolation. Cytokine production was approach allowed us to identify a transcriptional program that distin- evaluated by intracellular staining of cells stimulated overnight with anti- guished TFH from the other effector T cell subsets. We identify nu- CD3/CD28 and incubated with Golgi Plug (BD PharMingen) for 4 h before merous new factors that are likely to play an important role in medi- staining. The most highly polarized cultures were selected for RNA isola- ating T cell help to B cells, several of which we have characterized in tion and gene-chip analysis. depth. Immunofluorescence on tissue sections Cryostat sections of human tonsils were fixed in 1% paraformaldehyde/ Materials and Methods sodium phosphate, blocked with 10% normal goat or horse serum, and Abs and other reagents stained with primary Abs to CD84, CD57, BCL6, CD3, and CD4, followed by an incubation with fluorochrome-conjugated secondary Abs.