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L.D.D. and J.C. wrote the manuscript.
CD4 T helper (Th) cells are central to regulation of immune responses, with separable effector subsets defined by their lineage-specific transcription factor expression, cytokine production, and subsequent immune function (1). Th1 cells express the transcription factor T-bet that enables production of interferon-γ (IFN-γ) for cell-mediated immunity, necessary for protection from intracellular bacterial infections and initiation of delayed-type hypersensitivity (DTH). Th2 cells express the lineage-specific transcription factor GATA-3 that drives synthesis of interleukin-4 (IL-4), IL-5, and IL-13, cytokines responsible for clearance of helminthes (worms) and other extracellular parasites, and in the setting of appropriate genetic and environmental triggers, principal contributors to atopy. A third effector subset, Th17, more recently identified and characterized, expresses RORγt that is needed for transcription and ultimate synthesis of IL-17, a cytokine that promotes neutrophil recruitment and activation as part of the inflammatory response, and in so doing, is critical for elimination of extracellular bacteria, such as streptococci, and fungi (2). Like Th1 and Th2 cells, Th17 cells when abnormally activated promote autoimmunity, contributing to tissue inflammation in classical T-cell mediated diseases such as multiple sclerosis (3) and inflammatory arthritis (4). Along with cytotoxic CD8 T cells that are necessary for viral immunity, these three CD4 T cell effector subsets direct responses to the range of pathogens - viruses, intracellular bacteria, worms, and extracellular bacteria and fungi - encountered by the mammalian host.
Another critical CD4 Th cell function is B cell help, with insight into the nature of the T cells that promote antibody responses long overshadowed by advances in the understanding of Th1, Th2, and Th17 biology. However, recent studies have demonstrated that an additional effector subset, follicular helper T (TFH) cells, is largely responsible for B cell help during an immune response. TFH cells are defined by their location within germinal centers (GC), the site at which high affinity, class switched, and long lived plasma cells and memory B cells emerge (5), and their expression of the costimulatory molecule ICOS (inducible costimulator), necessary for their development, and CXCR5, a chemokine receptor that upon engagement of its ligand CXCL13 permits trafficking of these cells to the B cell follicle, the site of GC formation. TFH cells produce IL-21, a cytokine that cooperates with IL-4 to promote robust class-switched antibody responses (6), with the latter cytokine and IFN-γ important in enabling TFH-driven immunoglobulin class switching (7) from IgM to isotypes appropriate to the invading pathogen; IL-17 may play a similar supporting role (8). IL-21 is critical for the lupus phenotype, in that its overproduction in mice with enhanced TFH cell development due to a mutation in control of ICOS expression (Sanroque mice) leads to systemic autoimmunity (9). Morevover, antibody or genetic blockade of IL-21 signaling abrogates disease in lupus-prone mice (10, 11).
The analysis of TFH cells in humans thus far has primarily involved utilization of tonsillar tissue, where large numbers of TFH cells are normally resident, with their study at alternate sites largely unexplored. Simpson and colleagues now demonstrate in this issue of Arthritis & Rheumatism that TFH cells can be found in the peripheral blood of patients with lupus and Sjögren syndrome, a population they refer to as circulating TFH (cTFH) (12). They used flow cytometry to demonstrate that the circulating cells express proteins found on the TFH subset in secondary lymphoid organs, among them ICOS, CXCR5, and PD-1 (the latter also upregulated on TFH cells) (13). The level of expression of these markers paralleled that found on tonsillar TFH cells, with a stringent cutoff value used to convincingly demonstrate that cTFH cells were expanded in approximately a third of lupus patients - their proportion was 4 standard deviations above that found in healthy controls. This work also revealed a correlation between expansion of this CD4 Th subset in lupus with elevated autoantibody titers and glomerulonephritis, although not with disease activity as measured by SLEDAI.
TFH cells from the spleen, tonsil and lymph nodes produce IL-21 and express the transcriptional repressor Bcl6 (14, 15), the latter recently shown by others and us to be necessary for development of this subset in secondary lymphoid organs in mice (16-18). Yet, cTFH cells in systemic autoimmunity did not express the mRNA transcripts for il21 and bcl6, and their expansion did not correlate with amounts of IL-21 in sera (12). This finding is not necessarily consistent with the identification of the circulating subset as TFH, since activated CD4 T cells per se, including those destined for other effector fates, can transiently express ICOS, CXCR5, and PD-1; nonetheless, sustained and/or substantial expression of these molecules is characteristic of TFH cells, so the circulating population found in patients with lupus and Sjögren syndrome seem appropriately defined as cTFH cells. Why then do not the latter express the full complement of markers of TFH cells in secondary lymphoid organs such as the tonsil? Perhaps cTFH cells are a follicular helper subset distinct from the TFH population resident in secondary lymphoid tissues, and/or they are follicular helper T cells that upon maturation and trafficking out of the spleen and lymph nodes downregulate molecules critical for their effector program in lymphoid tissues. There are precedents for such plasticity: germinal center B cells upon maturation to plasma cells downregulate molecules critical for their function in splenic follicles, including suppression of Bcl6 under the influence of the transcriptional repressor Blimp-1 (19). Under the appropriate environmental signals, regulatory T cells in the gut convert to TFH cells needed to drive IgA production by resident B cells (20). While the converse is not known to be true, plasticity nonetheless is a defining feature of CD4 Th effector differentiation (1). Additional studies of the circulating T cells taken from the peripheral blood of humans or autoimmune Sanroque or other lupus-prone mice - Simpson et al. found these cells in the blood of such mice (12) -- would help further clarify their function, as would the search for a tonsillar or splenic population with a phenotype identical to that of cTFH (ICOShi, CXCR5hi, PD-1hi, IL-21lo, Bcl6lo). Culture and stimulation of the cTFH population ex vivo, and/or re-acclimatization to a secondary lymphoid organ or a site of inflammation such as the nephritic kidney, may be needed for resumption of the full TFH cell program.
Expansion of the cTFH subset in lupus patients correlated with increased serum titers of autoantibodies, and the incidence of glomerulonephritis, thromboembolic disease, and thrombocytopenia. Thus, while Simpson et al. found that such expansion cannot be used as a measure of SLE activity, it did correlate with features of disease that may ultimately result in organ damage. Given the correlation between high cTFH numbers and the incidence of glomerulonephritis, it would be important to determine whether the T cells found in the kidneys of nephritic lupus-prone mice (21) and humans (22) have the phenotype of TFH cells; this seems possible, as kidney-infiltrating cells express ICOS and are found in proximity to antibody-producing B cells (22). If they express molecules needed for tissue trafficking, cTFH cells might infiltrate the kidneys of lupus patients and acquire (or re-acquire) B cell helper function, influencing the local maturation of B cells, with tissue injury the consequence. An additional question is whether cTFH cells are found during normal immune responses, or is their presence restricted to autoimmunity. A search for cTFH cells post-vaccination or during pathogen challenge could address this issue.
We have learned much about the function and autoimmune potential of Th1, Th2, and Th17 cells following their isolation from the blood of normal and diseased humans. The identification of circulating TFH cells seems an equally important step in the study of this fourth CD4 T cell effector subset in humans. While additional experiments are needed for functional analyses, the work of Simpson and colleagues is a critical step in the emergence of this effector CD4 T cell subset from the shadows of its well-studied Th1, Th2, and Th17 counterparts, and an important beginning to understanding the role that these cells play in systemic autoimmunity.
We thank the members of the Craft laboratory for helpful discussions, and in particular, Amanda Poholek for critical review of the manuscript. We also acknowledge those authors whose work we could not cite because of space limitations.
This work was supported by NIH Grants AR40072 and AR44076, and grants from the Arthritis Foundation, Rheuminations, Inc., and the Connecticut Chapter of the Lupus Foundation.