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High affinity antibody responses against protein antigens critically depend on T cell help during the germinal center reaction. So called follicular helper T cells (TFH), present in the germinal centers of human tonsils, have been characterised by the expression of CXCR5; however, only subgroups of the heterogeneous CXCR5 T cell population strongly support antibody production. A paper in this issue of the European Journal of Immunology demonstrates that high expression of the inducible co-stimulator (ICOS) molecule, rather than CD57, correlates with the follicular helper function. This result represents another step towards a better understanding of the complexity of the different T cell subpopulations participating in the germinal center reaction.
Follicular helper T cells
High affinity antibody responses against protein antigens critically depend on T cell help during the germinal center reaction. The discovery of the chemokine receptor CXCR5 (BLR1) almost 10 years ago by Forster et al.1 allowed an activated CD4 effector T cell subpopulation, which was present in germinal centers of human tonsils and was shown to strongly support antibody production in vitro, to be defined 2, 3. These CXCR5+ follicular helper T (TFH) cells produced low amounts of cytokines, had lost their proliferative capacity and thereby clearly differed from other CD45RO+ memory cell populations such as the polarized Th1 or Th2 T cells 2, 4, 5. During the following years, various groups demonstrated the heterogeneity of the CXCR5+ CD4 T cells.
In humans, CXCR5+ T cells consist of at least four different subpopulations in secondary lymphoid tissues plus an additional circulating CXCR5+ T cell population in the peripheral blood: besides the bona fide germinal center B helper CD57+ CXCR5+ CD69+ CD25– CD4 T cell 5 defined by the expression of CD57, a CD57– CXCR5+ CD69– CD25– CD4 T cell located mostly in the outer zone of germinal centers provides no spontaneous B cell help but may be involved in B cell support after further activation 6; Lim et al. have discovered a regulatory T cell population which can locate to B cell follicles due to the expression of CXCR5 7; and another CXCR5+ CD4 T cell population resembles Th2 T cells by its cytokine production and expression of CRTH2 8. In this issue of the European Journal of Immunology, Rasheed et al.9 identify a terminally differentiated effector stage of TFH cells characterized by the high levels of expression of ICOS and CXCR5, while low expression of both molecules characterized a potential precursor stage; however, there is substantial overlap between the CD57+ CXCR5+ and the ICOShi CXCR5hi T cell populations since the majority of CD57+ CXCR5+ T cells also express high levels of ICOS. There is also possibly an overlap between the ICOSint CXCR5lo and the CD57– CXCR5+ T cells of the outer zone. Of all the reported markers, however, the high expression of ICOS seems to be associated the closest with the B cell helper function of TFH cells.
The gene expression profiles of tonsillar CD57+ CXCR5+ or ICOShi CXCR5hi T cell populations confirm the uniqueness of the TFH population 9–11. All three studies found high expression levels of CXCL13, a chemokine and the ligand of CXCR5, suggesting that TFH cells create their own microenvironment within germinal centers by recruiting B cells into their environment. The unique expression signature of TFH cells with regard to co-stimulatory molecules, cytokines and their receptors, and components of Notch and wnt signalling identify important pathways regulating the development and function of these cells. Among these, the high expression of ICOS stands out. ICOS belongs to the CD28 family of co-stimulatory molecules 12 and shares many common functions with CD28 13. The dramatic effect of ICOS deficiency on the germinal center reaction and secondary humoral immune responses was revealed by three different murine knock out lines 14–16, demonstrating a specific effect of ICOS co-stimulation not appreciated by the comparative gene profiling following CD28 and ICOS stimulation of isolated circulating human CD4 T cells 13. The critical mechanism by which, as well as the time points when, ICOS contributes to the humoral immune response still remain speculative. Besides the crucial influence of ICOS co-stimulation in the light zone of germinal centers 17, ICOS probably represents a critical co-factor during the encounter of freshly activated T and B cells at the T–B border in secondary lymphoid tissues, a central step in the initiation of the germinal center reaction. Consistent with this proposal, ICOS deficiency in mice not only disturbs the immune response downstream of the engagement of TFH cells in germinal centers but it also leads to a severe reduction in germinal center and, most remarkable in this context, TFH cell numbers 18. ICOS is the first molecule shown to be critical for the development of TFH cells while the requirement of OX40 signals 19 seems to be less stringent 18. It remains uncertain whether the effect of ICOS deficiency on TFH cell development is direct or secondary to disturbed germinal center formation.
Three years ago, ICOS deficiency was identified as the underlying genetic defect in nine patients with an antibody deficiency syndrome 20. Like the murine counterpart, the development of germinal centers and B cell memory was severely disturbed in these patients 21. Concomitantly, the number of circulating CXCR5+ T cells was significantly reduced and the few CD57+ CXCR5+ T cells normally present in the blood of healthy individuals were completely absent (Bossaller et al., submitted). Unfortunately, the origin and function of these circulating CXCR5+ T cells has not been well defined. The original suggestion that circulating CXCR5+ T cells represent recently activated T cells 4 may be correct for a minority but not most circulating CXCR5+ T cells which show no other activation markers and persist at more or less constant levels over time. A more intriguing concept (indicated in Fig. 1) supported by the absence of circulating CXCR5+ T cells in ICOS, as well as CD40L deficiency (Bossaller et al., submitted), is the possibility that circulating CD45RO+ CXCR5+ CD4 T cells are a germinal center-derived memory population, which can be readily recruited into humoral immune responses at the initiation of germinal centers.
Figure 1. A model for the differentiation of CXCR5+ T cells. Activation of T cells in the T cell zone or periphery will transiently upregulate the chemokine receptor CXCR5 and the costimulatory molecule ICOS. The change of chemokine receptor expression allows activated T cells to migrate along chemokine gradients towards the border between the T and B cell zone (A). Cognate interaction with activated B cells and contact with accessory cells, most likely involving ICOS, will allow antigen-specific T cells to retain CXCR5 expression, downregulate CCR7 expression and migrate into B cell follicles. These cells may represent the ICOSint CXCR5lo and the CD57– CXCR5+ T cells of the outer zone. After migration into the B cell follicle and formation of a germinal center CXCR5+ ICOS+ T cells differentiate into CXCR5hi ICOShi effector TFH cells (B). A circulating memory population (C), which remains CXCR5+ but regains the expression of CCR7, develops either from, or more likely in parallel to, the TFH population. This memory population might then recirculate and be recruited directly to the T–B border upon re-encounter with the specific antigen.
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Unlike tonsillar CD45RO+ CXCR5+ CD4 T cells, circulating CD45RO+ CXCR5+ CD4 T cells express CCR7 and no activation markers. Also expression arrays confirmed major differences between the circulating and the tonsillar effector CXCR5+ T cell population 9 but the analysis of surface marker expression and RNA expression profiles are limited in determining the relationship of various populations, since the development of lymphocytes is associated with major steps of differentiation. Thus, during B cell differentiation, it is well accepted that memory B cells and the terminal effector stage of B cell differentiation, the plasma cell, derive from germinal center B cells despite very distinct patterns of gene expression.
In conclusion, the study by Rasheed et al.9 defines high expression levels of ICOS as the best marker for the terminally differentiated effector function of TFH cells. The authors suggest that ICOSint CXCR5lo T cells represent a precursor population which may also be the precursor of circulating CXCR5+ T cells. Following on from this study, our task in the future will be to dissolve the complexity of T cell help during antibody responses by defining the interrelation and function of the different follicular and circulating T cell populations. Such an improved comprehension will potentially provide a better therapeutic approach to antibody deficiency syndromes 21 as well as autoantibody mediated autoimmunity 22.