Why are Tfh cell dynamics so fundamentally
Why are Tfh cell dynamics so fundamentally different in naive and immune animals? Initially, GC Dacarbazine must pass stringent affinity and specificity checkpoints to ensure only high-affinity non-self-reactive cells are selected. Therefore, restricting primary Tfh cells with the greatest helper capacity to the GC might serve to direct help to cognate B cells and avoid the activation of bystander B cells in the follicle. Accordingly, expression of the genes encoding the B helper cytokines IL-21 and IL-4 were restricted to primary GC Tfh cells. In the secondary response, memory B cells have already passed these checkpoints and therefore have less stringent activation thresholds. Correspondingly, secondary Tfh do not express the same high amounts of Il21 and Il4 transcripts as primary GC Tfh cells. Furthermore, memory B cells are widely distributed in persistent GC remnants within the follicle (Dogan et al., 2009, Talay et al., 2012) and extrafollicular sites including the bone marrow (Dogan et al., 2009, Paramithiotis and Cooper, 1997), tonsillar mucosal epithelium (Liu et al., 1995) and splenic marginal zone (Liu et al., 1988). Therefore, protective secondary antibody responses may depend on the rapid extrafollicular export of secondary Tfh cells to these sites. Thus, unlike primary responses where it takes 7 days or more for Tfh cells to mature, the stereotypic expansion of CXCR5hiPD-1hi cell with a “mature” Tfh cell phenotype that peaks by day 3 in our system may be a part of a pre-wired memory program (Hale et al., 2013). Nevertheless, some memory B cells do enter GCs (Dogan et al., 2009, Pape et al., 2011) and Tfh cells are still required in this location in the secondary response. In this respect, it is notable that the NMF analysis of single cell gene expression by secondary Tfh cells showed that there was a hidden subpopulation of cells with high expression of Bcl6 and Pdcd1 that might be destined to later colonize and persist in secondary GCs. Thus, follicular memory T cells also appear to bifurcate into two responding populations upon rechallenge. However, at the peak of the secondary Tfh response these responding cells are equally likely to be in the FM or GC. What are the molecular signals that guide Tfh cells as they navigate around the lymph node in the course of the immune response? It was recently reported that Tfh cells inside GCs have high expression of SIPR2, which acts to repel them from the S1P-rich lymph in the SCS and promote their retention in the GC (Moriyama et al., 2014). While S1pr2 was also upregulated in our single cell gene-expression analysis by primary GC Tfh cells, there were more striking changes in Gpr183 gene and EBI2 protein expression. Moreover, gene-function analysis using retroviral transduction and knockout mice confirmed a role for EBI2 in primary GC localization. These data also closely parallel the role of EBI2 in the positioning of GC B cells (Gatto et al., 2009, Pereira et al., 2009). Conspicuously, there was no differential expression of a number of chemokine receptors including Cxcr5, Ccr7, S1pr2, and Gpr183 in secondary Tfh cells located in the FM and GC, and this may explain the lack of spatial confinement upon antigen recall. Accordingly, we did not detect a defect in GC localization by EBI2-deficient KD OT2 T cells in secondary responses. Furthermore, Rgs16 was induced in primary, but not secondary, GC Tfh cells, suggesting an additional layer of control in chemokine receptor signaling allows the cells to integrate the changes in expression of these and possibly other chemokine receptors to determine their global positioning. Our finding that follicular memory T cells are located in the periphery of the draining lymph node is reminiscent of the CXCR3-dependent positioning of memory CD8+ T cells in this location (Kastenmüller et al., 2013). This positioning of follicular memory T cells at the lymph-tissue interface might facilitate quick and efficient surveillance of SCS macrophages which act as “fly paper” (Junt et al., 2007) to capture lymph-borne antigens (Carrasco and Batista, 2007, Junt et al., 2007, Phan et al., 2007, Roozendaal et al., 2009), particularly the antigen-antibody immune complexes that are generated upon secondary exposure (Phan et al., 2007, Roozendaal et al., 2009). While SCS macrophages are slow to phagocytose (Phan et al., 2009), they are nevertheless still capable of processing and presenting protein antigen to CD8+ T cells (Chtanova et al., 2009, Hickman et al., 2008) and lipid antigens to iNKT cells (Barral et al., 2010). Thus, the finding that follicular memory T cells are activated to proliferate in the subcapsular region also resolves the question of where the secondary antibody response is initiated. Memory B cells were recently shown to induce rapid BCL6 upregulation by “memory Tfh cells” in the spleen in the absence of dendritic cells (Ise et al., 2014). However, these experiments examined recall responses in the spleen of naive recipient mice following adoptive transfer of FACS-sorted “memory Tfh cells.” In contrast, we examined the in situ recall responses made by persistent antigen-specific cells in the lymph node of immune animals without any ex vivo manipulation. It should also be noted that other groups have been able to generate robust memory responses in naive recipient mice without the need for co-transfer of memory B cells (Lüthje et al., 2012, Weber et al., 2012). Hence, while memory B cells might support secondary Tfh responses at the T-B border under some circumstances, it is likely that local responses in draining lymph nodes can also be generated in the subcapsular region. Importantly, this temporospatial organization of memory responses bypasses the need for shuttling of primary Tfh cells from the T cell zone to the T-B border and finally into the follicle, and ensures rapid intrafollicular generation of secondary Tfh cells. Taken together, these data show that Tfh cell heterogeneity and complexity can be resolved by temporospatial dissection of their behavior and the molecular cues that guide this behavior. It is hoped that future studies using non-linear optical marking and single cell genomics will reveal further insights into Tfh cell biology and thereby provide cellular and molecular targets that could be manipulated to augment or dampen the antibody response to treat human diseases.