Expression of Foxp3 by T follicular helper cells in end-stage germinal centers

Regulating germinal center contraction Germinal centers (GCs) in secondary lymphoid organs are where mature B cells expand and differentiate. Although GC formation is well studied, the control of GC duration and contraction is less well understood. Using intravital imaging of mouse GCs and single-cell RNA sequencing, Jacobsen et al. report that T follicular helper (TFH) cells are a critical player in this process. They found that some late-GC TFH cells upregulate the transcription factor FOXP3 and acquire a regulatory T cell–like phenotype. These cells are distinct from T follicular regulatory (TFR) cells and, unlike TFR cells, are needed to shut down the GC reaction. Tweaking this process may be key to extending GC lifetimes and enhancing antibody responses in the context of vaccination. Science, abe5146, this issue p. eabe5146 Functional eggs were successfully produced in a mouse ovarian environment reconstituted from pluripotent stem cells. INTRODUCTION To produce antibodies that protect effectively against pathogens, B cells must undergo a Darwinian process of somatic hypermutation of their immunoglobulin genes followed by selective proliferation of variants with improved affinity for antigen. This process, known as affinity maturation, can dramatically increase the affinity and potency of antibodies elicited by infection and vaccination. Affinity maturation takes place in germinal centers (GCs), structures that form within secondary lymphoid organs in response to infection or immunization. Because somatic mutations accumulate progressively with time, the duration of the GC reaction is an important determinant of the extent to which antibodies can mutate and mature. Despite the importance of this parameter, our understanding of the factors that determine the duration of a GC and the timing of its contraction remains limited. RATIONALE In addition to B cells, a minor but critical component of the GC reaction are T follicular helper (TFH) cells characterized by expression of chemokine receptor CXCR5, inhibitory receptor PD-1, and transcription factor Bcl6. TFH cells provide mitogenic signals essential for the proliferative expansion of high-affinity B cells and thus play a key role in antibody affinity maturation. The size of the TFH cell population closely correlates with, and likely determines, the magnitude of the GC reaction, and interruption of the signals provided by TFH cells to B cells leads to GC dissolution, making TFH cells likely candidates for regulators of GC duration. The positive effects of TFH cells are counterbalanced by a population of GC-resident T cells that express Foxp3, the master transcription factor of the regulatory T cell (Treg) lineage. The best characterized of these Foxp3 populations are T follicular regulatory (TFR) cells, which arise through acquisition of a TFH-like phenotype by thymic-derived Tregs and control aspects of the B cell response ranging from B cell foreign and self-antigen specificity to isotype switching. Given the well-established role of CD4 T cells in sustaining GCs and the emerging role of Foxp3 in suppressing this reaction, we sought to determine whether expression of Foxp3 by GC-resident T cells could also play a role in GC longevity. RESULTS Imaging of the GC reactions in mice over time showed that the density and number of Foxp3 T cells within these structures surged acutely in the days preceding the onset of GC contraction. In contrast to their early- or peak-GC counterparts, late-GC Foxp3 T cells expressed high levels of TFH cell surface markers CXCR5 and PD-1 and engaged in long-lived dynamic interactions with GC B cells that resembled those observed for TFH cells, suggesting late-GC Foxp3 T cells may differ in ontogeny from the canonical TFR population abundant at earlier time points. T cell receptor sequencing suggested that, unlike TFR cells, late-GC Foxp3 T cells likely arise through up-regulation of Foxp3 by TFH cells in the days immediately preceding GC regression. This conversion was confirmed by adoptive transfer experiments, in which transferred Foxp3 naïve T cells acquired Foxp3 expression in late but not in early GCs. Transcriptionally, late-GC Foxp3 T cells closely resembled TFH cells while showing shifts in expression of Treg-related signatures that included loss of key T cell help-related genes. A gain-of-function experiment showed that ectopic Foxp3 expression by TFH cells, although incapable of completely shifting these cells toward a TFR phenotype, was sufficient to change expression of Treg-related gene signatures and trigger GC contraction. CONCLUSION Our data indicate that, in addition to canonical TFR cells, there exists a second population of Foxp3 GC T cells that arises immediately before GC contraction, through the up-regulation of Foxp3 and limited acquisition of Treg-like features by TFH cells. Functional experiments support a model in which the contraction, and eventual shutdown, of late-stage GCs is promoted by acquisition of Foxp3 by this TFH cell population. These findings raise the possibility that GC shutdown is an active process rather than simply a result of the progressive consumption of antigen by GC B cells. Manipulating this process may provide an avenue toward extending GC lifetime, potentially contributing to the induction of highly mutated antibodies by vaccination. A surge in Foxp3+ T cells precedes GC contraction. GCs are usually temporary structures that emerge upon infection or immunization, contracting and eventually disappearing upon cessation of antigenic stimulation. At their peak, GCs contain two major populations of T cells, TFH cells and TFR cells. Of these, only the latter population expresses the Treg-associated transcription factor Foxp3. We find that the days immediately preceding GC contraction are characterized by a surge in the numbers of Foxp3-expressing T cells, arising at least in part from up-regulation of Foxp3 by the TFH population. Gain-of-function experiments suggest that Foxp3 expression by TFH cells may aid in the process of GC shutdown. Germinal centers (GCs) are the site of immunoglobulin somatic hypermutation and affinity maturation, processes essential to an effective antibody response. The formation of GCs has been studied in detail, but less is known about what leads to their regression and eventual termination, factors that ultimately limit the extent to which antibodies mature within a single reaction. We show that contraction of immunization-induced GCs is immediately preceded by an acute surge in GC-resident Foxp3+ T cells, attributed at least partly to up-regulation of the transcription factor Foxp3 by T follicular helper (TFH) cells. Ectopic expression of Foxp3 in TFH cells is sufficient to decrease GC size, implicating the natural up-regulation of Foxp3 by TFH cells as a potential regulator of GC lifetimes.