**< 0

**< 0.01, ***< 0.001, unpaired Students test. To determine whether DCs are necessary for the generation of circulating Tfr and Tfh cells, we used BM chimeric mice (CD11c-DTR strain) to deplete DCs. suppressive function of this population was not dependent on specific antigen. Much like LN effector Tfr cells, circulating Tfr cells also suppressed B and Tfh cells, but with a much lower capacity. Our data show that circulating memory-like Tfr cells are less suppressive than LN Tfr cells and circulating memory-like Tfh cells are more potent than LN effector Tfh cells; therefore, these circulating populations can provide quick and strong systemic B cell help during secondary antigen exposure. Introduction Follicular Th cells (Tfh cells), a subset of CD4+ T cells, stimulate and maintain the germinal center (GC) reaction, enabling B cells to produce high-affinity antibodies. Tfh cells are defined by CXCR5, which directs them to the B cell zone via gradients of the chemokine CXCL13 (1, 2). Tfh cells express the transcription factor BCL6, which facilitates CXCR5 expression and stimulates IL-21 production, helping B Peptide 17 cells to undergo affinity maturation and produce antibody (3C5). Tfh cells also can produce other cytokines, including IFN-, IL-17, and IL-4, which may help with selection of antibody isotypes during class switch recombination. Follicular Tregs (Tfr cells) are a newly defined populace of CXCR5+ CD4+ T cells. Like Tfh cells, Tfr cells express high levels of CXCR5, ICOS, and PD-1 (6C9). However, Tfr cells are thought to originate in the periphery HRY from thymic-derived Treg (tTreg) precursors, in contrast to Tfh cells, which develop from naive FOXP3C T cells (7, 9). Importantly, Tfr and Tfh cells have opposing functions in regulating humoral immunity: whereas Tfr cells potently suppress humoral immune responses, Tfh cells stimulate them (6C9). The mechanisms by which Tfr cells suppress the GC reaction are still unclear. It is not known whether Tfr cells suppress Tfh cells, GC B cells, or both. Moreover, whether specific antigen is required for Tfr suppression is also not known. Understanding how Tfr cells inhibit humoral immunity has the potential to enable improved vaccination strategies. Tfr and Tfh cells are present not only in lymph nodes (LNs), but also in the blood circulation (9). Circulating Tfh cells from humans can provide help to B cells in Peptide 17 vitro (10, 11), and circulating Tfh cells from mice can stimulate B cells in vivo (9). A subset of human blood Tfh cells has been postulated to represent memory cells (1, 10, 12). This putative memory Tfh cell subset expresses CXCR5 comparably to LN Tfh cells, Peptide 17 but expresses less PD-1 and ICOS. However, bona fide Tfh cell memory has not been exhibited in vivo. It is possible that circulating Tfh cells may give rise to memory Tfh cells (9, 13, 14). Similarly, circulating Tfr cells also may have memory potential. Elucidating the associations between LN Tfr and Tfh cells and circulating Tfr and Tfh cells may provide insights into their memory cell development and function (2). Although LN Tfr and Tfh cells depend on CD28, ICOS, and B cells for development, the specific cues for blood Tfr and Tfh cell development and maintenance are not yet obvious (9). It has been suggested that blood Tfh cells do not require the GC reaction for differentiation, but whether this is true for Tfr cells is usually unknown (15). Circulating Tfh cells in humans appear to differ from LN Tfh cells, as assessed by microarray analysis; however, these differences may be due to decreased activation in the blood or contaminating Tfr cells (12). The most straightforward explanation for Tfr and Tfh cells in the blood circulation is usually that some Tfr and Tfh cells in the GC leave the LN. If this hypothesis were true, then circulating Tfr and Tfh cells would require LN Tfr and Tfh cells for their development. In support of this hypothesis, Tfr and Tfh cells are almost completely missing from your LNs and blood of CD28- and ICOS-deficient mice (9, 16, 17). However, PD-1Cdeficient mice have.