SUMMARY The nature of follicular helper CD4+ T (Tfh) cell differentiation remains controversial, including the minimal signals required for Tfh differentiation, and the time at which Tfh differentiation occurs. Here we determine that Tfh development initiates immediately during dendritic cell (DC) priming in vivo. We demonstrate that inducible costimulator (ICOS) provides a critical early signal to induce the transcription factor Bcl6, and Bcl6 then induces CXCR5, the canonical feature of Tfh cells. Strikingly, a bifurcation between Tfh and effector Th cells was measurable by the second cell division of CD4+ T cells, at day 2 after an acute viral infection: IL2Rαint cells expressed Bcl6 and CXCR5 (Tfh cell program), whereas IL2Rαhi cells exhibited strong Blimp1 expression that repressed Bcl6 (effector Th cell program). Virtually complete polarization between Bcl6+ Tfh cells and Blimp1+ effector Th cell populations developed by 72 hours, even without B cells. Tfh cells were subsequently lost in the absence of B cells, demonstrating a B cell requirement for maintenance of Bcl6 and Tfh cell commitment via sequential ICOS signals.

Immunotherapies that block inhibitory checkpoint receptors on T cells have transformed the clinical care of cancer patients 1 . However, whether the T cell response to checkpoint blockade relies on reinvigoration of pre-existing tumor infiltrating T cells (TILs) or on recruitment of novel T cells remains unclear 2 – 4 . Here, we performed paired single-cell RNA (scRNA) and T cell receptor (TCR)- sequencing on 79,046 cells from site-matched tumors from patients with basal cell carcinoma (BCC) or squamous cell carcinoma (SCC) pre- and post-anti-PD-1 therapy. Tracking TCR clones and transcriptional phenotypes revealed a coupling of tumor-recognition, clonal expansion, and T cell dysfunction marked by clonal expansions of CD8 + CD39 + T cells, which co-expressed markers of chronic T cell activation and exhaustion. However, this expansion did not derive from pre-existing TIL clones; rather, it was comprised of novel clonotypes not previously observed in the same tumor. Clonal replacement of T cells was preferentially observed in exhausted CD8 + T cells and evident in BCC and SCC patients. These results demonstrate that pre-existing tumor-specific T cells may have limited reinvigoration capacity, and that the T cell response to checkpoint blockade derives from a distinct repertoire of T cell clones that may have just recently entered the tumor.

Preclinical studies suggest that treatment with neoadjuvant immune checkpoint blockade is associated with enhanced survival and antigen-specific T cell responses over adjuvant treatment 1 ; however, optimal regimens have not been defined. Herein, we report results from a randomized phase II study of neoadjuvant nivolumab versus combined ipilimumab with nivolumab in 23 patients with high-risk resectable melanoma (NCT02519322). RECIST overall response rates (ORR), pathologic complete response rates (pCR), treatment-related adverse events (trAEs), and immune correlates of response were assessed. Treatment with combined ipilimumab and nivolumab yielded high response rates (RECIST ORR 73%, pCR 45%) but substantial toxicity (73% grade 3 trAEs), whereas treatment with nivolumab monotherapy yielded modest responses (ORR 25%, pCR 25%) and low toxicity (8% grade 3 trAEs). Immune correlates of response were identified, demonstrating higher lymphoid infiltrates in responders to both therapies and a more clonal and diverse T cell infiltrate in responders to nivolumab monotherapy. These results are the first to describe the feasibility of neoadjuvant immune checkpoint blockade in melanoma and emphasize the need for additional studies to optimize treatment regimens and to validate putative biomarkers.

Cytokines are important modulators of lymphocytes, and both interleukin-21 (IL-21) and IL-6 have proposed roles in T follicular helper (Tfh) differentiation, and directly act on B cells. Here we investigated the absence of IL-6 alone, IL-21 alone, or the combined lack of IL-6 and IL-21 on Tfh differentiation and the development of B cell immunity in vivo. C57BL/6 or IL-21−/− mice were treated with a neutralizing monoclonal antibody against IL-6 throughout the course of an acute viral infection (lymphocytic choriomeningitis virus, LCMV). The combined absence of IL-6 and IL-21 resulted in reduced Tfh differentiation and reduced Bcl6 protein expression. In addition, we observed that these cytokines had a large impact on antigen-specific B cell responses. IL-6 and IL-21 collaborate in the acute T-dependent antiviral antibody response (90% loss of circulating antiviral IgG in the absence of both cytokines). In contrast, we observed reduced germinal center formation only in the absence of IL-21. Absence of IL-6 had no impact on germinal centers, and combined absence of both IL-21 and IL-6 revealed no synergistic effect on germinal center B cell development. Studying CD4 T cells in vitro, we found that high IL-21 production was not associated with high Bcl6 or CXCR5 expression. TCR stimulation of purified naïve CD4 T cells in the presence of IL-6 also did not result in Tfh differentiation, as determined by Bcl6 or CXCR5 protein expression. Cumulatively, our data indicates that optimal Tfh formation requires IL-21 and IL-6, and that cytokines alone are insufficient to drive Tfh differentiation.

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