Preclinical mouse models suggest that the gut microbiome modulates tumor response to checkpoint blockade immunotherapy; however, this has not been well-characterized in human cancer patients. Here we examined the oral and gut microbiome of melanoma patients undergoing anti–programmed cell death 1 protein (PD-1) immunotherapy (n = 112). Significant differences were observed in the diversity and composition of the patient gut microbiome of responders versus nonresponders. Analysis of patient fecal microbiome samples (n = 43, 30 responders, 13 nonresponders) showed significantly higher alpha diversity (P < 0.01) and relative abundance of bacteria of the Ruminococcaceae family (P < 0.01) in responding patients. Metagenomic studies revealed functional differences in gut bacteria in responders, including enrichment of anabolic pathways. Immune profiling suggested enhanced systemic and antitumor immunity in responding patients with a favorable gut microbiome as well as in germ-free mice receiving fecal transplants from responding patients. Together, these data have important implications for the treatment of melanoma patients with immune checkpoint inhibitors.

Summary Interactions with antigen-presenting cells (APCs) interrupt T cell migration through tissues and trigger signaling pathways that converge on the activation of transcriptional regulators, including NFAT, which control T cell function and differentiation. Both stable and unstable modes of cognate T cell-APC interactions have been observed in vivo, but the functional significance of unstable, serial contacts has remained unclear. Here we used multiphoton intravital microscopy in lymph nodes and tumors to show that while NFAT nuclear import was fast (t1/2 max~1min), nuclear export was slow (t1/2~20min) in T cells. During delayed export, nuclear NFAT constituted a short-term imprint of transient TCR signals and remained transcriptionally active for the T cell tolerance gene Egr2, but not for the effector gene Ifng, which required continuous TCR triggering for expression. This provides a potential mechanistic basis for the observation that a predominance of unstable APC interactions correlates with the induction of T cell tolerance.

CD8+ T cells are master effectors of antitumor immunity, and their presence at tumor sites correlates with favorable outcomes. However, metabolic constraints imposed by the tumor microenvironment (TME) can dampen their ability to control tumor progression. We describe lipid accumulation in the TME areas of pancreatic ductal adenocarcinoma (PDA) populated by CD8+ T cells infiltrating both murine and human tumors. In this lipid-rich but otherwise nutrient-poor TME, access to using lipid metabolism becomes particularly valuable for sustaining cell functions. Here, we found that intrapancreatic CD8+ T cells progressively accumulate specific long-chain fatty acids (LCFAs), which, rather than provide a fuel source, impair their mitochondrial function and trigger major transcriptional reprogramming of pathways involved in lipid metabolism, with the subsequent reduction of fatty acid catabolism. In particular, intrapancreatic CD8+ T cells specifically exhibit down-regulation of the very-long-chain acyl-CoA dehydrogenase (VLCAD) enzyme, which exacerbates accumulation of LCFAs and very-long-chain fatty acids (VLCFAs) that mediate lipotoxicity. Metabolic reprogramming of tumor-specific T cells through enforced expression of ACADVL enabled enhanced intratumoral T cell survival and persistence in an engineered mouse model of PDA, overcoming one of the major hurdles to immunotherapy for PDA.

Granulocyte colony-stimulating factor (G-CSF) is routinely used to collect peripheral blood stem cells (PBSCs) from healthy donors for allogeneic hematopoietic stem cell transplantation (allo-HSCT). We show that, in both humans and mice, G-CSF mobilizes a subset of CD34(+) cells with mature monocyte features. These cells, which are phenotypically and functionally conserved in mice and humans, are transcriptionally distinct from myeloid and monocytic precursors but similar to mature monocytes and endowed with immunosuppressive properties. In response to interferon-γ released by activated T cells, these cells produce nitric oxide, which induces allogeneic T cell death both in vitro and in vivo. These apoptotic T cells are engulfed by macrophages that release transforming growth factor-β and promote regulatory T cell expansion. Indeed, the fraction of CD34(+) monocytes in peripheral blood CD34(+) cells inversely correlates with the incidence of acute graft-versus-host disease (GVHD) in humans. Therefore, G-CSF-mobilized cells are an attractive candidate population to be expanded ex vivo for cellular therapy against GVHD.