Immune checkpoint blockade (ICB) of PD-1 and CTLA-4 to treat metastatic melanoma (MM) has variable therapeutic benefit. To explore this in peripheral samples we characterized CD8 + T cell gene expression across a cohort of MM patients receiving anti-PD-1 alone (sICB) or in combination with anti-CTLA-4 (cICB). Whereas CD8 + transcriptional responses to sICB and cICB involve a shared gene set, the magnitude of cICB response is over four-fold greater, with preferential induction of mitosis and interferon related genes. Early samples from patients with durable clinical benefit demonstrated over-expression of T cell receptor (TCR) encoding genes. By mapping TCR clonality we find responding patients have more large clones (those occupying >0.5% of repertoire) post-treatment than non-responding patients or controls, and this correlates with effector memory T cell percentage. Single-cell RNA-sequencing of eight post-treatment samples demonstrates large clones over-express genes implicated in cytotoxicity and characteristic of effector memory T cells including CCL4, GNLY, and NKG7 . The six-month clinical response to ICB in MM patients is associated with the large CD8 + T cell clone count 21 days after treatment and agnostic to clonal specificity, suggesting that post-ICB peripheral CD8 + clonality can provide information regarding long-term treatment response and potentially facilitate treatment stratification.

SummaryNeutrophils are critical and short-lived mediators of innate immunity that require constant replenishment. Their differentiation in the bone marrow requires extensive cytoplasmic and nuclear remodeling, but the processes governing these energy-consuming changes are unknown. While previous studies show that autophagy is required for differentiation of other blood cell lineages, its function during granulopoiesis has remained elusive. Here, we have shown that metabolism and autophagy are developmentally programmed and essential for neutrophil differentiation in vivo. Atg7-deficient neutrophil precursors had increased glycolytic activity but impaired mitochondrial respiration, decreased ATP production, and accumulated lipid droplets. Inhibiting autophagy-mediated lipid degradation or fatty acid oxidation alone was sufficient to cause defective differentiation, while administration of fatty acids or pyruvate for mitochondrial respiration rescued differentiation in autophagy-deficient neutrophil precursors. Together, we show that autophagy-mediated lipolysis provides free fatty acids to support a mitochondrial respiration pathway essential to neutrophil differentiation.