Purpose: Response rates to immune checkpoint blockade (ICB; anti-PD-1/anti-CTLA-4) correlate with the extent of tumor immune infiltrate but the mechanisms underlying the recruitment of T cells following therapy are poorly characterized. A greater understanding of these processes may see the development of therapeutic interventions that enhance T cell recruitment and, consequently, improved patient outcomes. We therefore investigated the chemokines essential for immune cell recruitment and subsequent therapeutic efficacy of these immunotherapies. Experimental design: The chemokines upregulated by dual PD-1/CTLA-4 blockade were assessed using nanosting-based analysis with results confirmed at the protein level by flow cytometry and cytometric bead array. Blocking/neutralizing antibodies confirmed the requirement for key chemokines/cytokines and immune effector cells. Results were confirmed in patients treated with immune checkpoint inhibitors using single-cell RNAseq and paired survival analyses. Results: The CXCR3 ligands, CXCL9 and CXCL10, were significantly upregulated following dual PD-1/CTLA-4 blockade and both CD8⁺ T cell infiltration and therapeutic efficacy were CXCR3 dependent. In both murine models and patients undergoing immunotherapy, macrophages were the predominant source of CXCL9 and their depletion abrogated CD8⁺ T cell infiltration and the therapeutic efficacy of dual immune checkpoint blockade. Single cell RNA-seq analysis of patient TILs revealed that CXCL9/10/11 was predominantly expressed by macrophages following ICB and we identified a distinct macrophage signature that was associated with positive responses to immune checkpoint blockade. Conclusions: These data underline the fundamental importance of macrophage-derived CXCR3 ligands for the therapeutic efficacy of ICB and highlight the potential of manipulating this axis to enhance patient responses.

Purpose: Therapeutic blockade of immune checkpoints has revolutionized cancer treatment. Durable responses, however, occur in less than half of those treated, and efforts to improve treatment efficacy are confounded by a lack of understanding of the characteristics of the cells that initiate antitumor immune response. Patients and Methods: We performed multiparameter flow cytometry and quantitative multiplex immunofluorescence staining on tumor specimens from immunotherapy-naïve melanoma patients and longitudinal biopsy specimen obtained from patients undergoing anti–PD-1 therapy. Results: Increased numbers of CD69+CD103+ tumor-resident CD8+ T cells were associated with improved melanoma-specific survival in immunotherapy-naïve melanoma patients. Local IL15 expression levels strongly correlated with these tumor-resident T-cell numbers. The expression of several immune checkpoints including PD-1 and LAG3 was highly enriched in this subset, and these cells significantly expanded early during anti–PD-1 immunotherapy. Conclusions: Tumor-resident CD8+ T-cell numbers are more prognostic than total CD8+ T cells in metastatic melanoma. In addition, they are likely to initiate response to anti–PD-1 and anti–LAG-3 treatments. We propose that the immune profile of these cells prior to treatment could inform strategies for immune checkpoint blockade. Clin Cancer Res; 24(13); 3036–45. ©2018 AACR.

Immune checkpoint inhibitors have revolutionized the treatment of patients with advanced-stage metastatic melanoma, as well as patients with many other solid cancers, yielding long-lasting responses and improved survival. However, a subset of patients who initially respond to immunotherapy, later relapse and develop therapy resistance (termed “acquired resistance”), whereas others do not respond at all (termed “primary resistance”). Primary and acquired resistance are key clinical barriers to further improving outcomes of patients with metastatic melanoma, and the known mechanisms underlying each involves various components of the cancer immune cycle, and interactions between multiple signaling molecules and pathways. Due to this complexity, current knowledge on resistance mechanisms is still incomplete. Overcoming therapy resistance requires a thorough understanding of the mechanisms underlying immune evasion by tumors. In this review, we explore the mechanisms of primary and acquired resistance to immunotherapy in melanoma and detail potential therapeutic strategies to prevent and overcome them. Clin Cancer Res; 24(6); 1260–70. ©2017 AACR.

Purpose: Combination PD-1 and CTLA-4 inhibitor therapy has dramatically improved the survival of patients with advanced melanoma but is also associated with significant immune-related toxicities. This study sought to identify circulating cytokine biomarkers of treatment response and immune-related toxicity. Experimental Design: The expression of 65 cytokines was profiled longitudinally in 98 patients with melanoma treated with PD-1 inhibitors, alone or in combination with anti-CTLA-4, and in an independent validation cohort of 49 patients treated with combination anti-PD-1 and anti-CTLA-4. Cytokine expression was correlated with RECIST response and immune-related toxicity, defined as toxicity that warranted permanent discontinuation of treatment and administration of high-dose steroids. Results: Eleven cytokines were significantly upregulated in patients with severe immune-related toxicities at baseline (PRE) and early during treatment (EDT). The expression of these 11 cytokines was integrated into a single toxicity score, the CYTOX (cytokine toxicity) score, and the predictive utility of this score was confirmed in the discovery and validation cohorts. The AUC for the CYTOX score in the validation cohort was 0.68 at PRE [95% confidence interval (CI), 0.51–0.84; P = 0.037] and 0.70 at EDT (95% CI, 0.55–0.85; P = 0.017) using ROC analysis. Conclusions: The CYTOX score is predictive of severe immune-related toxicity in patients with melanoma treated with combination anti-CTLA-4 and anti-PD-1 immunotherapy. This score, which includes proinflammatory cytokines such as IL1a, IL2, and IFNα2, may help in the early management of severe, potentially life-threatening immune-related toxicity. See related commentary by Johnson and Balko, p. 1452