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.

No abstract

Chemotherapy combined with immunotherapy has improved the treatment of certain solid tumors, but effective regimens remain elusive for pancreatic ductal adenocarcinoma (PDAC). We conducted a randomized phase 2 trial evaluating the efficacy of nivolumab (nivo; anti-PD-1) and/or sotigalimab (sotiga; CD40 agonistic antibody) with gemcitabine/nab-paclitaxel (chemotherapy) in patients with first-line metastatic PDAC (NCT03214250). In 105 patients analyzed for efficacy, the primary endpoint of 1-year overall survival (OS) was met for nivo/chemo (57.7%, P = 0.006 compared to historical 1-year OS of 35%, n = 34) but was not met for sotiga/chemo (48.1%, P = 0.062, n = 36) or sotiga/nivo/chemo (41.3%, P = 0.223, n = 35). Secondary endpoints were progression-free survival, objective response rate, disease control rate, duration of response and safety. Treatment-related adverse event rates were similar across arms. Multi-omic circulating and tumor biomarker analyses identified distinct immune signatures associated with survival for nivo/chemo and sotiga/chemo. Survival after nivo/chemo correlated with a less suppressive tumor microenvironment and higher numbers of activated, antigen-experienced circulating T cells at baseline. Survival after sotiga/chemo correlated with greater intratumoral CD4 T cell infiltration and circulating differentiated CD4 T cells and antigen-presenting cells. A patient subset benefitting from sotiga/nivo/chemo was not identified. Collectively, these analyses suggest potential treatment-specific correlates of efficacy and may enable biomarker-selected patient populations in subsequent PDAC chemoimmunotherapy trials.

For at least 300 years the immune system has been targeted to improve human health. Decades of work advancing immunotherapies against infection and autoimmunity paved the way for the current explosion in cancer immunotherapies. Pathways targeted for therapeutic intervention in autoimmune diseases can be modulated in the opposite sense in malignancy and infectious disease. We discuss the basic principles of the immune response, how these are co-opted in chronic infection and malignancy, and how these can be harnessed to treat disease. T cells are at the center of immunotherapy. We consider the complexity of T cell functional subsets, differentiation states, and extrinsic and intrinsic influences in the design, success, and lessons from immunotherapies. The integral role of checkpoints in the immune response is highlighted by the rapid advances in FDA approvals and the use of therapeutics that target the CTLA-4 and PD-1/PD-L1 pathways. We discuss the distinct and overlapping mechanisms of CTLA-4 and PD-1 and how these can be translated to combination immunotherapy treatments. Finally, we discuss how the successes and challenges in cancer immunotherapies, such as the collateral damage of immune-related adverse events following checkpoint inhibition, are informing treatment of autoimmunity, infection, and malignancy.

No curative treatment options are available for advanced hepatocellular carcinoma (HCC). Anti‐PD1 antibody therapy can induce tumor regression in 20% of advanced HCC patients, demonstrating that co‐inhibitory immune checkpoint blockade has therapeutic potential for this type of cancer. However, whether agonistic targeting of co‐stimulatory receptors might be able to stimulate anti‐tumor immunity in HCC is as yet unknown. We investigated whether agonistic targeting of the co‐stimulatory receptor GITR could reinvigorate ex vivo functional responses of tumor‐infiltrating lymphocytes (TIL) freshly isolated from resected tumors of HCC patients. In addition, we compared GITR expression between TIL and paired samples of leukocytes isolated from blood and tumor‐free liver tissues, and studied the effects of combined GITR and PD1 targeting on ex vivo TIL responses. In all three tissue compartments, CD4 + FoxP3 + regulatory T cells (Treg) showed higher GITR − expression than effector T‐cell subsets. The highest expression of GITR was found on CD4 + FoxP3 hi CD45RA − activated Treg in tumors. Recombinant GITR‐ligand as well as a humanized agonistic anti‐GITR antibody enhanced ex vivo proliferative responses of CD4 + and CD8 + TIL to tumor antigens presented by mRNA‐transfected autologous B‐cell blasts, and also reinforced proliferation, IFN‐γ secretion and granzyme B production in stimulations of TIL with CD3/CD28 antibodies. Combining GITR ligation with anti‐PD1 antibody nivolumab further enhanced tumor antigen‐specific responses of TIL in some, but not all, HCC patients, compared to either single treatment. In conclusion, agonistic targeting of GITR can enhance functionality of HCC TIL, and may therefore be a promising strategy for single or combinatorial immunotherapy in HCC.

Recent advances in cancer immunotherapy have completely revolutionized cancer treatment strategies. Nonetheless, the increasing incidence of immune-related adverse events (irAEs) is now limiting the overall benefits of these treatments. irAEs are well-recognized side effects of some of the most effective cancer immunotherapy agents, including antibody blockade of the cytotoxic T-lymphocyte-associated protein 4 and programmed death protein 1/programmed-death ligand 1 pathways. To develop an action plan on the key elements needed to unravel and understand the key mechanisms driving irAEs, the Society for Immunotherapy for Cancer and the American Association for Cancer Research partnered to bring together research and clinical experts in cancer immunotherapy, autoimmunity, immune regulation, genetics and informatics who are investigating irAEs using animal models, clinical data and patient specimens to discuss current strategies and identify the critical next steps needed to create breakthroughs in our understanding of these toxicities. The genetic and environmental risk factors, immune cell subsets and other key immunological mediators and the unique clinical presentations of irAEs across the different organ systems were the foundation for identifying key opportunities and future directions described in this report. These include the pressing need for significantly improved preclinical model systems, broader collection of biospecimens with standardized collection and clinical annotation made available for research and integration of electronic health record and multiomic data with harmonized and standardized methods, definitions and terminologies to further our understanding of irAE pathogenesis. Based on these needs, this report makes a set of recommendations to advance our understanding of irAE mechanisms, which will be crucial to prevent their occurrence and improve their treatment.

Purpose: Determine the differential effect of a FcgR-binding, mIgG2a anti-GITR antibody in mouse tumor models, and characterize the tumor microenvironment for the frequency of GITR expression in T-cell subsets from seven different human solid tumors.Experimental Design: For mouse experiments, wild-type C57BL/6 mice were subcutaneously injected with MC38 cells or B16 cells, and BALB/c mice were injected with CT26 cells. Mice were treated with the anti-mouse GITR agonist antibody 21B6, and tumor burden and survival were monitored. GITR expression was evaluated at the single-cell level using flow cytometry (FC). A total of 213 samples were evaluated for GITR expression by IHC, 63 by FC, and 170 by both in seven human solid tumors: advanced hepatocellular carcinoma, non-small cell lung cancer (NSCLC), renal cell carcinoma, pancreatic carcinoma, head and neck carcinoma, melanoma, and ovarian carcinoma.Results: The therapeutic benefit of 21B6 was greatest in CT26 followed by MC38, and was least in the B16 tumor model. The frequency of CD8 T cells and effector CD4 T cells within the immune infiltrate correlated with response to treatment with GITR antibody. Analysis of clinical tumor samples showed that NSCLC, renal cell carcinoma, and melanoma had the highest proportions of GITR-expressing cells and highest per-cell density of GITR expression on CD4 þ Foxp3 þ T regulatory cells. IHC and FC data showed similar trends with a good correlation between both techniques.Conclusions: Human tumor data suggest that NSCLC, renal cell carcinoma, and melanoma should be the tumor subtypes prioritized for anti-GITR therapy development.

BackgroundThere are no validated biomarkers that can aid clinicians in selecting who would best benefit from anticytotoxic T lymphocyte-associated antigen 4 monotherapy versus combination checkpoint blockade in patients with advanced melanoma who have progressive disease after programmed death 1 (PD-1) blockade.MethodsWe conducted a randomized multicenter phase II trial in patients with advanced melanoma. Patients were randomly assigned to receive either 1 mg/kg of nivolumab plus 3 mg/kg of ipilimumab or 3 mg/kg of ipilimumab every 3 weeks for up to four doses. Patients were stratified by histological subtype and prior response to PD-1 therapy. The primary clinical objective was overall response rate by week 18. Translational biomarker analyses were conducted in patients with blood and tissue samples.ResultsObjective responses were seen in 5 of 9 patients in the ipilimumab arm and 2 of 10 patients in the ipilimumab+nivolumab arm; disease control rates (DCRs) (66.7% vs 60.0%) and rates of grade 3–4 adverse events (56% vs 50%) were comparable between arms. In a pooled analysis, patients with clinical benefit (CB), defined as Response Evaluation Criteria in Solid Tumors response or progression-free for 6 months, showed increased circulating CD4+ T cells with higher polyfunctionality and interferon gamma production following treatment. Tumor profiling revealed enrichment of NRAS mutations and activation of transcriptional programs associated with innate and adaptive immunity in patients with CB.ConclusionsIn patients with advanced melanoma that previously progressed on PD-1 blockade, objective responses were seen in both arms, with comparable DCRs. Findings from biomarker analyses provided hypothesis-generating signals for validation in future studies of larger patient cohorts.Trial registration numberNCT02731729.