Inflammatory B cells correlate with failure to checkpoint blockade in melanoma patients.

The understanding of the role of B cells in patients with solid tumors remains insufficient. We found that circulating B cells produced TNFα and/or IL-6, associated with unresponsiveness and poor overall survival of melanoma patients treated with anti-CTLA4 antibody. Transcriptome analysis of B cells from melanoma metastases showed enriched expression of inflammatory response genes. Publicly available single B cell data from the tumor microenvironment revealed a negative correlation between TNFα expression and response to immune checkpoint blockade. These findings suggest that B cells contribute to tumor growth via the production of inflammatory cytokines. Possibly, these B cells are different from tertiary lymphoid structure-associated B cells, which have been described to correlate with favorable clinical outcome of cancer patients. Further studies are required to identify and characterize B cell subsets and their functions promoting or counteracting tumor growth, with the aim to identify biomarkers and novel treatment targets

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

LAG-3 and PD-1+LAG-3 inhibition promote anti-tumor immune responses in human autologous melanoma/T cell co-cultures.

Despite the success of immunotherapy using checkpoint blockade, many patients with solid tumors remain refractory to these treatments. In human cancer, the experimental options to investigate the specific effects of antibodies blocking inhibitory receptors are limited and it is still unclear which cell types are involved. We addressed the question whether the direct interaction between T cells and tumor cells can be enforced through blocking a set of inhibitory receptors including PD-1, TIM-3, BTLA and LAG-3, blocked either individually or in dual combinations with the anti-PD-1 antibody, and to determine the condition that induces maximal T cell function preventing tumor cell proliferation. Using short-term Melan-A-specific or autologous re-stimulations, checkpoint blockade did not consistently increase cytokine production by tumor-derived expanded T cells. We next set up a 5-day co-culture assay with autologous melanoma cell lines and expanded tumor infiltrating T cells, originating from tumor specimens obtained from 6 different patients. Amongst all combos tested, we observed that blockade of LAG-3 alone, and more strongly when combined with PD-1 blockade, enforced T cell responses and tumor cell growth control. The combination of anti-LAG-3 plus anti-PD-1 acted through CD8 T cells and led to increased IFNγ production and cytotoxic capacity. Our results show that LAG-3 and PD-1 are regulating the direct interaction between tumor cells and autologous T cells, suggesting that therapy effects may be promoted by enhanced access of the corresponding blocking reagents to the tumor microenvironment

CD40 Agonist Restores the Antitumor Efficacy of Anti-PD1 Therapy in Muscle-Invasive Bladder Cancer in an IFN I/II-Mediated Manner.

Bladder cancer is one of the most common malignancies and has poor prognosis for patients with locally advanced, muscle-invasive, disease despite the efficacy of immune checkpoint blockade. To develop more effective immunotherapy strategies, we studied a genetic mouse model carrying deletion of Tp53 and Pten in the bladder, which recapitulates bladder cancer tumorigenesis and gene expression patterns found in patients. We discovered that tumor cells became more malignant and the tumor immune microenvironment evolved from an inflammatory to an immunosuppressive state. Accordingly, treatment with anti-PD1 was ineffective, but resistance to anti-PD1 therapy was overcome by combination with a CD40 agonist (anti-CD40), leading to strong antitumor immune responses. Mechanistically, this combination led to CD8 &lt;sup&gt;+&lt;/sup&gt; T-cell recruitment from draining lymph nodes. CD8 &lt;sup&gt;+&lt;/sup&gt; T cells induced an IFNγ-dependent repolarization toward M1-like/IFNβ-producing macrophages. CD8 &lt;sup&gt;+&lt;/sup&gt; T cells, macrophages, IFN I, and IFN II were all necessary for tumor control, as demonstrated in vivo by the administration of blocking antibodies. Our results identify essential cross-talk between innate and adaptive immunity to control tumor development in a model representative of anti-PD1-resistant human bladder cancer and provide scientific rationale to target CD40 in combination with blocking antibodies, such as anti-PD1/PD-L1, for muscle-invasive bladder cancer

Broad and Conserved Immune Regulation by Genetically Heterogeneous Melanoma Cells.

Although mutations drive cancer, it is less clear to what extent genetic defects control immune mechanisms and confer resistance to T-cell-based immunotherapy. Here, we studied the reactions of malignant and benign melanocyte lines to cytotoxic CD8(+) T cells (CTL) using flow cytometry and gene expression analyses. We found rapid and broad upregulation of immune-regulatory genes, essentially triggered by CTL-derived IFNγ and augmented by TNFα. These reactions were predominantly homogenous, independent of oncogenic driver mutations, and similar in benign and malignant cells. The reactions exhibited both pro- and antitumorigenic potential and primarily corresponded to mechanisms that were conserved, rather than acquired, by mutations. Similar results were obtained from direct ex vivo analysis of the tumor microenvironment. Thus, immune regulation in the tumor landscape may often be driven by conserved mechanisms, which may explain why T-cell-based immunotherapy can provide durable benefits with relatively infrequent escape. Cancer Res; 77(7); 1623-36. ©2017 AACR