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Cytotoxic CD8+ T cells (CTL) are a crucial component of the immune system notable for their ability to eliminate rapidly proliferating malignant cells. However, the T-cell intrinsic factors required for human CTLs to accomplish highly efficient antitumor cytotoxicity are not well defined. By evaluating human CD8+ T cells from responders versus nonresponders to treatment with immune checkpoint inhibitors, we sought to identify key factors associated with effective CTL function. Single-cell RNA-sequencing analysis of peripheral CD8+ T cells from patients treated with anti–PD-1 therapy showed that cells from nonresponders exhibited decreased expression of the cytolytic granule-associated molecule natural killer cell granule protein-7 (NKG7). Functional assays revealed that reduced NKG7 expression altered cytolytic granule number, trafficking, and calcium release, resulting in decreased CD8+ T-cell–mediated killing of tumor cells. Transfection of T cells with NKG7 mRNA was sufficient to improve the tumor-cell killing ability of human T cells isolated from nonresponders and increase their response to anti–PD-1 or anti–PD-L1 therapy in vitro. NKG7 mRNA therapy also improved the antitumor activity of murine tumor antigen–specific CD8+ T cells in an in vivo model of adoptive cell therapy. Finally, we showed that the transcription factor ETS1 played a role in regulating NKG7 expression. Together, our results identify NKG7 as a necessary component for the cytotoxic function of CD8+ T cells and establish NKG7 as a T-cell–intrinsic therapeutic target for enhancing cancer immunotherapy. See related article by Li et al., p. 154.

Background: Tumor surface PD-L1 (B7-H1) signals through T-cell surface receptor PD-1 to suppress antitumor immunity. PD-1 inhibitors like pembrolizumab block this pathway to prevent tumor escape from immunosurveillance. These PD-1 checkpoint inhibitors achieve objective response in 20-30% of patients with melanoma (1-3). As the majority of these patients are resistant to this treatment, there is a critical need to combat PD-1 checkpoint inhibitor resistance in melanoma. What is the mechanism of PD-1 checkpoint inhibitor resistance in melanoma? Our group has reported that soluble PD-L1 (sPD-L1, sB7-H1) is elevated in the serum of patients with PD-1 inhibitor-resistant melanoma and non-small cell lung cancer (NSCLC) (4). We have also shown that soluble PD-L1 is produced by cleavage from other tumor types and leads to apoptosis in CD8+ T-cells (5). The aim of this study is to confirm the source of sPD-L1 in human melanoma and its relation to outcomes. Methods: B7H1-expressing transgenic melanoma cells (Mel-B7H1) were treated with an array of protease inhibitors over 24 hours. Cells were then pelleted and supernatants were analyzed by ELISA for sPD-L1 (sB7-H1) production. Tumor serum samples were also analyzed by ELISA for sPD-L1 production.Tumor pathology slides from patients with melanoma (serum donors from the above study) were stained by immunohistochemistry with antibodies against proteases ADAM10 or ADAM17. Slides were visualized by microscopy for the presence of these antigens. Activated human CD8+ T-cells were cultured with recombinant human B7-H1 (PD-L1) protein, sPD-L1-rich, or sPD-L1-depleted supernatants from a tumor cell line in the presence of placebo or PD-1 checkpoint inhibitor antibodies. Cell survival was measured at 48 hours by flow cytometry.Results: ADAM10 inhibitor GI254023X, ADAM17 inhibitor TAPI-0, and broad metalloprotease inhibitor TAPI-2 inhibited Mel-B7H1 production of sB7H1 (p Citation Format: Jacob J. Orme, Khalid Jazieh, Susan Harrington, Matthew Ball, Tariq U. Azam, Xin (Cindy) Liu, Tiancheng Xie, Aaron Mansfield, Roxana S. Dronca, Haidong Dong. Tumor-associated ADAM10 and ADAM17 produce soluble PD-L1 (sPD-L1, sB7-H1) and affect downstream tumor immunity – A resistance mechanism to PD-1 checkpoint blockade in melanoma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A210.

Background: Clinical management of metastatic melanoma (MM) after PD-1 blockade failure remains a challenge and lacks a standard of care. Immunotherapy or chemotherapy alone provides limited benefit in this setting. Chemo-immunotherapy (CIT) combinations have demonstrated favorable efficacy and safety profiles in lung cancer patients. Our preclinical study in MM has shown that the addition of chemotherapy to PD-1 blockade can reshape a subset of therapy-responsive CD8+ T-cells with resultant enhanced antitumor activities, suggesting the potential clinical benefits of CIT in MM patients whose disease have progressed after anti-PD-1 therapy. Further understanding of the clinical benefits and immunoregulatory mechanisms of CIT in this setting is crucial for the development of optimal combinatorial chemo-immunotherapeutic strategies to improve clinical outcomes in patients with advanced cancer. Methods: MM patients (n=22) who have failed PD-1 blockade therapy were subsequently treated with CIT (paclitaxel and carboplatin in combination with pembrolizumab). The overall survival (OS), objective response rate (ORR), time-to-next therapy (TTNT), and toxicities were assessed. Using peripheral blood (PB) from MM patients, the phenotypic and functional changes induced by chemotherapy in therapy-responsive T-cells, in the setting of anti-PD1 therapy, were examined. The immunoregulatory effects of CIT were also examined in melanoma mouse model. Results: MM patients who have received subsequent CIT had a median OS of 5 years (95% CI: 2-NR) (median follow up of 3.9 years), with ORR of 61% (CR of 23%). The median TTNT was 8 months (95% CI: 6-15). No additional toxicities were identified. CX3CR1+CD8+ therapy-responsive T-cells are low in MM patients who have failed to respond to anti-PD-1 monotherapy. However, in MM patients who responded to subsequent CIT, this subset of therapy-responsive T-cells survived the chemotherapy with increased frequency and enhanced function. The clinical benefit of CIT is only observed in CX3CR1 wild type mice, not in KO mice, and ongoing PD-1 blockade is necessary to improve its anti-tumor activities. Conclusion: In MM patients who have failed anti-PD-1 therapy, the chemo-immunotherapy combination showed favorable clinical outcomes and an acceptable toxicity profile. CX3CR1+ CD8+ effector T-cells are responsible for the clinical benefit of CIT. This novel therapy-responsive population underlies the key cellular and molecular immunoregulatory mechanisms of chemotherapy. It serves as a meaningful marker to measure these collaborative effects and to develop the optimal chemo-immunotherapy strategy to improve clinical responses to current immune checkpoint blocking agents. Citation Format: Yiyi Yan, Haidong Dong, Roxana Dronca, Svetomir Markovic. CX3CR1+CD8+ T-cells are responsible to the clinical benefit of chemoimmunotherapy in metastatic melanoma patients after disease progression on PD-1 blockade [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr PR11.

Despite unprecedented successes with immune checkpoint blockade in advanced cancers, these agents provide durable clinical benefit in only a subset of patients. In addition, the clinical management of patients receiving immune checkpoint blockade remains quite challenging due to the unpredictable and kinetically heterogeneous responses which can manifest as late responses, pseudoprogression, or hyperprogression in subsets of patients. The dynamics and heterogeneity of programmed cell death protein ligand 1 (PD-L1, aka B7-H1) expression confound its use as a predictive biomarker in cancer immunotherapy, but blood-based biomarkers have the potential to predict responders and detect mechanisms of resistance to immunotherapy. We previously reported that Bim (BH3-only protein) is a downstream signaling molecule of the PD-1 pathway, and that Bim levels in circulating tumor-reactive T-cells reflected patient responses to anti-PD-1 therapy in melanoma. We also discovered the existence of soluble PD-L1 (sPD-L1) in the plasma of cancer patients and showed a correlation of sPD-L1 with cancer stages in renal cell carcinoma and with prognosis in metastatic melanoma. Here we evaluated the frequency of tumor-reactive T cells with Bim expression along with effector memory profile and sPD-L1 as biomarkers of response in a new cohort of patients with metastatic melanoma and lung cancer undergoing anti-PD-1 therapy. We recruited 100 cancer patients treated with anti-PD-1 therapy who had peripheral blood collected at baseline and at each subsequent radiographic tumor evaluation. Frequencies of Bim+ and effector memory CD8+ T cells were measured by flow cytometry in gated tumor-reactive CD11a high PD1+ CD8+ T cells. We also measured levels of sPD-L1 in plasma at baseline and serially during treatment with ELISA. Baseline T cell markers and sPD-L1 levels and their percent changes in patients who had a radiographic response were compared to those who had progressive disease (PD). We found that patients with objective response after 4 cycles of anti-PD-1 therapy had higher frequency of Bim+ CD8+ T cells at baseline compared to patients with PD (mean 43% vs. 30%, P=0.0484). The frequencies of Bim+ T cells decreased significantly after the first 3 months of treatment in responders compared with progressors (mean -16% vs. + 40% P=0.0111). The frequency of effector memory CD8+ T cells also dramatically increased in responders in 3-6 months after anti-PD-1 therapy (mean 89% vs. 9.56%, p=0.002). High baseline sPD-L1 was associated with progression on anti-PD-1 therapy (mean 2.8 ng/mL vs. 0.7 ng/mL, p=0.07) or development of immune-related toxicities and the levels increased by 12 weeks in patients progressing on therapy. Conclusion: Measurements of T cell biomarkers (Bim and effector memory) and sPD-L1 levels provide a new noninvasive way to predict and monitor patient responses to anti-PD-1 monotherapy in melanoma and lung cancers. Note: This abstract was not presented at the conference. Citation Format: Roxana S. Dronca, Aaron Mansfield, Xin Liu, Susan Harrington, Elizabeth Enninga, Svetomir Markovic, Lisa Kottschade, Rob Mcwilliams, Matthew Block, Wendy Nevala, Michael Thompson, Haidong Dong. Blood-based T cell biomarkers and soluble PD-L1 predict responses and immune-related toxicity to PD-1 blockade in melanoma and lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A38.