Purpose: Immune checkpoint inhibitors (ICIs) are used for the treatment of various cancers, but clinical trials of anti-PD-1 with recurrent glioblastoma patients have failed to show clinical benefits. In this study, we examined the differentiation status of CD8⁺ tumor-infiltrating lymphocytes (TILs) from primary glioblastoma patients and their reinvigoration by ICIs to understand the nature of T-cell exhaustion in glioblastoma. Methods: We isolated TILs from 98 patients with newly diagnosed glioblastoma and examined the expression of immune checkpoint receptors and T-cell transcription factors using flow cytometry. TILs were ex vivo stimulated with anti-CD3 in the presence of anti-PD-1 and/or anti-CTLA-4 and their proliferation assessed. Results: CD8⁺ TILs had significantly increased expression of immune checkpoint receptors, including PD-1 and CTLA-4, compared to peripheral blood CD8⁺ T cells. Among CD8⁺ TILs, PD-1⁺ cells exhibited more terminally differentiated phenotypes (i.e., Eomes^hiT-bet^lo) than PD-1^- cells. These data were confirmed by analyzing NY-ESO-1₁₅₇-specific CD8⁺ TILs. Evaluating the proliferation of CD8⁺ TILs after ex vivo stimulation with anti-CD3 and anti-PD-1, we found that proliferation inversely correlated with the percentage of Eomes^hiT-bet^lo cells among PD-1⁺CD8⁺ TILs. When anti-CTLA-4 was used in combination with anti-PD-1, an additional increase in CD8⁺ TIL proliferation was observed in patients with low percentages of Eomes^hiT-bet^lo CD8⁺ TILs, who responded well to anti-PD-1 in ex vivo assays, but not in patients with high percentages of Eomes^hiT-bet^lo CD8⁺ TILs, who did not respond to anti-PD-1. Conclusions: In primary glioblastoma, the differentiation status of CD8⁺ TILs determines their reinvigoration ability upon ICI treatment.

Purpose: Immune checkpoint inhibitors have shown therapeutic efficacy in various malignant diseases. However, anti-programmed death (PD)-1 therapy has not shown clinical efficacy in multiple myeloma (MM). Experimental Design: Bone marrow (BM) mononuclear cells were obtained from 77 newly diagnosed MM patients. We examined the expression of immune checkpoint receptors in BM CD8⁺ T cells and their functional restoration by ex vivo treatment with anti-PD-1 and TGF-b inhibitors. Results: We confirmed the upregulation of PD-1 and PD-L1 expression in CD8⁺ T cells and myeloma cells, respectively, from the BM of MM patients. PD-1-expressing CD8⁺ T cells from the BM of MM patients co-expressed other checkpoint inhibitory receptors and exhibited a terminally differentiated phenotype. These results were also observed in BM CD8⁺ T cells specific to myeloma antigens NY-ESO-1 and HM1.24. BM CD8⁺ T cells from MM patients exhibited reduced proliferation and cytokine production upon T-cell receptor stimulation. However, anti-PD-1 did not increase the proliferation of BM CD8⁺ T cells from MM patients, indicating that T-cell exhaustion in MM is hardly reversed by PD-1 blockade alone. Intriguingly, anti-PD-1 significantly increased the proliferation of BM CD8⁺ T cells from MM patients in the presence of inhibitors of TGF-b, which was overexpressed by myeloma cells. Conclusions: Our findings indicate that combined blockade of PD-1 and TGF-b may be useful for the treatment of MM.

The dynamic changes in the tumor immune microenvironment (TIME) triggered by neoadjuvant chemotherapy (NAC) have not been clearly defined in advanced-stage ovarian cancer. We analyzed the immunologic changes induced by NAC to correlate them with clinical outcomes. We compared the changes in the immune infiltration of high-grade serous carcinoma biopsies before and after NAC via immunohistochemistry (147 paired samples) and whole transcriptome sequencing (35 paired samples). Immunohistochemistry showed significantly increased PD-L1 levels and TIL levels after NAC. Whole transcriptome sequencing revealed that the stromal score, immune score, and cytolytic activity score significantly increased after NAC. An increased tumor-infiltrating lymphocyte (TIL) level in response to NAC was associated with shorter progression-free survival compared with decreased TIL level after NAC. In tumors with increased TIL levels after NAC, the relative fraction of CD8 T cells and regulatory T cells significantly increased with immunohistochemistry. Post-NAC tumors were enriched in gene sets associated with immune signaling pathways, such as regulatory T cell and JAK/STAT signaling pathways. NAC induced dynamic changes in the TIME that increased TIL levels, but their high abundance did not impart any survival benefit. Our data may provide therapeutic strategies to improve the survival benefit from immunotherapies in ovarian cancer.

Background This study assessed the antitumor activity and safety of durvalumab plus tremelimumab combined with neoadjuvant chemotherapy (NAC) in patients newly diagnosed with advanced ovarian cancer. Here, we report the primary endpoint of the original cohort of the KGOG 3046/TRU-D study. Methods In this investigator-initiated single-arm, phase II trial, patients with stage IIIC-IVB ovarian cancer were administered three cycles of durvalumab (1500 mg) and tremelimumab (75 mg) with NAC, followed by interval debulking surgery (IDS). After surgery, three cycles of durvalumab (1120 mg) and adjuvant chemotherapy followed by durvalumab maintenance (1120 mg [total 12 cycles]) were administered. The primary endpoint of the study was 12-month progression-free survival (PFS) rate. Results Twenty-three patients were enrolled. The median patient age was 60 years (range 44–77 years), and most patients presented with high-grade serous carcinoma (87.0%) and stage IV disease (87.0%). At the time of data cut-off on January 17, 2023, the median follow-up duration was 29.2 months (range 12.0–42.2). The 12-month, 24-month, and 30 month PFS rates were 63.6%, 45.0%, and 40.0%, respectively. All patients underwent IDS, with an R0 resection rate of 73.9%, and 17.4% achieved pathological complete response. Skin rashes were the most common treatment-related adverse events (TRAEs, 69.6%). However, all TRAEs completely resolved after steroid use. Conclusion This study showed promising activity with a durable clinical response, supporting the potential of NAC with dual immune checkpoint blockade in advanced-stage ovarian cancer. Trial registration number NCT03899610.

Park J, et al. J Gynecol Oncol. 2021 Dec;33:e12. https://doi.org/10.3802/jgo.2022.33.e12

Background: We aimed to investigate the distinct immunological characteristics of the tumor immune microenvironment in epithelial ovarian cancer (EOC) according to BRCA1/2 mutations status and differential PD-1 expression levels. Methods: Tumor-infiltrating lymphocytes (TILs) were collected from patients with newly diagnosed advanced-stage EOC (YUHS cohort, n=117). This YUHS cohort was compared with The Cancer Genome Atlas (TCGA) data for ovarian serous cystadenocarcinoma (n=482), in terms of survival outcomes and immune-related gene profiles according to BRCA1/2 status. We used multicolor flow cytometry to characterize the immune phenotypes and heterogeneity of TILs with or without BRCA1/2 mutations. In vitro functional assays were conducted to evaluate the reinvigorating ability of CD8⁺ TILs on anti-PD-1 treatment. Results: We found that EOC patients with BRCA1/2 mutations (BRCA1/2mt) exhibited better survival outcomes and significantly higher tumor mutation burden (TMB), compared with BRCA1/2 non-mutated (BRCA1/2wt) patients. Furthermore, CD8⁺ TILs within BRCA1/2mt tumors displayed characteristics indicating more severe T-cell exhaustion than their BRCA1/2wt counterparts. Notably, the capacity for anti-PD-1-mediated reinvigoration of CD8⁺ TILs was significantly greater in BRCA1/2wt tumors compared with BRCA1/2mt tumors. Additionally, within the BRCA1/2wt group, the frequency of PD-1^highCD8⁺ TILs was positively correlated with the reinvigoration capacity of CD8⁺ TILs after anti-PD-1 treatment. Conclusion: Our results highlight unique immune features of CD8⁺ TILs in EOC and a differential response to anti-PD-1 treatment, contingent on BRCA1/2 mutation status. These findings suggest that immune checkpoint blockade may be a promising frontline therapeutic option for selected BRCA1/2wt EOC patients.