Unique immune characteristics and differential anti-PD-1-mediated reinvigoration potential of CD8(+) TILs based on BRCA1/2 mutation status in epithelial ovarian cancers.

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. 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. 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-1highCD8+ TILs was positively correlated with the reinvigoration capacity of CD8+ TILs after anti-PD-1 treatment. 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.

Park J ，Kim JC ，Lee YJ ，Kim S ，Kim SW ，Shin EC ，Lee JY ，Park SH ... -  《Journal for ImmunoTherapy of Cancer》

Association and prognostic significance of BRCA1/2-mutation status with neoantigen load, number of tumor-infiltrating lymphocytes and expression of PD-1/PD-L1 in high grade serous ovarian cancer.

Strickland KC ，Howitt BE ，Shukla SA ，Rodig S ，Ritterhouse LL ，Liu JF ，Garber JE ，Chowdhury D ，Wu CJ ，D'Andrea AD ，Matulonis UA ，Konstantinopoulos PA ... -  《Oncotarget》

4-1BB co-stimulation further enhances anti-PD-1-mediated reinvigoration of exhausted CD39(+) CD8 T cells from primary and metastatic sites of epithelial ovarian cancers.

Responses to immunotherapy vary between different cancer types and sites. Here, we aimed to investigate features of exhaustion and activation in tumor-infiltrating CD8 T cells at both the primary and metastatic sites in epithelial ovarian cancer. Tumor tissues and peripheral blood were obtained from 65 patients with ovarian cancer. From these samples, we isolated tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells. These cells were used for immunophenotype using multicolor flow cytometry, gene expression profile using RNA sequencing and ex vivo functional restoration assays. We found that CD39+ CD8 TILs were enriched with tumor-specific CD8 TILs, and that the activation status of these cells was determined by the differential programmed cell death protein 1 (PD-1) expression level. CD39+ CD8 TILs with high PD-1 expression (PD-1high) exhibited features of highly tumor-reactive and terminally exhausted phenotypes. Notably, PD-1high CD39+ CD8 TILs showed similar characteristics in terms of T-cell exhaustion and activation between the primary and metastatic sites. Among co-stimulatory receptors, 4-1BB was exclusively overexpressed in CD39+ CD8 TILs, especially on PD-1high cells, and 4-1BB-expressing cells displayed immunophenotypes indicating higher degrees of T-cell activation and proliferation, and less exhaustion, compared with cells not expressing 4-1BB. Importantly, 4-1BB agonistic antibodies further enhanced the anti-PD-1-mediated reinvigoration of exhausted CD8 TILs from both primary and metastatic sites. Severely exhausted PD-1high CD39+ CD8 TILs displayed a distinctly heterogeneous exhaustion and activation status determined by differential 4-1BB expression levels, providing rationale and evidence for immunotherapies targeting co-stimulatory receptor 4-1BB in ovarian cancers.

Leem G ，Park J ，Jeon M ，Kim ES ，Kim SW ，Lee YJ ，Choi SJ ，Choi B ，Park S ，Ju YS ，Jung I ，Kim S ，Shin EC ，Lee JY ，Park SH ... -  《Journal for ImmunoTherapy of Cancer》

Immune Checkpoint Inhibitor-induced Reinvigoration of Tumor-infiltrating CD8(+) T Cells is Determined by Their Differentiation Status in Glioblastoma.

Immune checkpoint inhibitors (ICI) are used for the treatment of various cancers, but clinical trials of anti-programmed cell death protein 1 (PD-1) with patients with recurrent glioblastoma (GBM) have failed to show clinical benefits. In this study, we examined the differentiation status of CD8+ tumor-infiltrating lymphocytes (TIL) from patients with primary GBM and their reinvigoration by ICIs to understand the nature of T-cell exhaustion in GBM. We isolated TILs from 98 patients with newly diagnosed GBM 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-cytotoxic T-lymphocyte antigen 4 (CTLA-4) and their proliferation assessed. CD8+ TILs had significantly increased expression of immune checkpoint receptors, including PD-1 and CTLA-4, compared with peripheral blood CD8+ T cells. Among CD8+ TILs, PD-1+ cells exhibited more terminally differentiated phenotypes (i.e., EomeshiT-betlo) 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 EomeshiT-betlo 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 EomeshiT-betlo CD8+ TILs, who responded well to anti-PD-1 in ex vivo assays, but not in patients with high percentages of EomeshiT-betlo CD8+ TILs, who did not respond to anti-PD-1. In primary GBM, the differentiation status of CD8+ TILs determines their reinvigoration ability upon ICI treatment.

Park J ，Kwon M ，Kim KH ，Kim TS ，Hong SH ，Kim CG ，Kang SG ，Moon JH ，Kim EH ，Park SH ，Chang JH ，Shin EC ... -  《-》

Distinct exhaustion features of T lymphocytes shape the tumor-immune microenvironment with therapeutic implication in patients with non-small-cell lung cancer.

Reinvigoration of T-cell exhaustion with antibodies has shown promising efficacy in patients with non-small-cell lung cancer (NSCLC). However, the characteristics of T-cell exhaustion with regard to tumor-infiltrating lymphocytes (TILs) are poorly elucidated in NSCLC. Here, we investigated the exhaustion status of TILs in NSCLC patients at the intraindividual and interindividual levels. We obtained paired peripheral blood, normal adjacent tissues, peritumoral tissues, and tumor tissues from 96 NSCLC patients. Features of T-cell exhaustion were analyzed by flow cytometry. T cells were categorized according to their programmed cell death-1 (PD-1) expression (PD-1high, PD-1int, and PD-1neg cells). Patients were classified based on the presence or absence of discrete PD-1high CD8+ TILs. Production of effector cytokines by CD8+ TILs was measured after T-cell stimulation with or without antibodies against immune checkpoint receptors. Progressive T-cell exhaustion with marked expression of exhaustion-related markers and diminished production of effector cytokines was observed in PD-1high CD8+ TILs compared with PD-1int and PD-1neg CD8+ TILs. Patients with distinct PD-1high CD8+ TILs (PD-1high expressers) exhibited characteristics associated with a favorable anti-PD-1 response compared with those without these lymphocytes (non-PD-1high expressers). Combined inhibition of dual immune checkpoint receptors further restored effector cytokine production by CD8+ TILs following T-cell stimulation. PD-1high CD8+ T lymphocyte populations in the peripheral blood and tumors were significantly correlated. T-cell exhaustion was differentially regulated among individual patients and was prominent in a subgroup of NSCLC patients who may benefit from PD-1 blockade or combined blockade of other immune checkpoint receptors.

Kim CG ，Kim G ，Kim KH ，Park S ，Shin S ，Yeo D ，Shim HS ，Yoon HI ，Park SY ，Ha SJ ，Kim HR ... -  《Journal for ImmunoTherapy of Cancer》