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PD‐1+ TIM‐3+ T cells in malignant ascites predict prognosis of gastrointestinal cancer

Abstract The liquid biopsy of ascites fluid could be an excellent source of tumor and microenvironment for the study of prognostic biomarkers because of its accessibility. Tumor‐infiltrating lymphocytes (TILs) can predict prognosis in multiple malignancies, including the response to immune checkpoint inhibitors, a breakthrough cancer therapy. However, TILs’ profiles from malignant ascites have not been extensively studied. Using flow cytometric analysis, we quantified the proportion of exhausted T cells and memory/naive/effector T‐cell subsets, among the CD4+ and CD8+ T‐cell populations of paired TILs and peripheral blood T cell samples (n = 22). The correlation between CD4+ and CD8+ subset profiles suggested that the combined analysis of CD4+ and CD8+ cells in malignant ascites was clinically significant. We found that cells positive for the exhaustion markers programmed cell death‐1 (PD‐1), and T‐cell immunoglobulin and mucin domain 3 (TIM‐3), and cells coexpressing PD‐1 and TIM‐3 abundantly exist among malignant ascites TILs. Furthermore, patients with high frequency of PD‐1+ TIM‐3+ cells among the CD4+ and CD8+ T‐cell population showed worse clinical outcome in multivariate analysis (n = 27). We propose that exhausted ascites TILs represent a clinically significant prognostic biomarker in advanced gastrointestinal cancer and represent an important target for immune checkpoint inhibitors.

abundant T cells, natural killer (NK) cells, B cells, macrophages, and neutrophils. Each of these cells produces various cytokines that regulate the phenotype and function of components, leading to formation of tumor microenvironment in malignant ascites. 1 However, the characteristics of tumor-infiltrating lymphocytes (TILs) within ascites, which should be the potential sampling resource for translational research because of its easy access, have not been fully understood. Accumulating evidence has shown the clinical significance of TILs of the primary tissues as a therapeutic target and biomarker. In this study, we evaluated the character of ascites TILs, to elucidate the cells that constitute ascites TILs and to clarify their function. Ascites represent an alternative resource for TILs of the primary tissues and distinct from lymphocytes in peripheral blood (PB).

Isolation of mononuclear cells from peripheral blood and ascites TILs
PB mononuclear cells were obtained by standard gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). Ascites TILs obtained from patients were immediately centrifuged for 5 minutes at 1500 rpm. Cell pellets were then washed and re-suspended in 20 mL of PBS. The cell suspension was then layered on 5 mL of Ficoll-Paque PLUS, before gradient centrifugation as performed for PB.

Statistical analysis
Statistical analysis was carried out with JMP software (version 11.0, SAS institute). Student's t test was performed to compare two groups. Correlation analysis was calculated using the Spearman's statistic. Cox proportional hazards model for univariate and multivariate analysis was performed to calculate adjusted hazard ratios (HR) and their 95% confidence intervals (CI). The cut-off value was determined by the median of the variables. Variables with a P value of less than 0.05 in univariate analysis were tested in the multivariate analysis. The Kaplan-Meier method, with logrank test, was used to evaluate overall survival. Statistically significant differences are indicated by asterisks (*P < .05, **P < .01, ***P < .001).
More detailed version of methods are included in Data S1.
To study the relationship between expression of the inhibitory molecules and memory status within ascites TILs, we quantified the frequencies of memory/naive/effector T cells within each fraction  (Table 1). In the multivariate analysis, PD-1+ TIM-3+ cells in both CD4 and CD8 T cells (P = 0.0325; HR, 0.3255; 95% CI, 0.1111-0.9105) were shown to be the only significant prognostic variable (Table 1)  T-cell exhaustion has been intensively discussed in regard to CD8+ T cells, whereas the role of CD4+ exhausted T cells in the tumor microenvironment has not been fully evaluated. 13 There are differences between exhausted CD4+ and CD8+ T cells regarding cytokine production and transcriptional networks; 5 however, both play an important role in tumor elimination, and they interact with each other.
Our data showing a correlation between CD4+ and CD8+ T cells in regard to the frequency of exhaustion marker and memory/naive/effector subsets, strongly suggests that there is a common phenotypic signature between CD4+ and CD8+ cells. Quantitative analysis of TILs by FACS enabled a detailed evaluation of each cell fraction and provided an opportunity for novel findings that might have been otherwise undetectable using conventional immunohistochemistry analysis.
The observed relationship between exhausted T cells expressing PD-1 and TIM-3 among CD4+ helper and CD8+ cytotoxic T cells suggests that CD4+ helper T-cell exhaustion is biologically significant.
Taken together, through immune phenotyping analyses of ascites TILs, we have shown that a large proportion of CD4+ and CD8+ T cells show an exhausted phenotype within gastrointestinal malignant ascites, and that this may therefore be both a therapeutic target and prognostic biomarker for the disease.

ACKNOWLEDG MENTS
This study was supported in part by Japan Society for the Promotion of Science, and by Kyushu Medical Oncology Group (KMOG), a nonprofit organization.

DISCLOSURE
The authors have no conflict of interest.