Despite being a standard treatment option in breast cancer, immune checkpoint inhibitors (ICIs) are only efficacious for a subset of patients. To gain a better understanding of the antitumor immune response in breast cancer, we examined the heterogeneity of CD8 + T cells in tumors, metastatic lymph nodes (mLNs), and peripheral blood from patients with early breast cancer ( n = 131). Among tissue-resident memory CD8 + T (T RM ) cells, including virus- and tumor-specific CD8 + T cells, CD39 expression was observed in a tumor-specific and exhausted subpopulation in both tumors and mLNs. CD39 + T RM cells from tumors and mLNs exhibited a phenotypic similarity and clonally overlapped with each other. Moreover, tumor or mLN CD39 + T RM cells clonally overlapped with CD39 − T RM and non-T RM cells in the same compartment, implying a tissue-specific differentiation process. These inter-subpopulationally overlapping CD39 + T RM clonotypes were frequently detected among effector memory CD8 + T cells in peripheral blood, suggesting a systemic clonal overlap. CD39 + T RM cell enrichment was heterogeneous among molecular subtypes of breast cancer, which is associated with the different role of antitumor immune responses in each subtype. In vitro blockade of PD-1 and/or CTLA-4 effectively restored proliferation of CD39 + T RM cells and enhanced cytokine production by CD8 + T cells from tumors or mLNs, particularly in the presence of CD39 + T RM enrichment. This suggests that CD39 + T RM cells have a capacity for functional restoration upon ICI treatment. Thus, our study indicates that CD39 + T RM cells with a clonal overlap across compartments are key players in antitumor immunity in breast cancer.
Tobacco etch virus (TEV) protease is a 27‐kDa catalytic domain of the polyprotein nuclear inclusion a (NIa) in TEV, which recognizes the specific amino acid sequence ENLYFQG/S and cleaves between Q and G/S. Despite its substrate specificity, its use is limited by its autoinactivation through self‐cleavage and poor solubility during purification. It was previously reported that T17S/N68D/I77V mutations improve the solubility and yield of TEV protease and S219 mutations provide protection against self‐cleavage. In this study, we isolated TEV proteases with S219N and S219V mutations in the background of T17S, N68D, and I77V without the inclusion body, and measured their enzyme kinetics. The kcat of two isolated S219N and S219V mutants in the background of T17S, N68D, and I77V mutations was highly increased compared to that of the control, and S219N was twofold faster than S219V without Km change. This result indicates that combination of these mutations can further enhance TEV activity.
Immune checkpoint inhibitors are effective for advanced hepatocellular carcinoma (HCC), but there remains a need for peripheral blood biomarkers to predict the clinical response. Here, we analyzed the peripheral blood of 45 patients with advanced HCC who underwent nivolumab. During treatment, frequency of classical monocytes (CD14 + CD16 − ) was increased on day 7, and the fold increase in the frequency on day 7 over day 0 (cMonocyte D7/D0 ) was significantly higher in patients with durable clinical benefit (DCB) than in patients with non-DCB (NDB). When we analyzed transcriptomes of classical monocytes, CD274, gene encoding PD-L1, was upregulated in NDB patients compared to DCB patients at day 7. Notably, gene signature of suppressive tumor-associated macrophages, or IL4l1 + PD-L1 + IDO1 + macrophages, was enriched after treatment in NDB patients, but not in DCB patients. Accordingly, the fold increase in the frequency of PD-L1 + classical monocytes at day 7 over day 0 (cMonocyte-PDL1 D7/D0 ) was higher in NDB patients than DCB patients. The combined biomarker cMonocyte D7/D0 /cMonocyte-PDL1 D7/D0 was termed the "monocyte index", which was significantly higher in DCB patients than NDB patients. Moreover, the monocyte index was an independent prognostic factor for survival. Overall, our results suggest that early changes of circulating classical monocytes, represented as a monocyte index, could predict clinical outcomes of advanced HCC patients undergoing anti-PD-1 therapy.
Purpose: Regulatory T cells (Tregs) exert immunosuppressive functions and hamper anti-tumor immune responses in the tumor microenvironment. Understanding the heterogeneity of intratumoral Tregs, and how it changes with tumor progression, will provide clues regarding novel target molecules of Treg-directed therapies. Experimental Design: From 42 patients with renal cell carcinoma and 5 patients with ovarian cancer, immune cells from tumor and peripheral blood were isolated. We performed multicolor flow cytometry and RNA-sequencing to characterize the phenotypes and heterogeneity of intratumoral Tregs. In vitro functional assays were performed to evaluate suppressive capacity of Tregs and effect of CEACAM1-mediated depletion. The CT26 tumor model was used to evaluate the association between intratumoral Tregs and tumor growth, and examine the in vivo role of CEACAM1+ intratumoral Tregs on anti-tumor immunity. Results: We found that carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) was selectively expressed on intratumoral Tregs, while its expression on peripheral Tregs or other immune cells was low. The CEACAM1+ intratumoral Tregs accumulated with tumor progression, while the CEACAM1– subset did not. Notably, we found that CEACAM1 marked intratumoral Tregs that exhibited highly suppressive and activated phenotypes with substantial clonal expansion. Depletion of CEACAM1-expressing cells from tumor-infiltrating leukocytes led to increased effector functions of tumor-infiltrating T cells. Moreover, CEACAM1+ cell depletion further enhanced anti-PD-1–mediated reinvigoration of exhausted CD8+ T cells. Conclusions: CEACAM1 marks highly suppressive subset of intratumoral Tregs, and can be a target for selective depletion of intratumoral Tregs. These results may inform future studies on CEACAM1-mediated depletion in cancer patients.
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