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. 2024 May 3;43(1):134.
doi: 10.1186/s13046-024-03046-3.

CXCR6-positive circulating mucosal-associated invariant T cells can identify patients with non-small cell lung cancer responding to anti-PD-1 immunotherapy

Jingjing Qu #  1   2 , Binggen Wu #  1   2 , Lijun Chen  3 , Zuoshi Wen  4 , Liangjie Fang  1   2 , Jing Zheng  1   2 , Qian Shen  1   2 , Jianfu Heng  5 , Jianya Zhou  6   7 , Jianying Zhou  1   2
Affiliations

CXCR6-positive circulating mucosal-associated invariant T cells can identify patients with non-small cell lung cancer responding to anti-PD-1 immunotherapy

Jingjing Qu et al. J Exp Clin Cancer Res. .

Abstract

Background: Mucosal-associated invariant T (MAIT) cells have been reported to regulate tumor immunity. However, the immune characteristics of MAIT cells in non-small cell lung cancer (NSCLC) and their correlation with the treatment efficacy of immune checkpoint inhibitors (ICIs) remain unclear.

Patients and methods: In this study, we performed single-cell RNA sequencing (scRNA-seq), flow cytometry, and multiplex immunofluorescence assays to determine the proportion and characteristics of CD8+MAIT cells in patients with metastatic NSCLC who did and did not respond to anti-PD-1 therapy. Survival analyses were employed to determine the effects of MAIT proportion and C-X-C chemokine receptor 6 (CXCR6) expression on the prognosis of patients with advanced NSCLC.

Results: The proportion of activated and proliferating CD8+MAIT cells were significantly higher in responders-derived peripheral blood mononuclear cells (PBMCs) and lung tissues before anti-PD-1 therapy, with enhanced expression of cytotoxicity-related genes including CCL4, KLRG1, PRF1, NCR3, NKG7, GZMB, and KLRK1. The responders' peripheral and tumor-infiltrating CD8+MAIT cells showed an upregulated CXCR6 expression. Similarly, CXCR6+CD8+MAIT cells from responders showed higher expression of cytotoxicity-related genes, such as CST7, GNLY, KLRG1, NKG7, and PRF1. Patients with ≥15.1% CD8+MAIT cells to CD8+T cells ratio and ≥35.9% CXCR6+CD8+MAIT cells to CD8+MAIT cells ratio in peripheral blood showed better progression-free survival (PFS) after immunotherapy. The role of CD8+MAIT cells in lung cancer immunotherapy was potentially mediated by classical/non-classical monocytes through the CXCL16-CXCR6 axis.

Conclusion: CD8+MAIT cells are a potential predictive biomarker for patients with NSCLC responding to anti-PD-1 therapy. The correlation between CD8+MAIT cells and immunotherapy sensitivity may be ascribed to high CXCR6 expression.

Keywords: CXCR6; Circulating mucosal-associated invariant T cells; Immunotherapy; Non-small cell lung cancer; Single-cell RNA-sequencing.

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Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figures

Fig. 1
Fig. 1
A higher proportion of T cells in responders. (A) The flowchart of scRNA-seq (peripheral blood samples of NSCLC patients were collected from 3 responders and 3 non-responders at baseline). (B) UMAP analysis plot showing cell clusters (left), classification according to anti-PD-1 therapy response (middle) and the derivation of samples (right). (C) The expression of canonical marker genes in different cell subtypes. (D) The proportion of cell subtypes in responders and non-responders. (E) Differentially expressed genes of different cell subtypes R: Responders; NR: Non-Responders. GMPs: Granulocyte-monocyte progenitor cells; NK: Natural killer cells; MPs: Mononuclear phagocytes; pDCs: Plasmacytoid dendritic cells
Fig. 2
Fig. 2
CD8+MAIT cells showed a significantly increased proportion in responders. (A) Differentially expressed genes between responders and non-responders-derived T cells. (B) Scenic analysis for responders and non-responders-derived T cells. (C) UMAP analysis plot showing T-cell subtypes. (D) The proportion of T-cell subtypes in responders and non-responders. (E) Differentially expressed genes between responders and non-responders in CD8+MAIT cells. (F) Scenic analysis for responders and non-responders-derived CD8+MAIT cells. (G) Flow cytometric analysis of responders and non-responders-derived PBMCs by labeling CD3, CD8, CD161, TCR α7.2(18 responders vs. 12 non-responders). (H) The percentage of CD8+MAIT cells in responders and non-responders-derived CD8+T cells from cytometric analysis (18.7% VS 10.0%, P = 0.0057). (I) Tumor tissues stained by immunofluorescence for CD8, TCR Vα7.2 in responders and non-responders. (J) Percentage of CD8+MAIT cells in CD8+T cells in responders and non-responders (30.1% VS 15.7%, P = 0.0347)
Fig. 3
Fig. 3
Responders in CD8+MAIT cells had higher expression of cytotoxicity-related genes. (A) Cytotoxicity scoring for responders and non-responders-derived CD8+MAIT cells. (B) Differentially expressed cytotoxic genes between responders and non-responders-derived CD8+MAIT cells. (C) The incoming and outgoing cell-cell interaction strength for responders and non-responders-derived cell subtypes. (D) The outgoing interaction pattern for responders and non-responders-derived CD8+MAIT.
Fig. 4
Fig. 4
CD8+MAIT in responders cohort with higher CXCR6 expression. (A) Pseudo-time analysis for CXCR6 expression in different T-cell subtypes. (B) UMAP analysis for CXCR6 expression in responders and non-responders-derived CD8+MAIT cells. (C) Violin plots for CXCR6 expression in responders and non-responders-derived different T-cell subtypes. (D) Flow cytometric analysis of responders and non-responders-derived PBMCs by labeling CD3, CD8, CD161, TCR α7.2 and CD186 (18 responders vs. 12 non-responders). (E) The percentage of CD186+ (CXCR6+) CD8+MAIT cells in CD8+MAIT cells in responders and non-responders from cytometric analysis(P = 0.041). (F-G) Tumor tissues stained by immunofluorescence for CD8, TCR Vα7.2, CXCR6 in responders and non-responders and the summary data (47.44% VS 26.72%, P = 0.0274).
Fig. 5
Fig. 5
CXCR6 in responders was associated with cytotoxicity of CD8+MAIT cells. (A) The components of CXCR6-high and CXCR6-low CD8+MAIT cells. (B) Differentially expressed genes between CXCR6-high and CXCR6-low CD8+MAIT cells. (C) GO analysis for upregulated (left) and downregulated (right) pathways in CXCR6-high CD8+MAIT cells. (D) Cytotoxicity scoring for CXCR6-high and CXCR6-low CD8+MAIT cells. (E) Differentially expressed cytotoxic genes between CXCR6-high and CXCR6-low CD8+MAIT cells (P <0.05)
Fig. 6
Fig. 6
Levels of CD8+MAIT and CXCR6+CD8+MAIT cells before anti-PD-1 therapy identify responder patients. (A) CD8+MAIT cells/CD8+T cells ratio ≥15.1% had a better PFS than those patients with CD8+MAIT cells/CD8+T cells ratio <15.1% (NR vs. 5.3 months, P = 0.0245). (B) CXCR6+CD8+MAIT cells/CD8+MAIT cells ratio ≥35.9% had a better PFS than those patients with CXCR6 + CD8+MAIT cells/CD8+MAIT cells ratio <35.9% (NR vs. 5.5 months, P = 0.0008). (C-D) Kaplan–Meier survival analysis of the OS in patients with CD8+MAIT cells/CD8+T cells ratio ≥15.1% and < 15.1% (C), CXCR6 + CD8+MAIT cells/CD8+MAIT cells ratio ≥35.9% and < 35.9% (D)
Fig. 7
Fig. 7
The role of CD8+MAIT cells in lung cancer immunotherapy may be achieved by CXCR6-CXCL16 axis. (A) The contributions of main ligand-receptor pairs in cell-cell interaction of responders and non-responders-derived CD8+MAIT cells. (B) Ligand-receptor pairs participated in cell-cell interaction of CD8+MAIT cells. (C) Ligand-receptor pairs participated in cell-cell interaction of responders and non-responders-derived CD8+MAIT cells
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