Mutual Exclusivity and Co-occurrence Patterns of Immune Checkpoints Indicate NKG2A Relates to Anti-PD-1 Resistance in Gastric Cancer

Background: An increasing number of clinical studies have begun to explore combination strategies with immune checkpoint inhibitors, aiming to present new opportunities for overcoming anti-PD-1 treatment resistance in gastric cancer. Unfortunately, the exploration of certain immune checkpoint inhibitor combination strategies has yielded suboptimal results. Therefore, it is necessary to comprehensively analyze the expression patterns of immune checkpoints and identify optimal combination regimens of anti-PD-1 inhibitors with other immune checkpoint inhibitors.

Methods: Leveraging single-cell RNA sequencing (scRNA-seq) and multivariate linear regression interaction models, we dissected the immune checkpoint expression characteristics of CD8 T cells in gastric cancer and the immune checkpoint expression pattern (ICEP) mediating anti-PD-1 treatment resistance. Furthermore, we employed transcription factor analysis and CellOracle to explore the transcriptional regulatory mechanisms governing CD8 T cell differentiation fates. Finally, we utilized Nichenet and spatial transcriptomic analysis to investigate the spatial expression patterns of immune checkpoints.

Results: Interaction analysis indicated that, among the known immune checkpoints, co-expression of NKG2A and PD-1 might exert a more profound inhibitory effect on the proliferative capacity of CD8 T cells. The co-expression analysis revealed differential co-expression pattern of PD-1 and NKG2A, defined as ICEP1 (CD8 T cells co-expressing PD-1, CTLA-4, TIGIT, LAG-3 or CD38) and ICEP2 (CD8 T cells solely expressing NKG2A or co-expressing with other immune checkpoints), reflecting the co-occurrence pattern of PD-1 and the mutual exclusivity of NKG2A. Further, these two ICEP CD8 T cell subsets represented distinct CD8 T cell differentiation fates governed by MSC and RUNX3. Notably, ICEP2 CD8 T cells were associated with anti-PD-1 therapy resistance in gastric cancer. This phenomenon may be attributed to the recruitment of LGMN macrophages mediated by the CXCL16-CXCR6 signaling pathway.

Conclusion: This study unveiled two distinct ICEPs and the mutually exclusivity and co-occurrence characteristics of CD8 T cells in gastric cancer. The ICEP2 CD8 T cell subset, highly expressed in gastric cancer patients resistant to anti-PD-1 therapy, may be recruited by LGMN macrophages through CXCL16-CXCR6 axis. These findings provide evidence for NKG2A as a novel immunotherapeutic target in gastric cancer and offer new insights into combination strategies for immune checkpoint inhibitors in gastric cancer.