HLA-A+ tertiary lymphoid structures with reactivated tumor infiltrating lymphocytes are associated with a positive immunotherapy response in esophageal squamous cell carcinoma

Abstract

Background: Immune checkpoint blockade (ICB) therapy provides remarkable clinical benefits for multiple cancer types. However, the overall response rate to ICB therapy remains low in esophageal squamous cell carcinoma (ESCC). This study aimed to identify biomarkers of ICB therapy for ESCC and interrogate its potential clinical relevance.

Methods: We investigated gene expression in 42 treatment-naïve ESCC tumor tissues and identified differentially expressed genes, tumor-infiltrating lymphocytes and immune-related genes signatures associated with differential immunotherapy responses. We systematically assessed the tumor microenvironment using the NanoString GeoMx digital spatial profiler, single-cell RNA-seq and multiplex immunohistochemistry in ESCC. Finally, we evaluated the associations between HLA-A-positive tertiary lymphoid structures (TLSs) and patients' responses to ICB in 60 ESCC patients.

Results: Tumor infiltrating B lymphocytes and several immune-related gene signatures, such as the antigen presenting machinery (APM) signature, are significantly elevated in ICB treatment responders. Multiplex immunohistochemistry identified the presence of HLA-A⁺ TLSs and showed that TLS-resident cells increasingly express HLA-A as TLSs mature. Most TLS-resident HLA-A⁺ cells are tumor-infiltrating T (TIL-T) or tumor-infiltrating B (TIL-B) lymphocytes. Digital spatial profiling of spatially distinct TIL-T lymphocytes and single-cell RNA-seq data from 60 ESCC tumor tissues revealed that CXCL13-expressing exhausted TIL-Ts inside TLSs are reactivated with elevated expression of the APM signature as TLSs mature. Finally, we demonstrated that HLA-A⁺ TLSs and their major cellular components, TIL-Ts and TIL-Bs, are associated with a clinical benefit from ICB treatment for ESCC.

Conclusions: HLA-A⁺ TLSs are present in ESCC tumor tissues. TLS-resident TIL-Ts with elevated expression of the APM signature may be reactivated. HLA-A⁺ TLSs and their major cellular components, TIL-Ts and TIL-Bs, may serve as biomarkers for ICB-treated ESCC patients.