Divergent outcomes of anti-PD-L1 treatment coupled with host-intrinsic differences in TCR repertoire and distinct T cell activation states in responding versus non-responding tumors.

Differential responses to immune checkpoint inhibitors (ICI) may be attributed to tumor-intrinsic factors or environmental cues; however, these mechanisms cannot fully explain the variable ICI responses in different individuals. Here, we investigate the potential contribution of immunological heterogeneity with a focus on differences in T-cell receptor (TCR) repertoire to ICI responses, which has not been defined previously. To reveal additional factors underlying heterogeneous responses to ICI, we employed a squamous cell carcinoma (SCC) mouse model in which tumor-bearing recipients unambiguously diverged into responders (R) or non-responders (NR) upon anti-PD-L1 treatment. Treatment efficacy absolutely required CD8 T-cells and correlated positively with effector functions of CD8 tumor-infiltrating lymphocytes (TILs). We showed that TCR repertoires exhibited a similar magnitude of clonal expansion in R vs. NR CD8 TILs. However, the top expanded TCR clonotypes appeared to be mutually exclusive between R and NR CD8 TILs, which also occurred in a recipient-specific manner, demonstrating preferential expansion of distinct TCR clonotypes against the same SCC tumor. Unexpectedly, R vs. NR CD8 TILs reached all activation clusters and did not exhibit substantial global differences in transcriptomes. By linking single-cell transcriptomic data with unique TCR clonotypes, CD8 TILs harboring top TCR clonotypes were found to occupy distinct activation clusters and upregulate genes favoring anti-tumor immunity to different extents in R vs. NR. We conclude that stochastic differences in CD8 TIL TCR repertoire and distinct activation states of top TCR clonotypes may contribute to differential anti-PD-L1 responses. Our study suggests that host-intrinsic immunological heterogeneity may offer a new explanation for differential ICI responses in different individuals, which could impact on strategies for personalized cancer immunotherapy.

Differential responses to immune checkpoint inhibitor dictated by pre-existing differential immune profiles in squamous cell carcinomas caused by same initial oncogenic drivers.

BACKGROUND: While immune checkpoint inhibitors (ICI) were approved for head and neck squamous cell carcinomas (HNSCCs), the response rate remains relatively low. Mechanisms underlying ICI unresponsiveness versus sensitivity are not fully understood. METHOD: To better delineate differential responses to ICI treatment, we employed mouse SCC models, termed KPPA tumors that were caused by deleting p53 and hyperactivating PIK3CA, two most frequently mutated genes in human HNSCCs. We transplanted two KPPA tumor lines (TAb2 versus TCh3) into C57BL/6 recipients and examined the immune tumor microenvironment using flow cytometry. Furthermore, we employed single-cell RNA sequencing to identify the difference in tumor infiltrating lymphocytes (TILs). RESULTS: We found that different KPPA tumors exhibited heterogeneous immune profiles pre-existing treatment that dictated their sensitivity or unresponsiveness to anti-PD-L1. Unresponsive TAb2 tumors were highly enriched with functional tumor-associated macrophages (TAMs), especially M2-TAMs. In contrast, sensitive TCh3 tumors contained more CD8 TILs with better effector functions. TAb2 tumor cells drastically expanded F4/80+ TAMs from bone marrow precursors, requiring CSF1 and VEGF. Consistently, a higher combined expression of VEGF-C and CSF1 predicts worse survival in PIK3CAAmp/TP53Mutated HNSCC patients. Unresponsive TAb2 tumors upregulated distinct signaling pathways that correlate with aggressive tumor phenotypes. While anti-PD-L1 did not affect the TME of TAb2 tumors, it significantly increased the number of CD8 TILs in TCh3 tumors. CONCLUSIONS: We uncovered tumor-intrinsic differences that may underlie the differential responses to ICI by establishing and employing two SCC tumor lines, TAb2 vs. TCh3, both of which harbor TP53 deletion and PIK3CA hyperactivation. Our study indicates the limitation of stratifying cancers according to their genetic alterations and suggests that evaluating HNSCC tumor-intrinsic cues along with immune profiles in the TME may help better predict ICI responses. Our experimental models may provide a platform for pinpointing tumor-intrinsic differences underlying an immunosuppressive TME in HNSCCs and for testing combined immunotherapies targeting either tumor-specific or TAM-specific players to improve ICI efficacy.