Background
While immune checkpoint inhibitors (ICI) have shifted the paradigm of cancer treatment, the mechanisms that drive tumor rejection remain elusive. Neoadjuvant ICI therapies provide a unique window of opportunity to study temporal changes of immune responses to ICI at the tumor site.1 We interrogated transcriptional and TCR clonal dynamics of head and neck squamous cell carcinoma (HNSCC) patients who received neoadjuvant nivolumab (anti-PD1) ± ipilimumab (anti-CTLA4) or relatlimab (anti-LAG3). The aim was to elucidate temporal changes of T cells associated with favorable clinical response.
Methods
Fresh pre- and post-treatment tumor and peripheral blood specimens from HNSCC patients (n=41) were processed by 10x Genomics 5’ single-cell RNA-seq, TCR-seq, and cellular indexing of transcriptomes and epitopes (CITE)-seq. Of 41 patients, 35 have so far completed sequencing and were analyzed using Seurat (v4.0.0). Canonical T cell marker genes, GSEA and RNA velocity were used to define cellular states, enriched signaling pathways, and estimate transcriptional dynamics. FFPE tissue sections were used to perform multispectral imaging to evaluate T cell composition changes in tumor. Pathologic response was scored using standard criteria.2
Results
A type I interferon (IFN-I) response gene signature was strongly enriched in CD8+ TIL from responders at baseline. Interestingly, this IFN-I response signature was lost solely in the CD8+ TIL from patients that responded to nivolumab+relatlimab therapy. Responder CD8+ TIL co-expressed tumor-reactive markers CD39/ENTPD1 and CD103/ITGAE at baseline and underwent clonal expansion post therapy. Clonal TCR tracing of expanded CD8+CD39high TIL from patients treated with nivolumab+relatlimab therapy displayed decreased gene sets associated with T cell exhaustion, including decreased LAG3 and CD39 expression, and increased effector gene programs post therapy. Novel TCR clonotypes identified from post-treatment, responder tumors had smaller clone and population size. LAG3 mRNA expression by CD8+ TIL at baseline correlated with pathological response only in patients treated with nivolumab+relatlimab (n= 11, R=0.59, p=0.058), similar to LAG3 immunohistochemistry. Multispectral imaging showed that intratumoral CD8+ T cell density was significantly increased after treatment in major responders (n=7, p=0.019).
Conclusions
Transcriptional reprogramming of tumor-reactive CD8+ TIL from a hyper IFN-I responsive cell state to effector cell phenotype, potentially driven by LAG3 signaling inhibition, is a novel mechanism that may be required for tumor rejection in the context of neoadjuvant nivolumab+relatlimab therapy. Novel TCR clonotypes from post-treatment, responder tumors only occupied minor fraction of CD8 TIL population, which suggests that reprogramming and maintenance of resident tumor-reactive cells present at baseline is a major driver of anti-tumor immunity.
Acknowledgements
This research was funded by BMS CA223049 (RLF), P50 CA097190 (RLF), R01 CA206517 (RLF), and R01 DE031947 (RLF, LV). This research was supported in part by the University of Pittsburgh Center for Research Computing through the resources provided by using the HTC cluster, supported by NIH award number S10OD028483. This research utilized the UPMC Hillman Cancer Center Flow Cytometry Core Facility, supported in part by award P30 CA047904 (RLF).
Trial Registration
NCT04080804
References
Tarhini, A.A., et al. Neoadjuvant immunotherapy of locoregionally advanced solid tumors. J Immunother Cancer, 2022; 10(8).
Cottrell, T.R., et al. Pathologic features of response to neoadjuvant anti-PD-1 in resected non-small-cell lung carcinoma: a proposal for quantitative immune-related pathologic response criteria (irPRC). Ann Oncol, 2018; 29(8): 1853–1860.
Ethics Approval
This study was approved by University of Pittsburgh’s Institutional Review Board; approval number HCC 18–139/CA224–056