878 The immunomodulatory impact of chemotherapy when combined with PD-1 blockade in resectable lung cancers

Background Neoadjuvant PD-1 blockade combined with chemotherapy(ICB+chemo) is now standard of care for the treatment of resectable non-small cell lung cancer(NSCLC).1–4 However, the mechanisms underpinning its therapeutic efficacy are largely unexplored, namely if chemotherapy has an immunomodulatory role when combined with checkpoint blockade agents(ICB).4–6 We and others have shown that CXCL13 is upregulated on tumor-reactive TIL. We use CXCL13 as a surrogate for tumor specificity to better understand the immunomodulatory impact of chemotherapy when combined with ICB. Methods TIL from resected lung tumors treated with neoadjuvant PD-1 blockade alone(ICB, n=19) or combined with chemotherapy(ICB+chemo, n=18; NCT02259621) underwent single-cell TCRseq/RNAseq using 10X Genomics’ Single Cell 5’ V(D)J and 5’ DGE kit. Major pathological response(MPR) was ≤10% viable tumor at surgery for neoadjuvant-treated NSCLC patients. After QC, 1.78e6 cells were integrated, a CD8A cutoff was used. Seurat was used to normalize, identify variable features, scaled data, and integrated samples. SAVER imputed dropouts by borrowing information across genes and cells. CXCL13 marked tumor-reactive TIL, imputed expression values >1 were defined as CXCL13+. Spatial transcriptomics using 10X Genomics’ Visium CytAssist Spatial Gene Expression kit was performed on FFPE tissue. Results Transcriptomic profiles for 693,603 CD8+TIL clustered into 13 subsets. Cluster proportions did not differ between ICB alone and ICB+chemo cohorts, nor did pseudobulk gene expression. The percent of CXCL13+ in CD8+ TIL did not significantly differ; however, the magnitude of its expression was higher in ICB+chemo TIL(p<2.2e-16). CXCL13 was used to select CD8+ TIL with potential tumor specificity. GZMK was notably upregulated in CXCL13+ TIL from the ICB+chemo-treated cohort. TCF7, IL7R, and ETS1, which maintains IL7R expression, were also enriched in ICB+chemo-treated CXCL13+ TIL. In contrast, ITGAE(CD103) and ENTPD1(CD39), indicative of prolonged exposure to/residence in the tumor microenvironment, were enriched in CXCL13+ TIL from ICB alone (p=8.7e-06 and 8.6e-05, respectively). Spatial transcriptomics on a region of regression in a ICB+chemo-treated tumor revealed co-localization of CXCL13+ CD8 TIL with CXCR5+ B cells. Interestingly, GZMK expression was pronounced in the regression area, while GZMB, a canonical marker of effector T cell function, was restricted to an adjacent tumor nest without evidence of regression. Conclusions Our findings suggest that CXCL13 may not only mark tumor-reactive TIL, but also plays a role in their anti-tumor function, promoting tumor regression, specifically when chemotherapy is combined with PD-1 blockade. This may operate through increased B cell recruitment. Further validation in ex vivo/in vivo models is needed to elucidate the mechanistic underpinnings of ICB+chemo treatment Trial Registration For the ICB treated NSCLC patients enrolled under NA_00092076 at JHU (NCT02259621), samples have already been collected, stored, and published. References Hellmann MD, et al. Pathological response after neoadjuvant chemotherapy in resectable non-small-cell lung cancers: proposal for the use of major pathological response as a surrogate endpoint. Lancet Oncol 2014;15(1):e42–50. Pataer A, et al. Histopathologic response criteria predict survival of patients with resected lung cancer after neoadjuvant chemotherapy. J Thorac Oncol 2012;7(5):825–32. Forde PM, et al. Neoadjuvant PD-1 blockade in resectable lung cancer. N Engl J Med 2018;378(21):1976–1986. Forde PM, et al. Neoadjuvant nivolumab plus chemotherapy in resectable lung cancer. N Engl J Med 2022;386(21):1973–1985. Caushi JX, et al. Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers. Nature 2021;596(7870):126–132. Danilova L, et al. The mutation-associated neoantigen functional expansion of specific T cells (MANAFEST) assay: a sensitive platform for monitoring antitumor immunity. Cancer Immunol Res 2018;6(8):888–899. Ethics Approval This study was approved by the Institutional Review Boards (IRB) at Johns Hopkins University (JHU), approval number NA_00092076. Consent Written informed consent was obtained from all patients included in this study. A copy of the written consent is available for review by the Editor of this journal.

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