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

<h3>Background</h3> 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. <h3>Methods</h3> 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 <i>CD8A</i> 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 &gt;1 were defined as CXCL13+. Spatial transcriptomics using 10X Genomics’ Visium CytAssist Spatial Gene Expression kit was performed on FFPE tissue. <h3>Results</h3> 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<i> CXCL13</i>+ in CD8+ TIL did not significantly differ; however, the magnitude of its expression was higher in ICB+chemo TIL(p&lt;2.2e-16). <i>CXCL13</i> was used to select CD8+ TIL with potential tumor specificity. <i>GZMK</i> 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, <i>ITGAE</i>(CD103) and <i>ENTPD1</i>(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 <i>CXCL13</i>+ CD8 TIL with <i>CXCR5</i>+ 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. <h3>Conclusions</h3> 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 <h3>Trial Registration</h3> For the ICB treated NSCLC patients enrolled under NA_00092076 at JHU (NCT02259621), samples have already been collected, stored, and published. <h3>References</h3> Hellmann MD, <i>et al</i>. Pathological response after neoadjuvant chemotherapy in resectable non-small-cell lung cancers: proposal for the use of major pathological response as a surrogate endpoint. <i>Lancet Oncol</i> 2014;<b>15</b>(1):e42–50. Pataer A, <i>et al</i>. Histopathologic response criteria predict survival of patients with resected lung cancer after neoadjuvant chemotherapy. <i>J Thorac Oncol</i> 2012;<b>7</b>(5):825–32. Forde PM, <i>et al</i>. Neoadjuvant PD-1 blockade in resectable lung cancer. <i>N Engl J Med</i> 2018;<b>378</b>(21):1976–1986. Forde PM, <i>et al</i>. Neoadjuvant nivolumab plus chemotherapy in resectable lung cancer. <i>N Engl J Med</i> 2022;<b>386</b>(21):1973–1985. Caushi JX, <i>et al</i>. Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers. <i>Nature</i> 2021;<b>596</b>(7870):126–132. Danilova L, <i>et al</i>. The mutation-associated neoantigen functional expansion of specific T cells (MANAFEST) assay: a sensitive platform for monitoring antitumor immunity.<i> Cancer Immunol Res</i> 2018;<b>6</b>(8):888–899. <h3>Ethics Approval</h3> This study was approved by the Institutional Review Boards (IRB) at Johns Hopkins University (JHU), approval number NA_00092076. <b>Consent</b> 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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