25 Enrichment of tertiary lymphoid structures provides novel insight into mediators of anti-tumor immune activity in sarcomatoid renal cell carcinoma
Authors: Salgia Mr Nicholas , Aubrecht Mr Wilhelm , Attwood Kristopher , Chow Jacky , Eng Kevin , Wang Jianmin , Xu Bo , Long Mark , Kauffman Eric , Muhitch Jason 
Published: August 2024

Abstract

Background
Renal cell carcinoma (RCC) comprises various histological subtypes, with clear cell RCC (ccRCC) being the most prevalent histotype. RCC with sarcomatoid features (sRCC) is a unique kidney cancer subtype associated with aggressive biological features and poor clinical outcomes that can arise from multiple RCC histologies, most commonly ccRCC. While clinically aggressive, sRCC paradoxically has also demonstrated preferential responsiveness to immune checkpoint blockade (ICB) therapies in subgroup analyses of multiple phase III trials. However, the mediators of this immune sensitivity are largely unknown. We therefore applied transcriptomic techniques to identify orchestrators of immune activity within the sRCC tumor microenvironment (TME).

Methods
Nephrectomy specimens from patients with sRCC and ccRCC were procured for single cell RNA sequencing (scRNAseq). Clustering and dimensionality reduction were performed, and cell populations were annotated based on expression of canonical lineage markers. Immune populations (CD8+ T cells, CD4+ T cells, B/plasma cells, myeloid cells) were computationally extracted and differential gene expression between sRCC- and ccRCC-derived cells within each subpopulation was performed. Gene expression programs enriched in sRCC samples by scRNAseq were validated on publicly available bulk gene expression data comparing sRCC to ccRCC. Spatial transcriptomics were performed on sRCC tumor sections using the 10X Visium platform.

Results
Across 18 RCC specimens (10 sRCC; 8 ccRCC), 73,123 cells were analyzed by scRNAseq. Within the CD8+ T cell compartment, CXCL13 was the most significantly enriched nuclear-encoded gene in sRCC samples (log 2-fold change=1.29; q&lt;0.001). CXCL13 was also significantly enriched in CD4+ T cells from sRCC (q&lt;0.001), suggesting enhanced presence of follicular T cells within the sRCC TME.
As follicular T cells function in support of B cells, we next interrogated the B lymphocyte population. sRCC samples were enriched for mature B cell and plasma cells, with a five-fold increase in the relative abundance of plasma cells compared to ccRCC samples. Immune deconvolution of patient-derived RNA sequencing from the IMmotion 151 trial revealed a significant increase in the predicted proportion of B lymphocytes (including plasma cells) within the sRCC TME relative to ccRCC (p=0.034). Further, over 20 B lymphocyte activation and maturation pathways were consistently enriched (q&lt;0.25) in sRCC across clinical datasets (Javelin101, CheckMate, and TCGA KIPAN), including Signaling by the B Cell Receptor, Positive Regulation of B Cell Activation, and Immunoglobulin Production Involved in Immunoglobulin Mediated Immune Response, amongst others. B lymphocytes mediate anti-tumor immunity through antibody dependent cellular cytotoxicity (ADCC), and thus the phagocytic effectors of ADCC were interrogated. Myeloid populations differentially expressed FCγR3A in sRCC vs ccRCC (q&lt;0.001), which was recapitulated in bulk gene expression populations (q=0.002, 0.12, and 1.08x10-4 in Javelin101, CheckMate, and TCGA cohorts, respectively).
Given the enrichment of follicular T cell and differentiated B lymphocyte programs, we explored the presence of tertiary lymphoid structures (TLS) in sRCC. Two distinct TLS signatures – the 12 Chemokine Score and the TLS Imprint Signature – were significantly enriched in sRCC vs ccRCC across RCC patient datasets (Figure 1). Furthermore, spatial transcriptomics were applied to H&E slides of sRCC to successfully identify the presence of TLS by expression of TLS-associated genes adjacent to sarcomatoid regions.

Conclusions
TLS, which have previously been associated with response to ICB in RCC (Meylan et al. Immunity. 2022), are transcriptomically enriched for in sRCC, paralleling an observed increase in CXCL13-expressing T cells and differentiated B lymphocytes. Together, TLS and their constituents offer a previously unexplored mediator of immunosurveillance in the sRCC TME that may underlie the paradoxical responsiveness to ICB seen within this population clinically.