Abstract LB064: B cells synergize with T cell regulation at immune hotspots in lung squamous cell carcinoma
Authors: Zhang Hanyun , AbdulJabbar Khalid , Moore David A. , Akarca Ayse , Enfield Katey , Jamal-Hanjani Mariam , Raza Shan E Ahmed , Veeriah Selvaraju , Biswas Dhruva , Salgado Roberto , McGranahan Nicholas ,   TRACERx Consortium, Quesne John Le , Swanton Charles , Marafioti Teresa , Yuan Yinyin 
Published: June 2022

Abstract
The interplay of immune cell subpopulations underpinning heterogeneous immune infiltration is poorly understood, hindering the establishment of robust prognostic markers and therapeutic targets.
To study the prognostic implications and constitution of immune cell aggregates in lung squamous cell carcinoma, we integrated transcriptomic data and deep-learning-based spatial analysis. With the automatically identified tumor and immune cells on hematoxylin and eosin (H&E) diagnostic whole slide images, we were able to map regions with lymphoid aggregates (immune hotspots - IHs) and tumor aggregates (cancer hotspots) using the Getis-Ord hotspot analysis (Getis and Ord, 1992; Nawaz et al., 2015), in 462 TCGA samples. IHs were further categorized into intratumoral (intra-IH) and peritumoral (peri-IH) based on whether they overlapped with cancer hotspots. The intra-IH score, quantifying the fraction of IHs at the cancer interface, was associated with poor overall survival independently of age, stage, and packs per year (p &lt; 0.01, Hazard Ratio (HR) = 2.1 [1.3-3.2]). The increased score was coupled with upregulated transcriptional signals of B cells and T regulatory cells, and expression of B cell-related genes CXCR5, MS4A1 (CD20), CD79b.
To investigate cellular compositions of IHs, we selected 10 TRACERX patients with high intra-IH score based on H&E and performed immunohistochemistry on two serial sections followed by a deep learning model to locate subpopulations of T cells (CD4+FOXP3-, CD4+FOXP3+, CD8+) and B cells (CD20+CXCR5-, CD20+CXCR5+, and CD79b+). By spatially aligning these IHC images with H&E, we observed higher abundances of CD20+CXCR5+ and CD79b+ B cells at peri-IHs than intra-IHs (p&lt;0.01; p&lt;0.05), whereas none of the T cell subtypes showed a difference in localization. Tertiary lymphoid structures (TLSs) accounted for a minor proportion of peri-IHs (5.04 +- 4.38%) and intra-IHs (0.43 +- 0.61%). Furthermore, percentages of CD20+CXCR5+ B cells were significantly higher at peri-IHs than intra-IHs when TLSs were excluded (p=0.007), suggesting that the enrichment of CD20+CXCR5+ B cells at peri-IHs was not fully explained by the presence of TLSs.
To test if IHs have a role in the regulation of anti-tumor activity of T cytotoxic cells, we further measured the ratio of CD8+ to CD4+FOXP3+ T cells infiltrating into the tumor nest. The ratio at tumor regions adjacent to intra-IH was lower than the rest of the tumor nest (p&lt;0.001), suggesting a suppressive effect of intra-IH on tumor-infiltrating CD8+ T cells.
Taken together, our results signified a protumor role of intratumoral lymphoid aggregates beyond TLSs, which may be attributed to the spatial interplay between CD8+ T cells, Tregs and B cells. Immune hotspots can serve as a prognostic indicator and potential therapeutic target in lung squamous cell carcinoma.
Citation Format: Hanyun Zhang, Khalid AbdulJabbar, David A. Moore, Ayse Akarca, Katey Enfield, Mariam Jamal-Hanjani, Shan E Ahmed Raza, Selvaraju Veeriah, Dhruva Biswas, Roberto Salgado, Nicholas McGranahan, TRACERx Consortium, John Le Quesne, Charles Swanton, Teresa Marafioti, Yinyin Yuan. B cells synergize with T cell regulation at immune hotspots in lung squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB064.