115 Spatial dissection of T cell clonotype identity, transcriptional profiles, and cell-cell interactions in the tumor microenvironment and tertiary lymphoid structures

<h3>Background</h3> Determining the spatial interactions of T cell clonotypes in tumor microenvironments and tertiary lymphoid structures (TLSs) is essential to understanding adaptive anti-tumoral immune responses, but existing spatial sequencing methods remain unable to profile the TCR repertoire at high resolution. <h3>Methods</h3> We previously described the development of Slide-TCR-seq, which integrates Slide-seqV2 (spatially-resolved RNA capture by a DNA-barcoded bead array with 10µm resolution) with rhTCRseq (highly sensitive targeted capture of TCR sequences)^2,3,4 to facilitate amplification of the TCR transcript from CDR3 to the 3’ end. This approach enables the simultaneous measurement of cellular transcriptomes, T cell clonotypes, and spatial location at 10µm resolution. We examined melanoma and renal cell carcinoma (RCC) tumors with Slide-TCR-seq to understand the spatial relationships between T cell clonotypes, tumor cells, and immune cells in the tumor microenvironment and TLSs. <h3>Results</h3> We applied Slide-TCR-seq to melanoma and RCC metastases because of the well-characterized roles played by T cell phenotype and TCR repertoire in their immune microenvironments.^5,6 By histology and Slide-TCR-seq, we identified TLSs in both melanoma and RCC metastases. We observed that T cells located within the tumor regions were more clonally expanded than those in the TLSs. Furthermore, T cells in TLSs tended to be CD4+ T cells, while those infiltrating into tumor tended to be CD8+ T cells with an exhausted phenotype (p&lt;0.05 by FDR-corrected t-test)—together suggesting unique immunological roles of TLSs in tumors. In the melanoma metastasis, one T cell clone (CDR3 sequence CASRASNEQFF) was preferentially enriched in one of the two tumor lobes that were examined (p=1×10–¹⁰²). Compared to other clonotypes, <i>GZMB</i> (p=1×10–⁸; associated with cytotoxic T cell function) and <i>STAT3</i> (p=7×10–⁷; associated with activated T cells’ survival) were prominently upregulated in CASRASNEQFF. CASRASNEQFF T cells additionally exhibited unique cell non-autonomous mechanisms: monocytes neighboring CASRASNEQFF T cells displayed elevated <i>CXCL10</i> chemokine expression (p=5×10–²¹), which can recruit tumor-reactive effector T cells.⁷ Notably, monocytic expression of <i>CXCL10</i> was higher in the same lobe that was enriched with CASRASNEQFF T cells, implicating a preferential interaction between the two. Melanoma cells neighboring the CASRASNEQFF T cells also displayed differential gene expression, including downregulated <i>MGST1</i> expression (p=3×10–¹⁵). <h3>Conclusions</h3> Slide-TCR-seq enables spatially-resolved transcriptomics and TCR clonotyping. Our findings suggest that TLS T cells’ phenotype and TCR repertoire are distinct from tumor-infiltrating T cells, and that the transcriptional profiles of T cells, monocytes, and tumor cells may depend on their spatial relationships to one another in the tumor microenvironment. <h3>References</h3> Liu S, Iorgulescu B, Li S, <i>et al</i>. 76 Spatial mapping of T cell receptors and transcriptomes in renal cell carcinoma following immune checkpoint inhibitor therapy. <i>Journal for ImmunoTherapy of Cancer</i> 2021;<b>9</b>:doi: 10.1136/jitc-2021-SITC2021.076 Stickels, R. R. <i>et al</i>. Highly sensitive spatial transcriptomics at near-cellular resolution with Slide-seqV2. <i>Nat. Biotechnol</i>. (2020) doi:10.1038/s41587-020-0739-1. Rodriques SG, Stickels RR, Goeva A, Martin CA, Murray E, Vanderburg CR, Welch J, Chen LM, Chen F, Macosko EZ. Slide-seq: A scalable technology for measuring genome-wide expression at high spatial resolution. <i>Science</i>. 2019 Mar 29;<b>363</b>(6434):1463–1467. doi: 10.1126/science.aaw1219. Epub 2019 Mar 28. PMID: 30923225; PMCID: PMC6927209. Li, S. <i>et al</i>. RNase H-dependent PCR-enabled T-cell receptor sequencing for highly specific and efficient targeted sequencing of T-cell receptor mRNA for single-cell and repertoire analysis. <i>Nat. Protoc</i>. <b>14</b>, 2571–2594 (2019). Braun, D. A. <i>et al</i>. Progressive immune dysfunction with advancing disease stage in renal cell carcinoma. <i>Cancer Cell</i> (2021) doi:10.1016/j.ccell.2021.02.013. Oliveira, G. <i>et al</i>. Phenotype, specificity and avidity of antitumour CD8+ T cells in melanoma. <i>Nature</i> (2021) doi:10.1038/s41586-021-03704-y. Spranger, S., Dai, D., Horton, B. &amp; Gajewski, T. F. Tumor-Residing Batf3 Dendritic Cells Are Required for Effector T Cell Trafficking and Adoptive T Cell Therapy. <i>Cancer Cell</i><b> 31</b>, 711–723.e4 (2017). <h3>Ethics Approval</h3> This study was approved by MGB/DFCI/Broad institution’s Ethics Board; approval number 2019P000017.