Background 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.

Methods 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.

Results 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<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, GZMB (p=1×10–⁸; associated with cytotoxic T cell function) and STAT3 (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 CXCL10 chemokine expression (p=5×10–²¹), which can recruit tumor-reactive effector T cells.⁷ Notably, monocytic expression of CXCL10 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 MGST1 expression (p=3×10–¹⁵).

Conclusions 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.

References

1. Liu S, Iorgulescu B, Li S, et al. 76 Spatial mapping of T cell receptors and transcriptomes in renal cell carcinoma following immune checkpoint inhibitor therapy. Journal for ImmunoTherapy of Cancer 2021;9:doi: 10.1136/jitc-2021-SITC2021.076

2. Stickels, R. R. et al. Highly sensitive spatial transcriptomics at near-cellular resolution with Slide-seqV2. Nat. Biotechnol. (2020) doi:10.1038/s41587-020-0739-1.

3. 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. Science. 2019 Mar 29;363(6434):1463–1467. doi: 10.1126/science.aaw1219. Epub 2019 Mar 28. PMID: 30923225; PMCID: PMC6927209.

4. Li, S. et al. 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. Nat. Protoc. 14, 2571–2594 (2019).

5. Braun, D. A. et al. Progressive immune dysfunction with advancing disease stage in renal cell carcinoma. Cancer Cell (2021) doi:10.1016/j.ccell.2021.02.013.

6. Oliveira, G. et al. Phenotype, specificity and avidity of antitumour CD8+ T cells in melanoma. Nature (2021) doi:10.1038/s41586-021-03704-y.

7. Spranger, S., Dai, D., Horton, B. & Gajewski, T. F. Tumor-Residing Batf3 Dendritic Cells Are Required for Effector T Cell Trafficking and Adoptive T Cell Therapy. Cancer Cell 31, 711–723.e4 (2017).

Ethics Approval This study was approved by MGB/DFCI/Broad institution’s Ethics Board; approval number 2019P000017.