Transcriptional profiling of tumor infiltrating and ciculating T lymphocytes of mice harboring MOC1 tumors

Combination TGF-ß neutralization and PD-L1 immune checkpoint blockade promotes anti-tumoral systemic immunity by enhancing tumor egress of CD8+ TILs. This enhanced egress associated with reduced expression of the tissue residency marker Itgae and its upstream regulator Znf683, with the circulating CD8+ T cells expressing higher Cxcr3, an observation made as well in samples from patients treated with dual TGF-? neutralization and ICB. Overall design: Tumor tissues from five individual mice were processed and cell suspensions were stained with anti-mouse TCR-ß and T cells were sorted to >99% purity. Two captures per treatment of 10,000 cells each were performed. Cells were mixed with barcoded gel beads and 5' GEM Kit v3 reagents and single cell capture was performed (10X Genomics). Following reverse transcription, cDNA and murine TCRs were amplified, and gene expression and TCR sequencing libraries were re-constructed. Each DNA library was loaded into a sequencing lane on a NovaSeq system. Spleens and tumor draining lymph nodes from individual mice were processed and T cells were isolated using the EasySep Mouse T cell Isolation Kit from StemCell Technologies. RNA was extracted from all sorted samples using the RNEasy Mini Kit and deep TCR sequencing was performed using the SMARTer Mouse TCR Profiling Kit from MiLaboratories.