Intratumoral antigen-signalling traps CD8+ T cells to confine exhaustion to the tumour site (bulk TCR-Seq)

Immunotherapy advances have been hindered by difficulties tracking the behaviours of lymphocytes following antigen-signalling. Here we present the antigen-receptor signalling reporter (AgRSR) mouse to fate-map lymphocytes responding to antigen at specific times and locations, to determine the behaviour of T cells involved in the tumour immune response. Contrary to reports of ready egress of T cells out of the tumour, we find that intratumoral antigen-signalling traps CD8+ T cells in the tumour. These clonal populations expand and become increasingly exhausted over time. In contrast, antigen- signalled regulatory T cell (Treg) clonal populations readily recirculate out of the tumour. Consequently, intratumoral antigen-signalling acts as a gatekeeper to compartmentalize CD8+ T cell responses – even within the same clonotype, enabling immunity to confine exhaustion to a specific tissue site. YUMMER1.7 bearing AgRSR mice were injected with tamoxifen (intraperitoneally or intratumorally). 8 or 18 days after injection, EYFP+ and EYFP- cells were sorted from the secondary lymphoid tissues and tumour, and processed through bulk TCR-seq. Library strategy: TCR-seq, VDJ from 5' RACE total mRNA. Technical details:-Sample sorting and storage: cells sorted in Buffer RLT Plus with DDT -RNA extraction kit: RNeasy Plus Micro Kit -RNA quantification and QC: Qubit RNA HS and Tapestation High Sensitivity RNA - Library preparation kit: Takara SMARTer Mouse TCR alpha-beta Profiling Kit - Library quantification and QC: Qubit DNA HS and Tapestation High Sensitivity D5000 - Sequencing instrument: Illumina NextSeq2000 -Sequencing: P2 600 cycles, 300+300 with 10%PhiX spike-in TCR-seq analysis: MiXCR.