Single-cell mRNA sequencing and TCR sequencing of CD4+ T cells in a mouse model of cutaneos B16 melanoma

This study sought to identify transcriptional states of CD4+ T cells from melanoma-challenged skin, primary tumours and tumour-draining lymph nodes. TCR sequencing was performed to identify expanded clonotypes to infer tumour-antigen specificity. This study identified heterogeneous populations of melanoma-specific CD4+ T cells that are distinct in the different anatomical locations. Overall design: C57B/6 mice were challenged with B16.gD melanoma using the recently developed epicutaneous melanoma model (Park et al, Nature 2019). Melanoma-challenged skin and draining (brachial) lymph nodes from the same mice were harvested 8 days following inoculation. Primary tumours and tumour-draining (brachial) lymph nodes from tumour-bearing mice were harvested 20 days following inoculation. As a batch control, a brachial lymph node was harvested from a naive C57BL/6 mouse. Samples were stained simultaneously with antibodies for flow cytometry and TotalSeq-C hashtag antibodies (Biolegend). Samples were sorted on CD45.2+CD3+CD8-CD4+CD44+ cells except for the naïve lymph node (batch control) which was sorted on CD45.2+CD3+CD8-CD4+CD44-CD62L+. Day 8 skin and lymph node samples were pooled from 32 mice across two experiments. Day 20 tumour and lymph node samples were pooled from 4 mice in one experiment.