Microbiota-induced CD4⁺ T cell plasticity remodels the tumor microenvironment to enhance PD-1 therapy

Single-cell RNA sequencing (scRNA-seq) and TCR sequencing (scTCR-seq) were performed using the Chromium Single Cell 5′ v2 reagent kit and Chromium Single Cell Mouse TCR Amplification Kit (10x Genomics). Tumor-infiltrating lymphocytes from B16-3340 tumors and lymphocytes from the SILP of SFB⁻ and SFB+ mice (n=5 in each group) were isolated as described above. CD4+ T cells were then sorted from pooled cells of either SFB⁻ or SFB+ tumor tissues or SILP of individual mice using FACS Aria II (BD Biosciences). Sorted CD4+ T cells from each group (Tumor SFB⁻, Tumor SFB+, SILP SFB⁻ and SILP SFB+) were resuspended in PBS containing 0.05% BSA and stained with cell hashing antibodies, TotalSeq-C0301 to C0304 (BioLegend, Cat#155861, Cat#155863, Cat#155865, Cat#155867)57 for 20 minutes on ice. Cells were then washed 3 times with MACS buffer. CD4+ T cells from both SFB⁻ and SFB+ tumors were combined at a 1:1 ratio. Similarly, CD4+ T cells from the SILP of SFB⁻ and SFB+ mice were combined at a 1:1 ratio. Approximately 1.5 × 104 cells per sample were loaded onto the Chromium Controller (10x Genomics) and libraries were prepared with the Chromium Single Cell 5′ kit following the manufacturer’s instructions. Libraries were sequenced on an Illumina HiSeq 6000 and sequencing reads were aligned to the mouse reference genome (mm10) using Cell Ranger (10x Genomics). Downstream data were processing, and analysis were performed with the R packages Seurat v558.