Single-cell RNA-seq for identification of B-cell specific checkpoint molecules regulating anti-tumor immunity

The role of B cells in anti-tumor immunity is still debated and accordingly, most therapies have focused on targeting T and NK cells to inhibit tumor growth1,2. Here, using high-throughput flow cytometry, bulk and single-cell RNA- and BCR-sequencing of B cells temporally during B16F10 melanoma growth, we identified a subset of B cells that expands specifically in the draining lymph node over time in tumor bearing-mice. The expanding B cell subset expresses the cell surface molecule T cell immunoglobulin and mucin domain 1 (TIM-1) and a unique transcriptional signature, including multiple co-inhibitory molecules such as PD-1, TIM-3, TIGIT and LAG-3. While conditional deletion of these co-inhibitory molecules on B cells had little or no effect on tumor burden, selective deletion of Havcr1 (the gene encoding TIM-1) in B cells both dramatically inhibited tumor growth and enhanced effector T cell responses. Loss of TIM-1 enhanced the type 1 interferon response in B cells, which augmented B cell activation and increased antigen presentation and co-stimulation, resulting in increased expansion of tumor-specific effector T cells. Our results demonstrate that manipulation of TIM-1-expressing B cells enables engagement of the second arm of adaptive immunity to promote anti-tumor immunity and inhibit tumor growth. For the B cell atlas analysis or the examination of CD19cre/+ and TIM-1BKO mice, viable leukocytes were sorted by FACS-sorted from tumors (70% CD3e+ and CD19+ cells, 30% total CD45+ cells), dLN and ndLN (100% CD45+ cells) at three different time points as depicted in Extended Date Figure 2a. For the analysis of TIM-1-expressing B cells, viable B220+ CD19+ CD138+ and - cells derived from dLN, ndLN and spleen from C57Bl6/J mice were sorted by FACS. Cells were resuspended in PBS containing 2% FCS and stained with oligo tagged TotalSeq antibodies (BioLegend) for 30 minutes on ice. Cells were washed and pooled accordingly, centrifuged at 1,200 rcf for 5 minutes at 4°C and resuspended in PBS + 2% FCS. For the B cell temporal profiling, 9 samples were combined into each channel of the Chromium system (10x Genomics): Tumor, dLN, ndLN from three different timepoints: days 7, 10 and 16 of one replicate. For the examination of CD19cre/+ and TIM-1BKO, 6 samples were combined into each channel: tumor, dLN, ndLN derived from one biological replicate of each genotype. For the TIM-1 positive cells analysis, cells derived from LN were loaded in separate channels and the splenic cells TIM-1 positive and TIM-1 negative were combined. For samples that did not include scBCR-seq and/or scTCR-seq and 5' feature barcoding, sorted cells were separated into droplet emulsions using the Chromium Single Cell 3′ Solution (v2) according to manufacturer’s instructions (10x Genomics).