Chronic infectious diseases have a substantial impact on the human B cell compartment including a notable expansion of B cells here termed atypical B cells (ABCs). Using unbiased single-cell RNA sequencing (scRNA-seq), we uncovered and characterized heterogeneities in naïve B cell, classical memory B cells, and ABC subsets. We showed remarkably similar transcriptional profiles for ABC clusters in malaria, HIV, and autoimmune diseases and demonstrated that interferon-γ drove the expansion of ABCs in malaria. These observations suggest that ABCs represent a separate B cell lineage with a common inducer that further diversifies and acquires disease-specific characteristics and functions. In malaria, we identified ABC subsets based on isotype expression that differed in expansion in African children and in B cell receptor repertoire characteristics. Of particular interest, IgD+IgMlo and IgD−IgG+ ABCs acquired a high antigen affinity threshold for activation, suggesting that ABCs may limit autoimmune responses to low-affinity self-antigens in chronic malaria.

Multiple ongoing clinical trials use site-specific nucleases to disrupt T cell receptor (TCR) genes in order to allow for allogeneic T cell therapy [1][2][3][4][5] . In particular, the first U.S. clinical trial using CRISPR-Cas9 entailed the targeted disruption of the TCR chains and programmed cell death protein 1 (PDCD1) in T cells of refractory cancer patients 6 . Here, we used the same guide RNA sequences and applied single-cell RNA sequencing (scRNAseq) to more than 7000 primary human T cells, transfected with CRISPR-Cas9. Four days post-transfection, we found a loss of chromosome 14, harboring the TCRα locus, in up to 9% of the cells, and a chromosome 14 gain in up to 1.4% of the cells. We further identified truncations of chromosome 7, harboring the TCRβ locus, in 9.9% of the cells. Loss of heterozygosity (LOH) was further validated using fluorescence in situ hybridization (FISH) and the temporal dynamics of cleavage and incomplete repair were monitored using digital droplet PCR (ddPCR). Aneuploidy was found among all T cell subsets and was associated with transcriptional signatures of reduced proliferation and metabolism as well as with induced p53 activation and cell death. We conclude that aneuploidy and chromosomal truncations are frequent outcomes of CRISPR-Cas9 cleavage in clinical protocols. Monitoring and minimizing these aberrant products is crucial for future applications of genome editing in T cell engineering and beyond.

Unleashing cytotoxic CD8+ T cells for effective cancer treatment requires understanding T cell states across different tumor microenvironments. Here, we developed an algorithm to recover both shared and tumor type specific programs and used it to analyze a scRNA-seq compendium of 38,852 CD8+ T cells from 141 patients spanning nine different human cancers. We uncovered a pan-cancer T cell dysfunction program that was predictive of clinical responses to immunotherapy and highlighted CXCR6 as a pan-cancer marker of dysfunctional T cells. In mouse models, CXCR6 increased following checkpoint blockade and was repressed by TCF1. Its ligand, CXCL16, was expressed primarily by myeloid cells, and was co-regulated with antigen presentation genes. CXCR6 deletion decreased Tox, CX3CR1, and Bcl2 expression, predisposing dysfunctional PD-1+Tim3+CD8+ T cells to apoptosis, and compromising tumor growth control. Our approach discovered a TCF1:CXCR6-CXCL16 regulatory axis essential for effective anti-tumor immunity, revealing a new perspective on T cell dysfunction and new opportunities for modulating this cell state.

SUMMARYMultiple ongoing clinical trials use site-specific nucleases to disrupt T cell receptor (TCR) genes in order to allow for allogeneic T cell therapy1–5. In particular, the first U.S. clinical trial using CRISPR-Cas9 entailed the targeted disruption of the TCR chains and programmed cell death protein 1 (PDCD1) in T cells of refractory cancer patients6. Here, we used the same guide RNA sequences and applied single-cell RNA sequencing (scRNAseq) to more than 7000 primary human T cells, transfected with CRISPR-Cas9. Four days post-transfection, we found a loss of chromosome 14, harboring the TCRα locus, in up to 9% of the cells, and a chromosome 14 gain in up to 1.4% of the cells. We further identified truncations of chromosome 7, harboring the TCRβ locus, in 9.9% of the cells. Loss of heterozygosity (LOH) was further validated using fluorescence in situ hybridization (FISH) and the temporal dynamics of cleavage and incomplete repair were monitored using digital droplet PCR (ddPCR). Aneuploidy was found among all T cell subsets and was associated with transcriptional signatures of reduced proliferation and metabolism as well as with induced p53 activation and cell death. We conclude that aneuploidy and chromosomal truncations are frequent outcomes of CRISPR-Cas9 cleavage in clinical protocols. Monitoring and minimizing these aberrant products is crucial for future applications of genome editing in T cell engineering and beyond.