Fig 2: Single-cell analysis reveals changes in intra-tumoral cell populations in tumors from Aire+/+ and Aire−/− mice.a UMAP plot depicting the clusters from splenic and tumor CD45+ cells. The cluster numbers correspond to individual cell populations described below the UMAP plot. b Individual UMAP plots from splenic and tumor CD45+ cells depicting the expression of cell markers associated with cell clusters identified in Fig. 5a. T cells (CD3e+), B cells (CD79a+), macrophage/myeloid (Msr1+ C1qb+), dendritic cells (Clec9a+), pDCs (Siglech+), NK cells (Ncr1+), and neutrophils (S100a8+). c UMAP plots depicting the unsupervised clustering of tumor CD45+ cells in Aire+/+ and Aire−/− treated with Isotype or anti-PD1. d Bar graph depicting the composition of each cluster identified in tumor CD45+ cells from Aire+/+ and Aire−/− mice treated with Isotype or anti-PD1. Transcriptional changes in all cell types identified across all conditions are included in Supplementary Data 6.

Fig 3: TIGIT+ cells display a distinct cytokine profile. (a) Representative dot plots showing intracellular cytokine production after cultivating CLL PBMCs for 24 h with CD3/CD28 activating beads. (b) Cytokine production of TIGIT- or TIGIT+CD4+ T cells in 14 samples. (c) Mean fluorescence intensity ratio (MFIR) of CD155 and CD112 on CD5+CD19+ CLL (top) or CD5+ T cells (bottom). The histograms show representative FACS plots of CLL cells (gated for CD5+CD19+cells) and T cells (CD5+ cells) stained with isotype controls (in gray) and CD112/CD155 specific antibodies (in black). The dot plots show results from n = 14 samples.

Fig 4: Tumors from Aire−/− mice have increased levels of cytotoxic genes.a, b Heatmaps depicting differentially regulated genes associated with T cell receptor signaling or chemokine signaling in tumors from Aire+/+ (n = 4) and Aire−/− (n = 3) mice treated with anti-PD1. Differentially regulated genes were defined with log2 fold changes of 1.5 in either up or down direction and with p values <0.01; TPM transcripts per million. The expression value of each gene was divided by the median expression of the same gene across all samples. c Transcript levels of Cd3e, Cd8α, Ifnγ, Tnfα, and FasL in tumors from Aire+/+ and Aire−/− mice. TPM transcripts per million. Data are represented as mean ± SEM, (*P < 0.05; **P < 0.01; ***P < 0.001), by Student’s t test. TPM values are provided in Supplementary Data 1.

Fig 5: CD4+ TIGIT+ cells provide a supportive microenvironment for CLL cells. (a) Representative dot plots showing gating strategy for flow cytometric cell sorting. (b) PBMCs have been depleted of TIGIT+, PD-1+ or TIGIT+PD-1+ CD4+ or CD8+ cells followed by incubation with CD3/CD28 activating beads. After 5 days in culture, CLL viability was measured and corresponding T/ CLL ratios were analysed (n = 6) (c).