Transcriptional landscape of CD4⁺ T cells in B-NHL and healthy donor tonsils. (A) Schematic representation of the workflow of scRNA-seq, TCR single-cell variable diversity joining sequencing (scVDJ-seq), and CITE-seq of sorted CD4⁺ T cells. (B) Single-cell data derived from sorted CD4⁺ T-cell populations from healthy donor tonsils (n = 3), FL (n = 3), and DLBCL (n = 3) projected onto uniform manifold approximation and projection (UMAP) by combining scRNA-seq and CITE-seq using weighted nearest neighbor (Wnn) method, provided 13 distinct clusters based on gene and protein expression differences for 18 771 cells passing the quality control. The clusters were annotated based on a combination of gene and protein expression as naive (KLF2, CCR7, LEF1, and protein expression of CD45RA), 3 memory clusters (CD69, SELL, and lack of CD45RA protein expression), GZM⁺ CD4⁺ T cells (GZMK/A, NKG7, and CST7), 2 T follicular helper (Tfh) clusters (PDCD1, CXCR5, IL21, TOX2, and protein expression of PD-1, CXCR5), a T helper cell cluster (CXCR4 and KLF6), 2 clusters of Tregs (FOXP3, IL2RA, CTLA4, and protein expression of CD25 but lack of CD127), and 1 cluster of unconventional FOXP3⁻LAG3⁺ Tregs (LAG3, CTLA4, IL10, and lack of CD127 protein). (C) UMAPs of the single-cell data shown in panel B, divided based on tissue origin and color coded based on patient sample. (D) Dot plots showing average expression of top 5 differentially expressed genes (DEGs) for each cluster. (E) Expression of selected genes (purple) and surface protein expression (green) overlaid onto the Wnn UMAP coordinates from panel B. (F) Pie charts showing the cellular abundance of each cell cluster per patient sample.