Journal: bioRxiv
Article Title: TCR signaling via NFATc1 constrains IL-15-induced NK-like activation of human memory CD8 + T cells
doi: 10.1101/2025.01.13.632884
Figure Lengend Snippet: ( A-K ) Total CD8 + T cells were isolated from patients at acute and convalescent stages of HAV infection ( n = 5) and healthy donors ( n = 3) by magnetic separation, and stained with DNA barcode-labeled MHC class I dextramers conjugated with PE (dCODE dextramers). dCODE dextramer-positive and -negative CD8 + T cells were flow-sorted, and then re-mixed at a 1:9 ratio and further stained with CITE-seq/antibody-derived tag (ADT) antibodies. Subsequently, these cells were processed for single-cell RNA sequencing. ( A ) Schematic overview of the experimental design. ( B ) UMAP projections of 33,291 dCODE-negative ( n = 16,295) and HAV-specific CD8 + T cells ( n = 2,940), and bystander CD8 + T cells, including HCMV-specific ( n = 11,456), EBV-specific ( n = 809) and IAV-specific ( n = 1,791) CD8 + T cells. ( C ) Violin plots showing ADT expression of CD38 and HLA-DR, and normalized gene expression of PRF1 and GZMB between bystander CD8 + T cells from acute HAV infection patients versus healthy donors. ( D ) Violin plot showing enrichment score for DEGs upregulated by stimulation with IL-15 compared to with IL-15 plus anti-CD3 in memory CD8 + T cells, compared between HAV-specific and bystander CD8 + T cells from patients with acute HAV infection. ( E ) GSEA of upregulated DEGs from IL-15-stimulated memory CD8 + T cells, using the transcriptomes of HAV-specific versus bystander CD8 + T cells from patients with acute HAV infection obtained using a pseudo bulk approach. ( F ) Violin plot showing enrichment score for DEGs upregulated by stimulation with IL-15 and IL-15 plus anti-CD3 in memory CD8 + T cells, compared between HAV-specific and bystander CD8 + T cells from patients with acute HAV infection. ( G ) Violin plot showing enrichment score for DEGs upregulated by stimulation with IL-15 compared to IL-15 plus ionomycin in memory CD8 + T cells, compared between HAV-specific and bystander CD8 + T cells from scRNA-seq analysis of patients with acute HAV infection. ( H ) Violin plots showing normalized expression of genes that are associated with IFN response in HAV-specific versus bystander CD8 + T cells from patients with acute HAV infection. ( I and J ) Violin plots showing (I) ADT expression of NKp46, NKp30, KIR2L1/S1/S3/S5, and GPR56 and (J) normalized gene expression of HCST, NCR3, CD244, and NKG7 in HAV-specific versus bystander CD8 + T cells from patients with acute HAV infection. ( K ) ADT expression of NKG2D and CCR5 in HAV-specific versus bystander CD8 + T cells from patients with acute HAV infection. Statistical analysis was performed using the Mann-Whitney U-test *P < 0.05, ***P < 0.001, ****P < 0.0001.
Article Snippet: Sorted CD8 + T cells from PBMCs of patients ( n = 5) at acute and convalescent stages of HAV infection and healthy donors ( n = 3) were stained with PE-conjugated MHC-I dCODE dextramers (Immudex) specific for HLA-A*0201 HAV 3D 2026 LLYNCCYHV, HLA-A*0201 HCMV pp65 495 NLVPMVATV, HLA-A*0201 EBV BMLF1 259 GLCTLVAML, and HLA-A*0201 IAV MP 58 GILGFVFTL.
Techniques: Isolation, Infection, Staining, Labeling, Derivative Assay, RNA Sequencing Assay, Expressing, MANN-WHITNEY
