cd8 memory t cells miltenyi biotech  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: Distinct mechanisms are employed by T-cell-instructed myeloid cells for IL-1β production in humans and mice

doi: 10.1101/2025.02.26.640341

Figure Lengend Snippet: Isolated monocytes, autologous memory CD4+ T-cells or memory CD8+ T-cells from human PBMCs were cultured alone or co-cultured in the absence/presence of anti-CD3 antibodies for 16 hours. Immunoblotting analysis of pro-IL-1β, β-actin, and cleaved-IL-1β in the whole cell lysates or the supernatants.

Article Snippet: Primary human monocytes, memory CD8+ T cells and memory CD4+ T cells were isolated from PBMCs using CD14 MicroBeadsTM, memory CD8+ isolation kit and memory CD4+ isolation kit according to the manufacturer’s instructions (Miltenyi Biotec), respectively.

Techniques: Isolation, Cell Culture, Western Blot

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 and B ) Peripheral blood mononuclear cells (PBMCs) from healthy donors ( n = 9) were stimulated for 48 hours with IL-2 (10 ng/ml), IL-4 (10 ng/ml), IL-7 (10 ng/ml), IL-9 (10 ng/ml), IL-15 (10 ng/ml) or IL-21 (50 ng/ml). (A) Representative figures and cumulative data presenting NKG2D expression on CCR7 − memory CD8 + T cells from healthy donor PBMCs following stimulation with indicated cytokines. (B) NKG2D expression on CCR7 − memory CD8 + T cells stimulated for 48 hours with varying doses of IL-15 (0.01, 0.1, 1, 10, and 100 ng/ml). ( C and D ) Sorted CCR7 − memory CD8 + T cells from healthy donors were stimulated with IL-15 (10 ng/ml), anti-CD3 (coated, 1 µg/ml), or the combination of both. Representative figures and cumulative data presenting the expression of (C) NKG2D ( n = 10) and (D) CD38 and HLA-DR ( n = 8) on CCR7 − memory CD8 + T cells upon stimulation for 48 hours and 96 hours, respectively. ( E and F ) Sorted CCR7 − memory CD8 + T cells from healthy donors ( n = 8) were stained with CellTrace Violet, and stimulated with IL-15 (10 ng/ml), anti-CD3 (coated, 1 µg/ml), or the combination of both. After 96 hours of stimulation, we measured the (E) Ki-67 expression and (F) proliferation of CCR7 − memory CD8 + T cells. Error bars represent mean ± SD. Statistical analysis was performed using the paired Student’s t test. NS, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Sorted CCR7 − memory CD8 + T cells were pre-treated with everolimus (10 μM; Selleckchem), FK506 (10 ng/ml; Selleckchem), CsA (100 ng/ml; Selleckchem), SP600125 (10 μM; Selleckchem), or SR11302 (10 μM; Selleckchem) for one hour, followed by stimulation with IL-15, anti-CD3, or the combination of both.

Techniques: Expressing, Staining

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-H ) Sorted CCR7 − memory CD8 + T cells from healthy donors ( n = 10) were stimulated for 48 hours with IL-15 (10 ng/ml), anti-CD3 (coated, 1 µg/ml), or the combination of both. Total RNA was isolated and RNA-seq was performed. ( A ) Principal component analysis (PCA) indicating segregation of the gene expression patterns of memory CD8 + T cells stimulated with IL-15, anti-CD3, or the combination of both. ( B ) Venn diagram of individual and overlapping differentially expressed genes of memory CD8 + T cells stimulated with IL-15, anti-CD3, or the combination of both, compared to unstimulated cells and ( C ) Heat-map showing the z scores of normalized read counts (adjusted P < 0.05, Log2 fold-change > 1). ( D ) Heat-map of differentially expressed genes between memory CD8 + T cells stimulated with IL-15 alone or IL-15 plus anti-CD3 (adjusted P < 0.05, Log2 fold-change > 1). ( E and F ) Bar- plots showing the enrichment scores (−log10[ p value]) of various pathways enriched in DEGs upregulated by (E) IL-15 or (F) IL-15 plus anti-CD3. ( G and H ) GSEA of (G) GOBP interferon-mediated signaling pathway (GO: 0140888) and (H) GOBP positive regulation of natural killer-cell-mediated cytotoxicity (GO: 0045954) in memory CD8 + T cells stimulated with IL-15 alone versus IL-15 plus anti-CD3.

Article Snippet: Sorted CCR7 − memory CD8 + T cells were pre-treated with everolimus (10 μM; Selleckchem), FK506 (10 ng/ml; Selleckchem), CsA (100 ng/ml; Selleckchem), SP600125 (10 μM; Selleckchem), or SR11302 (10 μM; Selleckchem) for one hour, followed by stimulation with IL-15, anti-CD3, or the combination of both.

Techniques: Isolation, RNA Sequencing Assay, Expressing

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-E ) Sorted CCR7 − memory CD8 + T cells from healthy donors ( n = 10) were stimulated for 12 hours with IL-15 (10 ng/ml), ionomycin (500 ng/ml), or the combination of both. Total RNA was isolated and RNA-seq was performed. ( A ) PCA indicating segregation of gene expression patterns of memory CD8 + T cells stimulated with IL-15, ionomycin, or the combination of both. ( B ) Venn diagram of individual and overlapping differentially expressed genes of memory CD8 + T cells stimulated with IL-15, ionomycin, or the combination of both compared to unstimulated cells and ( C ) Heat-map showing the z scores of normalized read counts (adjusted P < 0.05, Log2 fold-change > 1). ( D ) Heat-map of differentially expressed genes between memory CD8 + T cells stimulated with IL-15 or with IL-15 plus ionomycin (adjusted P < 0.05, Log2 fold-change > 1). ( E ) Bar-plots showing the enrichment scores (−log10[ p value]) of various pathways enriched in DEGs upregulated by IL-15 and down-regulated by IL-15 plus ionomycin. Statistical analysis was performed using the paired Student’s t test. **** P < 0.0001.

Article Snippet: Sorted CCR7 − memory CD8 + T cells were pre-treated with everolimus (10 μM; Selleckchem), FK506 (10 ng/ml; Selleckchem), CsA (100 ng/ml; Selleckchem), SP600125 (10 μM; Selleckchem), or SR11302 (10 μM; Selleckchem) for one hour, followed by stimulation with IL-15, anti-CD3, or the combination of both.

Techniques: Isolation, RNA Sequencing Assay, Expressing

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 and B ) Sorted CCR7 − memory CD8 + T cells from healthy donors were stimulated with IL-15 (10 ng/ml), ionomycin (500 ng/ml), or the combination of both. Representative figures and cumulative data presenting the expression of (A) NKG2D ( n = 10) and (B) CD38 and HLA-DR ( n = 8) on CCR7 − memory CD8 + T cells after 48 hours and 96 hours of stimulation, respectively. ( C ) Sorted CCR7 − memory CD8 + T cells from healthy donors ( n = 7) were pretreated with FK506 (10 ng/ml) for 1 hour, and then stimulated with IL-15 (10 ng/ml) and anti-CD3 (coated, 1 µg/ml). After 48 hours of stimulation, NKG2D expression was measured on CCR7 − memory CD8 + T cells. ( D ) PBMCs from patients with acute HAV infection were stimulated for 24 hours with IL-15 (10 ng/ml) or a combination of IL-15 plus ionomycin (500 ng/ml). After 48 hours of stimulation, NKG2D expression was measured on CCR7 − memory ( n = 10), HCMV-specific ( n = 6), and EBV- specific ( n = 6) CD8 + T cells. ( E ) Sorted CCR7 − memory CD8 + T cells from patients with acute HAV infection ( n = 6) were cultured as in (C) and NKG2D expression was measured on CCR7 − memory CD8 + T cells. ( F ) Sorted total CD8 + T cells from patients with acute HAV infection ( n = 6) were stimulated with IL-15 (10 ng/ml) or a combination of IL-15 plus ionomycin (500 ng/ml) for 24 hours, and co-cultured with PKH-26-labeled K562 cells at a 20:1 effector:target cell (E:T) ratio for 12 hours. Cytotoxicity was analyzed by flow cytometry following staining of the dead cells with TO-PRO-3. Error bars represent mean ± SD. Statistical analysis was performed using the paired Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Sorted CCR7 − memory CD8 + T cells were pre-treated with everolimus (10 μM; Selleckchem), FK506 (10 ng/ml; Selleckchem), CsA (100 ng/ml; Selleckchem), SP600125 (10 μM; Selleckchem), or SR11302 (10 μM; Selleckchem) for one hour, followed by stimulation with IL-15, anti-CD3, or the combination of both.

Techniques: Expressing, Infection, Cell Culture, Labeling, Flow Cytometry, Staining

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: PBMCs from healthy donors ( n = 6) were pretreated with ( A ) SP600125 (10 μM) or ( B ) SR11302 (10 μM) for 1 hour, and then stimulated with IL-15 (10 ng/ml). After 48 hours of stimulation, NKG2D expression was measured on CCR7 − memory CD8 + T cells. ( C ) PBMCs from healthy donors ( n = 9) were transfected with c-Jun siRNA (200 nM), c-Fos siRNA (200 nM) or the combination of both siRNAs and rested for 4 days. Subsequently, PBMCs were stimulated with IL-15 (10 ng/ml) for 48 hours and NKG2D expression was measured on CCR7 − memory CD8 + T cells. ( D ) PBMCs from healthy donors ( n = 5) were transfected with pcDNA3.1 plasmids containing wild-type or T551A mutant NFATc1. Subsequently, PBMCs were stimulated with IL-15 (10 ng/ml) for 48 hours and NKG2D expression was measured on CCR7 - memory CD8 + T cells. ( E ) Sorted CCR7 − memory CD8 + T cells from healthy donors ( n = 8) were stimulated with PMA (10 ng/ml), ionomycin (500 ng/ml), or the combination of both for 48 hours and NKG2D expression was measured. Error bars represent mean ± SD. Statistical analysis was performed using the paired Student’s t test. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Sorted CCR7 − memory CD8 + T cells were pre-treated with everolimus (10 μM; Selleckchem), FK506 (10 ng/ml; Selleckchem), CsA (100 ng/ml; Selleckchem), SP600125 (10 μM; Selleckchem), or SR11302 (10 μM; Selleckchem) for one hour, followed by stimulation with IL-15, anti-CD3, or the combination of both.

Techniques: Expressing, Transfection, Mutagenesis

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 CCR7 − memory CD8 + T cells were pre-treated with everolimus (10 μM; Selleckchem), FK506 (10 ng/ml; Selleckchem), CsA (100 ng/ml; Selleckchem), SP600125 (10 μM; Selleckchem), or SR11302 (10 μM; Selleckchem) for one hour, followed by stimulation with IL-15, anti-CD3, or the combination of both.

Techniques: Isolation, Infection, Staining, Labeling, Derivative Assay, RNA Sequencing Assay, Expressing, MANN-WHITNEY