Journal: Cell Death & Disease

Article Title: PD-1 protects expanding human T cells from premature restimulation-induced cell death by modulating TCR and CD28 signaling

doi: 10.1038/s41419-026-08530-6

Figure Lengend Snippet: T cells isolated from healthy human donors were activated with anti-CD3/28 beads and IL-2 was added on Day 3. A PD-1, PD-L1, and PD-L2 expression were measured via flow cytometry over 15 days for N = 8 in CD4+ and N = 5 in CD8 + T cells. B Day 4 purified CD8 + T cells were restimulated with autologous irradiated PBMC and anti-CD3 (1 μg/mL) ± anti-CD28 (1 μg/mL) in the presence of control antibody, anti-PD-1, or anti-PD-L1 at 10 μg/mL for 20 hr. Death was assessed via flow cytometry by propidium iodide staining. Single dot correlates with one human donor. Connecting line compares change in untreated and treated samples within the same donor. N = 3. C Diagram illustrating the proxy APC (pAPC) design created with a 1:1:1 ratio of anti-CD3, anti-CD28, and IgG1κ or recombinant human Fc-chimera PD-L1. D Freshly isolated CD4+ or CD8+ human T cells were activated at a 1:1 cell:bead ratio with IgG or PD-L1 beads and monitored via flow cytometry for CD69 expression after 24 hr ( N = 3 CD4 + , N = 5 CD8 + ) and CD25 expression after 72 hr ( N = 3 CD4 + , N = 6 CD8 + ). E Blastogenesis patterns of unactivated, IgG, or PD-L1 activated cells on Day 3 are demonstrated by dot plots assessing forward scatter (FSC) (x-axis) and side scatter (SSC) (y-axis). The progressive changes in FSC monitored via flow cytometry over 3 days are summarized for CD4+ and CD8 + T cells. N = 3 CD4 + , N = 5 CD8 + .

Article Snippet: Isotype control antibodies were paired with the corresponding blocking antibody at 10 μg/mL: IgG1κ (BioXCell, #BP0297) or IgG4 (BioXCell, #CP148).

Techniques: Isolation, Expressing, Flow Cytometry, Purification, Irradiation, Control, Staining, Recombinant

Journal: Cell Death & Disease

Article Title: PD-1 protects expanding human T cells from premature restimulation-induced cell death by modulating TCR and CD28 signaling

doi: 10.1038/s41419-026-08530-6

Figure Lengend Snippet: A CD8+ or CD4 + T cells were restimulated on Day 4 or Day 11 with p328-IgG of p328-L1 beads at a 1:1 bead:cell ratio and monitored for cell loss via flow cytometry 24 hr later using propidium iodide. N = 8 CD4+ early, N = 11 CD4+ late, N = 9 CD8+ early, N = 18 CD8+ late. B The difference in % cell loss between p328-IgG and p328-L1 beads in ( A ) is plotted for each donor, cell type, and cell stage evaluated. (same N as ( A ). C RICD assays set-up in ( A ) were examined on the flow cytometer using propidium iodide to calculate the percentage of live cells remaining after 24 hr of restimulation. N = 7 CD4+ early, N = 9 CD4+ late, N = 12 CD8+ early, N = 8 CD8+ late. D PD-L1 dose response was determined using the standard RICD setup amd a new bead set that gradually decreased PD-L1 concentration on the bead from 33% to 0%. Day 4 CD4+ and CD8 + T cells were utilized in this experiment. N = 12 CD4 + , N = 10 CD8 + , statistical analysis was calculated with two-way ANOVA. E Diagram representing RICD setup and creation of secondary bead set that was ligated 100% to IgG1κ (control secondary bead) or 67% IgG1κ and 33% recombinant PD-L1, matching the PD-L1 protein concentration on the p328-L1 primary bead. F Day 4 CD8+ and CD4 + T cells were restimulated with a 2:1 bead:cell ratio comprising a 1:1 ratio of the primary bead and 1:1 ratio of the secondary bead. RICD was monitored as in ( A ) N = 12.

Article Snippet: Isotype control antibodies were paired with the corresponding blocking antibody at 10 μg/mL: IgG1κ (BioXCell, #BP0297) or IgG4 (BioXCell, #CP148).

Techniques: Flow Cytometry, Concentration Assay, Control, Recombinant, Protein Concentration

Journal: Cell Death & Disease

Article Title: PD-1 protects expanding human T cells from premature restimulation-induced cell death by modulating TCR and CD28 signaling

doi: 10.1038/s41419-026-08530-6

Figure Lengend Snippet: A Representative histogram of data displaying demarcation of AnnexinV hi cells -/+ bead restimulation. B Day 4 and Day 11 CD4+ and CD8 + T cells were left unstimulated or were restimulated with p328-IgG or p328-L1 beads for 6 hr before being stained with AnnexinV and propidium iodide and read on the flow cytometer. N = 6. C Representative flow cytometric histogram of PI cell cycle phases based on DNA content. D Day 4 and Day 11 CD4+ and CD8 + T cells were left unstimulated or were restimulated with p328-IgG or p328-L1 beads for 18 hr before being permeabilized and stained with propidium iodide to determine % of cells in the subG1 phase. N = 8 CD4+ early, N = 8 CD4+ late, N = 10 CD8+ early, N = 9 CD8+ late. E Analysis using gated cells from ( D ) was performed to quantify changes in the G1 phase. F Cell phase characterization from ( D ) was analyzed to determine the PD-L1 induced difference in subG1 and G1 phases.

Article Snippet: Isotype control antibodies were paired with the corresponding blocking antibody at 10 μg/mL: IgG1κ (BioXCell, #BP0297) or IgG4 (BioXCell, #CP148).

Techniques: Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: PD-1 protects expanding human T cells from premature restimulation-induced cell death by modulating TCR and CD28 signaling

doi: 10.1038/s41419-026-08530-6

Figure Lengend Snippet: A CD8+ or CD4 + T cells were restimulated on Day 4 with soluble anti-CD3 (OKT3) at 100 ng/mL or anti-CD3/CD28 coated beads at a 1:1 bead:cell ratio. Cell loss/apoptosis was quantified 18–24 hr later after propidium iodide staining and flow cytometry. N = 5 CD4+ early, N = 6 CD4+ late, N = 5 CD8+ early, N = 6 CD8+ late. B Day 4 and Day 11 CD4+ and CD8 + T cells from N = 5 individual donors were stained for CD28 and PD-1 expression and compared via flow cytometry. C Diagram illustrating restimulation beads conjugated with or without anti-CD28, with p328-IgG used to maintain total protein concentration. D CD8+ or CD4 + T cells were restimulated on Day 4 with beads illustrated in ( C ) at a 1:1 bead:cell ratio, monitored for cell loss 24 hr later using propidium iodide using flow cytometry. E The difference in cell loss induced by restimulation with beads -/+ anti-CD28 (leaving anti-CD3 + /- PD-L1-Fc constant) was plotted for every donor ( N = 10).

Article Snippet: Isotype control antibodies were paired with the corresponding blocking antibody at 10 μg/mL: IgG1κ (BioXCell, #BP0297) or IgG4 (BioXCell, #CP148).

Techniques: Staining, Flow Cytometry, Expressing, Protein Concentration

Journal: Cell Death & Disease

Article Title: PD-1 protects expanding human T cells from premature restimulation-induced cell death by modulating TCR and CD28 signaling

doi: 10.1038/s41419-026-08530-6

Figure Lengend Snippet: A Day 4 CD4+ and CD8 + T cells ( N = 4 donors each) were restimulated with p328-IgG or p328-L1 beads at a 1:1 bead:cell ratio for 5 hr prior to lysate collection. Control cells were left unstimulated or treated with 1 μM STX. After cell lysis and protein standardization, donor lysates were pooled for each stimulation condition and analyzed on the apoptosis protein array. Representative spots for differentially expressed proteins are shown. Protein signal intensity from membrane imaging was analyzed and normalized using QuickSpots software. T cells were restimulated as in ( A ), lysed in RIPA buffer and separated by SDS-PAGE to quantify FASL ( B ) and survivin ( C ). Data are representative of experiments for 3–4 separate donors. ( D ) Day 4 CD4+ and CD8 + T cells were treated with 10 μg/mL anti-FASL blocking antibody for 1 hr prior to restimulation with the quad bead set. Cells were restimulated for 24 hr and stained with propidium iodide; % viability was assessed by flow cytometry. N = 7 CD4 + , N = 5 CD8 + .

Article Snippet: Isotype control antibodies were paired with the corresponding blocking antibody at 10 μg/mL: IgG1κ (BioXCell, #BP0297) or IgG4 (BioXCell, #CP148).

Techniques: Control, Lysis, Protein Array, Membrane, Imaging, Software, SDS Page, Blocking Assay, Staining, Flow Cytometry