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: Soluble anti-CD3 mAb (clone: OKT3, Biogems, #05121-20) was administered in complete RPMI + rIL-2 to a final concentration of 100 ng/mL.

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 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: Soluble anti-CD3 mAb (clone: OKT3, Biogems, #05121-20) was administered in complete RPMI + rIL-2 to a final concentration of 100 ng/mL.

Techniques: Staining, Flow Cytometry, Expressing, Protein Concentration

Journal: Molecular Therapy Oncology

Article Title: Highly efficient gene knockout in tumor-infiltrating lymphocytes by adenine base editing

doi: 10.1016/j.omton.2025.201041

Figure Lengend Snippet: Adenine base editing for TIL engineering (A) Schematic overview of the study design. (1) After resection, tumor lesions were minced into small fragments. For the Pre-REP, tumor fragments were either digested or cultured in medium supplemented with high dose of IL-2, or directly seeded to allow the outgrowth of TILs. Pre-REP TILs were cryopreserved at this stage. (2) Frozen Pre-REP TILs were thawed and activated for two days. Activated TILs were electroporated (EP) with the desired combination of adenine base editor and guide (g)RNA using the MaxCyte ExPERT ATx device. (3) For the REP, one hour after EP the TILs were transferred to a 24-well G-Rex plate with serum-free medium supplemented with anti-CD3 (OKT3) monoclonal antibody, IL-2, and irradiated feeder cells. The phenotype and function of Mock-EP and ABE-TILs were analyzed at the end of the REP protocol. (B) Screening of activation and exhaustion markers in unstimulated and post-REP TILs stimulated for 24 h with anti-CD3/CD28 beads. Both CD4 + and CD8 + subsets of two Ova- and two Mel-TILs post-REP were analyzed ( n = 4). (C) Diagram of TIM3 and TIGIT locus showing relative positions for splice-disrupting gRNAs. (D) Confirmation of A to G conversion in edited TILs. Chromatograms obtained by Sanger sequencing demonstrate the point mutation introduced by ABE in exemplary TIGIT-edited TILs. (E) Engineering single gene KO TILs. Exemplary histogram for TIM3 and TIGIT expressions in Mock-EP and ABE Ova-and Mel-TILs. Violin plots show quantification of KO efficiency in REP-TILs ( n = 3). Mock-EP TILs were transfected with ABE mRNA alone and were used as a control in all experiments. The Illustration was created with BioRender.com.

Article Snippet: For electroporation, frozen pre-REP TILs thawed and activated in serum-free X-VIVO15 supplemented with 3000 IU/mL of recombinant human IL-2 (Proleukin, Novartis, Basel, Switzerland) and 30 ng/ml GMP-grade soluble anti-CD3 (OKT3) antibody (Miltenyi Biotech) for 1–2 days before electroporation.

Techniques: Cell Culture, Irradiation, Activation Assay, Sequencing, Mutagenesis, Transfection, Control

Journal: Molecular Therapy Oncology

Article Title: Highly efficient gene knockout in tumor-infiltrating lymphocytes by adenine base editing

doi: 10.1016/j.omton.2025.201041

Figure Lengend Snippet: Dual adenine base editing of TIM3 and TIGIT improves cytokine production and serial killing by TILs (A) Left: exemplary plots show the percentage of IFN-γ producing TILs activated with anti-CD3/CD28 beads. Right: Bar plots represent pooled data from 4 different TILs. (B) Left: exemplary plots show the percentage of IFN-γ producing TILs co-cultured with Mel-traget1. To evaluate tumor-specific effector function, REP-TILs were co-cultured at an effector-to-target ratio of 1:1 with autologous melanoma target cells overnight. Intracellular IFN-γ and TNF-α were measured using flow cytometry. Right: Bar plots represent pooled data from 3 independent experiments for each Melanoma-specific TIL. (C) Experimental design for tumor rechallenge experiment. The Illustration was created with BioRender.com . (D) Exemplary plot showing serial cytolytic ability of T cells determined by a real-time killing assay. The plot is exemplary of 3 independent experiments. Edited and non-edited TILs were co-cultured at an effector-to-target ratio of 2:1 with autologous melanoma target cells. T cell-mediated cytotoxicity was determined by measuring GFP positive tumor cells at 2-h intervals for approximately 90 h post T cell addition using the Incucyte S3 system. (E) Evaluation of exhaustion markers in TILs after repeated tumor challenge. Each plot represents pooled data from 2 independent experiments for melanoma-specific TIL. Mock-electroporated T cells were transfected with ABE mRNA alone and were used as a control in all experiments. For Statistical analysis paired t test was performed. Statistical significance indicated as ∗ p < 0.05 and ∗∗ p < 0.01. ns; not significant.

Article Snippet: For electroporation, frozen pre-REP TILs thawed and activated in serum-free X-VIVO15 supplemented with 3000 IU/mL of recombinant human IL-2 (Proleukin, Novartis, Basel, Switzerland) and 30 ng/ml GMP-grade soluble anti-CD3 (OKT3) antibody (Miltenyi Biotech) for 1–2 days before electroporation.

Techniques: Cell Culture, Flow Cytometry, Transfection, Control