cd8 media  (R&D Systems)

Journal: bioRxiv

Article Title: HIV-1 infection does not confer intrinsic resistance to cell death induced by cytotoxic T lymphocytes

doi: 10.64898/2026.03.23.713717

Figure Lengend Snippet: Experimental method to assess resistance to CTL-mediated killing. (A) Co-culture setup: primary CD4 + T cells from PLWH are pulsed with mutant p53 peptide and cultured with autologous, pre-expanded CD8 + T cells and p53-specific scDb. IPDA is performed on the viable CD4 + T cell population isolated before and after the co-culture to assess for enrichment of infected cells, indicative of resistance. (B) scDb mechanism of action: scDbs bind to cognate peptide:MHC complexes on the target cell surface and also to the CD3ε subunit of the TCR complex to induce localized cytotoxicity. (C) Flow cytometry gating strategy to determine frequency of viable target cells. (D) Representative histograms of co-cultures with and without scDbs. The reduction in the viable CD4 + T cell count (marked region) was used to determine the amount of CTL-mediated lysis. I Representative viability data for study participant OM5418 for the experimental condition and seven control conditions.

Article Snippet: Effector cells for co-cultures were expanded through stimulation with soluble anti-CD3 mAb (15 ng/mL, clone OKT3, BioLegend, Cat. # 317302) in CD8 media (base media + 250 U/uL human IL-2 (R&D Systems, Cat. # 202-IL-050/CF) + 5 ng/mL recombinant human IL-7 (BioLegend, Cat. # 581906)) for 72 hours.

Techniques: Co-Culture Assay, Mutagenesis, Cell Culture, Isolation, Infection, Flow Cytometry, Cell Characterization, Lysis, Control

Journal: bioRxiv

Article Title: HIV-1 infection does not confer intrinsic resistance to cell death induced by cytotoxic T lymphocytes

doi: 10.64898/2026.03.23.713717

Figure Lengend Snippet: Effect of active viral expression on cell death resistance. (A) Experimental setup: Healthy donor CD4 + T cells were activated for 72 hours with PHA and infected with different HIV-1 GFP-reporter constructs. Cells were then pulsed with exogenous p53 R175H peptide and co-cultured with pre-expanded autologous CD8 + T cells and p53-specific scDb or irrelevant control scDb. Viability and GFP-expression were analyzed through flow cytometry. (B) Genome maps of the HIV-1 reporter constructs used to infect target cells. Each row represents one reading frame. Striped segments indicate gene segments that are no longer translated due to insertion of a STOP-codon. (C) Representative flow plot showing viable uninfected (GFP-negative) and HIV-1-expressing (GFP-positive) cell populations. (D) Viability of uninfected target cells or cells infected with indicated HIV-1 reporter constructs after co-culture with the HLA-A2:p53 R175H -restricted scDb. Each dot shows the average of three technical replicates per condition. Symbols represent individual donors; assay was performed 2 independent times for each of the n=5 healthy donors, except for the full-length condition, where one donor was only tested once for technical reasons. A p-value less than 0.05 was considered significant. Abbreviations: PHA, phytohemagglutinin; LTR, long terminal repeat; IRES, internal ribosome entry site. ns: no significance (p > 0.05).

Article Snippet: Effector cells for co-cultures were expanded through stimulation with soluble anti-CD3 mAb (15 ng/mL, clone OKT3, BioLegend, Cat. # 317302) in CD8 media (base media + 250 U/uL human IL-2 (R&D Systems, Cat. # 202-IL-050/CF) + 5 ng/mL recombinant human IL-7 (BioLegend, Cat. # 581906)) for 72 hours.

Techniques: Expressing, Infection, Construct, Cell Culture, Control, Flow Cytometry, Co-Culture Assay