PMC

Triple combination vector protects CD8⁺ T cells from HIV-1 infection. PBMC-derived CD8⁺ T cells were stimulated by anti-CD3 and anti-CD28 antibodies for 3 days and transduced with EGFP-P2A-CDζ (CDζ), Triple combination vector (Triple CDζ) or control EGFP vector (EGFP). On day 4 post vector transduction, CDζ-positive populations were purified using anti-CD4 microbeads, and infected with HIV-1_NL4-3 or HIV-1_{NFNSX SL9} at MOI = 0.2 or 1.0, respectively. Cells were plated at 0.2 × 10⁵ cells/200 200 ml in a 96-well plate, and the amount of HIV-1 p24 production in culture supernatant was measured at days4,8 and 12 post infection(A. HIV-1_NL4-3 B. HIV-1_NFNSX SL9). (A&B) HIV-1 p24^Gag production in the culture supernatants was determined by ELISA. Numbers are mean ± SD from 3 indepedent wells.

CDζ CAR-transduced CD8⁺ T cells are susceptible to HIV-1 infection. A) CD8⁺ T cells were transduced with EGFP-P2A-CDζ (CDζ) or control EGFP (EGFP) vector. The cells transduced with the vector were sorted based on their EGFP expression and infected with one of three different HIV-1 strains, HIV-1_NL4-3, HIV-1_{NFNSX SL9} or HIV-1_JR-CSF at MOI = 0.2, 1.0 or 1.0, respectively. Cells were plated at 1 × 10⁵ cells/500 ml in a 48-well plate, and HIV-1 p24^Gag production in the culture supernatants was determined by ELISA at day 7 post infection. CD4⁺ T cells transduced with control EGFP were used as a positive control for HIV-1 infection. B) Intracellular HIV-1 p24^Gag staining by KC57 antibody. CD8⁺ T cells transduced with EGFP-P2A-CDζ (CDζ) or control EGFP (EGFP) vector were infected with HIV-1_NL4-3, HIV-1_{NFNSX SL9} or HIV-1_JR-CSF. Intracellular HIV-1 p24^Gag was analyzed by fiow cytometry after staining PE-conjugated KC57 antibody 7 days post infection.

Protecting CD8⁺ T cells from HIV-1 infection minimizes impairment of cell proliferation mediated by HIV-1 infection via CDζ CAR. CD8⁺ T cells modified with EGFP-P2A-CDζ (CDζ), Triple combination vector (Triple CDζ) or control EGFP vector (EGFP) described in were infected with HIV-1^NL4-3 or HIV-1^{NFNSX SL9} at MOI = 0.2 or 1.0, respectively. The cells were plated at 0.2 × 105 cells/200 ml in a 96 well plate, and total cell numbers in the wells were determined by MACSQuant analyzer. Numbers are mean ± SD from 3 independent wells. A two-tailed paired t test was used to assess a significant difference in CDζ or Triple CDζ group versus EGFP control group. *: statistically significant (p value less than or equal to 0.05). NS: not significant (p value greater than 0.1). p values: HIV-1^NL4-3/EGFP vs HIV-1^NL4-3/CDζ = 0.005, HIV-1^NL4-3/EGFP vs HIV-^1NL4-3/Triple CDζ = 0.0026, HIV-1NFNSX/EGFP vs HIV-1NFNSX/CDζ = 0.006, HIV-1NFNSX/EGFP vs HIV-N^1FNSX/Triple CDζ = 0.798, Mock/EGFP vs Mock/CDζ = 0.233, Mock/EGFP vs Mock/Triple CDζ = 0.606.

Triple combination vector exerts identical anti-HIV-1 effect with prototype CDζ CAR vector. A & B) Expression profile of Triple combination vector in CD8⁺ T cells. PBMC- derived CD8⁺ T cells were stimulated by anti-CD3 and anti-CD28 antibodies for 3 days, and transduced with EGFP-P2A-CDζ (CDζ), Triple combination vector (Triple CDζ) or control EGFP vector (EGFP). Cells were then cultured for 4 days and stained with anti-CD4 antibody (A) and anti-CCR5 antibody (B). C) HIV-1-dependent cytokine production by CDζ CAR. Cells transduced with Triple combination vector were incubated with T2 cells infected with or without HIV-1_NL4-3 for 16 h at a 10:1 E:T ratio. Intracellular cytokines (IFN-γ and TNF-α) were analyzed by flow cytometry. D) Triple CDζ induces HIV-1-dependent T2 cell killing. Cells transduced with EGFP-P2A-CDζ (CDζ), Triple combination vector (Triple CDζ) or control EGFP (EGFP) vector were fiow cytometry-sorted based on their EGFP expression, and incubated with T2 cells infected with or without HIV-1_NL4-3 labeled with Na₂(⁵¹CrO₄) for 3.5 h at a 10:1 E:T ratio. Cytolytic activity was determined by analysis of ⁵¹Cr release. Numbers are mean ± SD from 3 independent reactions. Total incorporated ⁵¹Cr was set as 100=. Two-tailed paired t test was used to calculate statistical significance. NS: not significant (p value greater than 0.1).