Figure 1.

Intracellular Costimulatory Domains Differentially Modulate HIV-Specific CAR₄ T Cell Cytokine Expression

(A) Schematic representation of each CAR construct and fluorescence-activated cell sorting (FACS) plots identifying HIV-specific CAR-modified CD4⁺ T cells (CAR₄) as GFP⁺ and CD4⁺ relative to untransduced CD4⁺ T cells (UTD₄). Purified human CD4⁺ T cells from a healthy human donor were activated with anti-CD3/CD28 Dynabeads and transduced with a lentiviral vector encoding one of six HIV-specific (CD4-based) CARs that express unique intracellular domains (ICDs), either CD3ζ (ζ), 4-1BBζ, CD28ζ, CD27ζ, OX40ζ, or ICOSζ. Each CAR was linked to GFP by an intervening T2A sequence to facilitate in vitro detection. (B) After 10 days of expansion, CAR₄ T cells were in vitro stimulated with HIV_YU2 GFP160⁺ K562 cells (K.Env), and intracellular cytokine analysis was performed. Data are representative of three donors. (C) Heatmap showing the percentage of responding CAR₄ T cells for each of the indicated cytokines. (D) Polyfunctionality profiles of combinatorial subsets for CAR₄ T cells producing 0–5 human cytokines: TNF, IL-2, IFN-γ, GM-CSF, and MIP-1β. (C and D) Data are the average of three donors. (E) Summary data of three donors per CAR₄ T cell population producing two or more cytokine functions after antigen stimulation. Lines indicate mean, and error bars show ±SEM.