Transcriptomic Analysis of αCD19 CAR T Cells Reveals Venetoclax-Induced Changes in T Cell Quality and Function
Authors: Mandeville Taylor K , Wright Jacob P , Mavis Cory , Cortese, MD, MPH Matthew J. , Rosario Spencer , Olejniczak Scott , Hernandez-Ilizaliturri Francisco 
Published: December 2024

Introduction
Bcl-2 expression is associated with poor outcomes in relapse/refractory (R/R) diffuse large B cell lymphoma (DLBCL) patients undergoing standard of care treatment, including patients that fail αCD19 CAR T therapy (CAR T). Venetoclax is a BH3 mimetic that selectively inhibits Bcl-2 protein. We previously observed Venetoclax-induced changes within CAR T cells via flow cytometry, noting heightened populations of TNaive (TN) and TStem Cell Memory (TSCM) memory subsets, decreased population frequency of TRegulatory (TReg) cells, increased cytotoxic effector function, and enhanced activation status. Here, we aim to validate these findings at the transcriptomic level, while elucidating the underlying mechanism supporting these Venetoclax-induced modifications to T cell quality and function.
Methods
Transduction was performed on T cells from three healthy donors using lentiviral vectors encoding for 2nd generation α-CD19 CAR constructs, containing CD28 (28ζ) or 4-1BB (BBζ) costimulatory domains, 24 hours after activation using αCD3/CD28 TransACT (Miltenyi). Untransduced T cells (UNT) were used as a nonspecific control. Following 10-14 days of expansion, CAR T cells and accompanying UNT T cells were harvested for downstream application. Briefly, either UNT, 28ζ, or BBζ CAR T cells were co-cultured with chemo-rituximab resistant CD19+ lymphoma model, Raji4RH, for 24 hours with or without Venetoclax-supplemented media. Cultures were then subjected to negative selection by Pan T cell Isolation Kit (Miltenyi) and submitted for bulk RNA sequencing (RNA-seq). RNA-seq data was analyzed using Spliced Transcripts Alignment to a Reference (STAR) algorithm, and a profile of differentially expressed genes (DEG) was created. Log2Fold Change of significant DEGs between Venetoclax-exposed and control (no exposure to Venetoclax) was then calculated for all genes of interest.
Results
Significant increase of IFNG (28ζ, Log2FC= 1.8; BBζ, Log2FC= 2.4) and GZMB (28ζ, Log2FC=0.12; BBζ, Log2FC=0.3) expression within Venetoclax-exposed CAR T cells was observed. DEGs governing T cell activation,CD69 (28ζ, Log2FC= 1.93; BBζ, Log2FC= 2.04), and proliferation and survival, IL2 (28ζ, Log2FC= 2.1; BBζ, Log2FC= 3.1) were significantly upregulated. To examine shifts in TN/TSCM and TReg subsets, TCF7, FOXP3, and IL12RB2 were analyzed.TCF7 (UNT, Log2FC= 0.13; 28ζ, Log2FC= -0.08; BBζ, Log2FC= 0.31) expression was significantly upregulated within UNT and BBζ Venetoclax-exposed cells, but not 28ζ. FOXP3 expression was significantly downregulated among all Venetoclax-exposed cells (UNT, Log2FC= -0.47; 28ζ, Log2FC= -0.3; BBζ, Log2FC= -0.4), and IL12RB2 expression was increased among all Venetoclax-exposed CAR T cells (28ζ, Log2FC= 0.35; BBζ, Log2FC= 0.41). BCL2 (28ζ, Log2FC= -0.13; BBζ, Log2FC= -0.14) expression was significantly decreased in all Venetoclax-exposed cells, with MCL1 (28ζ, Log2FC= 0.35; BBζ, Log2FC= 0.14) expression increasing among Venetoclax-exposed CAR T cells. Genes encoding for endoplasmic reticulum (ER) and mitochondria-associated proteins ITPR3 (28ζ, Log2FC= 0.32; BBζ, Log2FC= 0.35), MCU (28ζ, Log2FC= 0.82; BBζ, Log2FC= 0.31), and VDAC1 (28ζ, Log2FC= 1.5; BBζ, Log2FC= 0.1) were significantly increased in all Venetoclax-exposed cells.
Conclusion
Our data suggest that Venetoclax induced significant changes in CAR T cell function related to cytotoxicity (IFNG, GZMB), activation and survival (CD69, IL2), memory and immunomodulation (TCF7, IL12RB2, FOXP3), Bcl-2 family expression (BCL2, MCL1), and mitochondrial function (ITPR3, MCU, VDAC1). We sought to define a compensatory mechanism that exists among Bcl-2 family proteins in T cells following Venetoclax exposure. In all Venetoclax-exposed CAR T cells, MCL1 was significantly upregulated, while BCL2 was decreased. Due to localization of Bcl-2 at the ER where it inhibits flux of calcium via IP3R, we posit that Venetoclax induces T cell-intrinsic changes via calcium signaling modulation. Further understanding of how Venetoclax exposure impacts CAR T cell function is vital to enhance the quality of CAR T therapy in R/R DLBCL patients.