Transcriptomic Analysis of αCD19 CAR T Cells Reveals Venetoclax-Induced Changes in T Cell Quality and Function

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 T_Naive (T_N) and T_{Stem Cell Memory} (T_SCM) memory subsets, decreased population frequency of T_Regulatory (T_Reg) 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 T_N/T­_SCM and T_Reg­ 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.