What question did this study set out to answer?

The research aims to understand SRC-3's role in maintaining FOXP3 expression and suppressive function in engineered Tregs.

April 5, 2026

Abstract 5960: CRISPR-Cas engineering of allogeneic regulatory T cells reveals an essential role for steroid receptor coactivator 3 (SRC-3) in maintaining FOXP3 expression and suppressive function

Key Points

The research aims to understand SRC-3's role in maintaining FOXP3 expression and suppressive function in engineered Tregs.
Generated hypoimmunogenic Tregs using CRISPR-Cas editing to knockout B2M, TCRα/β, and SRC-3.
Analyzed the impact of SRC-3 deletion on Treg suppressive function and lineage commitment.
Performed RNA sequencing to assess transcript diversity and expression levels in knockout Tregs.
SRC-3 knockout Tregs showed impaired suppressive function and reduced FOXP3 levels.
Triple knockout of TCR+B2M+SRC-3 resulted in greater transcript diversity and altered chemokine expression.
Co-culture with unedited Tregs restored some suppressive function, highlighting a compensatory mechanism.

Abstract

Abstract Genetically engineered allogeneic regulatory T cells (Tregs) represent a promising off-the-shelf therapy to prevent graft-versus-host disease and transplant rejection, but their clinical utility is constrained by immune rejection and alloreactivity. Using CRISPR-Cas editing, we generated hypoimmunogenic human Tregs through multiplex knockout of B2M (reducing HLA class I), TCRα/β (eliminating receptor-mediated alloreactivity), and SRC-3 (a transcriptional coactivator linked to Treg stability). In prior murine studies, SRC-3 disruption enhanced tumor clearance, but in human Tregs, its deletion impaired suppressive function, underscoring SRC-3’s role in maintaining lineage identity. Functionally, SRC-3 knockout Tregs failed to suppress effector T cell proliferation, a defect mirrored in triple knockout (TCR+B2M+SRC-3) cells. Co-culture with unedited Tregs restored suppression, suggesting functional compensation. FOXP3 protein levels were markedly reduced in SRC-3 and triple knockout Tregs, consistent with destabilized lineage commitment. These findings highlight SRC-3, TCR, and B2M as central regulators of transcriptional and antigen-presentation pathways shaping Treg identity. RNA sequencing revealed that triple knockout Tregs exhibited increased transcript diversity compared to controls. SRC-1/NCOA1 and SRC-2/NCOA2 were upregulated in SRC-3 and triple knockout samples, while B2M and TRAC were strongly downregulated in triple knockouts. Chemokine transcripts were elevated in SRC-3 and triple knockout Tregs, with triple knockouts also showing diverse cytokine upregulation. Collectively, these results demonstrate that while genome editing can enhance allogeneic compatibility, SRC-3 is indispensable for human Treg stability and suppressive function. Effective engineering of universal Tregs must therefore balance hypoimmunogenic design with preservation of functional integrity for adoptive cell therapy. Citation Format: Subhashree Pradhan, Bryan Nikolai, Aiden Lynn Moser, Davis Alexander Graham, Lanz Rainer, Bert W O'Malley, David Lonard. CRISPR-Cas engineering of allogeneic regulatory T cells reveals an essential role for steroid receptor coactivator 3 (SRC-3) in maintaining FOXP3 expression and suppressive function abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5960.

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