Abstract B063: A gene-editable human regulatory T-cell platform reveals USP22 as a regulator of FOXP3 stability and IFN-γ production

Abstract

Regulatory T cells (Tregs) are key modulators of immune responses and are increasingly targeted for their therapeutic potential in hematologic malignancies, autoimmune diseases, and immune-mediated complications of hematopoietic cell transplantation. Robust human Treg assays are therefore essential to evaluate novel targets and therapeutic modalities. However, due to experimental challenges in isolation, expansion, and optimization of reliable functional readouts, progress has been slow and results remain variable. In this study, we established a human Treg platform enabling high-purity isolation, expansion, functional profiling, and gene modification, using the oncogenic deubiquitinase USP22 as a reference target. In our hands, Tregs achieved >90% purity (CD4+CD25+FOXP3+) with 92% viability and maintained a stable phenotype through Days 7, 15, and 18, reaching approximately 60–80-fold expansion before plateauing. Expanded Tregs demonstrated potent suppressive activity, with reduced responder T-cell proliferation as Tresp:Treg ratios decreased from 32:1 to 2:1, accompanied by increased anti-inflammatory cytokine production. Genome editing of USP22 was efficient and compatible with downstream proliferation, phenotypic stability, and cytokine analyses. Approximately 60% knockdown of USP22 led to a ∼4-fold increase in IFN-γ mRNA expression and loss of FOXP3 at both mRNA and protein levels, accompanied by reduced IL-2 mRNA expression, while IL-2RA (CD25) and CTLA-4 mRNA levels remained unchanged. We developed a human Treg workflow that integrates high-purity isolation, robust expansion, and functional characterization with gene modification capability. USP22 perturbation demonstrates the utility of this system in dissecting molecular regulators of Treg function. This flexible platform can be adapted for diverse gene targets, including deubiquitinases such as USP22, to support preclinical immunology and cell therapy research relevant to hematologic and autoimmune diseases. Shereen Kesserwan, Sarju Patel, Madison Greer, Sukanya Jadhav, Palin Biner, Ajita V. Singh. A gene-editable human regulatory T-cell platform reveals USP22 as a regulator of FOXP3 stability and IFN-γ production [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr B063.