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Abstracts Session 1

Sunday April 30, 2023 - 16:40 to 18:00

Room: Grand Georgian

2.11 Development of a potency assay for regulatory T cell therapy applications

Macyn L Leung, Canada

Masters of Science student
Department of Medicine
University of British Columbia
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Development of a potency assay for regulatory T cell therapy applications

Macyn Leung1,2, Sabine Ivison2,3, Katherine N MacDonald2,4, Lieke Sanderink2,3, Megan K Levings2,3,4.

1Department of Medicine, University of British Columbia, Vancouver, BC, Canada; 2Childhood Diseases, BC Children’s Hospital Research Institute, Vancouver, BC, Canada; 3Department of Surgery, University of British Columbia, Vancouver, BC, Canada; 4School of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada

Introduction: Regulatory T cells (Tregs) are a promising cell therapy for the prevention of rejection and graft versus host disease after transplantation. Clinical trials have demonstrated Treg adoptive cell transfer to be safe, however the lack of a standardized functional assay to measure and compare the potency of different Treg products makes it difficult to compare study results and refine products for optimal in vivo efficacy. Moreover, potency assays are highly desirable for regulatory agencies. In vitro suppression of T cell proliferation is the current gold standard for measuring Treg function, but the current protocols are highly variable and the key points of technical versus biological variation are undefined. We thus aimed to standardize and optimize a Treg suppression assay.
Methods: Tregs were isolated and expanded from discarded human thymus. Responder targets of suppression were allogeneic peripheral blood mononuclear cells (PBMCs) or CD3-enriched PBMCs. We first tested how commonly varied steps and reagents – such as time of co-culture, activation reagents, media, and types of responder cells – affected assay reproducibility and feasibility. We then tested the assay’s capacity to distinguish between suppression mediated by activated Tregs versus conventional T cells, and incorporated transendocytosis of CD80 and CD86 from B cells as one of the assay readouts.
Results: We found the optimal conditions for reproducible responder T cell proliferation was activation for 3 days with Dynabeads Human T-Expander CD3/CD28. Media comparisons revealed that optimal Treg suppression required the addition of human serum to serum-free media; suppression assays in the absence of serum showed poor Treg suppression even at high Treg-to-PBMC ratios (e.g., 1:2.5). Measuring suppression of T cell proliferation was poorly able to distinguish between suppression mediated by activated Tregs versus inhibitory effects of activated conventional T cells. In contrast, transendocytosis of CD80 and CD86 on B cells (gated as CD19+ cells in PBMCs) was only mediated by Tregs and not by activated CD4+ conventional T cells.  Interestingly, assay reproducibility was more strongly affected by intra-experimental factors (i.e. different experimental days) than a biological variation due to the use of Tregs or PBMCs from different individuals.
Conclusion: Measurement of CD80 and CD86 transendocytosis can be readily incorporated into Treg suppression assays and is a more specific measure of Treg function compared to suppression of T cell proliferation. Suppression assay variation can be reduced by standardizing media, activation reagents, and responder cell type, thus bringing us closer to fulfilling the critical need for a potency assay to test the efficacy of Treg therapies.

The authors are thankful for the volunteers, patients and their parents for contribution of samples along with the surgical and cardiac clinic staff at the British Columbia Children's Hospital who made this study possible, with special thanks to Allison Jamieson, Melanie Ganshorn, Aliyah Hassan, Lyn Nguyen and Colleen Ring.


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