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Leung, Macyn

University of British Columbia

Regulatory T cells (Tregs) are a promising cell therapy for the prevention of immune-mediated pathologies including autoimmune diseases, allergy, transplant rejection and graft versus host disease. Early phase clinical trials have demonstrated Treg adoptive cell transfer to be safe, however the lack of a standardized potency assay to measure the suppressive function of Treg products makes it difficult to compare study results and refine products for optimal in vivo efficacy. Moreover, as clinical trials progress to later phases, potency assays become increasingly desirable by 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. I thus aimed to standardize and optimize a Treg suppression assay and expand on the mechanisms being measured within the in vitro assay. I first optimized the commonly varied steps and reagents such as time of co-culture, activation reagents, media, and types of responder cells that affected assay reproducibility and feasibility. Using Tregs isolated and expanded from discarded human thymus, I then tested the assay’s capacity to distinguish between suppression by activated Tregs versus conventional T cells, and incorporated suppression of B cell CD80 and CD86 expression as one of the assay readouts. I found that the optimal conditions for reproducible responder T cell proliferation was activation for 3 days with Dynabeads Human T-Expander CD3/CD28. Human serum supplementation in serum-free media was necessary for Treg suppression. Measuring suppression of T cell proliferation alone was poorly able to distinguish between suppression mediated by activated Tregs versus competition for resources by activated conventional T cells. However, suppression of CD80 and CD86 on B cells within the assay was Treg-specific. The measurement of CD80 and CD86 suppression on B cells can be readily incorporated into the classic Treg suppression assay and is a more specific measure of Treg function compared to suppression of T cell proliferation. By standardizing the media, activation reagents, and responder cell type, I have generated a robust, Treg-specific suppression assay that brings the field closer to fulfilling the critical need for a potency assay in Treg cell therapies.

Text

2023-08-09

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/85425

Experimental Medicine

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/