Purification and Expansion of Antigen-Specific Human Tregs for Clinical Application Using a Single Step CD25 Positive Selection

Regulatory T cells (Tregs) play a role in maintaining T cell homeostasis and self-tolerance. Tregs immunotherapy has great potential for the prevention of allograft rejection and graft-vs-host disease (GVHD), but GMP reagents and procedures are required for clinical use. We have shown that allo-specific Tregs can be isolated by positive CD25 selection and negative selection of CD127, CD8 and CD19 by Miltenyi immunobeads, and then expanded in amounts sufficient for potential clinical use. Such a number of immunobeads would make a GMP procedure so costly to be clinically unaffordable. Therefore, in this study we tested whether a single step of CD25 positive selection using magnetic micro beads followed by expansion with allogeneic DC, rapamycin, IL-2, and IL-15 for 12 days generates Tregs that retain their phenotype (Foxp3) expression to a high level, are minimally contaminated with non-Tregs, and are highly suppressive.

We explored a simplified approach that employs only CD25 positive selection, followed by culture. Concerns still remain on the purity and contamination of the expanded population, in particular contamination with cytotoxic CD8⁺T cells as these cells could have unfavorable outcomes in immunotherapy trials.

We purified Tregs using the ‘clinical grade’ CD25 human positive selection magnetic beads from Miltenyi; we then labeled the Tregs with CFSE and cultured with allogeneic monocyte-derived DC as stimulus in the presence of rapamycin, IL-2 and IL-15. At the end of 12 days expanded allo-specific Tregs were analyzed for their phenotype, sorted and tested for alloreactive and suppressive functions. We performed a total of 10 experiments:

The purity of CD4⁺CD25⁺CD127dim Foxp3⁺after the initial separation ranged from 20–83%, median-44%.

To assess whether the non-Tregs in the culture were allo-reactive we sorted the expanded Tregs into: CFSE⁺CD25⁺, CFSE⁻CD25⁻, CFSE⁺CD25⁻, CFSE⁻CD25⁺, we then set up a 3 and 6 days mixed leukocyte reaction (MLR) against the initial stimulator or self. Non-Tregs in the culture did not respond to antigen with or without IL-2, whereas the allo-reactive Tregs, CFSE⁻CD25⁺, responded to allogeneic DC only in the presence of IL-2. CFSE⁺ Tregs did not respond even in the presence of IL-2. To determine whether the entire expanded population was enriched with antigen specific suppression, we culture the enriched ‘Tregs’ at various ratios with naïve autologous CD4⁺CD25⁻responder T cells and allogeneic DC from original stimulator, and found that a 50% suppression was achieved with a 1:2000 Treg:Tconv ratio. To analyze fidelity of cytokine secretion profile, we re-stimulated cultured ‘Tregs’ with allogeneic DC or PMA/Iononycin and compared data to expanded Tconv from the same individual; Tregs had significantly more TGF-beta and less TNF-alpha secretion than Tconv.

Our data demonstrate that Tregs purified with the GMP-compatible CD25 Miltenyi immunobeads can be expanded, retain high levels of Foxp3 expression, and maintain high suppressive potency. The frequency of contaminating cells after culture was small and we did not observed residual alloreactivity.

No relevant conflicts of interest to declare.