Restimulation After Cryopreservation and Thawing Preserves the Phenotype and Function of Expanded Baboon Regulatory T Cells

Abstract

Background: Regulatory T cells (Treg) are being explored for their tolerance-inducing capabilities. Freezing and banking Treg for future use makes this strategy more clinically applicable. We aimed to devise an improved method of expanding and cryopreserving Treg to maximize yield, purity, and function for use in xenotransplantation.

Methods: Baboon peripheral blood mononuclear cells (PBMC) were isolated from whole blood. CD4+/CD25^hi cells were isolated by flow cytometric sorting and expanded for 26 days in culture with IL-2, anti-CD3 antibody, artificial APCs transfected with human CD58, CD32, and CD80, and rapamycin with weekly restimulations. Expanded Treg were frozen for 2 months then thawed and cultured for 48 hours in medium plus 1) no additives, 2) IL-2, 3) anti-CD3 antibody, 4) IL-2 + anti-CD3 antibody, and 5) IL-2 + anti-CD3 antibody + L cells. Phenotype and suppression were assessed after expansion, immediately after thawing, and after culturing.

Results: We expanded purified baboon Treg more than 10,000-fold. Expanded Treg exhibited excellent suppression in functional assays. Cryopreservation decreased suppressive function without changing phenotype, but increasing amounts of reactivation after thawing produced significantly better viability and suppressive function with a trend towards greater Treg purity.

Conclusions: We produced numbers of expanded Tregs consistent with clinical use. In contrast to some previous reports, both Treg phenotype and suppressive function were preserved or even enhanced by increasing amounts of restimulation after thawing. Thus, banking of expanded recipient Tregs for in vivo infusion should be possible.

FIGURE 1

Expanded Treg lines. Treg expansion in five lines between 2 animals using the protocol described in this paper. Expansion levels were as high as 10,000-fold.

FIGURE 2

Cell numbers after reactivation. Four million thawed Treg were plated for 48 hours in each of 5 conditions: media plus (1) no additives, (2) IL-2, (3) anti-CD3, (4) IL-2 + anti-CD3, and (5) IL-2 + anti-CD3 + L cells (artificial APCs transfected with human CD58, CD32, and CD80). Average cell numbers and standard deviation for each condition after 48 hours are shown. This experiment was performed 5 times using 3 unique Treg lines. NS: non-significant. *: P < 0.05 by ANOVA. ANOVA, analysis of variance.

FIGURE 3

Phenotypes after cryopreservation. Pooled phenotypes as determined by flow cytometry immediately after thawing and after restimulation for 48 hours in the 3 conditions that permitted cell survival. Shown are average percentages and standard deviation for 5 separate experiments using 3 unique Treg lines. Phenotypic differences are not statistically significant (P ≥ 0.05), but there was a trend toward significance for CD4 (P = 0.13) and CD8 (P = 0.059). Gates for CD25 and Foxp3 were established using isotype controls.

FIGURE 4

Suppression after cryopreservation. Pooled suppression data from suppression assays carried out using Treg before freezing, immediately after thawing, and after restimulation for 48 hours in the 3 conditions that permitted cell survival. Shown are average percentages and standard deviation for responder cell proliferation stimulated by anti-CD3/CD28 beads at various titrations of Treg from 2 different lines. There is a significant difference in suppression under different conditions at each titer by ANOVA (P = 0.0007 at 1:2, otherwise P ≤ 0.0001). The specific conditions that differ by Tukey post hoc analysis are specified above each titer. For post hoc analysis, *P ≤ 0.05; **P ≤0.01; ***P ≤ 0.001.