Human Leukocyte Antigen (HLA) Class II Expression on Regulatory T Cells (Tregs) Isolated from Discarded Human Thymus is Induced by In Vitro Expansion Conditions

Abstract

Tregs modulate immune responses and are a promising therapeutic tool to limit graft rejection in organ transplantation. We showed that potent Tregs can be isolated and expanded from human thymuses, routinely removed during pediatric cardiac surgery, indicating potential for an 'off-the-shelf' tolerogenic therapy. Upregulation of HLA on expanded Tregs may increase their immunogenicity, which is a possible obstacle for clinical purpose. Here, we studied HLA class II expression on thymic Tregs under various expansion conditions. FOXP3+CD25+ Tregs were isolated from infant thymuses (n=5) by mechanical dissociation and magnetic-bead-based cell separation and cultured under various conditions for 14 days (TABLE 1). Anti-IFN-γ antibodies were added to condition B to study the role of IFN-γ. Treg phenotype and HLA class II expression were assessed by flow cytometry. Cytokine production was analyzed by multiplex assay. All culture conditions expanded the Treg population, except condition H. Cultures with artificial antigen-presenting cells (conditions A, B, C, D) contained fewer HLA class II-expressing Tregs than T Cell Activator cultures (conditions E, F, G) [range: 2-45% vs. 18-84%, respectively; p<0.001]. Additionally, CST OpTmizer medium (conditions C, D) induced less class II-expressing Tregs than ImmunoCult-XF medium (conditions A, B) [range: 2-29% vs. 17-45%, respectively; p=0.004]. Addition of rapamycin had no effect. The % class II+ Tregs was positively correlated to IFN-γ production (p=0.02) and decreased in the presence of anti-IFN-γ antibodies in a dose-dependent manner. The conditions used for Treg expansion affected the percentage of thymic Tregs that upregulate HLA class II, which at least in part was due to IFN-γ production by the cells. Expansion protocols that limit IFN-γ production may therefore generate less immunogenic Tregs for tolerogenic therapy in organ transplantation.