Human HLA-DR+CD27+ regulatory T cells show enhanced antigen-specific suppressive function
Xiaoqian Ma, Lu Cao, Martina Raneri, Hannah Wang, Qi Cao, Yuanfei Zhao, Naiara G. Bediaga, Gaetano Naselli, Leonard C. Harrison, Wayne J. Hawthorne, Min Hu, Shounan Yi, Philip J. O’Connell
Xiaoqian Ma, Lu Cao, Martina Raneri, Hannah Wang, Qi Cao, Yuanfei Zhao, Naiara G. Bediaga, Gaetano Naselli, Leonard C. Harrison, Wayne J. Hawthorne, Min Hu, Shounan Yi, Philip J. O’Connell
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Research Article Immunology Transplantation

Human HLA-DR+CD27+ regulatory T cells show enhanced antigen-specific suppressive function

Abstract

Regulatory T cells (Tregs) have potential for the treatment of autoimmune diseases and graft rejection. Antigen specificity and functional stability are considered critical for their therapeutic efficacy. In this study, expansion of human Tregs in the presence of porcine PBMCs (xenoantigen-expanded Tregs, Xn-Treg) allowed the selection of a distinct Treg subset, coexpressing the activation/memory surface markers HLA-DR and CD27 with enhanced proportion of FOXP3+Helios+ Tregs. Compared with their unsorted and HLA-DR+CD27+ double-positive (DP) cell–depleted Xn-Treg counterparts, HLA-DR+CD27+ DP-enriched Xn-Tregs expressed upregulated Treg function markers CD95 and ICOS with enhanced suppression of xenogeneic but not polyclonal mixed lymphocyte reaction. They also had less Treg-specific demethylation in the region of FOXP3 and were more resistant to conversion to effector cells under inflammatory conditions. Adoptive transfer of porcine islet recipient NOD/SCID IL2 receptor γ–/– mice with HLA-DR+CD27+ DP-enriched Xn-Tregs in a humanized mouse model inhibited porcine islet graft rejection mediated by 25-fold more human effector cells. The prolonged graft survival was associated with enhanced accumulation of FOXP3+ Tregs and upregulated expression of Treg functional genes, IL10 and cytotoxic T lymphocyte antigen 4, but downregulated expression of effector Th1, Th2, and Th17 cytokine genes, within surviving grafts. Collectively, human HLA-DR+CD27+ DP-enriched Xn-Tregs expressed a specific regulatory signature that enabled identification and isolation of antigen-specific and functionally stable Tregs with potential as a Treg-based therapy.

Authors

Xiaoqian Ma, Lu Cao, Martina Raneri, Hannah Wang, Qi Cao, Yuanfei Zhao, Naiara G. Bediaga, Gaetano Naselli, Leonard C. Harrison, Wayne J. Hawthorne, Min Hu, Shounan Yi, Philip J. O’Connell

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Figure 3

In vitro suppression assay of HLA-DR+CD27+ DP-enriched Xn-Tregs.

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In vitro suppression assay of HLA-DR+CD27+ DP-enriched Xn-Tregs. (A) MLR...
(A) MLR assay of Xn-Treg suppressive capacity compared with Pc-Treg. Carboxyfluorescein diacetate succinimidyl ester–labeled (CFSE-labeled) autologous human PBMCs (CD4+CD25+CD127–/lo depleted) were stimulated with irradiated xenogeneic porcine (Xn-MLR) or allogeneic (Allo MLR) PBMCs or anti-CD3/CD28 dynabeads (Poly MLR), in the presence or absence of serial dilutions of unsorted Xn-Treg or HLA-DR+CD27+ DP-enriched Xn-Treg or HLA-DR+CD27+ DP-depleted Xn-Treg or anti-CD3/CD28 dynabead–expanded Pc-Treg for 7 days, prior to measurement of PBMC proliferation by CFSE dilution. (B) Alloantigen-expanded Treg (Al-Treg) suppressive capacity in Xn-MLR. CFSE-labeled autologous human PBMCs were stimulated with irradiated xenogeneic porcine PBMCs, in the presence or absence of serial dilutions of unsorted Al-Treg or HLA-DR+CD27+ DP-enriched Al-Treg or HLA-DR+CD27+ DP-depleted Al-Treg or Pc-Treg for 7 days, prior to measurement of PBMC proliferation by CFSE dilution. (C) Assessment of IFN-γ concentration in supernatants of the Xn-MLR assay of Xn-Treg suppression. IFN-γ secretion in supernatants collected from xenogeneic MLR assay as described in A was measured by ELISA. Data are presented as mean ± SD of 4 independent experiments with Tregs from 4 individual donors (A) except in Poly MLR with 6 individual donors for Xn-Treg, DP-depleted Xn-Treg, and DP-enriched Xn-Treg, from 3 individual donors (C) and 6 independent experiments with Treg from 6 individual donors (B). Two-way ANOVA: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.