Absence of Stat1 in donor CD4+ T cells promotes the expansion of Tregs and reduces graft-versus-host disease in mice
Huihui Ma, … , Suzanne Lentzsch, Markus Y. Mapara
Huihui Ma, … , Suzanne Lentzsch, Markus Y. Mapara
Published June 13, 2011
Citation Information: J Clin Invest. 2011;121(7):2554-2569. https://doi.org/10.1172/JCI43706.
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Research Article Hematology

Absence of Stat1 in donor CD4+ T cells promotes the expansion of Tregs and reduces graft-versus-host disease in mice

Abstract

STAT1 is the main signal transducer for type I and II IFNs and plays a central role in the regulation of innate and adaptive immune responses. We used Stat1-deficient mice to test the role of donor Stat1 in MHC-matched minor histocompatibility antigen–mismatched (mHA-mismatched) and fully MHC-mismatched models of bone marrow transplantation. Lack of Stat1 in donor splenocytes reduced graft-versus-host disease (GVHD) in both immunogenetic disparities, leading to substantially attenuated morbidity and mortality. Donor Stat1 deficiency resulted in reduced alloantigen-induced activation and expansion of donor T cells and correlated with the expansion of CD4+CD25+Foxp3+ Tregs in vivo. This expansion of Tregs was further confirmed by studies showing that Stat1 deficiency promoted the proliferation, while inhibiting the apoptosis, of natural Tregs, and that absence of Stat1 enhanced the induction of inducible Tregs both in vitro and in vivo. Ex vivo expanded Stat1–/– Tregs were superior to wild-type Tregs in suppressing alloantigen-driven expansion of T cells in vitro and in inhibiting the development of GVHD. These observations demonstrate that Stat1 is a regulator of Tregs and that targeting Stat1 in CD4+ T cells may facilitate in vitro and in vivo expansion of Tregs for therapeutic use.

Authors

Huihui Ma, Caisheng Lu, Judith Ziegler, Ailing Liu, Antonia Sepulveda, Hideho Okada, Suzanne Lentzsch, Markus Y. Mapara

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Figure 7

Absence of Stat1 in CD4+ T cells leads to reduced cell death and enhanced proliferation of in vitro–cultured nTregs.

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Absence of Stat1 in CD4+ T cells leads to reduced cell death and enhance...
(A–D) Freshly purified Stat1+/+ and Stat1–/– CD4+CD25+ cells (1 × 105) were labeled with CFSE and stimulated for 3 days with α-CD3/α-CD28 (1 μg/ml) antibodies and IL-2 (10 ng/ml) in the absence (A–C) or presence (D) of 10 ng/ml human TGF-β1. Representative results from 3 independent experiments are shown. (A) Absolute numbers of Foxp3+ Tregs were calculated by multiplying viable cell numbers by the percentage of CD25+Foxp3+ cells. (B) Viability was determined by Trypan blue staining. Proliferation status of Tregs was analyzed by CFSE dilution in CD4+Foxp3+ cells in the absence (C) or presence (D) of human TGF-β1. Numbers represent the percentages of cells present in the given quadrants. (E and F) Freshly purified Stat1+/+ and Stat1–/– CD4+CD25+ cells were cultured for 3 days with α-CD3/α-CD28 antibodies, IL-2, and TGF-β in the absence and presence of α–IFN-γ (10 μg/ml) antibodies. (E) Apoptotic cell death was determined by Annexin V staining using FCM. Top: One representative experiment of 3 independent experiments is shown. Numbers represent the percentages of Annexin V+ cells. Bottom: Bar graph summary of the data. Data are mean ± SEM. (F) Phosphorylated Tyr701 Stat1 and Tyr705 Stat3 and total Stat1 and Stat3 expression in purified natural Tregs was analyzed by Western blot; β-actin was used as loading control. Blots are representative of 3 independent experiments.