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Background: Survival after lung transplantation (LTx) is hampered by uncontrolled inflammation and alloimmunity. Regulatory T-cells (Tregs) are being studied as a cellular therapy in solid organ transplantation. Whether these systemically administered Tregs can function at the appropriate location and time is an important concern. We hypothesised that in vitro -expanded recipient-derived Tregs can be delivered to donor lungs prior to LTx via ex vivo lung perfusion (EVLP), maintaining their immunomodulatory ability., Methods: In a rat model, Wistar Kyoto (WKy) CD4 + CD25 high Tregs were expanded in vitro prior to EVLP. Expanded Tregs were administered to Fisher 344 (F344) donor lungs during EVLP; left lungs were transplanted into WKy recipients. Treg localisation and function post-transplant were assessed. In a proof-of-concept experiment, cryopreserved expanded human CD4 + CD25 + CD127 low Tregs were thawed and injected into discarded human lungs during EVLP., Results: Rat Tregs entered the lung parenchyma and retained suppressive function. Expanded Tregs had no adverse effect on donor lung physiology during EVLP; lung water as measured by wet-to-dry weight ratio was reduced by Treg therapy. The administered cells remained in the graft at 3 days post-transplant where they reduced activation of intra-graft effector CD4 + T-cells; these effects were diminished by day 7. Human Tregs entered the lung parenchyma during EVLP where they expressed key immunoregulatory molecules (CTLA4 + , 4-1BB + , CD39 + and CD15s + )., Conclusions: Pre-transplant Treg administration can inhibit alloimmunity within the lung allograft at early time points post-transplant. Our organ-directed approach has potential for clinical translation., Competing Interests: Conflict of interest: E. Miyamoto reports fellowships from The Kyoto University Foundation, Japan; the Cell Science Research Foundation, Japan; and the International Society for Heart and Lung Transplantation, during the conduct of the study. Conflict of interest: A. Takahagi has nothing to disclose. Conflict of interest: A. Ohsumi has nothing to disclose. Conflict of interest: T. Martinu has nothing to disclose. Conflict of interest: D. Hwang has nothing to disclose. Conflict of interest: K.M. Boonstra has nothing to disclose. Conflict of interest: B. Joe has nothing to disclose. Conflict of interest: J.M. Umana has nothing to disclose. Conflict of interest: K.F. Bei has nothing to disclose. Conflict of interest: D. Vosoughi has nothing to disclose. Conflict of interest: M. Liu has nothing to disclose. Conflict of interest: M. Cypel reports being co-founder of Perfusix Canada and Traferox Technologies Inc. Toronto, and reports consultancy for Lung Bioengineering and United Therapeutics, during the conduct of the study. Conflict of interest: S. Keshavjee reports being co-founder of Perfusix Canada and Traferox Technologies Inc. Toronto, and reports consultancy for Lung Bioengineering and United Therapeutics, during the conduct of the study. Conflict of interest: S.C. Juvet reports grants from Ontario Institute for Regenerative Medicine and Cystic Fibrosis Canada, during the conduct of the study., (Copyright ©The authors 2022. For reproduction rights and permissions contact permissions@ersnet.org.)

Regulatory T cell (Treg) therapy is a potential curative approach for a variety of immune-mediated conditions, including autoimmunity and transplantation, in which there is pathological tissue damage. In mice, IL-33R (ST2)-expressing Tregs mediate tissue repair by producing the growth factor amphiregulin, but whether similar tissue-reparative Tregs exist in humans remains unclear. We show that human Tregs in blood and multiple tissue types produced amphiregulin, but this was neither a unique feature of Tregs nor selectively upregulated in tissues. Human Tregs in blood, tonsil, synovial fluid, colon, and lung tissues did not express ST2, so ST2 + Tregs were engineered via lentiviral-mediated overexpression, and their therapeutic potential for cell therapy was examined. Engineered ST2 + Tregs exhibited TCR-independent, IL-33-stimulated amphiregulin expression and a heightened ability to induce M2-like macrophages. The finding that amphiregulin-producing Tregs have a noneffector phenotype and are progressively lost upon TCR-induced proliferation and differentiation suggests that the tissue repair capacity of human Tregs may be an innate function that operates independently from their classical suppressive function., (Copyright © 2019 by The American Association of Immunologists, Inc.)

T lymphocytes bearing the αβ T cell receptor (TCR) but lacking CD4, CD8, and markers of natural killer (NK) cell differentiation are known as 'double-negative' (DN) T cells and have been described in both humans and rodent models. We and others have shown that DN T cells can act as regulatory T cells (Tregs) that are able to prevent allograft rejection, graft-versus-host disease, and autoimmune diabetes. In the last few years, new data have revealed evidence of DN Treg function in vivo in rodents and humans. Moreover, significant advances have been made in the mechanisms by which DN Tregs target antigen-specific T cells. One major limitation of the field is the lack of a specific marker that can be used to distinguish truly regulatory DN T cells (DN Tregs) from non-regulatory ones, and this is the central challenge in the coming years. Here, we review recent progress on the role of DN Tregs in transplantation and autoimmunity, and their mechanisms of action. We also provide some perspectives on how DN Tregs compare with Foxp3(+) Tregs.

TCRαβ(+) CD3(+) CD4(-) CD8(-) NK1.1(-) double negative (DN) Tregs comprise 1-3% of peripheral T lymphocytes in mice and humans. It has been demonstrated that DN Tregs can suppress allo-, xeno- and auto-immune responses in an Ag-specific fashion. However, the mechanisms by which DN Tregs regulate immune responses remain elusive. Whether DN Tregs can regulate DCs has not been investigated previously. In this study, we demonstrate that DN Tregs express a high level of CTLA4 and are able to down-regulate costimulatory molecules CD80 and CD86 expressed on Ag-expressing mature DCs (mDCs). DN Tregs from CTLA4 KO mice were not able to downregulate CD80 and CD86 expression, indicating that CTLA4 is critical for DN Treg-mediated downregulation of costimulatory molecule expression on Ag-expressing mature DCs. Furthermore, DN Tregs could kill both immature and mature allogeneic DCs, as well as Ag-loaded syngeneic DCs, in an Ag-specific manner in vitro and in vivo, mainly through the Fas-FasL pathway. These data demonstrate, for the first time, that DN Tregs are potent regulators of DCs and may have the potential to be developed as a novel immune suppression treatment., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Peripheral αβTCR(+)CD3(+)CD4(-)CD8(-) NK1.1/CD56(-) double-negative (DN) Treg cells are a relatively rare subset of regulatory cells found in both humans and mice, typically comprising less than 5% of the total peripheral T-cell pool. Numerous studies have shown that DN Tregs can inhibit CD4(+) and CD8(+) T-cell responses in vitro and in vivo using a variety of model systems [Zhang et al., Nature Medicine 6:782, 2000; Young et al., Blood 100:3408, 2002; Ford et al., Experimental Medicine 196:261, 2002; Young et al., Journal of Immunology 171:134, 2003; Ford et al., European Journal of Immunology 37:2234, 2007; Zhang et al., Blood 109:4071, 2007; Fischer et al., Blood 105:2828, 2005]. This chapter describes published methods for the phenotypic identification of DN Tregs, their isolation from secondary lymphoid organs of mice or human peripheral blood, activation and expansion, and assays for their ability to suppress T-cell proliferation, induce apoptosis, and promote tolerance to allografts in vivo.

Limited data are available on the mechanisms that constrain the function of regulatory populations of T cells. Prostaglandin E2 (PGE2) is an endogenous membrane phospholipid metabolite that has important immunomodulatory effects on T cell function. Our previous microarray data indicated that E prostanoid receptor 2 (EP2), a receptor for PGE2, is expressed by regulatory alphabetaTCR(+) CD4(-) CD8(-) NK1.1(-) double negative T (DN Treg) cell clones but not by their non-regulatory natural mutants. Hence, the hypothesis that PGE2 may influence DN Treg cell proliferation and/or regulatory function was tested in this study. Our data indicate that PGE2 acts via the EP2 receptor on DN Treg cells to inhibit their proliferation, an effect reproduced by the EP2-specific agonist butaprost and abrogated by the EP2 antagonist AH6809. In contrast, PGE2 did not affect the ability of DN Treg cells to kill syngeneic CD8(+) T cells activated by allogeneic stimulation. Together, these findings suggest a role for PGE2 in limiting the expansion of DN Treg cells.