Chronic graft-versus-host disease (cGVHD) remains the main cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (AHSCT). cGVHD is a multisystem, immune-mediated disease characterized by tissue fibrosis and immune dysregulation of T and B cells (MacDonald et al., 2017MacDonald K.P. Hill G.R. Blazar B.R. Chronic graft-versus-host disease: biological insights from preclinical and clinical studies.Blood. 2017; 129: 13-21Crossref PubMed Scopus (120) Google Scholar). The pathophysiology of cGVHD involves homeostatic abnormalities of thymus-derived regulatory T cells (tTregs, described as CD4+ FOXP3+ IL-2Ra/CD25hi IL-7Ra/CD127low), including low tTreg numbers, decreased thymic export, and increased susceptibility to Fas-mediated apoptosis (Matsuoka et al., 2010Matsuoka K. Kim H.T. McDonough S. Bascug G. Warshauer B. Koreth J. et al.Altered regulatory T cell homeostasis in patients with CD4+ lymphopenia following allogeneic hematopoietic stem cell transplantation.J Clin Invest. 2010; 120: 1479-1493Crossref PubMed Scopus (166) Google Scholar). In addition, cGVHD is associated with elevated levels of IL-7 and functional IL-2 deficiency (Matsuoka et al., 2013Matsuoka K. Koreth J. Kim H.T. Bascug G. McDonough S. Kawano Y. et al.Low-dose interleukin-2 therapy restores regulatory T cell homeostasis in patients with chronic graft-versus-host disease.Sci Transl Med. 2013; 5: 179ra43Crossref PubMed Scopus (294) Google Scholar). Treatment of cGVHD with low-dose subcutaneous IL-2 has proven to be safe and increase tTreg numbers (Koreth et al., 2011Koreth J. Matsuoka K. Kim H.T. McDonough S.M. Bindra B. Alyea 3rd, E.P. et al.Interleukin-2 and regulatory T cells in graft-versus-host disease.N Engl J Med. 2011; 365: 2055-2066Crossref PubMed Scopus (731) Google Scholar). The role of other cell subpopulations with regulatory functions and their possible usage for the treatment of cGVHD is under investigation (Schneidawind et al., 2013Schneidawind D. Pierini A. Negrin R.S. Regulatory T cells and natural killer T cells for modulation of GVHD following allogeneic hematopoietic cell transplantation.Blood. 2013; 122: 3116-3121Crossref PubMed Scopus (65) Google Scholar). Type 1 adaptive regulatory T cells (Tr1s) are FOXP3-negative CD4 T cells that secrete high amounts of IL-10, have immunosuppressive properties, and are found more frequently in AHSCT recipients with persistent mixed chimerism than in those with complete donor chimerism (Gagliani et al., 2013Gagliani N. Magnani C.F. Huber S. Gianolini M.E. Pala M. Licona-Limon P. et al.Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells.Nat Med. 2013; 19: 739-746Crossref PubMed Scopus (473) Google Scholar). In addition to IL-10, Tr1s produce high amounts of TGF-β and variable levels of IFN-γ, but no IL-2 or IL-4 (Groux et al., 1997Groux H. O'Garra A. Bigler M. Rouleau M. Antonenko S. de Vries J.E. et al.A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis.Nature. 1997; 389: 737-742Crossref PubMed Scopus (3068) Google Scholar). Coexpression of CD49b and LAG-3 was described as a specific surrogate marker of Tr1s (Gagliani et al., 2013Gagliani N. Magnani C.F. Huber S. Gianolini M.E. Pala M. Licona-Limon P. et al.Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells.Nat Med. 2013; 19: 739-746Crossref PubMed Scopus (473) Google Scholar), facilitating their study in humans. A phase I clinical study demonstrated the efficacy of in vitro engineered Tr1-like cells (host antigen–specific IL-10–anergized T cells) after AHSCT (Bacchetta et al., 2014Bacchetta R. Lucarelli B. Sartirana C. Gregori S. Lupo Stanghellini M.T. Miqueu P. et al.Immunological outcome in haploidentical-HSC transplanted patients treated with IL-10-anergized donor T cells.Front Immunol. 2014; 5: 16Crossref PubMed Scopus (102) Google Scholar, Zhang and Hill, 2019Zhang P. Hill G.R. Interleukin-10 mediated immune regulation after stem cell transplantation: mechanisms and implications for therapeutic intervention.Semin Immunol. 2019; 44: 101322Crossref PubMed Scopus (3) Google Scholar), but the role of Tr1s in human cGVHD needs to be further investigated. Herein, we prospectively analyzed Tr1 and tTreg frequency, phenotype, and function in the peripheral blood of 14 AHSCT patients with active cGVHD, 11 with cGVHD in remission, 12 without cGVHD, and 12 healthy donors. All samples were collected following written informed consent according to the Declaration of Helsinki. This study received the agreement of the local ethics committee. Tr1s were defined as CD3+CD4+CD45RA−CD49b+LAG3+ and tTregs as CD3+CD4+CD25hiCD127−. Patients’ characteristics are summarized in Supplementary Table S1. Tr1 frequencies were higher in AHSCT patients with active cGVHD than in patients with cGVHD in remission (10% and 2.3%, respectively; P < 0.001), patients without cGVHD (3.8%, P < 0.001), and healthy donors (4.0%, P < 0.001) (Figure 1a and b). Conversely, tTreg frequencies were lower in patients with active cGVHD than patients with cGVHD in remission (5.5% and 9.5%, respectively; P = 0.03), patients without cGVHD (11.2%, P = 0.02), and healthy donors (10.9%, P = 0.01) (Figure 1b). Therefore, the Tr1:tTreg ratio was reversed in patients with active cGVHD compared with patients with cGVHD in remission (2.0 and 0.4, respectively; P < 0.001), patients without cGVHD (0.4, P < 0.001), and healthy donors (0.5, P < 0.001) (Figure 1b). Three patients experienced partial remission of cGVHD after low-dose IL-2 treatment and showed an early drop in Tr1 frequencies (from 24% to 13% on average) and increase in tTreg frequencies (from 6% to 13.2% on average) one week after treatment. Two of these patients were further analyzed 12 and 24 months later while they had ongoing remission of cGVHD. Notably, the frequencies of Tr1s and tTregs and the Tr1:tTreg ratio remained at values comparable to those in healthy donors (Figure 1c). In all study subjects, Tr1s expressed lower levels of IL-2Rα than tTregs (P < 0.001) but higher levels than conventional T cells (P < 0.001). Conversely, levels of IL-7Rα in Tr1s were higher than in tTregs (P < 0.001) but similar to those in conventional T cells (Figure 1d). These results are consistent with the previously observed increased levels of IL-7 in patients with cGVHD (Matsuoka et al., 2013Matsuoka K. Koreth J. Kim H.T. Bascug G. McDonough S. Kawano Y. et al.Low-dose interleukin-2 therapy restores regulatory T cell homeostasis in patients with chronic graft-versus-host disease.Sci Transl Med. 2013; 5: 179ra43Crossref PubMed Scopus (294) Google Scholar), which could stimulate Tr1 expansion. They are also in line with the absence of expansion of Tr1s after IL-2 treatment. The mechanisms that lead to Tr1 drop after IL-2 treatment remain to be understood but could involve IL-7 deprivation, as IL-7 levels were shown to decrease in the serum of patients treated with IL-2 (Matsuoka et al., 2013Matsuoka K. Koreth J. Kim H.T. Bascug G. McDonough S. Kawano Y. et al.Low-dose interleukin-2 therapy restores regulatory T cell homeostasis in patients with chronic graft-versus-host disease.Sci Transl Med. 2013; 5: 179ra43Crossref PubMed Scopus (294) Google Scholar). Next, we measured IL-10 secretion of CD4 T cells following anti-CD3/CD28 stimulation, as described previously (Gagliani et al., 2013Gagliani N. Magnani C.F. Huber S. Gianolini M.E. Pala M. Licona-Limon P. et al.Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells.Nat Med. 2013; 19: 739-746Crossref PubMed Scopus (473) Google Scholar). As shown in Figure 2a and b, patients with active cGVHD had increased frequencies of IL-10–secreting CD4 T cells compared with patients with cGVHD in remission (2.6% vs. 0.5%, respectively; P = 0.02), patients without cGVHD (1.0%, P = 0.03), and healthy donors (1.4%, P = 0.19). The proportions of IL-10–secreting cells correlated with those of Tr1s across all patient groups (r = 0.7, P = 0.003, Figure 2c), and IL-10–secreting cells were significantly enriched within the CD45RA−CD49b+LAG3+ subset of CD4 T cells (Figure 2d). Finally, we assessed the suppressive capacity of bead-purified IL-10–secreting CD4 T cells on autologous CD4 T-cell proliferation in response to anti-CD3/CD28 stimulation. As shown in Figure 2e, IL-10+ CD4 T cells significantly suppressed autologous CD4 T-cell proliferation as compared with IL-10− CD4 T cells. Altogether, these data indicate that CD3+CD4+CD45RA−CD49b+LAG3+ cells have the hallmark functional properties of Tr1s, and that Tr1s are expanded in active cGVHD. Several papers reported an impaired recovery of circulating functional CD4+CD25+ tTregs in patients with active cGVHD compared with patients without cGVHD after AHSCT or healthy donors (Alho et al., 2016Alho A.C. Kim H.T. Chammas M.J. Reynolds C.G. Matos T.R. Forcade E. et al.Unbalanced recovery of regulatory and effector T cells after allogeneic stem cell transplantation contributes to chronic GVHD.Blood. 2016; 127: 646-657Crossref PubMed Scopus (91) Google Scholar, Matsuoka et al., 2013Matsuoka K. Koreth J. Kim H.T. Bascug G. McDonough S. Kawano Y. et al.Low-dose interleukin-2 therapy restores regulatory T cell homeostasis in patients with chronic graft-versus-host disease.Sci Transl Med. 2013; 5: 179ra43Crossref PubMed Scopus (294) Google Scholar, Matsuoka et al., 2010Matsuoka K. Kim H.T. McDonough S. Bascug G. Warshauer B. Koreth J. et al.Altered regulatory T cell homeostasis in patients with CD4+ lymphopenia following allogeneic hematopoietic stem cell transplantation.J Clin Invest. 2010; 120: 1479-1493Crossref PubMed Scopus (166) Google Scholar, Zorn et al., 2005Zorn E. Kim H.T. Lee S.J. Floyd B.H. Litsa D. Arumugarajah S. et al.Reduced frequency of FOXP3+ CD4+CD25+ regulatory T cells in patients with chronic graft-versus-host disease.Blood. 2005; 106: 2903-2911Crossref PubMed Scopus (366) Google Scholar). The role of Tr1s in the maintenance of immune tolerance to non–self-antigens after AHSCT may be complex (Gagliani et al., 2013Gagliani N. Magnani C.F. Huber S. Gianolini M.E. Pala M. Licona-Limon P. et al.Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells.Nat Med. 2013; 19: 739-746Crossref PubMed Scopus (473) Google Scholar, Roncarolo et al., 2011Roncarolo M.G. Gregori S. Lucarelli B. Ciceri F. Bacchetta R. Clinical tolerance in allogeneic hematopoietic stem cell transplantation.Immunol Rev. 2011; 241: 145-163Crossref PubMed Scopus (63) Google Scholar), especially as the functional plasticity of these cell subsets has been demonstrated. In inflammatory conditions, IL-10–producing Tr1s can derive from the differentiation of tTregs (Le Buanec et al., 2011Le Buanec H. Gougeon M.L. Mathian A. Lebon P. Dupont J.M. Peltre G. et al.IFN-α and CD46 stimulation are associated with active lupus and skew natural T regulatory cell differentiation to type 1 regulatory T (Tr1) cells.Proc Natl Acad Sci USA. 2011; 108: 18995-19000Crossref PubMed Scopus (35) Google Scholar). Immunoregulatory mechanisms are generally induced in the context of immune activation and inflammation. We may then speculate that increased Tr1 levels could represent a compensatory mechanism to (i) the inflammatory environment and (ii) the impaired Treg recovery in these patients. Notably, the increase in Tr1 levels may not be sufficient to circumvent ongoing cGVHD. Thus, our data do not preclude that Tr1s may play a beneficial role to prevent cGVHD and be used in an adoptive transfer therapy. Additional in situ studies of Tr1 localization and proportion in skin tissue would improve our understanding of the regulatory properties of Tr1s in the setting of cGVHD. Future in vivo studies should allow us to optimize adoptive transfer strategies to treat this disease. Datasets related to this article can be found at https://flowrepository.org/experiments/2596, hosted at Flow Repository. Nana Talvard-Balland: http://orcid.org/0000-0001-8590-6351 Aurélien Sutra Del Galy: http://orcid.org/0000-0002-4716-1472 David Michonneau: http://orcid.org/0000-0003-4553-3065 Helene Le Buanec: http://orcid.org/0000-0002-2139-0357 Francois Chasset: http://orcid.org/0000-0003-2580-8607 Marie Robin: http://orcid.org/0000-0001-7655-4083 Régis Peffault de Latour: http://orcid.org/0000-0001-6222-4753 Alienor Xhaard: http://orcid.org/0000-0002-4449-989X Flore Sicre de Fontbrune: http://orcid.org/0000-0003-2000-1556 Nathalie Parquet: http://orcid.org/0000-0002-4254-9857 Sophie Duchez: http://orcid.org/0000-0003-1629-1810 Valérie Schiavon: http://orcid.org/0000-0001-8466-774X Michel Rybojad: http://orcid.org/0000-0002-6276-7261 Anne Bergeron-Lafaurie: http://orcid.org/0000-0003-2156-254X Martine Bagot: http://orcid.org/0000-0002-0400-1954 Armand Bensussan: http://orcid.org/0000-0002-0409-2497 Sophie Caillat-Zucman: http://orcid.org/0000-0002-4535-3550 Gérard Socié: http://orcid.org/0000-0002-2114-7533 Jean-David Bouaziz: http://orcid.org/0000-0002-4993-2461 Adèle de Masson: http://orcid.org/0000-0001-7828-6211 The authors state no conflict of interest. The authors thank Christelle Doliger and Niclas Setterblad (Plateforme Technologique de l’Institut de Recherche Saint-Louis). This work was supported by Société Française de Dermatologie and Association d’Entraide des Greffés de Moëlle Osseuse. Nana Talvard-Balland was supported by the Ligue Nationale contre le Cancer. Adèle de Masson was supported by the Institut National du Cancer/Institut Thématique Multi-Organismes Cancer. Conceptualization: ADM, JDB, GS, SCZ, HLB; Data Curation: ADM, HLB, SD, VS; Formal Analysis: ADM, NTB, JDB; Investigation: ADM, NTB, JDB; Writing - Original Draft Preparation: ADM, JDB, NTB, GS, SCZ; Writing - Review and Editing: NTB, ASDG, DM, HLB, FC, MR, RPDL, AX, FSDF, NP, SD, VS, AB, MB, AB, SCZ, GS, JDB, ADM This prospective study was conducted at Saint-Louis Hospital from January 2014 to June 2015. The diagnosis and staging of chronic graft-versus-host disease were made at study inclusion using the National Institutes of Health criteria (Lee et al., 2015Lee S.J. Wolff D. Kitko C. Koreth J. Inamoto Y. Jagasia M. et al.Measuring therapeutic response in chronic graft-versus-host diseaseNational Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: IV. The 2014 Response Criteria Working Group report.Biol Blood Marrow Transplant. 2015; 21: 984-999Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). All samples were collected following written informed consent. This study received the agreement of the local ethics committee. Patients’ characteristics are shown in Supplementary Table S1. Peripheral blood mononuclear cells were isolated by density gradient centrifugation (LSM, PAA Laboratories) and stained using combinations of the following antibodies: anti-CD3-Krome Orange (clone UCHT1), anti-CD4 TITC (SFCI12T4D11), and anti-CD25 PE-Cy7 (B1.49.9) (all from Beckman Coulter, Brea, CA); anti-CD45RA PerCP-Cy5.5 (HI100, Biolegend, San Diego, CA); anti-CD127 APC-eFluor 780 (eBio-RDR5), anti-CD49b APC (P1H5), and anti-Foxp3 PE (PCH101) (all from eBioscience, San Diego, CA); anti-LAG3 PE or PerCP (polyclonal goat IgG, R&D Systems, Minneapolis, MN); and Fixable Viability Stain 450 (BD Biosciences, San Jose, CA). Fluorescence minus one was used as control. Data were acquired on a BD Canto II cytometer (Becton Dickinson, Franklin Lakes, NJ) and analyzed using FlowJo Software version X. Thymus-derived regulatory T cells, conventional T cells, and type 1 adaptive regulatory T cells were identified as live CD3+CD4+CD25hiCD127lo cells, CD3+CD4+CD25loCD127+ cells, and CD3+CD4+CD45RA−CD49b+LAG3+ cells, respectively. CD4 T cells were isolated from freshly isolated peripheral blood mononuclear cells using the CD4 T-cell isolation kit (Miltenyi Biotec). Cell purity was shown to be >95% by flow cytometry. CD4 T cells were plated at 106 cells/ml in complete RMPI 1640 medium and stimulated for 72 hours at 37 °C using plate-bound anti-CD3 antibody (1 μg/ml, OKT3, eBioscience) and soluble anti-CD28 antibody (10 μg/ml, CD28.2, BD Biosciences). After 72 hours, cells were washed and assessed for IL-10 production by flow cytometry or further enriched where indicated, using the IL-10 secretion assay (Miltenyi Biotec) according to provider’s recommendations. Purified CD4 T cells were labeled with CellTrace CFSE (0.5 μM, Life Technologies, Carlsbad, CA). IL-10–secreting CD4 T cells (suppressor cells) were enriched as described previously and added to autologous carboxyfluorescein succinimidyl ester–labeled CD4 T cells (responder cells) in a 1:1 ratio. IL-10–negative CD4 T cells were used as negative control. Cells were then stimulated for 5 days as described previously and proliferation of CD4 T cells was assessed by carboxyfluorescein succinimidyl ester dilution. Statistical analyses were performed using Prism (GraphPad) for Windows. Medians were compared between two groups using nonparametric Mann-Whitney U tests. P-value < 0.05 was considered significant. Abbreviations: ALL, acute lymphocytic leukemia; AML, acute myelogenous leukemia; cGVHD, chronic graft-versus-host disease; HSCT, hematopoietic stem cell transplantation; MAC, myeloablative conditioning; MDS, myelodysplastic syndrome; MM, multiple myeloma; MMF, mycophenolate mofetil; MPS, myeloproliferative syndrome; MTX, methotrexate; MUD, matched unrelated donor; NA, not applicable; NHL, non-Hodgkin lymphoma; RIC, reduced-intensity conditioning.

Expansion of Circulating CD49b+LAG3+ Type 1 Regulatory T Cells in Human Chronic Graft-Versus-Host Disease