Treatment with agonistic DR3 antibody results in expansion of donor Tregs and reduced graft-versus-host disease
2015; 126 (4): 546-557
Donor Requirements for Regulatory T Cell Suppression of Murine Graft-versus-Host Disease
JOURNAL OF IMMUNOLOGY
2015; 195 (1): 347-355
The paucity of regulatory T cells limits clinical translation to control aberrant immune reactions including graft-versus-host disease. Recent studies showed that the agonistic antibody to DR3 (αDR3) expanded CD4(+)FoxP3(+) Tregs in vivo. We investigated whether treating donor mice with a single dose of αDR3 could alleviate acute GVHD in a MHC-mismatched bone marrow transplantation model. αDR3 induced selective proliferation of functional Tregs. CD4(+) T cells isolated from αDR3-treated mice contained higher numbers of Tregs and were less proliferative to allogeneic stimuli. In vivo GVHD studies confirmed that Tregs from αDR3-treated donors expanded robustly and higher frequencies of Tregs within donor CD4(+) T cells were maintained, resulting in improved survival. Conventional T cells derived from αDR3-treated donors showed reduced activation and proliferation. Serum levels of pro-inflammatory cytokines (IFNγ, IL-1β and TNFα) and infiltration of donor T cells into GVHD target tissues (gastrointestinal tract and liver) were decreased. T cells from αDR3-treated donors retained graft-versus-tumor effects. In conclusion, a single dose of αDR3 alleviates acute GVHD while preserving GVT effects by selectively expanding and maintaining donor Tregs. This novel strategy will facilitate the clinical application of Treg based therapies.
View details for DOI 10.1182/blood-2015-04-637587
View details for Web of Science ID 000358869300021
View details for PubMedID 26063163
Third-party CD4(+) invariant natural killer T cells protect from murine GVHD lethality
2015; 125 (22): 3491-3500
Adoptive transfer of freshly isolated natural occurring CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) prevents graft-versus-host disease (GVHD) in several animal models and following hematopoietic cell transplantation (HCT) in clinical trials. Donor-derived Treg have been mainly used, as they share the same MHC with CD4(+) and CD8(+) conventional T cells (Tcon) that are primarily responsible for GVHD. Third party-derived Treg are a promising alternative for cellular therapy, as they can be prepared in advance, screened for pathogens and activity, and banked. We explored MHC disparities between Treg and Tcon in HCT to evaluate the impact of different Treg populations in GVHD prevention and survival. Third-party Treg and donor Treg are equally suppressive in ex vivo assays, whereas both donor and third-party but not host Treg protect from GVHD in allogeneic HCT, with donor Treg being the most effective. In an MHC minor mismatched transplantation model (C57BL/6 → BALB/b), donor and third-party Treg were equally effective in controlling GVHD. Furthermore, using an in vivo Treg depletion mouse model, we found that Treg exert their main suppressive activity in the first 2 d after transplantation. Third-party Treg survive for a shorter period of time after adoptive transfer, but despite the shorter survival, they control Tcon proliferation in the early phases of HCT. These studies provide relevant insights on the mechanisms of Treg-mediated protection from GVHD and support for the use of third-party Treg in clinical trials.
View details for DOI 10.4049/jimmunol.1402861
View details for Web of Science ID 000358064500042
Haploidentical hematopoietic transplantation from KIR ligand-mismatched donors with activating KIRs reduces nonrelapse mortality
2015; 125 (20): 3173-3182
Graft-versus-host disease (GVHD) is driven by extensive activation and proliferation of alloreactive donor T cells causing significant morbidity and mortality following allogeneic hematopoietic cell transplantation (HCT). Invariant natural killer T (iNKT) cells are a potent immunoregulatory T-cell subset in both humans and mice. Here, we explored the role of adoptively transferred third party CD4(+) iNKT cells for protection from lethal GVHD in a murine model of allogeneic HCT across major histocompatibility barriers. We found that low numbers of CD4(+) iNKT cells from third party mice resulted in a significant survival benefit with retained graft-versus-tumor (GVT) effects. In vivo expansion of alloreactive T cells was diminished while displaying a Th2-biased phenotype. Notably, CD4(+) iNKT cells from third party mice were as protective as CD4(+) iNKT cells from donor mice although third party CD4(+) iNKT cells were rejected early after allogeneic HCT. Adoptive transfer of third party CD4(+) iNKT cells resulted in a robust expansion of donor CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) that were required for protection from lethal GVHD. However, in vivo depletion of myeloid-derived suppressor cells (MDSCs) abrogated both Treg expansion and protection from lethal GVHD. Despite the fact that iNKT cells are a rare cell population, the almost unlimited third party availability and feasibility of in vitro expansion provide the basis for clinical translation.
View details for DOI 10.1182/blood-2014-11-612762
View details for Web of Science ID 000355979800021
Engraftment of Embryonic Stem Cells and Differentiated Progeny by Host Conditioning with Total Lymphoid Irradiation and Regulatory T Cells
2015; 10 (11): 1793-1802
CD4(+) invariant natural killer T cells protect from murine GVHD lethality through expansion of donor CD4(+)CD25(+)FoxP3(+) regulatory T cells
2014; 124 (22): 3320-3328
Because activating killer cell immunoglobulinlike receptors (KIRs) are heterogeneously expressed in the population, we investigated the role of donor activating KIRs in haploidentical hematopoietic transplants for acute leukemia. Transplants were grouped according to presence vs absence of KIR-ligand mismatches in the graft-vs-host direction (ie, of donor-vs-recipient natural killer [NK]-cell alloreactivity). In the absence of donor-vs-recipient NK-cell alloreactivity, donor activating KIRs had no effects on outcomes. In the 69 transplant pairs with donor-vs-recipient NK-cell alloreactivity, transplantation from donors with KIR2DS1 and/or KIR3DS1 was associated with reduced risk of nonrelapse mortality, largely infection related (KIR2DS1 present vs absent: hazard ratio [HR], 0.25; P = .01; KIR3DS1 present vs absent: HR, 0.18; P = .006), and better event-free survival (KIR2DS1 present vs absent: HR, 0.31; P = .011; KIR3DS1 present vs absent: HR, 0.30; P = .008). Transplantation from donors with KIR2DS1 and/or KIR3DS1 was also associated with a 50% reduction in infection rate (P = .003). In vitro analyses showed that KIR2DS1 binding to its HLA-C2 ligand upregulated inflammatory cytokine production by alloreactive NK cells in response to infectious challenges. Because ∼40% of donors able to exert donor-vs-recipient NK-cell alloreactivity carry KIR2DS1 and/or KIR3DS1, searching for them may become a feasible, additional criterion in donor selection.
View details for DOI 10.1182/blood-2014-09-599993
View details for Web of Science ID 000355689000022
View details for PubMedID 25769621
"Designed" grafts for HLA-haploidentical stem cell transplantation.
2014; 123 (7): 967-973
Dysregulated donor T cells lead to destruction of host tissues resulting in graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). We investigated the impact of highly purified (>95%) donor CD4(+) invariant natural killer T (iNKT) cells on GVHD in a murine model of allogeneic HCT. We found that low doses of adoptively transferred donor CD4(+) iNKT cells protect from GVHD morbidity and mortality through an expansion of donor CD4(+)CD25(+)FoxP3(+) regulatory T cells (Treg). These Treg express high levels of the Ikaros transcription factor Helios and expand from the Treg pool of the donor graft. Furthermore, CD4(+) iNKT cells preserve T cell-mediated graft-versus-tumor (GVT) effects. Our studies reveal new aspects of the cellular interplay between iNKT cells and Treg in the context of tolerance induction after allogeneic HCT and set the stage for clinical translation.
View details for DOI 10.1182/blood-2014-05-576017
View details for Web of Science ID 000347463100021
HLA-haploidentical transplantation with regulatory and conventional T cell adoptive immunotherapy prevents acute leukemia relapse.
Today human leukocyte antigen-haploidentical transplantation is a feasible option for patients with high-risk acute leukemia who do not have matched donors. Whether it is T-cell replete or T-cell depleted, it is still, however, associated with issues of transplant-related mortality and posttransplant leukemia relapse. After reports that adoptive immunotherapy with T-regulatory cells controls the alloreactivity of conventional T lymphocytes in animal models, tomorrow's world of haploidentical transplantation will focus on new "designed" grafts. They will contain an appropriate ratio of conventional T lymphocytes and T-regulatory cells, natural killer cells, γ δ T cells, and other accessory cells. Preliminary results of ongoing clinical trials show the approach is feasible. It is associated with better immune reconstitution and a quite powerful graft-versus-leukemia effect with a low incidence of graft-versus-host disease and no need for posttransplant pharmacological prophylaxis. Future strategies will focus on enhancing the clinical benefit of T-regulatory cells by increasing their number and strengthening their function.
View details for DOI 10.1182/blood-2013-10-531764
View details for PubMedID 24363403
Mast cells suppress murine GVHD in a mechanism independent of CD4(+)CD25(+) regulatory T cells
2013; 122 (22): 3659-3665
Post-transplant relapse is still the major cause of treatment failure in high-risk acute leukemia. Attempts to manipulate alloreactive T cells to spare normal cells while killing leukemic cells have been unsuccessful. In HLA-haploidentical transplantation we reported that donor-derived T regulatory cells (Tregs), co-infused with conventional T cells (Tcons), protected recipients against graft versus host disease (GvHD). The present phase II study investigated whether Treg-Tcon adoptive immunotherapy prevents post-transplant leukemia relapse. 43 adults with high-risk acute leukemia (AML 33; ALL 10) were conditioned with a TBI-based regimen. Grafts included CD34(+) cells (mean 9.7x10(6)/kg), Tregs (mean 2.5x10(6)/kg) and Tcons (mean 1.1x10(6)/kg). No post-transplant immunosuppression was given. 95% patients achieved full-donor type engraftment. 15% developed ≥ grade II acute GvHD. The probability of disease-free survival was 0.56 at a median follow-up of 46 months. The very low cumulative incidence of relapse (0.05) was significantly better than in historical controls. These results demonstrate the immunosuppressive potential of Tregs can be used to suppress GvHD without loss of the benefits of GvL activity. Humanized murine models provided insights into the mechanisms underlying separation of GvL from GvHD, suggesting the GvL effect is due to largely unopposed Tcon alloantigen recognition in bone marrow.
View details for DOI 10.1182/blood-2014-03-564401
View details for PubMedID 24923299
Regulatory T cells and natural killer T cells for modulation of GVHD following allogeneic hematopoietic cell transplantation
2013; 122 (18): 3116-3121
To investigate the role of mast cells in hematopoietic cell transplantation, we assessed graft-versus-host disease (GVHD) in C57BL/6-Kit(W-sh/W-sh) recipients, which virtually lack mast cells, compared with C57BL/6 WT recipients. GVHD was severely exacerbated in C57BL/6-Kit(W-sh/W-sh) mice (median survival time = 13 vs 60 days in wild-type [WT] mice; P < .0001). The increased mortality risk in C57BL/6-Kit(W-sh/W-sh) hosts correlated with increased T-cell numbers in lymph nodes, liver, and gastrointestinal tract sites, as indicated by bioluminescence imaging (P < .001). We did not detect any deficit in the number or function of CD4(+)CD25(+) regulatory T cells (Tregs) in C57BL/6-Kit(W-sh/W-sh) mice. Furthermore, Tregs were equally effective at reducing GVHD in C57BL/6-Kit(W-sh/W-sh) recipients compared with WT recipients containing mast cells. Furthermore, we found that survival of C57BL/6-Kit(W-sh/W-sh) mice during GVHD was significantly improved if the mice were engrafted with bone marrow-derived cultured mast cells from WT C57BL/6 mice but not from interleukin (IL)-10-deficient C57BL/6 mice. These data indicate that the presence of mast cells can significantly reduce GVHD independently of Tregs, by decreasing conventional T-cell proliferation in a mechanism involving IL-10. These experiments support the conclusion that mast cells can mediate a novel immunoregulatory role during hematopoietic cell transplantation.
View details for DOI 10.1182/blood-2013-08-519157
View details for Web of Science ID 000329726200022
T regulatory cell separation for clinical application
TRANSFUSION AND APHERESIS SCIENCE
2012; 47 (2): 213-216
Alloreactivity of donor lymphocytes leads to graft-versus-host disease (GVHD) contributing to significant morbidity and mortality following allogeneic hematopoietic cell transplantation (HCT). Within the past decade, significant progress has been made in elucidating the mechanisms underlying the immunologic dysregulation characteristic of GVHD. The recent discoveries of different cell subpopulations with immune regulatory function has led to a number of studies aimed at understanding their role in allogeneic HCT and possible application for the prevention and treatment of GVHD and a host of other immune-mediated diseases. Preclinical animal modeling has helped define the potential roles of distinct populations of regulatory cells that have progressed to clinical translation with promising early results.
View details for DOI 10.1182/blood-2013-08-453126
View details for Web of Science ID 000326503200010
View details for PubMedID 24068494
TLR3 essentially promotes protective class I-restricted memory CD8(+) T-cell responses to Aspergillus fumigatus in hematopoietic transplanted patients
2012; 119 (4): 967-977
We selected T regulatory cells (Tregs) from standard leukapheresis using double-negative selection (anti-CD8 and anti-CD19) followed by positive selection (anti-CD25) and 72 procedures were performed. A median of 263×10(6)cells (range 143-470×10(6)) were recovered with a mean of CD4(+)/CD25(+) cells of 94.5±2.4% (36.5±18.6% CD4(+)/CD25(+hi)). FoxP3(+) cells were equal to 79.8%±22.2. CD127(+) cells were 12.5%±8.2. The inhibition assay showed an inhibition rate of 67±22. Cells isolated by means of this approach can be used in allogeneic hematopoietic stem cell transplantation to reduce the incidence and severity of GvHD without bystander inhibition of general immunity.
View details for DOI 10.1016/j.transci.2012.06.007
View details for Web of Science ID 000308849100018
View details for PubMedID 22795999
FISH analysis reveals frequent co-occurrence of 4q24/TET2 and 5q and/or 7q deletions
2012; 36 (1): 37-41
Aspergillus fumigatus is a model fungal pathogen and a common cause of severe infections and diseases. CD8⁺ T cells are present in the human and murine T-cell repertoire to the fungus. However, CD8⁺ T-cell function in infection and the molecular mechanisms that control their priming and differentiation into effector and memory cells in vivo remain elusive. In the present study, we report that both CD4⁺ and CD8⁺ T cells mediate protective memory responses to the fungus contingent on the nature of the fungal vaccine. Mechanistically, class I MHC-restricted, CD8⁺ memory T cells were activated through TLR3 sensing of fungal RNA by cross-presenting dendritic cells. Genetic deficiency of TLR3 was associated with susceptibility to aspergillosis and concomitant failure to activate memory-protective CD8⁺ T cells both in mice and in patients receiving stem-cell transplantations. Therefore, TLR3 essentially promotes antifungal memory CD8⁺ T-cell responses and its deficiency is a novel susceptibility factor for aspergillosis in high-risk patients.
View details for DOI 10.1182/blood-2011-06-362582
View details for Web of Science ID 000299860700011
View details for PubMedID 22147891
Genetically-Determined Hyperfunction of the S100B/RAGE Axis Is a Risk Factor for Aspergillosis in Stem Cell Transplant Recipients
2011; 6 (11)
We investigated TET2 deletion in 418 patients with hematological malignancies. Overall interphase FISH detected complete or partial TET2 monoallelic deletion (TET2(del)) in 20/418 cases (4.7%). TET2(del) was very rare in lymphoid malignancies (1/242 cases; 0.4%). Among 19 positive myeloid malignancies TET2(del) was associated with a 4q24 karyotypic abnormality in 18 cases. In AML, TET2(del) occurred in CD34-positive hematopoietic precursors and preceded established genomic abnormalities, such as 5q- and -7/7q-, which were the most frequent associated changes (Fisher's exact test P=0.000).
View details for DOI 10.1016/j.leukres.2011.08.004
View details for Web of Science ID 000298149100025
View details for PubMedID 21920603
Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation
2011; 117 (14): 3921-3928
Invasive aspergillosis (IA) is a major threat to the successful outcome of hematopoietic stem cell transplantation (HSCT), although individual risk varies considerably. Recent evidence has established a pivotal role for a danger sensing mechanism implicating the S100B/receptor for advanced glycation end products (RAGE) axis in antifungal immunity. The association of selected genetic variants in the S100B/RAGE axis with susceptibility to IA was investigated in 223 consecutive patients undergoing HSCT. Furthermore, studies addressing the functional consequences of these variants were performed. Susceptibility to IA was significantly associated with two distinct polymorphisms in RAGE (-374T/A) and S100B (+427C/T) genes, the relative contribution of each depended on their presence in both transplantation counterparts [patient SNP(RAGE), adjusted hazard ratio (HR), 1.97; P = 0.042 and donor SNP(RAGE), HR, 2.03; P = 0.047] or in donors (SNP(S100B), HR, 3.15; P = 7.8e-(4)) only, respectively. Functional assays demonstrated a gain-of-function phenotype of both variants, as shown by the enhanced expression of inflammatory cytokines in RAGE polymorphic cells and increased S100B secretion in vitro and in vivo in the presence of the S100B polymorphism. These findings point to a relevant role of the danger sensing signaling in human antifungal immunity and highlight a possible contribution of a genetically-determined hyperfunction of the S100B/RAGE axis to susceptibility to IA in the HSCT setting.
View details for DOI 10.1371/journal.pone.0027962
View details for Web of Science ID 000297555800046
View details for PubMedID 22114731
Dectin-1 Y238X polymorphism associates with susceptibility to invasive aspergillosis in hematopoietic transplantation through impairment of both recipient- and donor-dependent mechanisms of antifungal immunity
2010; 116 (24): 5394-5402
Hastening posttransplantation immune reconstitution is a key challenge in human leukocyte antigen (HLA)-haploidentical hematopoietic stem-cell transplantation (HSCT). In experimental models of mismatched HSCT, T-regulatory cells (Tregs) when co-infused with conventional T cells (Tcons) favored posttransplantation immune reconstitution and prevented lethal graft-versus-host disease (GVHD). In the present study, we evaluated the impact of early infusion of Tregs, followed by Tcons, on GVHD prevention and immunologic reconstitution in 28 patients with high-risk hematologic malignancies who underwent HLA-haploidentical HSCT. We show for the first time in humans that adoptive transfer of Tregs prevented GVHD in the absence of any posttransplantation immunosuppression, promoted lymphoid reconstitution, improved immunity to opportunistic pathogens, and did not weaken the graft-versus-leukemia effect. This study provides evidence that Tregs are a conserved mechanism in humans.
View details for DOI 10.1182/blood-2010-10-311894
View details for Web of Science ID 000289265500028
View details for PubMedID 21292771
The C-type lectin receptor Dectin-1 plays a pivotal role in antifungal immunity. In this study, the recently characterized human DECTIN1 Y238X early stop codon polymorphism leading to diminished Dectin-1 receptor activity was studied in relation to invasive aspergillosis susceptibility and severity in patients receiving hematopoietic stem cell transplantation. We found that the presence of the DECTIN1 Y238X polymorphism in either donors or recipients of hematopoietic stem cell transplantation increased susceptibility to aspergillosis, with the risk being highest when the polymorphism was present simultaneously in both donors and recipients (adjusted hazard ratio = 3.9; P = .005). Functionally, the Y238X polymorphism impaired the production of interferon-γ and interleukin-10 (IL-10), in addition to IL-1β, IL-6, and IL-17A, by human peripheral mononuclear cells and Dectin-1 on human epithelial cells contributed to fungal recognition. Mechanistically, studies on preclinical models of infection in intact or bone marrow-transplanted Dectin-1 knockout mice revealed that protection from infection requires a distinct, yet complementary, role of both donor and recipient Dectin-1. This study discloses Dectin-1 deficiency as a novel susceptibility factor for aspergillosis in high-risk patients and identifies a previously unsuspected role for Dectin-1 in antifungal immunity that is the ability to control both resistance and tolerance to the fungus contingent on hematopoietic/nonhematopoietic compartmentalization.
View details for DOI 10.1182/blood-2010-04-279307
View details for Web of Science ID 000285141200040
View details for PubMedID 20807886