Nrf2 regulates CD4+ T cell-induced acute graft-versus-host disease in mice.

Nuclear factor erythroid-derived 2-like 2 (Nrf2) is a ubiquitously expressed transcription factor that is well known for its role in regulating the cellular redox pathway. Although there is mounting evidence suggesting a critical role for Nrf2 in hematopoietic stem cells and innate leukocytes, little is known about its involvement in T-cell biology. In this study, we identified a novel role for Nrf2 in regulating alloreactive T-cell function during allogeneic hematopoietic cell transplantation (allo-HCT). We observed increased expression and nuclear translocation of Nrf2 upon T-cell activation in vitro, especially in CD4+ donor T cells after allo-HCT. Allo-HCT recipients of Nrf2 -/- donor T cells had significantly less acute graft-versus-host disease (GVHD)-induced mortality, morbidity, and pathology. This reduction in GVHD was associated with the persistence of Helios+ donor regulatory T cells in the allograft, as well as defective upregulation of the gut-homing receptor LPAM-1 on alloreactive CD8+ T cells. Additionally, Nrf2 -/- donor CD8+ T cells demonstrated intact cytotoxicity against allogeneic target cells. Tumor-bearing allo-HCT recipients of Nrf2 -/- donor T cells had overall improved survival as a result of preserved graft-versus-tumor activity and reduced GVHD activity. Our findings characterized a previously unrecognized role for Nrf2 in T-cell function, as well as revealed a novel therapeutic target to improve the outcomes of allo-HCT.

Corrigendum: Suppression of luteinizing hormone enhances HSC recovery after hematopoietic injury.

This corrects the article DOI: 10.1038/nm.4470.

Suppression of luteinizing hormone enhances HSC recovery after hematopoietic injury.

There is a substantial unmet clinical need for new strategies to protect the hematopoietic stem cell (HSC) pool and regenerate hematopoiesis after radiation injury from either cancer therapy or accidental exposure. Increasing evidence suggests that sex hormones, beyond their role in promoting sexual dimorphism, regulate HSC self-renewal, differentiation, and proliferation. We and others have previously reported that sex-steroid ablation promotes bone marrow (BM) lymphopoiesis and HSC recovery in aged and immunodepleted mice. Here we found that a luteinizing hormone (LH)-releasing hormone antagonist (LHRH-Ant), currently in wide clinical use for sex-steroid inhibition, promoted hematopoietic recovery and mouse survival when administered 24 h after an otherwise-lethal dose of total-body irradiation (L-TBI). Unexpectedly, this protective effect was independent of sex steroids and instead relied on suppression of LH levels. Human and mouse long-term self-renewing HSCs (LT-HSCs) expressed high levels of the LH/choriogonadotropin receptor (LHCGR) and expanded ex vivo when stimulated with LH. In contrast, the suppression of LH after L-TBI inhibited entry of HSCs into the cell cycle, thus promoting HSC quiescence and protecting the cells from exhaustion. These findings reveal a role of LH in regulating HSC function and offer a new therapeutic approach for hematopoietic regeneration after hematopoietic injury.

Production of BMP4 by endothelial cells is crucial for endogenous thymic regeneration.

The thymus is not only extremely sensitive to damage but also has a remarkable ability to repair itself. However, the mechanisms underlying this endogenous regeneration remain poorly understood, and this capacity diminishes considerably with age. We show that thymic endothelial cells (ECs) comprise a critical pathway of regeneration via their production of bone morphogenetic protein 4 (BMP4) ECs increased their production of BMP4 after thymic damage, and abrogating BMP4 signaling or production by either pharmacologic or genetic inhibition impaired thymic repair. EC-derived BMP4 acted on thymic epithelial cells (TECs) to increase their expression of Foxn1, a key transcription factor involved in TEC development, maintenance, and regeneration, and its downstream targets such as Dll4, a key mediator of thymocyte development and regeneration. These studies demonstrate the importance of the BMP4 pathway in endogenous tissue regeneration and offer a potential clinical approach to enhance T cell immunity.

Loss of thymic innate lymphoid cells leads to impaired thymopoiesis in experimental graft-versus-host disease.

Graft-versus-host disease (GVHD) and posttransplant immunodeficiency are frequently related complications of allogeneic hematopoietic transplantation. Alloreactive donor T cells can damage thymic epithelium, thus limiting new T-cell development. Although the thymus has a remarkable capacity to regenerate after injury, endogenous thymic regeneration is impaired in GVHD. The mechanisms leading to this regenerative failure are largely unknown. Here we demonstrate in experimental mouse models that GVHD results in depletion of intrathymic group 3 innate lymphoid cells (ILC3s) necessary for thymic regeneration. Loss of thymic ILC3s resulted in deficiency of intrathymic interleukin-22 (IL-22) compared with transplant recipients without GVHD, thereby inhibiting IL-22-mediated protection of thymic epithelial cells (TECs) and impairing recovery of thymopoiesis. Conversely, abrogating IL-21 receptor signaling in donor T cells and inhibiting the elimination of thymic ILCs improved thymopoiesis in an IL-22-dependent fashion. We found that the thymopoietic impairment in GVHD associated with loss of ILCs could be improved by restoration of IL-22 signaling. Despite uninhibited alloreactivity, exogenous IL-22 administration posttransplant resulted in increased recovery of thymopoiesis and development of new thymus-derived peripheral T cells. Our study highlights the role of innate immune function in thymic regeneration and restoration of adaptive immunity posttransplant. Manipulation of the ILC-IL-22-TEC axis may be useful for augmenting immune reconstitution after clinical hematopoietic transplantation and other settings of T-cell deficiency.

Donor CD19 CAR T cells exert potent graft-versus-lymphoma activity with diminished graft-versus-host activity.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for hematological malignancies. However, graft-versus-host disease (GVHD) and relapse after allo-HSCT remain major impediments to the success of allo-HSCT. Chimeric antigen receptors (CARs) direct tumor cell recognition of adoptively transferred T cells. CD19 is an attractive CAR target, which is expressed in most B cell malignancies, as well as in healthy B cells. Clinical trials using autologous CD19-targeted T cells have shown remarkable promise in various B cell malignancies. However, the use of allogeneic CAR T cells poses a concern in that it may increase risk of the occurrence of GVHD, although this has not been reported in selected patients infused with donor-derived CD19 CAR T cells after allo-HSCT. To understand the mechanism whereby allogeneic CD19 CAR T cells may mediate anti-lymphoma activity without causing a significant increase in the incidence of GVHD, we studied donor-derived CD19 CAR T cells in allo-HSCT and lymphoma models in mice. We demonstrate that alloreactive T cells expressing CD28-costimulated CD19 CARs experience enhanced stimulation, resulting in the progressive loss of both their effector function and proliferative potential, clonal deletion, and significantly decreased occurrence of GVHD. Concurrently, the other CAR T cells that were present in bulk donor T cell populations retained their anti-lymphoma activity in accordance with the requirement that both the T cell receptor (TCR) and CAR be engaged to accelerate T cell exhaustion. In contrast, first-generation and 4-1BB-costimulated CAR T cells increased the occurrence of GVHD. These findings could explain the reduced risk of GVHD occurring with cumulative TCR and CAR signaling.

Relationship between spontaneous γH2AX foci formation and progenitor functions in circulating hematopoietic stem and progenitor cells among atomic-bomb survivors.

Accumulated DNA damage in hematopoietic stem cells is a primary mechanism of aging-associated dysfunction in human hematopoiesis. About 70 years ago, atomic-bomb (A-bomb) radiation induced DNA damage and functional decreases in the hematopoietic system of A-bomb survivors in a radiation dose-dependent manner. The peripheral blood cell populations then recovered to a normal range, but accompanying cells derived from hematopoietic stem cells still remain that bear molecular changes possibly caused by past radiation exposure and aging. In the present study, we evaluated radiation-related changes in the frequency of phosphorylated (Ser-139) H2AX (γH2AX) foci formation in circulating CD34-positive/lineage marker-negative (CD34+Lin-) hematopoietic stem and progenitor cells (HSPCs) among 226Hiroshima A-bomb survivors. An association between the frequency of γH2AX foci formation in HSPCs and the radiation dose was observed, but the γH2AX foci frequency was not significantly elevated by past radiation. We found a negative correlation between the frequency of γH2AX foci formation and the length of granulocyte telomeres. A negative interaction effect between the radiation dose and the frequency of γH2AX foci was suggested in a proportion of a subset of HSPCs as assessed by the cobblestone area-forming cell assay (CAFC), indicating that the self-renewability of HSPCs may decrease in survivors who were exposed to a higher radiation dose and who had more DNA damage in their HSPCs. Thus, although many years after radiation exposure and with advancing age, the effect of DNA damage on the self-renewability of HSPCs may be modified by A-bomb radiation exposure.

Increased GVHD-related mortality with broad-spectrum antibiotic use after allogeneic hematopoietic stem cell transplantation in human patients and mice.

Intestinal bacteria may modulate the risk of infection and graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Allo-HSCT recipients often develop neutropenic fever, which is treated with antibiotics that may target anaerobic bacteria in the gut. We retrospectively examined 857 allo-HSCT recipients and found that treatment of neutropenic fever with imipenem-cilastatin and piperacillin-tazobactam antibiotics was associated with increased GVHD-related mortality at 5 years (21.5% for imipenem-cilastatin-treated patients versus 13.1% for untreated patients, P = 0.025; 19.8% for piperacillin-tazobactam-treated patients versus 11.9% for untreated patients, P = 0.007). However, two other antibiotics also used to treat neutropenic fever, aztreonam and cefepime, were not associated with GVHD-related mortality (P = 0.78 and P = 0.98, respectively). Analysis of stool specimens from allo-HSCT recipients showed that piperacillin-tazobactam administration was associated with perturbation of gut microbial composition. Studies in mice demonstrated aggravated GVHD mortality with imipenem-cilastatin or piperacillin-tazobactam compared to aztreonam (P < 0.01 and P < 0.05, respectively). We found pathological evidence for increased GVHD in the colon of imipenem-cilastatin-treated mice (P < 0.05), but no difference in the concentration of short-chain fatty acids or numbers of regulatory T cells. Notably, imipenem-cilastatin treatment of mice with GVHD led to loss of the protective mucus lining of the colon (P < 0.01) and the compromising of intestinal barrier function (P < 0.05). Sequencing of mouse stool specimens showed an increase in Akkermansia muciniphila (P < 0.001), a commensal bacterium with mucus-degrading capabilities, raising the possibility that mucus degradation may contribute to murine GVHD. We demonstrate an underappreciated risk for the treatment of allo-HSCT recipients with antibiotics that may exacerbate GVHD in the colon.

Circulating Hematopoietic Stem and Progenitor Cells in Aging Atomic Bomb Survivors.

It is not yet known whether hematopoietic stem and progenitor cells (HSPCs) are compromised in the aging population of atomic bomb (A-bomb) survivors after their exposure nearly 70 years ago. To address this, we evaluated age- and radiation-related changes in different subtypes of circulating HSPCs among the CD34-positive/lineage marker-negative (CD34(+)Lin(-)) cell population in 231 Hiroshima A-bomb survivors. We enumerated functional HSPC subtypes, including: cobblestone area-forming cells; long-term culture-initiating cells; erythroid burst-forming units; granulocyte and macrophage colony-forming units; and T-cell and natural killer cell progenitors using cell culture. We obtained the count of each HSPC subtype per unit volume of blood and the proportion of each HSPC subtype in CD34(+)Lin(-) cells to represent the lineage commitment trend. Multivariate analyses, using sex, age and radiation dose as variables, showed significantly decreased counts with age in the total CD34(+)Lin(-) cell population and all HSPC subtypes. As for the proportion, only T-cell progenitors decreased significantly with age, suggesting that the commitment to the T-cell lineage in HSPCs continuously declines with age throughout the lifetime. However, neither the CD34(+)Lin(-) cell population, nor HSPC subtypes showed significant radiation-induced dose-dependent changes in counts or proportions. Moreover, the correlations of the proportions among HSPC subtypes in the survivors properly revealed the hierarchy of lineage commitments. Taken together, our findings suggest that many years after exposure to radiation and with advancing age, the number and function of HSPCs in living survivors as a whole may have recovered to normal levels.

Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration.

Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch and epidermal growth factor (EGF) signals supporting Lgr5(+) crypt base columnar ISCs for normal epithelial maintenance. However, little is known about the regulation of the ISC compartment after tissue damage. Using ex vivo organoid cultures, here we show that innate lymphoid cells (ILCs), potent producers of interleukin-22 (IL-22) after intestinal injury, increase the growth of mouse small intestine organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both mouse and human intestinal organoids, increasing proliferation and promoting ISC expansion. IL-22 induced STAT3 phosphorylation in Lgr5(+) ISCs, and STAT3 was crucial for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after mouse allogeneic bone marrow transplantation enhanced the recovery of ISCs, increased epithelial regeneration and reduced intestinal pathology and mortality from graft-versus-host disease. ATOH1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independently of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support the intestinal epithelium, activating ISCs to promote regeneration.

Enhanced hematopoietic stem cell function mediates immune regeneration following sex steroid blockade.

Mechanisms underlying age-related defects within lymphoid-lineages remain poorly understood. We previously reported that sex steroid ablation (SSA) induced lymphoid rejuvenation and enhanced recovery from hematopoietic stem cell (HSC) transplantation (HSCT). We herein show that, mechanistically, SSA induces hematopoietic and lymphoid recovery by functionally enhancing both HSC self-renewal and propensity for lymphoid differentiation through intrinsic molecular changes. Our transcriptome analysis revealed further hematopoietic support through rejuvenation of the bone marrow (BM) microenvironment, with upregulation of key hematopoietic factors and master regulatory factors associated with aging such as Foxo1. These studies provide important cellular and molecular insights into understanding how SSA-induced regeneration of the hematopoietic compartment can underpin recovery of the immune system following damaging cytoablative treatments. These findings support a short-term strategy for clinical use of SSA to enhance the production of lymphoid cells and HSC engraftment, leading to improved outcomes in adult patients undergoing HSCT and immune depletion in general.

Sex steroid blockade enhances thymopoiesis by modulating Notch signaling.

Paradoxical to its importance for generating a diverse T cell repertoire, thymic function progressively declines throughout life. This process has been at least partially attributed to the effects of sex steroids, and their removal promotes enhanced thymopoiesis and recovery from immune injury. We show that one mechanism by which sex steroids influence thymopoiesis is through direct inhibition in cortical thymic epithelial cells (cTECs) of Delta-like 4 (Dll4), a Notch ligand crucial for the commitment and differentiation of T cell progenitors in a dose-dependent manner. Consistent with this, sex steroid ablation (SSA) led to increased expression of Dll4 and its downstream targets. Importantly, SSA induced by luteinizing hormone-releasing hormone (LHRH) receptor antagonism bypassed the surge in sex steroids caused by LHRH agonists, the gold standard for clinical ablation of sex steroids, thereby facilitating increased Dll4 expression and more rapid promotion of thymopoiesis. Collectively, these findings not only reveal a novel mechanism underlying improved thymic regeneration upon SSA but also offer an improved clinical strategy for successfully boosting immune function.

Linkage between dendritic and T cell commitments in human circulating hematopoietic progenitors.

The relationships between commitments of dendritic cells (DCs) and T cells in human hematopoietic stem cells are not well understood. In this study, we enumerate and characterize conventional DC and plasmacytoid DC precursors in association with T cell and thymus-derived types of NK cell precursors among CD34(+) hematopoietic progenitor cells (HPCs) circulating in human peripheral blood. By limiting-dilution analyses using coculture with stroma cells expressing Notch1 ligand, the precursor frequencies (PFs) of DCs in HPCs were found to significantly correlate with T cell PFs, but not with NK cell PFs, among healthy donors. Clonal analyses showed that the majority of T/NK dual- and T single-lineage precursors-but only a minority of NK single-lineage precursors-were associated with the generation of DC progenies. All clones producing both DC and T cell progenies were found with monocyte and/or granulocyte progenies, suggesting DC differentiation via myeloid DC pathways. Analyses of peripheral blood HPC subpopulations revealed that the lineage split between DC and T/NK cell progenitor occurs at the stage prior to bifurcation into T and NK cell lineages. The findings suggest a strong linkage between DC and T cell commitments, which may be imprinted in circulating lymphoid-primed multipotent progenitors or in more upstream HPCs.

PLZF confers effector functions to donor T cells that preserve graft-versus-tumor effects while attenuating GVHD.

Efforts to limit GVHD mediated by alloreactive donor T cells after allogeneic bone marrow transplantation are limited by a concomitant decrease in graft-versus-tumor (GVT) activity and increased possibilities of tumor relapse. Using a novel approach, we adoptively transferred conventional T cells expressing the transcription factor promyelocytic leukemia zinc finger (PLZF), which confers effector properties resembling invariant natural killer T cells, such as copious production of cytokines under suboptimal stimulation. PLZF expression in T-cell allografts attenuates expansion of alloreactive T cells, leading to lower GVHD. Intact alloreactivity-driven antitumor cytokine responses result in preserved GVT effects, leading to improved survival. Our findings suggest that therapy with PLZF-overexpressing T cells would result in overall improved outcomes due to less GVHD and intact GVT effects.

Age-associated changes in the differentiation potentials of human circulating hematopoietic progenitors to T- or NK-lineage cells.

Age-associated changes of T and NK cell (T/NK) potential of human hematopoietic stem cells are unknown. In this study, we enumerate and characterize T/NK precursors among CD34(+)Lin(-) cell populations circulating in normal human adult peripheral blood (PB) by a limiting-dilution assay using coculture with OP9-DL1 stroma cells expressing Notch 1 ligand, Delta-like 1. The frequency of T cell precursors in CD34(+)Lin(-) cells was found to decrease with donor age, whereas the ratio of NK to T cell precursor frequency (NK/T ratio) increased with age, suggesting that lymphoid differentiation potential of PB progenitors shifts from T to NK cell lineage with aging. Clonal analyses of CD34(+)Lin(-) cells showed that differences in the NK/T ratio were attributable to different distributions of single- and dual-lineage T/NK precursor clones. Because nearly all of the clones retained monocyte and/or granulocyte differentiation potentials in coculture with OP9-DL1 cells, T/NK precursors in PB are considered to be contained in the pool of T/NK/myeloid multipotent progenitors. The age-associated increase in NK over T cell commitment might occur in precursor cells with T/NK/myeloid potential.

Adoptively transferred TRAIL+ T cells suppress GVHD and augment antitumor activity.

Current strategies to suppress graft-versus-host disease (GVHD) also compromise graft-versus-tumor (GVT) responses. Furthermore, most experimental strategies to separate GVHD and GVT responses merely spare GVT function without actually enhancing it. We have previously shown that endogenously expressed TNF-related apoptosis-inducing ligand (TRAIL) is required for optimal GVT activity against certain malignancies in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In order to model a donor-derived cellular therapy, we genetically engineered T cells to overexpress TRAIL and adoptively transferred donor-type unsorted TRAIL+ T cells into mouse models of allo-HSCT. We found that murine TRAIL+ T cells induced apoptosis of alloreactive T cells, thereby reducing GVHD in a DR5-dependent manner. Furthermore, murine TRAIL+ T cells mediated enhanced in vitro and in vivo antilymphoma GVT response. Moreover, human TRAIL+ T cells mediated enhanced in vitro cytotoxicity against both human leukemia cell lines and against freshly isolated chronic lymphocytic leukemia (CLL) cells. Finally, as a model of off-the-shelf, donor-unrestricted antitumor cellular therapy, in vitro-generated TRAIL+ precursor T cells from third-party donors also mediated enhanced GVT response in the absence of GVHD. These data indicate that TRAIL-overexpressing donor T cells could potentially enhance the curative potential of allo-HSCT by increasing GVT response and suppressing GVHD.

Nrf2 regulates haematopoietic stem cell function.

Coordinating the balance between haematopoietic stem cell (HSC) quiescence and self-renewal is crucial for maintaining haematopoiesis lifelong. Equally important for haematopoietic function is modulating HSC localization within the bone marrow niches, as maintenance of HSC function is tightly controlled by a complex network of intrinsic molecular mechanisms and extrinsic signalling interactions with their surrounding microenvironment. In this study we demonstrate that nuclear factor erythroid 2-related factor 2 (Nfe2l2, or Nrf2), well established as a global regulator of the oxidative stress response, plays a regulatory role in several aspects of HSC homeostasis. Nrf2 deficiency results in an expansion of the haematopoietic stem and progenitor cell compartment due to cell-intrinsic hyperproliferation, which was accomplished at the expense of HSC quiescence and self-renewal. We further show that Nrf2 modulates both migration and retention of HSCs in their niche. Moreover, we identify a previously unrecognized link between Nrf2 and CXCR4, contributing, at least partially, to the maintenance of HSC function.

Interleukin-22 protects intestinal stem cells from immune-mediated tissue damage and regulates sensitivity to graft versus host disease.

Little is known about the maintenance of intestinal stem cells (ISCs) and progenitors during immune-mediated tissue damage or about the susceptibility of transplant recipients to tissue damage mediated by the donor immune system during graft versus host disease (GVHD). We demonstrate here that deficiency of recipient-derived IL-22 increased acute GVHD tissue damage and mortality, that ISCs were eliminated during GVHD, and that ISCs as well as their downstream progenitors expressed the IL-22 receptor. Intestinal IL-22 was produced after bone marrow transplant by IL-23-responsive innate lymphoid cells (ILCs) from the transplant recipients, and intestinal IL-22 increased in response to pretransplant conditioning. However, ILC frequency and IL-22 amounts were decreased by GVHD. Recipient IL-22 deficiency led to increased crypt apoptosis, depletion of ISCs, and loss of epithelial integrity. Our findings reveal IL-22 as a critical regulator of tissue sensitivity to GVHD and a protective factor for ISCs during inflammatory intestinal damage.

Regulation of intestinal inflammation by microbiota following allogeneic bone marrow transplantation.

Despite a growing understanding of the link between intestinal inflammation and resident gut microbes, longitudinal studies of human flora before initial onset of intestinal inflammation have not been reported. Here, we demonstrate in murine and human recipients of allogeneic bone marrow transplantation (BMT) that intestinal inflammation secondary to graft-versus-host disease (GVHD) is associated with major shifts in the composition of the intestinal microbiota. The microbiota, in turn, can modulate the severity of intestinal inflammation. In mouse models of GVHD, we observed loss of overall diversity and expansion of Lactobacillales and loss of Clostridiales. Eliminating Lactobacillales from the flora of mice before BMT aggravated GVHD, whereas reintroducing the predominant species of Lactobacillus mediated significant protection against GVHD. We then characterized gut flora of patients during onset of intestinal inflammation caused by GVHD and found patterns mirroring those in mice. We also identified increased microbial chaos early after allogeneic BMT as a potential risk factor for subsequent GVHD. Together, these data demonstrate regulation of flora by intestinal inflammation and suggest that flora manipulation may reduce intestinal inflammation and improve outcomes for allogeneic BMT recipients.