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.

Autophagy gene Atg16L1 prevents lethal T cell alloreactivity mediated by dendritic cells.

Atg16L1 mediates the cellular degradative process of autophagy and is considered a critical regulator of inflammation based on its genetic association with inflammatory bowel disease. Here we find that Atg16L1 deficiency leads to an exacerbated graft-versus-host disease (GVHD) in a mouse model of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Atg16L1-deficient allo-HSCT recipients with GVHD displayed increased T cell proliferation due to increased dendritic cell (DC) numbers and costimulatory molecule expression. Reduced autophagy within DCs was associated with lysosomal abnormalities and decreased amounts of A20, a negative regulator of DC activation. These results broaden the function of Atg16L1 and the autophagy pathway to include a role in limiting a DC-mediated response during inflammatory disease, such as GVHD.

A small-molecule c-Rel inhibitor reduces alloactivation of T cells without compromising antitumor activity.

Preventing unfavorable GVHD without inducing broad suppression of the immune system presents a major challenge of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We developed a novel strategy to ameliorate GVHD while preserving graft-versus-tumor (GVT) activity by small molecule-based inhibition of the NF-κB family member c-Rel. Underlying mechanisms included reduced alloactivation, defective gut homing, and impaired negative feedback on interleukin (IL)-2 production, resulting in optimal IL-2 levels, which, in the absence of competition by effector T cells, translated into expansion of regulatory T cells. c-Rel activity was dispensable for antigen-specific T-cell receptor (TCR) activation, allowing c-Rel-deficient T cells to display normal GVT activity. In addition, inhibition of c-Rel activity reduced alloactivation without compromising antigen-specific cytotoxicity of human T cells. Finally, we were able to demonstrate the feasibility and efficacy of systemic c-Rel inhibitor administration. Our findings validate c-Rel as a promising target for immunomodulatory therapy and demonstrate the feasibility and efficacy of pharmaceutical inhibition of c-Rel activity.

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.

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.

The central nervous system is a target of acute graft versus host disease in mice.

Despite significant advances in prevention and management, graft versus host disease (GVHD) is still a leading complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although skin, gut, liver, thymus, and lung are GVHD targets, neurological complications (NC) have also been reported following allo-HSCT. We demonstrate that the central nervous system (CNS) can be a direct target of alloreactive T cells following allo-HSCT in mice. We found significant infiltration of the CNS with donor T lymphocytes and cell death of neurons and neuroglia in allo-HSCT recipients with GVHD. We also found that allo-HSCT recipients with GVHD had deficits in spatial learning/memory and demonstrated increased anxious behavior. These findings highlight CNS sensitivity to damage caused by alloreactive donor T cells and represent the first characterization of target cell subsets and NC during GVHD. Therefore, these clinically relevant studies offer a novel and rational explanation for the well-described neurological symptoms observed after allo-HSCT.

Extrathymic development of murine T cells after bone marrow transplantation.

Restoring T cell competence is a significant clinical challenge in patients whose thymic function is severely compromised due to age or cytoreductive conditioning. Here, we demonstrate in mice that mesenteric LNs (MLNs) support extrathymic T cell development in euthymic and athymic recipients of bone marrow transplantation (BMT). Furthermore, in aged murine BMT recipients, the contribution of the MLNs to the generation of T cells was maintained, while the contribution of the thymus was significantly impaired. Thymic impairment resulted in a proportional increase in extrathymic-derived T cell progenitors. Extrathymic development in athymic recipients generated conventional naive TCRαß T cells with a broad Vß repertoire and intact functional and proliferative potential. Moreover, in the absence of a functional thymus, immunity against known pathogens could be augmented using engineered precursor T cells with viral specificity. These findings demonstrate the potential of extrathymic T cell development for T cell reconstitution in patients with limited thymic 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.

Interleukin-22 drives endogenous thymic regeneration in mice.

Endogenous thymic regeneration is a crucial function that allows for renewal of immune competence after stress, infection, or immunodepletion. However, the mechanisms governing this regeneration remain poorly understood. We detail such a mechanism, centered on interleukin-22 (IL-22) and triggered by the depletion of CD4(+)CD8(+) double-positive thymocytes. Intrathymic levels of IL-22 were increased after thymic insult, and thymic recovery was impaired in IL-22-deficient mice. IL-22, which signaled through thymic epithelial cells and promoted their proliferation and survival, was up-regulated by radio-resistant RORγ(t)(+)CCR6(+)NKp46(-) lymphoid tissue inducer cells after thymic injury in an IL-23-dependent manner. Administration of IL-22 enhanced thymic recovery after total body irradiation. These studies reveal mechanisms of endogenous thymic repair and offer innovative regenerative strategies for improving immune competence.