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Allogeneic hematopoietic transplantation is a powerful treatment for hematologic malignancies. Posttransplant immune incompetence exposes patients to disease relapse and infections. We previously demonstrated that donor alloreactive natural killer (NK) cells ablate recipient hematopoietic targets, including leukemia. Here, in murine models, we show that infusion of donor alloreactive NK cells triggers recipient dendritic cells (DCs) to synthesize β-2-microglobulin (B2M) that elicits the release of c-KIT ligand and interleukin-7 that greatly accelerate posttransplant immune reconstitution. An identical chain of events was reproduced by infusing supernatants of alloreactive NK/DC cocultures. Similarly, human alloreactive NK cells triggered human DCs to synthesize B2M that induced interleukin-7 production by thymic epithelial cells and thereby supported thymocyte cellularity in vitro. Chromatography fractionation of murine and human alloreactive NK/DC coculture supernatants identified a protein with molecular weight and isoelectric point of B2M, and mass spectrometry identified amino acid sequences specific of B2M. Anti-B2M antibody depletion of NK/DC coculture supernatants abrogated their immune-rebuilding effect. B2M knock-out mice were unable to undergo accelerated immune reconstitution, but infusion of (wild-type) NK/DC coculture supernatants restored their ability to undergo accelerated immune reconstitution. Similarly, silencing the B2M gene in human DCs, before coculture with alloreactive NK cells, prevented the increase in thymocyte cellularity in vitro. Finally, human recombinant B2M increased thymocyte cellularity in a thymic epithelial cells/thymocyte culture system. Our studies uncover a novel therapeutic principle for treating posttransplant immune incompetence and suggest that, upon its translation to the clinic, patients may benefit from adoptive transfer of large numbers of cytokine-activated, ex vivo–expanded donor alloreactive NK cells.

Endogenous thymic regeneration is a crucial process that allows for the renewal of immune competence following stress, infection or cytoreductive conditioning. Fully understanding the molecular mechanisms driving regeneration will uncover therapeutic targets to enhance regeneration. We previously demonstrated that high levels of homeostatic apoptosis suppress regeneration and that a reduction in the presence of damage-induced apoptotic thymocytes facilitates regeneration. Here we identified that cell-specific metabolic remodeling after ionizing radiation steers thymocytes towards mitochondrial-driven pyroptotic cell death. We further identified that a key damage-associated molecular pattern (DAMP), ATP, stimulates the cell surface purinergic receptor P2Y2 on cortical thymic epithelial cells (cTECs) acutely after damage, enhancing expression ofFoxn1, the critical thymic transcription factor. Targeting the P2Y2 receptor with the agonist UTPγS promotes rapid regeneration of the thymusin vivofollowing acute damage. Together these data demonstrate that intrinsic metabolic regulation of pyruvate processing is a critical process driving thymus repair and identifies the P2Y2 receptor as a novel molecular therapeutic target to enhance thymus regeneration.SUMMARYThymocytes rapidly and transiently undergo pyroptosis after acute thymic damage and promote regeneration.Damage-induced redirection of pyruvate acutely enhances mitochondrial OXPHOS in thymocytes.Elevated mitochondrial ROS promotes pyroptosis in thymocytes after acute insult by driving caspase 1 cleavage.Extracellular ATP release promotesFoxn1expression in cTECs via activation of P2Y2Therapeutic targeting of the P2Y2 receptor promotes thymic regeneration.

Following periods of haematopoietic cell stress, such as after chemotherapy, radiotherapy, infection and transplantation, patient outcomes are linked to the degree of immune reconstitution, specifically of T cells. Delayed or defective recovery of the T cell pool has significant clinical consequences, including prolonged immunosuppression, poor vaccine responses and increased risks of infections and malignancies. Thus, strategies that restore thymic function and enhance T cell reconstitution can provide considerable benefit to individuals whose immune system has been decimated in various settings. In this Review, we focus on the causes and consequences of impaired adaptive immunity and discuss therapeutic strategies that can recover immune function, with a particular emphasis on approaches that can promote a diverse repertoire of T cells through de novo T cell formation., Reconstitution of the immune system after depletion by chemotherapy, radiotherapy, infection or transplantation is crucial to maintain protection from infection and to respond to immune-based therapy. Here the authors describe the ways in which a diverse T cell compartment can be restored, focusing on therapeutic strategies that drive the production of new T cells.

SUMMARYThe thymus is essential for establishing adaptive immunity yet undergoes age-related atrophy leading to compromised immune responsiveness. The thymus is also extremely sensitive to acute insult and although capable of regeneration, this capacity declines with age. Focusing on non-hematopoietic stromal cells, and using single-cell and spatial transcriptomics, lineage-tracing, and advanced imaging, we discovered two atypical thymic epithelial cell (TEC) states that emerged with age. Age-associated (aa)TECs formed atypical high-density epithelial clusters that were devoid of thymocytes, an accretion of non-functional thymic tissue that worsened with age and exhibited features of partial epithelial-to-mesenchymal transition (EMT).In silicointeraction analysis revealed that aaTEC emergence drew tonic signals from other TEC populations at baseline, acting as a sink for TEC growth factors. Following damage, aaTEC expanded substantially, further perturbing trophic pathways, and correlating with defective regeneration of the involuted thymus. These findings define a unique feature of thymic involution linked to immune aging.

The capacity of T cells to recognize and mount an immune response against tumor antigens depends on the large diversity of the T-cell receptor (TCR) repertoire generated in the thymus during the process of T-cell development. However, this process is dramatically impaired by immunological insults, such as that caused by cytoreductive cancer therapies and infections, and by the physiological decline of thymic function with age. Defective thymic function and a skewed TCR repertoire can have significant clinical consequences. The presence of an adequate pool of T cells capable of recognizing specific tumor antigens is a prerequisite for the success of cancer immunotherapy using checkpoint blockade therapy. However, while this approach has improved the chances of survival of patients with different types of cancer, a large proportion of them do not respond. The limited response rate to checkpoint blockade therapy may be linked to a suboptimal TCR repertoire in cancer patients prior to therapy. Here, we focus on the role of the thymus in shaping the T-cell pool in health and disease, discuss how the TCR repertoire influences patients’ response to checkpoint blockade therapy and highlight approaches able to manipulate thymic function to enhance anti-tumor immunity.

Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma characterized by an impaired myogenic differentiation despite the expression of myogenic master genes MYOD and MYOG. Therefore, the restoration of differentiation is considered an anti-cancer therapy. SKP2 is an oncogenic E3-ubiquitin ligase that promotes cell proliferation by targeting the CDKi p21Cip1 and p27Kip1. Previous works showed that SKP2 overexpression is induced by the fusion oncoprotein PAX3-FOXO1 expressed in fusion positive (FP)-RMS cells, and promotes tumor cell proliferation through p27kip1 degradation. However, the role of SKP2 in fusion negative (FN)-RMS cells, devoid of any fusion gene, remains unclear. We report here that SKP2 transcript and protein levels are up-regulated in RMS patients and cell lines compared to normal tissue. Accordingly, we observed increased acetylation of H3K27 histone mark in RMS patients and cell lines compared to myoblasts and muscle tissue. We then show that in RMS cell lines SKP2 expression is induced by MYOD, which binds two SKP2 regulatory regions, an intronic and a distal enhancers, identified by Hi-C and 3C experiments. SKP2 knockdown in FN-RMS cells leads to p21Cip1 and p27Kip1 protein levels up-regulation coupled with G1/S cell cycle arrest. Rescue experiments showed that SKP2 promotes cell proliferation directly targeting p27Kip1. Moreover, SKP2 binds and promotes degradation of p57Kip2 and its silencing restores myogenic differentiation associated to MYOG and de novo MyHC expression in FN-RMS cells. SKP2 depletion also induces cell senescence and prevents anchorage-independent growth and stemness in vitro, and tumor growth in vivo. In turn, SKP2 forced expression partially rescued the anti-cancer effects preventing the increase of p21Cip1, p27Kip1, p57Kip2 and MYOG, promoting re-entry into cell cycle, inhibiting human myoblasts cell differentiation and restoring the tumorigenic potential in FN-RMS. Since neddylation is an essential step for the activity of SKP2, we used MLN4924, an inhibitor of the Nedd8 Activating Enzyme (NAE), under clinical investigation, to resume SKP2 knockdown features. MLN4924 induces p21Cip1 and p27Kip2 expression, promotes senescence and apoptosis, and hampers cell growth in vitro and in vivo both in FP- and FN-RMS. These results unveil an unprecedented role for SKP2 in governing both proliferation and myogenic differentiation in RMS, suggesting that targeting SKP2 functions through MLN4924 treatment might have clinical relevance in FP- and FN-RMS. The study has been founded by AIRC and 5xmille 2021/Ministero della Salute to RR. Citation Format: Silvia Pomella, Matteo Cassandri, Doris Phelps, Clara Perrone, Michele Pezzella, Marco Wachtel, Benjamin Sunkel, Antonella Cardinale, Zoe Walters, Cristina Cossetti, Sonia Rodriguez, Nadia Carlesso, Janet Shipley, Lucio Miele, Beat Schafer, Enrico Velardi, Peter Houghton, Berkley Gryder, Benjamin Stanton, Concetta Quintarelli, Biagio De Angelis, Franco Locatelli, Rossella Rota. A MYOD-SKP2 axis boosts oncogenic properties of fusion negative rhabdomyosarcoma and is counteracted by neddylation inhibition in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 668.

As the thymus represents the primary site of T-cell development, optimal thymic function is of paramount importance for the successful reconstitution of the adaptive immunity after allogeneic hematopoietic stem cell transplantation. Thymus involutes as part of the aging process and several factors, including previous chemotherapy treatments, conditioning regimen used in preparation to the allograft, occurrence of graft-versus-host disease, and steroid therapy that impair the integrity of the thymus, thus affecting its role in supporting T-cell neogenesis. Although the pathways governing its regeneration are still poorly understood, the thymus has a remarkable capacity to recover its function after damage. Measurement of both recent thymic emigrants and T-cell receptor excision circles is valuable tools to assess thymic output and gain insights on its function. In this review, we will extensively discuss available data on factors regulating thymic function after allogeneic hematopoietic stem cell transplantation, as well as the strategies and therapeutic approaches under investigation to promote thymic reconstitution and accelerate immune recovery in transplanted patients, including the use of cytokines, sex-steroid ablation, precursor T-cells, and thymus bioengineering. Although none of them is routinely used in the clinic, these approaches have the potential to enhance thymic function and immune recovery, not only in patients given an allograft but also in other conditions characterized by immune deficiencies related to a defective function of the thymus.

Medulloblastoma (MB) is a childhood malignant brain tumour comprising four main subgroups characterized by different genetic alterations and rate of mortality. Among MB subgroups, patients with enhanced levels of the c-MYC oncogene (MBGroup3) have the poorest prognosis. Here we identify a previously unrecognized role of the pro-autophagy factor AMBRA1 in regulating MB. We demonstrate that AMBRA1 expression depends on c-MYC levels and correlates with Group 3 patient poor prognosis; also, knockdown of AMBRA1 reduces MB stem potential, growth and migration of MBGroup3 stem cells. At a molecular level, AMBRA1 mediates these effects by suppressing SOCS3, an inhibitor of STAT3 activation. Importantly, pharmacological inhibition of autophagy profoundly affects both stem and invasion potential of MBGroup3 stem cells, and a combined anti-autophagy and anti-STAT3 approach impacts the MBGroup3 outcome. Taken together, our data support the c-MYC/AMBRA1/STAT3 axis as a strong oncogenic signalling pathway with significance for both patient stratification strategies and targeted treatments of MBGroup3.

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.

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.

The success of allogeneic hematopoietic stem cell transplantation, a key treatment for many disorders, is intertwined with T cell immune reconstitution. The thymus plays a key role post allogeneic hematopoietic stem cell transplantation in the generation of a broad but self-tolerant T cell repertoire, but it is exquisitely sensitive to a range of insults during the transplant period, including conditioning regimens, corticosteroids, infections, and graft-versus-host disease. Although endogenous thymic repair is possible it is often suboptimal, and there is a need to develop exogenous strategies to help regenerate the thymus. Therapies currently in clinical trials in the transplant setting include keratinocyte growth factor, cytokines (IL-7 and IL-22), and hormonal modulation including sex steroid inhibition and growth hormone administration. Such regenerative strategies may ultimately enable the thymus to play as prominent a role after transplant as it once did in early childhood, allowing a more complete restoration of the T cell compartment.

Waldenström's macroglobulinemia (WM) is a B-cell non-Hodgkin's lymphoma (B-NHL) characterized by immunoglobulin M (IgM) monoclonal gammopathy and the medullary expansion of clonal lymphoplasmacytic cells. Neoplastic transformation has been partially attributed to hyperactive MYD88 signaling, secondary to the MYD88 L265P mutation, occurring in the majority of WM patients. Nevertheless, the presence of chronic active B-cell receptor (BCR) signaling, a feature of multiple IgM+ B-NHL, remains a subject of speculation in WM. Here, we interrogated the BCR signaling capacity of primary WM cells by utilizing multiparametric phosphoflow cytometry and found heightened basal phosphorylation of BCR-related signaling proteins, and augmented phosphoresponses on surface IgM (sIgM) crosslinking, compared with normal B cells. In support of those findings we observed high sIgM expression and loss of phosphatase activity in WM cells, which could both lead to signaling potentiation in clonal cells. Finally, led by the high-signaling heterogeneity among WM samples, we generated patient-specific phosphosignatures, which subclassified patients into a 'high' and a 'healthy-like' signaling group, with the second corresponding to patients with a more indolent clinical phenotype. These findings support the presence of chronic active BCR signaling in WM while providing a link between differential BCR signaling utilization and distinct clinical WM subgroups.

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.

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.

Velardi et al. show that sex steroids regulate thymopoiesis by directly modulating Notch signaling, and provide a novel clinical strategy to boost immune regeneration., 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.

Despite its critical role in supplying naïve T cells to the circulation, the thymus is particularly sensitive to immune injury, such as that caused by cytoreductive chemo- or radiation therapy, shock, infection and graft versus host disease (GVHD). Crucially, insufficient thymic recovery has been directly correlated with increased risk of opportunistic infections and poor clinical outcomes in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Prolonged immune deficiency is particularly pronounced in older patients whose thymi are already significantly impaired due to age-related thymic involution. Previous preclinical studies have revealed several strategies that can enhance thymic function and immune reconstitution after transplant, including sex steroid ablation (SSA), growth factors (growth hormone, GH, keratinocyte growth factor, KGF, insulin-like growth factor 1, IGF-1, interleukin-7, IL-7) and ex vivo generated precursor T cells (preT). In addition, recent studies have shown that other approaches, such as interleukein-22 (IL-22) and nutritional changes, may represent additional candidates to enhance thymic regeneration. In this review we provide updates on these strategies and comment on their potential to be translated into clinical therapies.

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.

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 molecular pathways that regulate the tissue repair function of type I interferon (IFN-I) during acute tissue damage are poorly understood. We describe a protective role for IFN-I and the RIG-I/MAVS signaling pathway during acute tissue damage in mice. Mice lacking mitochondrial antiviral-signaling protein (MAVS) were more sensitive to total body irradiation- and chemotherapy-induced intestinal barrier damage. These mice developed worse graft-versus-host disease (GVHD) in a preclinical model of allogeneic hematopoietic stem cell transplantation (allo-HSCT) than did wild-type mice. This phenotype was not associated with changes in the intestinal microbiota but was associated with reduced gut epithelial integrity. Conversely, targeted activation of the RIG-I pathway during tissue injury promoted gut barrier integrity and reduced GVHD. Recombinant IFN-I or IFN-I expression induced by RIG-I promoted growth of intestinal organoids in vitro and production of the antimicrobial peptide regenerating islet-derived protein 3 g (RegIIIg). Our findings were not confined to RIG-I/MAVS signaling because targeted engagement of the STING (stimulator of interferon genes) pathway also protected gut barrier function and reduced GVHD. Consistent with this, STING-deficient mice suffered worse GVHD after allo-HSCT than did wild-type mice. Overall, our data suggest that activation of either RIG-I/MAVS or STING pathways during acute intestinal tissue injury in mice resulted in IFN-I signaling that maintained gut epithelial barrier integrity and reduced GVHD severity. Targeting these pathways may help to prevent acute intestinal injury and GVHD during allogeneic transplantation. 2017

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.

After allogeneic hematopoietic stem cell transplantation (allo-HSCT), intestinal bacteria modulate risks of infection and graft-versus-host disease (GVHD). Neutropenic fever is common and treated with a choice of clinically equivalent antibiotics that target obligately anaerobic bacteria (anaerobes) to varying degrees. We retrospectively examined 857 allo-HSCT recipients and found that treatment of neutropenic fever with imipenem-cilastatin and piperacillin-tazobactam was associated with increased GVHD-related mortality at 5 years (21.5% in imipenem-cilastatin-treated patients vs. 13.1% in untreated patients, p=0.025, and 19.8% in piperacillin-tazobactam-treated patients vs. 11.9% in 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 microbiota composition showed that piperacillin-tazobactam administration was associated with increased compositional perturbation. Studies in mouse models demonstrated similar effects of these antibiotics, as well as aggravated GVHD mortality with imipenem-cilastatin or piperacillin-tazobactm compared to aztreonam (p

Silymarin, a mixture of bioactive flavonolignans isolated from Silybum marianum, exhibits anti-carcinogenic, anti-inflammatory and cytoprotective effects. In this study, the in vitro immunomodulatory activity of silymarin was investigated using CD4+ splenocytes from C57/Bl6 mice. Proliferation assay revealed that silymarin, at 50 microM concentration, significantly inhibited CD4+ cells proliferation. ELISA analyses indicated that silymarin significantly inhibited IL-2 and IFN-gamma production. Immunofluorescence staining performed on the mouse hybridoma T cell line (3DO) revealed a block of nuclear translocation of transcription factor kappaB (NF-kappaB), which is known to be responsible for IL-2 transcriptional activation. Moreover, silymarin inhibited p65/NF-kappaB phosphorylation in CD4+ T cell. These results suggest that silymarin is able to inhibit T cell activation and proliferation, notably acting on pathways of NF-kappaB activation/translocation.

There is a substantial unmet clinical need for new strategies to protect the hematopoietic stem cell (HSC) pool and regenerate hematopoiesis after radiation injury, either from cancer therapy or accidental exposure1,2. In addition to their role in promoting sexual dimorphisms, increasing evidence suggests that sex hormones regulate HSC self-renewal, differentiation, and proliferation3–5. We and others previously reported that sex steroid ablation promotes bone marrow (BM) lymphopoiesis and HSC recovery in aged and immunodepleted mice5–7. Here we show that a luteinizing hormone-releasing hormone-antagonist (LHRH-Ant), currently used widely in the clinic 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, but instead relied on suppression of luteinizing hormone (LH) levels. Human and mouse long-term self-renewing HSCs (LT-HSCs) expressed high levels of the luteinizing hormone/choriogonadotropin receptor (LHCGR) and expand ex vitro when stimulated with LH. In contrast, suppression of LH after L-TBI inhibited entry of HSCs into the cell cycle, thus promoting quiescence of HSCs and protecting them from exhaustion. These findings reveal a role for LH in regulating HSC function and offer a new therapeutic approach for hematopoietic regeneration after injury.

Summary Bone marrow transplantation (BMT) offers curative potential for patients with high-risk hematologic malignancies, but the post-transplantation period is characterized by profound immunodeficiency. Recent studies indicate that the intestinal microbiota not only regulates mucosal immunity, but can also contribute to systemic immunity and hematopoiesis. Using antibiotic-mediated microbiota depletion in a syngeneic BMT mouse model, here we describe a role for the intestinal flora in hematopoietic recovery after BMT. Depletion of the intestinal microbiota resulted in impaired recovery of lymphocyte and neutrophil counts, while recovery of the hematopoietic stem and progenitor compartments and the erythroid lineage were largely unaffected. Depletion of the intestinal microbiota also reduced dietary energy uptake and visceral fat stores. Caloric supplementation through sucrose in the drinking water improved post-BMT hematopoietic recovery in mice with a depleted intestinal flora. Taken together, we show that the intestinal microbiota contribute to post-BMT hematopoietic reconstitution in mice through improved dietary energy uptake.

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 maintenance1,2. However, little is known about the regulation of the ISC compartment after tissue damage. Utilizing ex vivo organoid cultures, we provide evidence that innate lymphoid cells (ILCs), potent producers of Interleukin-22 (IL-22) after intestinal injury3,4, increased the growth of murine small intestine (SI) organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both murine and human intestinal organoids, increasing proliferation, and promoting ISC expansion. IL-22 induced Stat3 phosphorylation in Lgr5+ ISCs, and Stat3 was critical for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after murine allogeneic bone marrow transplantation (BMT) enhanced recovery of ISCs, increased epithelial regeneration, and reduced intestinal pathology and mortality from graft vs. host disease (GVHD). Atoh1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independent of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support intestinal epithelium, activating ISCs to promote regeneration.

Endogenous thymic regeneration is a crucial function that allows for renewal of immune competence following immunodepletion caused by common cancer therapies such as cytoreductive chemotherapy or radiation; however, the mechanisms governing this regeneration remain poorly understood. Moreover, despite this capacity, prolonged T cell deficiency is a major clinical hurdle in recipients of hematopoietic stem cell transplantation (HSCT) and can precipitate high morbidity and mortality from opportunistic infections, and may even facilitate malignant relapse. We have recently described a central role for group 3 innate lymphoid cells (ILC) in a complex cellular and molecular network that drives endogenous thymic regeneration (Dudakov 2012 Science 336:91). Although IL-22 contributes considerably towards thymic regeneration and mice deficient for IL-22 lag behind WT controls in their recovery of thymic function, there is still some tissue regeneration in these mice, suggesting that other regeneration pathways also contribute to thymic repair. Unbiased transcriptome analysis on the damage-resistant non-hematopoietic compartemtn of the thymus revealed significant upregulation of Bmp4 and its downstream signalling targets (Fig. 1a). Further interrogation revealed that while thymic expression of BMP4 was restricted to fibroblasts and endothelial cells (ECs), only ECs increase their expression of Bmp4 after damage; and specific and inducible deletion of BMP4 in ECs led to significantly worse regeneration (Fig. 1b). Thymopoiesis is dependent on the close interaction between developing thymocytes and the non-hematopoietic stromal microenvironment, which includes highly specialized thymic epithelial cells (TECs) and ECs. While the role of TECs has been well studied, the contribution of ECs to thymopoiesis and thymic regeneration has thus far remained largely unclear. Careful interrogation of ECs after damage revealed that, much like ILCs, ECs are extremely resistant to multiple clinically relevant models of acute tissue injury including corticosteroids, chemotherapy and TBI. However, whole organ imaging analysis using light sheet field microscopy suggested that even though the number of ECs remain unchanged after damage, there is considerable structural changes to the vasculature including shortening of the vessels and reduced branching. Although BMP4 receptors are widely expressed in the thymus, there was enriched expression for BMP4 receptor subunits on TECs, which is consistent with the role of BMP4 in thymus ontogeny by promoting TEC development, at least partially due to its ability to induce expression of Foxn1 (Fig. 1c), a key transcription factor for the development and maintenance of TECs. Consistent with these findings, after thymic damage we observed a significant increase in the expression of Foxn1 after damage as well as GSEA enrichment for downstream FOXN1 target genes (Fig. 1d); including Dll4, the Notch ligand critical for T cell development and whose concentration we have previously shown can directly regulate thymic size (Velardi 2014 J Exp Med 211:2341). Finally, using a technique whereby ECs are transduced with the adenoviral gene E4ORF1 - ECs could be expanded ex vivo (exEC) and, when administered to mice after SL-TBI, significantly boost recovery of thymic function; but only when the exEC were derived from the thymus but not from heart or kidney (Fig. 1e). Consistent with endogenous regeneration, in vivo administration of exEC(Thy) induced the expression by TECs of Foxn1 and Dll4 . Here we demonstrate that rather than just being passive conduits that deliver oxygen and nutrients, ECs are active participants in organ function producing distinct paracrine factors that orchestrate thymic renewal. These studies thus not only detail a novel pathway promoting endogenous thymic regeneration, but also offer an innovative clinical approach to enhance T cell immunity in recipients of allo-HSCT and for individuals with T cell deficiencies due to aging, infectious disease, and common cancer treatments such as chemo- and radiation-therapy. Figure 1 Figure 1. Disclosures van den Brink: PureTech Health: Consultancy; Therakos Institute: Other: Speaking engagement; Seres: Research Funding; Jazz Pharmaceuticals: Consultancy.

The thymus is extremely sensitive to exogenous insults but has a remarkable capacity to regenerate which is lost with age. Reactive oxygen species (ROS) accumulate early after tissue damage and despite their toxic potential, ROS and their byproducts (such as lipid peroxidation products-LPPs) can act as regeneration signals by activating membrane or intracellular sensors and subsequent stress-response signalling pathways. Using Sublethal Total Body Irradiation (SL-TBI) as a model of acute thymic injury, we found a rapid accumulation of thymic ROS as well as lipid peroxidation products on cell membranes after SLTBI (Figure 1A&B). The damage-sensing ion channel Transient Receptor Potential cation channel family A member 1 (TRPA1) represents one of the major damage sensing receptors that can mediate cellular responses to oxidative stress mediators, such as LPPs. Using immunofluorescence (IF) microscopy we found that TRPA1 is enriched in the thymic medulla. Interestingly, although TRPA1 has been classically identified in nociceptive fibers, the major TRPA1 expressing structures in the thymus were not nerve fiber terminals, but primarily thymic endothelial cells (Figure1C), fibroblasts and subsets of epithelial cells. We have recently demonstrated that thymic endothelial cells can regulate regeneration through secretion of BMP-4, which can enhance Foxn1 expression and proliferation of thymic epithelial cells. In order to assess the functional role of TRPA1 in thymic regeneration after injury, we utilized TRPA1 knockout (TRPA1-/-) mice and quantified thymic reconstitution after SL-TBI. TRPA1-/- mice had significantly lower thymic cellularity compared to their age- and sex-matched WT controls, suggesting an association between TRPA1 deficiency and delayed endogenous thymic recovery (Figure 1D). The major deficit in thymocyte counts primarily affected double negative-4 (DN4), double positive (DP) and CD4+ single positive (SP-CD4+) thymocyte numbers. The thymic stroma of TRPA1-/- mice had lower endothelial cell and fibroblast counts (Figure 1D). In accordance with these findings drinking water administration of the TRPA1 agonist Allyl-Isothiocyanate (AITC), resulted in enhanced thymic regeneration after radiation exposure. Besides its positive effects on thymocyte counts, AITC significantly augmented endothelial cell counts after irradiation (Figure 1E). In conclusion these results suggest that TRPA1 plays a non-redundant role in thymic regeneration and that exogenous TRPA1 stimulation can enhance immune recovery after damage. Disclosures van den Brink: Seres: Research Funding; Jazz Pharmaceuticals: Consultancy; PureTech Health: Consultancy; Therakos Institute: Other: Speaking engagement.

Human thymus is completely developed in late fetal stages and its function peaks in newborns. After the first year of life, the thymus undergoes a progressive atrophy that dramatically decreases de novo T-lymphocyte maturation. Hormonal signaling and changes in the microRNA expression network are identified as underlying causes of human thymus involution. However, specific pathways involved in the age-related loss of thymic function remain unknown. In this study, we analyzed differential gene-expression profile and microRNA expression in elderly (70 years old) and young (less than 10 months old and 11 years old) human thymic samples. Our data have shown that WNT pathway deregulation through the overexpression of different inhibitors by the nonadipocytic component of the human thymus stimulates the age-related involution. These results are of particular interest because interference of WNT signaling has been demonstrated in both animal models and in vitro studies, with the three major hallmarks of thymic involution: (i) epithelial structure disruption, (ii) adipogenic process, and (iii) thymocyte development arrest. Thus, our results suggest that secreted inhibitors of the WNT pathway could be explored as a novel therapeutical target in the reversal of the age-related thymic involution.

Innate lymphoid cells (ILCs) are a newly described heterogeneous population of immune cells that can be defined by their expression of specific transcription factors (Tbet, GATA3 or RORgt) and their production of cytokines (IFNg, IL-13, or IL-22). Group 3 ILCs (which can be identified by expression of RORgt and production of IL-22) have been implicated in the maintenance and function of tissues as diverse as liver, gut, lung, spleen and lymph nodes. We have recently described a central role for intrathymic group 3 ILCs (tILC3) in a complex network of endogenous thymic regeneration (Dudakov et al. 2012 Science 336:91-95); a crucial function that allows for renewal of immune competence following infection or immune depletion caused by cytoreductive chemotherapy or radiation injury. In this model, 1) loss of thymic cellularity (and in particular the depletion of CD4+CD8+ double positive, DP, thymocytes) triggers, 2) upregulation of IL-23 by dendritic cells (DCs) which induces, 3) the production of IL-22 by tILC3. Given that IL-22 promotes the survival and proliferation of thymic epithelial cells (TECs), this cascade of events leads to regeneration of the supporting epithelial microenvironment and, ultimately, to rejuvenation of thymopoiesis. In our previous studies we had demonstrated that, unlike other lymphoid cells, tILC3 were extremely radio-resistant with little if any depletion of cells after even lethal doses of total body irradiation (TBI). Consistent with these findings, here we show that a considerable proportion of tILC3 were non-cycling in steady-state conditions and expressed high endogenous levels of the anti-apoptotic protein Bcl-2 (Fig. 1a). Perhaps unsurprising given their resistance to proliferation-targeted damage, a residual population of host-derived tILC3s could be identified for up to 12 months after syngeneic hematopoietic stem cell transplantation (HSCT). Although at this stage it is unclear if this is because they are very long-lived or if they have the capacity for self-renewal, residual host tILC3 were almost exclusively non-proliferating and expressed high levels of Bcl-2, indicating a quiescent state. Transcriptome analysis of IL-22 target cells revealed two mechanisms by which IL-22 mediates its effects on TECs; 1) by directly promoting the upregulation of proliferation-associated molecules such as E2f2; and 2) by reducing expression of negative signalling regulators such as Socs3 (an inhibitor of cytokine signalling) and Tnfrsf11b (Osteoprotegerin, a RANKL decoy receptor). This suggests a possible secondary role for IL-22 in promoting enhanced responsiveness to other regenerative factors, such as KGF, BMP4 and RANKL, all of which are increased in the thymus as part of the regenerative response after TBI (Fig. 1b). In our previous studies we found that increased production of IL-22 by tILC3 in response to immune injury was strikingly consistent across several mouse models with lesions in T cell development, including TBI, exposure to corticosteroids, and in mice with genetic mutations. However, one model where this increase in IL-22 does not occur is in the setting of graft versus host disease (GVHD), where tILC3s are profoundly depleted in the thymus (Fig. 1c), likely contributing towards reduced rejuvenation of thymic cellularity and failure to recover during GVHD. Intriguingly, although IL-22 appears to play a considerable role in the regenerative capacity of tILC3, preliminary studies suggest that depletion of tILC3 in IL-22 deficient mice leads to significantly worse recovery compared to Il22-/- mice replete with tILC3 (Fig. 1d). Consistent with this hypothesis of an alternate role in regeneration beyond IL-22 production, production of RANKL is also increased by tILC3 after thymic damage. Thus, we have identified that tILC3 are highly radio-resistant and long-lived owing largely to their quiescent nature and resistance to apoptosis. These pre-clinical studies focusing on tILC3 biology not only help to identify the mechanisms that allow this nascent cell population to mediate its regenerative effects, but also offer a tantalising glimpse into an alternate pathway mediating their regeneration in the thymus. Taken together, these studies could have the potential to result in novel clinical approaches to enhance T cell immunity in individuals with T cell deficiencies due to aging, infectious disease, chemotherapy or radiation injury. Disclosures: No relevant conflicts of interest to declare.

Mechanisms regulating host tissue recovery from immune-mediated damage in gastrointestinal graft vs. host disease (GI GVHD) remain incompletely understood. Prophylactic strategies selectively promoting epithelial regeneration after allogeneic hematopoietic stem/progenitor cell transplantation (allo-HCT) have the potential to reduce GVHD without limiting therapeutic graft vs. leukemia/lymphoma (GVL) responses. We have previously shown that IL-22 produced by recipient-derived innate lymphoid cells (ILCs) provides a critical signal for epithelial recovery following experimental allo-HCT. IL-22-deficient recipients demonstrated increased GVHD mortality and significantly worse loss of crypt base intestinal stem cells (ISCs) during GVHD. Paradoxically, GVHD led to reduced GI IL-22 levels in wild-type (WT) recipients due to the elimination of radioresistant intestinal ILCs. We therefore sought to determine if IL-22 administration after allo-HCT could negate the effect of ILC elimination and reduce GVHD pathology without impairing GVL. We utilized a clinically modeled LP into C57BL/6 (B6) minor antigen mismatched model with T cell-depleted marrow and MACS-purified T cells transplanted into lethally irradiated mice. Recipients were treated daily with PBS or 4ug murine recombinant (r)IL-22 delivered via intraperitoneal (IP) injection starting day 7 post-HCT. This schedule was based on the results of rIL-22 pharmacokinetics tested in untransplanted mice. We found that daily IP administration with rIL-22 led to decreased GVHD pathology in recipient small intestine, large intestine, and liver three weeks post-HCT (Figure 1, p To assess the effects of IL-22 administration on the ISC compartment, we performed LP into B6 allo-HCT using Lgr5-LacZ ISC reporter mice. Recipients treated with rIL-22 demonstrated increased numbers of Lgr5+ ISC three weeks post-HCT during active GVHD with no immunosuppression (Figure 2, p Given the lack of IL-22R expression in hematopoietic cells, we hypothesized that IL-22 administration would not limit GVL. This was confirmed by monitoring luciferase+ A20 bioluminescence in B6 into BALB/c tumor challenge recipients treated with rIL-22. Finally, we have previously shown that rIL-22 administration can increase the number of double positive thymocytes post-HCT by protecting thymic epithelium from radiation injury and from GVHD. We hypothesized that this could translate into improved peripheral T cell reconstitution even during active GVHD. Indeed, FVB into BALB/c MHC-mismatched transplant with Rag2-GFP marrow and WT T cells indicated that IL-22 administration increased the development of donor marrow-derived CD4 and CD8+ thymic emigrants four weeks post-HCT (Figure 3, p In summary, we found that IL-22 administration could reduce intestinal pathology, improve ISC recovery, and promote donor marrow-derived T cell development during GVHD. Importantly, IL-22 administration did not impair GVL. These results suggest that post-transplant IL-22 administration represents a novel strategy to protect intestinal epithelium and improve immune reconstitution after allo-HCT. Disclosures: No relevant conflicts of interest to declare.

Stroke is the third leading cause of death and the leading cause of long-term disability in adults. Current therapeutic strategies for stroke, including thrombolytic drugs, such as tissue plasminogen activator offer great promise for the treatment, but complimentary neuroprotective treatments are likely to provide a better outcome. To counteract the ischemic brain injury in mice, a new therapeutic approach has been employed by using palmitoylethanolamide (PEA). PEA is one of the members of N-acyl-ethanolamine family maintain not only redox balance but also inhibit the mechanisms of secondary injury on ischemic brain injury. Treatment of the middle cerebral artery occlusion (MCAo)-induced animals with PEA reduced edema and brain infractions as evidenced by decreased 2,3,5-triphenyltetrazolium chloride (TTC) staining across brain sections. PEA-mediated improvements in tissues histology shown by reduction of lesion size and improvement in apoptosis level (assayed by Bax and Bcl-2) further support the efficacy of PEA therapy. We demonstrated that PEA treatment blocked infiltration of astrocytes and restored MCAo-mediated reduced expression of PAR, nitrotyrosine, iNOS, chymase, tryptase, growth factors (BDNF and GDNF) and GFAP. PEA also inhibited the MCAo-mediated increased expression of pJNK, NF-κB, and degradation of IκB-α. PEA-treated injured animals improved neurobehavioral functions as evaluated by motor deficits. Based on these findings we propose that PEA would be useful in lowering the risk of damage or improving function in ischemia-reperfusion brain injury-related disorders.

This study was designed to assess a protective effect of palmitoylethanolamide (PEA) in the development of inflammation after ischemia-reperfusion injury of the kidney. Moreover, to suggest a possible mechanism, renal ischemia-reperfusion was performed in mice with targeted disruption of peroxisome proliferator-activated receptor α (PPAR-α) gene (PPAR-αKO) to explain whether the observed PEA effect was dependent on PPAR-α pathway. Peroxisome proliferator-activated receptor-αKO and littermate wild-type controls (PPAR-αWT) were subjected to bilateral renal artery occlusion (30 min) and reperfusion (6 h) and received PEA (10 mg/kg i.p.) 15 min before release of clamps. Serum and urinary indicators of renal dysfunction and tubular and reperfusion injury were measured, specifically serum urea, creatinine, aspartate aminotransferase and γ-glutamyl transferase, and creatinine clearance. In addition, renal sections were used for histological scoring of renal injury and for immunologic evidence of nitrotyrosine formation, poly[adenosine diphosphate-ribose] (PAR), and adhesion molecules expression. The oxidative stress-sensitive nuclear factor κB signaling pathway was also investigated by Western blot analysis. Kidney myeloperoxidase activity and malondialdehyde levels were measured for assessment of polymorphonuclear leukocyte cell infiltration and lipid peroxidation, respectively. Apoptotic mechanisms were also investigated. Moreover, the infiltration and activation of mast cells were explored. In vivo, PEA administration during ischemia significantly reduced the increase in (i) creatinine, γ-glutamyl transferase, aspartate aminotransferase; (ii) nuclear translocation of nuclear factor κB p65; (iii) kidney myeloperoxidase activity and malondialdehyde levels; (iv) nitrotyrosine, PAR, and adhesion molecules expression; (v) the infiltration and activation of mast cells; and (vi) apoptosis. Our results clearly demonstrate that PEA significantly attenuated the degree of renal dysfunction, injury, and inflammation caused by ischemia-reperfusion injury. Moreover, the positive effects of PEA were at least in part dependent on PPAR-α pathway.

Spinal cord injury (SCI) is a traumatic event that causes a secondary and extended inflammation characterized by infiltration of immune cells, including T lymphocytes, release of pro-inflammatory mediators in the lesion site, and tissue degeneration. Current therapeutic approaches for SCI are limited to glucocorticoids (GC) due to their potent anti-inflammatory activity. GC efficacy resides, in part, in the capability to inhibit NF-κB, T lymphocyte activation, and the consequent cytokine production. In this study, we performed experiments aimed to test the susceptibility of glucocorticoid-induced leucine zipper (GILZ) transgenic (GILZ(TG)) mice, in which GILZ is selectively over-expressed in T lymphocytes, to SCI induction. Consistent with a decreased inflammatory response, GILZ(TG) were less susceptible to SCI as compared to wild-type littermates. Notably, inhibition of NF-κB activation and nuclear translocation, diminished T lymphocytes activation and tissue infiltration, as well as decreased release of cytokines were evident in GILZ(TG) as compared to wild-type mice. Moreover, GILZ(TG) showed a reduced tumor necrosis factor-α, IL-1β, Inductible nitric oxide synthase (iNOS) and nytrotyrosine production, apoptosis, and neuronal tissue damage. Together these results indicate that GILZ mimics the anti-inflammatory effect of GC and represents a potential pharmacological target for modulation of T lymphocyte-mediated immune response in inflammatory disorders, such as SCI.

Correct function of spermatogonia is critical for the maintenance of spermatogenesis throughout life, but the cellular pathways regulating undifferentiated spermatogonia proliferation, differentiation, and survival are only partially known. We show here that long glucocorticoid-induced leucine zipper (L-GILZ) is highly expressed in spermatogonia and primary spermatocytes and controls spermatogenesis. Gilz deficiency in knock-out (gilz KO) mice leads to a complete loss of germ cell lineage within first cycles of spermatogenesis, resulting in male sterility. Spermatogenesis failure is intrinsic to germ cells and is associated with increased proliferation and aberrant differentiation of undifferentiated spermatogonia and with hyperactivity of Ras signaling pathway as indicated by an increase of ERK and Akt phosphorylation. Spermatogonia differentiation does not proceed beyond the prophase of the first meiotic division due to massive apoptosis associated with accumulation of unrepaired chromosomal damage. These results identify L-GILZ as a novel important factor for undifferentiated spermatogonia function and spermatogenesis.

SummaryLittle 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.