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Background Graft-versus-host disease (GvHD) presents a major cause for morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Rabbit-derived antithymocyte globulin (rATG) treatment reduces the incidence of GvHD after allogeneic hematopoietic stem cell transplantation. However, delayed immune reconstitution following rATG treatment, partly caused by hampered thymic function, is being discussed. The present study aims at elucidating possible cytotoxic effects of 2 commonly used rATG preparations on cultured human thymic stroma, especially thymic epithelial cells (TECs). Methods A primary TEC culture was established and the binding and cytotoxicity of 2 rATG preparations to the aforementioned cells were assessed by flow cytometry and immunofluorescence analyses. The release of several cytokines by cultured thymic stroma cells in response to rATG was analyzed via multiplex enzyme-linked immunosorbent assays. Results Both preparations showed a comparable dose-dependent binding to TECs and exerted a similar complement-independent, dose-dependent cytotoxicity. rATG exposure further resulted in hampered secretion of interleukin (IL)-7, IL-15, and IL-6, cytokines being involved in thymic T cell development and proliferation. Pretreatment with keratinocyte growth factor diminished rATG-induced cytotoxicity of TECs and restored their IL-7 and IL-15 secretion. Conclusions Cytotoxic effects on TECs link the rATG-induced thymic damage to the delayed T cell reconstitution, witnessed after rATG treatment. Our data support a combination treatment of rATG and thymus-protective strategies such as keratinocyte growth factor to simultaneously offer sufficient GvHD prophylaxis and overcome delayed T cell reconstitution caused by thymic damage.

T cell recognition of unknown antigens relies on the tremendous diversity of the T cell receptor (TCR) repertoire; generation of which can only occur in the thymus. TCR repertoire breadth is thus critical for not only coordinating the adaptive response against pathogens but also for mounting a response against malignancies. However, thymic function is exquisitely sensitive to negative stimuli, which can come in the form of acute insult, such as that caused by stress, infection, or common cancer therapies; or chronic damage such as the progressive decline in thymic function with age. Whether it be prolonged T cell deficiency after hematopoietic cell transplantation (HCT) or constriction in the breadth of the peripheral TCR repertoire with age; these insults result in poor adaptive immune responses. In this review, we will discuss the importance of thymic function for generation of the TCR repertoire and how acute and chronic thymic damage influences immune health. We will also discuss methods that are used to measure thymic function in patients and strategies that have been developed to boost thymic function.

Even though the thymus is exquisitely sensitive to acute insults like infection, shock, or common cancer therapies such as cytoreductive chemo- or radiation-therapy, it also has a remarkable capacity for repair. This phenomenon of endogenous thymic regeneration has been known for longer even than its primary function to generate T cells, however, the underlying mechanisms controlling the process have been largely unstudied. Although there is likely continual thymic involution and regeneration in response to stress and infection in otherwise healthy people, acute and profound thymic damage such as that caused by common cancer cytoreductive therapies or the conditioning regimes as part of hematopoietic cell transplantation (HCT), leads to prolonged T cell deficiency; precipitating high morbidity and mortality from opportunistic infections and may even facilitate cancer relapse. Furthermore, this capacity for regeneration declines with age as a function of thymic involution; which even at steady state leads to reduced capacity to respond to new pathogens, vaccines, and immunotherapy. Consequently, there is a real clinical need for strategies that can boost thymic function and enhance T cell immunity. One approach to the development of such therapies is to exploit the processes of endogenous thymic regeneration into novel pharmacologic strategies to boost T cell reconstitution in clinical settings of immune depletion such as HCT. In this review, we will highlight recent work that has revealed the mechanisms by which the thymus is capable of repairing itself and how this knowledge is being used to develop novel therapies to boost immune function.

T cells play a critical role in mediating antigen-specific and long-term immunity against viral and bacterial pathogens, and their development relies on the highly specialized thymic microenvironment. T cell immunodeficiency can be acquired in the form of inborn errors, or can result from perturbations to the thymus due to aging or irradiation/chemotherapy required for cancer treatment. Hematopoietic stem cell transplant (HSCT) from compatible donors is a cornerstone for the treatment of hematological malignancies and immunodeficiency. Although it can restore a functional immune system, profound impairments exist in recovery of the T cell compartment. T cells remain absent or low in number for many months after HSCT, depending on a variety of factors including the age of the recipient. While younger patients have a shorter refractory period, the prolonged T cell recovery observed in older patients can lead to a higher risk of opportunistic infections and increased predisposition to relapse. Thus, strategies for enhancing T cell recovery in aged individuals are needed to counter thymic damage induced by radiation and chemotherapy toxicities, in addition to naturally occurring age-related thymic involution. Preclinical results have shown that robust and rapid long-term thymic reconstitution can be achieved when progenitor T cells, generated in vitro from HSCs, are co-administered during HSCT. Progenitor T cells appear to rely on lymphostromal crosstalk via receptor activator of NF-κB (RANK) and RANK-ligand (RANKL) interactions, creating chemokine-rich niches within the cortex and medulla that likely favor the recruitment of bone marrow-derived thymus seeding progenitors. Here, we employed preclinical mouse models to demonstrate that in vitro-generated progenitor T cells can effectively engraft involuted aged thymuses, which could potentially improve T cell recovery. The utility of progenitor T cells for aged recipients positions them as a promising cellular therapy for immune recovery and intrathymic repair following irradiation and chemotherapy, even in a post-involution thymus.

The use of prior exercise training has shown promise in minimizing doxorubicin (DOX)-induced physical impairments. The purpose of this study was to compare changes in thymus mass, thymocyte (T-cell) number, and tissue peroxidation following chronic endurance exercise and DOX treatment in the rat. The thymus mass, number of viable T-cells, and levels of malondialdehyde and 4-hydroxyalkenals (MDA+4-HAE) were compared 3 days post-injection between rats assigned to the following treatment conditions: ( a) 10 weeks of endurance training, followed by a saline injection 24 hours after the last training session (TM+SAL); ( b) treadmill training as above, followed by a single, bolus 10-mg/kg injection of DOX (TM+10); ( c) treadmill training with 12.5 mg/kg of DOX (TM+12.5); ( d) sedentary (without exercise) and a saline injection (SED+SAL); ( e) sedentary with 10 mg/kg of DOX (SED+10); and ( f) sedentary with 12.5 mg/kg (SED+12.5). Thymic mass and T-cell numbers significantly decreased following DOX injections. TM rats exhibited significantly less lipid peroxidation compared with paired-dose SED groups. TM+10 did not significantly differ from SED+SAL in thymic levels of lipid peroxidation. We conclude that chronic endurance exercise decreases levels of lipid peroxidation in the thymus seen with acute DOX treatment.

Posttransplant cyclophosphamide (PTCy) has been found to be effective in ameliorating acute graft-versus-host disease (GVHD) in patients following allogeneic hematopoietic stem cell transplantation (aHSCT). Adoptive transfer of high numbers of donor Tregs in experimental aHSCT has shown promise as a therapeutic modality for GVHD regulation. We recently described a strategy for in vivo Treg expansion targeting two receptors: TNFRSF25 and CD25. To date, there have been no direct comparisons between the use of PTCy and Tregs regarding outcome and immune reconstitution within identical groups of transplanted mice. Here, we assessed these two strategies and found both decreased clinical GVHD and improved survival long term. However, recipients transplanted with Treg-expanded donor cells (TrED) exhibited less weight loss early after HSCT. Additionally, TrED recipients demonstrated less thymic damage, significantly more recent thymic emigrants, and more rapid lymphoid engraftment. Three months after HSCT, PTCy-treated and TrED recipients showed tolerance to F1 skin allografts and comparable immune function. Overall, TrED was found superior to PTCy with regard to weight loss early after transplant and initial lymphoid engraftment. Based on these findings, we speculate that morbidity and mortality after transplant could be diminished following TrED transplant into aHSCT recipients, and, therefore, that TrED could provide a promising clinical strategy for GVHD prophylaxis.

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.

Background The delay in immune reconstitution after hematopoietic stem cell transplantation (HSCT), especially a delay in central immune reconstitution, leads to opportunistic infections and disease relapse after transplantation and affects the long-term outcome of HSCT. This delay is mainly attributable to thymic damage after myeloablative chemotherapy and radiotherapy. Methods We established a model of allogeneic bone marrow transplantation (BMT) in mice and administered ghrelin (GRL) 7 days before the conditioning regimen or the day after BMT to explore the effect of GRL on thymus. Results All the GRL-treated mice, especially those administered GRL before the conditioning regimen, exhibited more intact thymic architecture and a more rapid restoration of CD4 T lymphocytes after BMT than those of the corresponding control mice. Moreover, the levels of T cell receptor excision circles were significantly higher in the mice treated with GRL before the conditioning regimen than in the control mice at 28 days after BMT. Conclusions Our findings suggest that GRL may be a novel potential therapeutic approach to protecting the thymic epithelium from conditioning regimen-induced damage and promoting rapid and durable thymic and peripheral CD4 T cell recovery after HSCT.

Prevention of chronic graft-versus-host disease (cGVHD) remains a major challenge in allogeneic hematopoietic cell transplantation (HCT) owing to limited understanding of cGVHD pathogenesis and lack of appropriate animal models. In this study, we report that, in classical acute GVHD models with C57BL/6 donors and MHC-mismatched BALB/c recipients and with C3H.SW donors and MHC-matched C57BL/6 recipients, GVHD recipients surviving for >60 d after HCT developed cGVHD characterized by cutaneous fibrosis, tissue damage in the salivary gland, and the presence of serum autoantibodies. Donor CD8+ T cells were more potent than CD4+ T cells for inducing cGVHD. The recipient thymus and de novo–generated, donor-derived CD4+ T cells were required for induction of cGVHD by donor CD8+ T cells but not by donor CD4+ T cells. Donor CD8+ T cells preferentially damaged recipient medullary thymic epithelial cells and impaired negative selection, resulting in production of autoreactive CD4+ T cells that perpetuated damage to the thymus and augmented the development of cGVHD. Short-term anti-CD4 mAb treatment early after HCT enabled recovery from thymic damage and prevented cGVHD. These results demonstrate that donor CD8+ T cells cause cGVHD solely through thymic-dependent mechanisms, whereas CD4+ T cells can cause cGVHD through either thymic-dependent or independent mechanisms.

The immune reconstitution after allogeneic hematopoietic stem cell transplantation comprises thymus-dependent and thymus-independent pathways. We wanted to improve the understanding of this complex process using two different measurements at definite checkpoints of T-cell neogenesis. We therefore assessed the thymus-dependent pathway by combining measurements of single joint T-cell receptor excision circles (sjTREC) and β T-cell receptor excision circles (βTREC) in an improved quantitative light-cycler hybridization polymerase chain reaction assay. In a subgroup of patients, we additionally assessed the proliferation kinetics of the CD31(+) thymic naïve cell population, which corresponds to recent thymic emigrants by six-color immunostaining. After the establishment of normal values in 22 healthy volunteers, we applied our polymerase chain reaction to 66 patients undergoing allogeneic hematopoietic stem cell transplantation at a median age of 44 years. It took more than 2 years after transplant to restore the pre-transplant thymic proliferation capacity. Only one third of the patients in our longitudinal study reached age-adjusted normal values for both sjTREC and βTREC at a median follow-up of 558 days, with acute graft-versus-host disease being the most prominent negative factor by univariate analysis. We observed several patterns of sjTREC and βTREC recovery suggesting different mechanisms of thymic damage in individual patients. In a comparison of CD31(+) thymic naïve cells between volunteers and patients after transplant we found a significantly higher peak proliferation rate within the latter population in the first year after transplantation. The combination of measurements of sjTREC and βTREC by our simplified polymerase chain reaction assay provides insight about the stage of T-cell development affected by different types of damage and may help to choose the correct therapeutic intervention. Besides the sole thymic T-cell neogenesis, proliferation within the CD31(+) thymic naïve cell compartment contributed to the replenishment of the naïve T-cell pool after transplantation.

Acute graft-versus-host disease (aGvHD) continues to be a major obstacle to allogeneic haematopoietic stem cell transplantation. Thymic damage secondary to aGvHD along with corticosteroids and other non-selective T lymphocyte-suppressive agents used in the treatment of aGvHD concurrently impair thymopoiesis and negatively impact on immunoreconstitution of the adaptive immune compartment and ultimately adversely affect clinical outcome. Extracorporeal photopheresis (ECP) is an alternative therapeutic strategy that appears to act in an immunomodulatory fashion, potentially involving regulatory T lymphocytes and dendritic cells. By promoting immune tolerance and simultaneously avoiding systemic immunosuppression, ECP could reduce aGvHD and enable a reduction in other immunosuppression, allowing thymic recovery, restoration of normal T lymphopoiesis, and complete immunoreconstitution with improved clinical outcome. Although the safety and efficacy of ECP has been demonstrated, further randomised controlled studies are needed as well as elucidation of the underlying mechanisms responsible and the effect of ECP on thymic recovery.

Delayed T cell recovery after allogeneic hematopoietic stem cell transplant (allo-HSCT) is an important contributor to transplant-related morbidity and mortality due to infection and malignant relapse. Optimal immune reconstitution requires a functional thymus, however, at the present time strategies to improve thymic reconstitution are limited. Zinc (Zn) is the second most abundant trace metal in the body, binding to more than 300 proteins. Zn deficiency is a clinical condition causing immunosuppression and thymic atrophy with a consequent reduction in the number of circulating recent thymic emigrants (RTEs). Furthermore, the role of Zn in tissue regeneration after damage has been clearly demonstrated in liver, skin, and intestinal diseases. In a pilot clinical trial, we previously demonstrated that patients receiving oral Zn supplementation after autologous HSCT showed increased thymic-dependent T cell reconstitution in the absence of adverse clinical events. Although a clear clinical benefit was observed, the mechanisms underlying this process are poorly understood. Thus, we used a murine model to evaluate the effect of Zn supplementation in thymic reconstitution after acute damage. Using a model of thymic damage caused by sub-lethal total body irradiation (SL-TBI, 550 cGy), we found that mice that received Zn supplementation demonstrated increased thymic cellularity when compared to untreated age-matched mice (Fig. 1a). Importantly, this finding was also confirmed in a clinically-applicable model of MHC-matched allogeneic HSCT (Fig. 1b). We have previously demonstrated endothelial cells (EC), which are extremely resistant to damaged, are able to trigger thymic endogenous reconstitution after damage by producing regenerative factors such as BMP4, which targets thymic epithelial cells (TECs), a key population crucial for T cell development. Interestingly, in our model of Zn administration, we found an increase in the number of regeneration-initiating ECs (Fig. 1c), and increased proliferation of TECs (Fig. 1d), which can occur in response to BMP4. Consistent with the hypothesis that Zn supplementation is activating the BMP4 pathway, thymic levels of BMP4 were increased at day +10 in zinc-treated mice (Fig. 2a, 2b), and, when stimulated in vitro for 24 hours with Zn sulfate, ex vivo propagated ECs (exECs) were directly induced to produce BMP4 (Fig. 2c), suggesting a likely mechanism by which Zn supplementation promotes thymic reconstitution. EC also express on their surface the Zn receptor GPR39 (Fig 3a), which is able to translate extracellular Zn signals in intracellular changes, and upregulate GPR39 expression after damage (Fig 3b). In conclusion, we demonstrate a mechanism by which zinc supplementation can improve thymic function and offers an innovative therapeutic strategy to improve T cell reconstitution in patients receiving allo-HSCT.

Thymic epithelial cells (TECs) and dendritic cells are essential for the maintenance of thymopoiesis. Because these stromal elements define the progenitor niche, provide critical survival signals and growth factors, and direct positive and negative selection, detailed study of these populations is necessary to understand important elements for thymic renewal after cytotoxic injury. Study of TEC is currently hindered by lengthy enzymatic separation techniques with decreased viability. We present a new rapid separation technique that yields consistent viable TEC numbers in a quarter of the prior preparation time. Using this new procedure, we identify changes in stromal populations following total body irradiation (TBI). By flow cytometry, we show that TBI significantly depletes UEA+ medullary TEC, while sparing Ly51+ CD45− cells. Further characterization of the Ly51+ subset reveals enrichment of fibroblasts (CD45− Ly51+ MHCII−), while cortical TECs (CD45− Ly51+ MHCII+) were markedly reduced. Dendritic cells (CD11c+ CD45+) were also decreased following TBI. These data suggest that cytotoxic preparative regimens may impair thymic renewal by reducing critical populations of cortical and medullary TEC, and that such thymic damage can be assessed by this new rapid separation technique, thereby providing a means of assessing optimal conditioning pretransplant for enhancing thymic‐dependent immune reconstitution posttransplant.

Hematopoietic cell transplantation (HCT) is frequently complicated by graft-versus-host disease (GVHD). During the past three decades, experimental studies and clinical observations have elucidated the pathophysiology of acute GVHD, but the biology of chronic GVHD is much less well understood. Recommendations of the NIH Consensus Development Project on Criteria for Clinical Trials in Chronic GVHD have begun to standardize the diagnosis and clinical assessment of the disease. These criteria have emphasized the importance of qualitative differences, as opposed to time of onset after HCT, in making the distinction between acute and chronic GVHD. Experimental studies have generated at least four theories to explain the pathophysiology of chronic GVHD. These theories include 1) thymic damage and defective negative selection of T cells generated from marrow progenitors after HCT, 2) aberrant production of transforming growth factor-beta, 3) auto-antibody production, and 4) deficiency of T-regulatory cells. Recent studies in humans have corroborated a possible role for each of these mechanisms in humans. No animal model fully replicates all of the features of chronic GVHD in humans, and it appears likely that multiple biological mechanisms account for the diverse features the disease. Chronic GVHD may represent a "syndrome" with diverse causes among individual patients. In the future, it might become possible to tailor specific therapeutic interventions for patients as individually needed for each distinct pathophysiologic mechanism involved in development of the disease.

Thymus Chemotherapy and radiation treatments in cancer patients damage a number of tissues and organs, including the thymus. Prolonged thymic damage can lead to T cell deficiency and increased susceptibility to opportunistic infections and malignancies. Wertheimer et al. examined thymic regeneration in mice after sublethal total body radiation. They found a critical role for bone morphogenetic protein 4 (BMP4) signaling in thymic regeneration. Endothelial cells provided a critical source of BMP4, which induces expression of the transcription factor FOXN1 in thymic epithelial cells to promote thymic regeneration. Sci. Immunol. 3 , eaal2736 (2018).

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.

BACKGROUND We studied whether T-cell clones, which appear in the periphery as a result of the failure of thymic negative selection during graft-versus-host disease (GVHD), have any in vivo anti-host reactivity and can cause GVHD in an adoptive transfer model. METHODS We performed our studies in a murine model (B10.BR into CBA/J) for allogeneic bone marrow transplantation with major histocompatibility complex-matched and minor histocompatibility antigen-mismatched unrelated donors and unique Vbeta T-cell deletion patterns in donors and recipients. RESULTS GVHD resulted in the appearance of Vbeta6+ T cells as a result of a loss of negative selection. We found that Vbeta6+ T cells from normal donors proliferated in vitro and in vivo. Depletion of Vbeta6+ T cells from the donor T-cell inoculum resulted in less GVHD morbidity and a decrease in the loss of thymic cellularity. To test the anti-host reactivity of de novo generated Vbeta6+ T cells in animals with GVHD, we developed an adoptive transfer model of splenic T cells from CBA/J host animals with GVHD into sublethally irradiated CBA/J recipients Depletion of Vbeta6+ T cells from the splenic T cells before adoptive transfer could significantly decrease the transient GVHD morbidity in the sublethally irradiated hosts. CONCLUSIONS Our data indicate that GVHD-associated thymic damage results in a loss of thymic negative selection, which leads to the appearance of T-cell clones with anti-host reactivity in vitro and in vivo.

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.

Chronic graft-versus-host disease (CGVHD) is one of the most significant complications of long-term survivors after allogeneic hematopoietic stem cell transplantation (allo-HSCT). CGVHD may have protean manifestations and can pose unique diagnostic and therapeutic challenges. New recommendations that emphasize the importance of qualitative differences, as opposed to time of onset after HSCT, are now being used to standardize the diagnosis and clinical assessment of CGVHD, but they require validation. During the past 3 decades, experimental studies and clinical observations have elucidated the mechanisms of acute GVHD, but its biology is much less well-understood. Experimental studies have generated at least 4 theories to explain the pathophysiology of CGVHD: (1) thymic damage and the defective negative selection of T cells, (2) regulatory T cell deficiencies, (3) auto-antibody production by aberrant B cells, and (4) the formation of profibrotic lesions. Mouse models have provided important insights into the pathophysiology of CGVHD, and these have helped improve clinical outcomes following allo-HSCT, but no animal model fully replicates all of the features of CGVHD in humans. In this article, recent clinical changes, the pathogenesis of CGHVD, the cellular and cytokine networks implicated in its pathogenesis, and the animal models used to devise strategies to prevent and treat CGVHD are reviewed.

The thymic stroma supports T lymphocyte development and consists of an epithelium maintained by thymic epithelial progenitors. The molecular pathways that govern epithelial homeostasis are poorly understood. Here we demonstrate that deletion of Rac1 in Keratin 5/Keratin 14 expressing embryonic and adult thymic epithelial cells leads to loss of the thymic epithelial compartment. Rac1 deletion led to an increase in c-Myc expression and a generalized increase in apoptosis associated with a decrease in thymic epithelial proliferation. Our results suggest Rac1 maintains the epithelial population, and equilibrium between Rac1 and c-Myc may control proliferation, apoptosis and maturation of the thymic epithelial compartment. Understanding thymic epithelial maintenance is a step toward the dual goals of in vitro thymic epithelial cell culture and T cell differentiation, and the clinical repair of thymic damage from graft-versus-host-disease, chemotherapy or irradiation.

T-cell production throughout life depends on efficient colonization and intrathymic expansion of BM-derived hematopoietic precursors. After irradiation-induced thymic damage, thymic recovery is facilitated by Flt3 ligand (FL), expressed by perivascular fibroblasts surrounding the thymic entry site of Flt3 receptor-positive progenitor cells. Whether intrathymic FL-Flt3 interactions play a role in steady-state replenishment of T cells remains unknown. Here, using competitive BM transplantation studies and fetal thymic organ cultures we demonstrated the continued numerical advantage of Flt3+ intrathymic T-cell precursors. Sub-kidney capsule thymic transplantation experiments, in which WT and FL-/- thymic lobes were grafted into FL-/- recipients, revealed that FL expression by the thymic microenvironment plays a role in steady-state thymopoiesis. The deficiency of the most immature thymic T-cell precursors correlated to upregulation of FL by thymic MTS15+ fibroblasts, suggesting that the number of Flt3+ progenitor cells may regulate the thymic expression of this cytokine. Together, these results show that FL expression by thymic stromal fibroblasts interacting with Flt3+ T-cell progenitors is important for the physiological maintenance of early T-cell development.

Acute and chronic graft-versus-host disease (GVHD) remain the major complications limiting the efficacy of allogeneic hemopoietic stem cell transplantation. Chronic GVHD can evolve from acute GVHD, or in some cases may overlap with acute GVHD, but how acute GVHD evolves to chronic GVHD is unknown. In this study, in a classical CD8+ T cell-dependent mouse model, we found that pathogenic donor CD4+ T cells developed from engrafted hemopoietic stem cells (HSCs) in C57BL/6SJL(B6/SJL, H-2b) mice suffering from acute GVHD after receiving donor CD8+ T cells and HSCs from C3H.SW mice (H-2b). These CD4+ T cells were activated, infiltrated into GVHD target tissues, and produced high levels of IFN-γ. These in vivo-generated CD4+ T cells caused lesions characteristic of chronic GVHD when adoptively transferred into secondary allogeneic recipients and also caused GVHD when administered into autologous C3H.SW recipients. The in vivo generation of pathogenic CD4+ T cells from engrafted donor HSCs was thymopoiesis dependent. Keratinocyte growth factor treatment improved the reconstitution of recipient thymic dendritic cells in CD8+ T cell-repleted allogeneic hemopoietic stem cell transplantation and prevented the development of pathogenic donor CD4+ T cells. These results suggest that de novo-generated donor CD4+ T cells, arising during acute graft-versus-host reactions, are key contributors to the evolution from acute to chronic GVHD. Preventing or limiting thymic damage may directly ameliorate chronic GVHD.

Cadmium immunotoxicity in rodents is primarily characterized by marked thymic damage and splenomegaly. To understand the toxicity of Cd on lymphoid cells in vivo, a single dose of Cd as CdCl2 (1.8 mg/kg, i.p.) was administered to male BALB/c mice and cytotoxicity (MTT assay), oxidative stress indicators (glutathione, reactive oxygen species) and apoptotic markers (mitochondrial membrane potential, caspase-3 activity, phosphatidylserine externalization, apoptotic DNA, intranucleosomal DNA fragmentation) were assessed in thymic and splenic single cell suspensions, at various time intervals. Lowering of body weight gain and cellularity and a loss in cell viability was seen in the Cd treated mice. The earliest significant increase in ROS at 18 h, followed by mitochondrial membrane depolarization, caspase-3 activation and GSH depletion at 24h in spleen and later at 48 h in thymus, strongly implicate the possible involvement of ROS. A pronounced inhibition of cell proliferative response at 48 h and 72 h may also be linked to Cd induced apoptosis. The morphological alterations including thymic cortical cell depletion and an increase in red pulp with diminished white pulp in spleen were observed at 48 h and beyond. The splenic cells appeared more susceptible than thymus cells to the adverse effects of Cd. The present study, therefore, demonstrates potentiation of oxidative stress followed by mitochondrial-caspase dependent apoptotic pathway. This may, in part, be responsible for causing suppression of cell proliferative response, thymic atrophy and splenomegaly.

Current models hold that CD4+ depletion occurs as a result of direct and indirect effects of HIV, which both kill peripheral CD4+ cells and prevent adequate regeneration. Although age-associated involution diminishes thymic reserve and HIV is clearly thymotoxic, clinical trials have nonetheless shown that large proportions of patients who sustain adequate control of viral replication with highly active antiretroviral therapy (HAART) will demonstrate some evidence for thymic-dependent immune reconstitution, which is associated with improved immune competence. Furthermore, patients with insufficient or absent immune reconstitution following HAART generally lack evidence for thymopoiesis. Current studies are focused on improving our understanding of the causes for thymic failure in HIV infection. Recent work has demonstrated that some HIV strains, especially those that are CXCR4 trophic, are more thymotoxic and may contribute to irreversible thymic damage in this population.

The thymus is the primary lymphoid organ supplying new lymphocytes to the periphery. Clinical and morphologic studies of HIV-infected children and adults indicate that the thymus is affected by HIV. Thymic dysfunction and thymic involution occur during HIV disease and have been associated with rapid progression in infants infected perinatally with HIV. In vitro information of thymic organ culture, thymic epithelial cell culture, the SCID-hu mouse system and SHIV infection of primates have supported HIV-induced thymic damage. The mechanisms underlying this could be many, including direct thymocyte killing by the virus, apoptosis, or disruption of thymic stromal architecture. T cell receptor excision circles (TREC) have been developed as a marker of new thymic emigrants. Decreases in TREC concentrations have been found in both HIV-infected pediatric and adult patients. Mathematical models have suggested that thymic infection in children is more severe than in adults, particularly during infection with strains that use CXCR4 as coreceptor. Recent evidence suggests that thymic recovery may be achieved in some patients as a result of potent antiretroviral therapy. Extensive thymic damage may, however, hamper immune reconstitution, particularly in pediatric patients. This review attempts to summarize evidence for thymic involvement during HIV infection in children and in adults, the role of thymic infection in disease progression, and the contribution of the thymus to immune restoration following potent antiviral therapy. Immunologic interventions aiming at restoring thymic function in AIDS patients are also reviewed.

Abstract 339 There is little understanding of the maintenance and regeneration of epithelial tissues after allogeneic transplant. Most clinical strategies to limit epithelial damage from graft vs. host disease (GVHD) also limit post-transplant immune function. Damage to the gastrointestinal (GI) tract from GVHD is a major cause of morbidity and mortality, and damage to the thymus from pre-transplant conditioning and GVHD can impair immune reconstitution, predispose patients to infection, and increase the risk of relapse. Therefore, understanding of tissue damage and recovery could lead to strategies selectively protecting epithelial tissues, improving intestinal barrier function, and promoting immune reconstitution without worsening post-transplant immunosuppression. We have recently identified that IL-22 from recipient-derived innate lymphoid cells (ILC) is critical for promoting intestinal recovery from GVHD and for promoting thymic recovery from radiation/pre-transplant conditioning. IL-22 deficient mice demonstrated significantly reduced thymopoiesis after total body irradiation (TBI), and IL-22 deficient murine bone marrow transplant (BMT) recipients demonstrated increased GVHD mortality and intestinal histopathology, deficiency of the antimicrobial molecules Reg3γ and Reg3β, and loss of intestinal stem cells needed for epithelial recovery. The source of thymic and intestinal IL-22 was RORγ+CD3−NKp46−IL-7R+CCR6+ lymphoid-tissue-inducer-like cells. Similar to as had been observed in the thymus, intestinal ILC produced IL-22 in response to IL-23, which was upregulated after TBI (p Although intestinal ILC could survive lethal TBI, they were significantly depleted by both MHC mismatched (B6BALB/c) and MHC matched (LPB6) GVHD. Similarly, GVHD led to depletion of thymic IL-22+ ILC and reduction in thymic IL-22 levels (p In summary, IL-22+ ILC are radioresistant and capable of regulating tissue-specific epithelial recovery after allogeneic BMT. However, recipient ILC are extremely sensitive to GVHD, leading to a loss of the IL-22-mediated recovery response. IL-21 blockade can prevent the elimination of recipient thymic ILC by donor T cells in GVHD, and IL-22 administration can restore the thymopoiesis that is lost in GVHD due to ILC elimination. Maintenance of epithelial function post-BMT is thus an active innate immune response requiring cooperation between both recipient stroma and recipient hematopoietic cells. Disclosures: No relevant conflicts of interest to declare.

234 Thymic GVHD (tGVHD) after allogeneic bone marrow transplantation (allo-BMT) is associated with prolonged immunodeficiency. We have previously shown that thymic output after allo-BMT is directly related to thymus size, and inversely related to donor T cell dose and GVHD severity. Additionally, radiation-containing preparative regimens upregulate the death receptors Fas and DR5 on thymic stroma (especially epithelium) while decreasing expression of the anti-apoptotic protein cFLIP, thereby sensitizing the thymus to GVHD. Moreover, small numbers of donor alloreactive T cells are sufficient to cause tGVHD, and they utilize the Fas/Fas ligand (FasL) and TRAIL/DR5 pathways to mediate damage thymic stroma, architecture and function. We performed experiments in both MHC-mismatched and MHC-matched minor antigen-disparate model systems, and demonstrated the exquisite sensitivity of the thymus to as few as 1–2.5×10 5 donor T cells, which mediated tGVHD without evidence of overt clinical disease or significant weight loss. Additionally, tGVHD is partially reversible in our model systems (contingent on a low T cell dose), such that mice with tGVHD exhibit a transient but partially reversible decrease in thymic cellularity when measured at days 28 vs. 60 post-transplant (Figure 1). To further study the role of TRAIL in tGVHD, we asked whether (1) alloreactive T cells and (2) the inflammation associated with conditioning and acute GVHD, were strictly required for TRAIL/DR5-mediated thymic damage. We treated recipients of T cell-depleted allo-BMT with the amDR5-1 agonistic antibody (0.2 mg i.p. per dose) either in the ‘early‘ peri-transplant period, or ‘late,‘ in the second week post-transplant. Allo-BMT recipients treated ‘early‘ with amDR5-1 had significantly decreased thymic cellularity and splenic BM-derived T cells as compared with controls. Furthermore, we observed similar BM cellularity and BM-derived lineage − sca-1 + ckit + (LSK), all in the absence of donor alloreactive T cells and GVHD (Figure 2). We observed similar results with mice treated ‘late‘ amDR5-1 using the later schedule, which indicates that the thymus has continued sensitivity to TRAIL throughout the post-transplant period, and that GVHD and/or conditioning-associated cytokines are not required to enable TRAIL-mediated damage to the thymus. We further assessed the expression of DR5 on donor BM-derived thymocytes to determine whether amDR5-1 acted directly on thymocytes. We observed that on day 28 after T cell-depleted allo-BMT, only 1-2% of donor thymocytes expressed DR5, suggesting that amDR5-1 (and potentially TRAIL) mediate their effects on thymic cellularity and function primarily via an indirect mechanism (Figure 3). These data suggest to us that significant damage to the thymus and thymopoiesis during allo-BMT: can be mediated by soluble TRAIL agonists, even without the presence of whole donor alloreactive T cells can occur, via the TRAIL pathway, without a full complement of the inflammatory processes typically associated with GVHD pathophysiology, including the cytokine storm, and can occur early and late post-transplant, and that early exposure of the thymus to TRAIL/DR5 signals are sufficient to cause a durable loss in thymic cellularity, donor CD4 + CD8 + thymocytes counts, and numbers of donor BM T cells exported to the periphery. ccurs via the TRAIL pathway, without significant direct damage (via TRAIL/DR5 interactions) to the bone marrow compartment, and thus by implication, thymic-seeding precursors derived from the BM. Together, our data in clinically-relevant mouse allo-BMT models suggests that the thymus is highly sensitive to GVHD and endures severe damage at relatively low levels of systemic GVHD. Moreover, post-transplant thymic atrophy is a partially-reversible process which depends on the T cell dose, and which occurs via the TRAIL pathway. Finally, we provide significant mechanistic insight which shows that TRAIL-mediated thymic damage can occur (1) throughout the early post-transplant period, and (2) does not strictly require alloreactive T cells, or the inflammatory processes associated with conditioning and GVHD. Disclosures: No relevant conflicts of interest to declare.

The thymic-dependent pathway that involves generation of new naive T cells from donor-derived precursor cells accounts for the more durable reconstitution of the T-cell compartment and generates a more diverse TCR repertoire. Thymic function and production of recent thymic emigrants (RTEs) may be directly evaluated through the quantification, by real-time polymerase chain reaction (PCR), of the T-cell receptor excision circles (TRECs). Following hematopoietic stem cell transplant (HSCT), there is a prolonged period of profound immune deficiency, which continues for years after HSCT. The factors that inhibit thymic function may include age, graft-versus-host disease (GVHD), and direct thymic damage from chemoradiotherapy. GVHD after HSCT also leads to thymic insufficiency, possibly by direct attack on the thymic stroma by allogeneic effector cells. The aim of our study is to analysis T cell receptor excision circles (TRECs) in patients with GVHD after allogeneic stem cell transplantation. We used real-time polymerase chain reaction (PCR) to quantify SjTRECs in 12 patients with GVHD(9 males, 3 females; median age 32 years old), who underwent HLA-matching sibling BMT and/or peripheral blood stem cell transplantation (PBSCT) at our department. Quantitative detection of sjTRECs in DNA of peripheral blood mononuclear cells from 13 normal individualals. The median value of sjTRECs copies P1 000 PBMCs was 4.37±3.64 in normal indiviuals. However, the decreased levels of TRECs were most profound in the group of patients with active chronic GVHD at the time of study. it was 0.26±0.22 copies P1 000 PBMCs in 12 patients with GVHD (P < 0. 00007). We conclude that measurement of sjTREC may provide an important tool for predicting thymus-dependent T-cell reconstitution in GVHD patients after transplantation.