ALBERT

Description: Thymopoiesis is a complex process involving crosstalk between developing thymocytes and the non-hematopoietic stromal microenvironment, which includes thymic epithelial cells (TECs), fibroblasts and endothelial cells (ECs). Despite its importance, the thymus is exquisitely sensitive to cellular insults, including cytoreductive chemo- and radiation therapy required for successful hematopoietic stem cell transplantation; therefore identification of thymic repair mechanisms will offer promising therapeutic targets for immune regeneration. Recent studies in tissues such as liver, lung and bone marrow have revealed that ECs not only passively deliver oxygen and nutrients to tissues, but also actively produce distinct paracrine factors that can orchestrate their repair. The role of thymic ECs in thymopoiesis beyond their contribution of local circulation and the importation of lymphoid progenitors has not been comprehensively studied. In order to evaluate the role of ECs in thymic regeneration, we closely examined the kinetics of thymic recovery following total body irradiation (TBI, 550cGy) in young C57BL/6 mice. Although we observed a dramatic decline in total thymic cellularity, we found no significant change in the number of thymic ECs, suggesting they are extremely radio-resistant (Fig. 1A). Interestingly, although thymic ECs appeared to be resistant to the cytoreductive effects of TBI, we revealed an increase in their proliferation as measured by expression of Ki67 shortly after injury, indicating a role in the endogenous regeneration of the thymus (Fig. 1B). Given their radioresistance and cycling after TBI, we hypothesized that thymic ECs can provide instructive signals supporting thymic regeneration. To explore potential regenerative signals stemming from ECs during thymic regeneration, we performed a transcriptome analysis of highly purified ECs of untreated mice and at days 4 and 7 following TBI. Among the 288 genes that were altered in ECs after TBI (131 upregulated and 157 downregulated), we found significant upregulation in the expression of BMP4 (which has previously been implicated in thymic regeneration) and was validated using quantitative PCR (Fig 1C). In addition, we have developed a novel light sheet fluorescence microscopy approach to visualize the thymic vasculature in 3D at high resolution (Fig. 1D). Consistent with their potential role in aiding thymic regeneration, administration of ex vivo expanded thymic ECs (ex-EC) could significantly enhance thymic regeneration when given after TBI. Intriguingly, this regenerative effect of ex-ECs was tissue specific as ex-EC derived from heart or kidney did not exhibit a similar regenerative effect (Fig. 1E). Similar to our findings in the endogenous regeneration setting, we found that thymic ex-ECs demonstrated a significantly higher BMP4 expression compared to cardiac and kidney ex-ECs (Fig. 1F). Previous reports have suggested that BMP4 signaling is capable of directly regulating the key transcription factor of TEC function, FOXN1. We found that incubation of the TEC cell line C9 with thymic ex-EC conditioned medium induced an increase in FOXN1 expression but was abrogated in the presence of Noggin, a potent BMP signaling inhibitor (Fig. 1G). These findings support the hypothesis that BMP4 mediates the regenerative effect of ECs in thymic regeneration. In summary, we found that thymic ECs are capable of mediating endogenous thymic regeneration, likely via their expression of BMP4. Adoptive transfer of ex-ECs leads to enhanced thymic regeneration after immune injury. Thus, adoptive transfer of thymic ECs represents a novel strategy to improve immune function in immunocompromised patients. Figure 1 Figure 1. Disclosures Ginsberg: Angiocrine Bioscience: Employment. Rafii:Angiocrine Biosciences: Founder Other.

Description: 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

Description: 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.

Description: 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.

Description: A tightly regulated network of intrinsic and extrinsic signaling pathways exists to preserve HSC pool size and function. This is particularly relevant during hematopoietic injuries when dormant HSCs transiently start to proliferate to replenish blood cells; as unbalanced HSC proliferation can lead to stem cell exhaustion, long-term myelosuppression and death. Although there has been growing interest in how circulating sex hormones influence HSC function (Nakada et al., 2014; Sanchez-Aguilera et al., 2014), this pathway remains poorly understood. Here we describe a heretofore unknown role for the upstream hormone regulator, luteinizing hormone (LH), in regulating HSC biology. We found that both human and mouse HSCs highly expressed the LH receptor, and its expression was decreased or nearly absent in downstream progenitors (Figure 1a). LH significantly promoted HSC colony forming potential in cobblestone area-forming cell and colony-forming cell assays that, together with expression of the receptor, suggested that LH increased HSC expansion in vitro by acting directly on the most primitive HSCs. To investigate whether LH levels could impact on HSC pool size during hematopoietic stress in vivo, we challenged mice using models that force HSCs out of their quiescent status, Poly I:C and sub-lethal dose of total body irradiation (SL-TBI, 550cGy). We found that ablation of LH production using a luteinizing hormone-releasing hormone-antagonist (LHRH-Ant) retained significantly more HSCs in G0 in both models (Figure 1b,c). Previous reports have shown that induction of HSC quiescence after high-dose irradiation correlates with increased hematopoietic recovery and enhanced mouse survival (Chen et al., 2008; Himburg et al., 2014; Johnson et al., 2010). Given its effectiveness in promoting HSC quiescence and the fact that LHRH-Ant are widely available and clinically approved, we hypothesized that LHRH-Ant could represent a rational non-cellular medical countermeasure for mitigating radiation injury and promoting hematopoietic regeneration when administered after hematopoietic insult. To test this hypothesis, we used a lethal TBI (L-TBI) dose of 840cGy that mediated lethality in more than 90% of B6 male mice. We found that pharmacological inhibition of LH using LHRH-Ant 24h after L-TBI spared the most primitive long term HSCs (Figure 1d) thus promoting hematopoietic recovery and mouse survival (Figure 1e). Consistent with our original hypothesis we also found a significantly higher proportion of Ki-67− quiescent HSCs in the LHRH-Ant-treated group with fewer proliferative HSCs compared to controls. Given the wide-ranging hormonal changes induced by LHRH suppression and the previously reported effects mediated by sex steroid ablation on hematopoietic stem/progenitor cell (HSPC) compartment (Khong et al., 2015), we next evaluated whether the LHRH-Ant effects on mouse survival after L-TBI were independent from the suppression of the downstream sex steroids. Administration of LHRH-Ant improved survival rates in surgically castrated mice following radiation injury, while surgical castration alone did not, indicating that the regenerative effects were independent from downstream sex steroids. To confirm whether the protective effects of LHRH-Ant treatment depended on suppression of LH, we administered the LH receptor agonist human chorionic gonadotropin (hCG) to LHRH-Ant treated mice that had been given L-TBI one-day prior. Consistent with our hypothesis, administration of hCG abrogated the beneficial effects of LHRH-Ant on survival after radiation injury. Taken together our studies showed that HSCs are a physiological target of LH, which promotes their proliferation. Furthermore, pharmacological inhibition of LH signaling using a single dose of an LHRH-Ant represents a rational and feasible approach to preserve the HSC pool after high dose radiation, thereby mitigating acute hematopoietic radiation syndrome. Disclosures Van Den Brink: Seres: Research Funding; Novartis: Consultancy; Regeneron: Consultancy; Flagship Ventures: Consultancy; Boehringer Ingelheim: Consultancy; Merck: Consultancy.

Description: Thymopoiesis is a complex process dependent on precise signals from the supporting thymic stromal microenvironment that orchestrates the progression of precursor T cells through well-defined maturation stages. It is well documented that the decline in thymic size and function with age is in part correlated with an increase in sex steroids. This age-related decline in function can be detrimental to the recovery of the thymus in patients receiving radio or chemo-therapy with hematopoietic stem cell transplantation (HSCT). Delayed immune reconstitution, especially in the T cell lineage, is associated with an increased risk of opportunistic infections and malignant relapses. Therefore strategies to enhance thymic reconstitution has the potential to decrease the period of T cell lymphopenia and increase overall clinical outcome. In the process of evaluating the effects of sex steroids in the decline of the thymic function, we found a decrease in the expression of the key thymopoietic factors IL-7, CCL25 and Delta-like 4 (DLL4) by thymic stromal cells after testosterone treatment (Figure 1A). We then addressed if these transcriptional changes were the result of a direct regulation by the androgen receptor (AR). Using a computational approach, and subsequently confirmed by ChIP studies, we found that AR directly bound and negatively regulated the promoter of DLL4, a critical gene involved in T cell commitment and differentiation. We and others have previously shown that sex steroid ablation (SSA) can regenerate young and aged immune system by promoting bone marrow and thymic lymphopoiesis and promoting recovery from autologous and allogeneic HSCT. However the mechanisms underlying the sex steroid-mediate thymic involution and its regeneration after SSA are poorly understood. Moreover, one of the main drawbacks to standard clinical methods of sex steroid ablation using luteinizing hormone releasing hormone (LHRH) agonists (LHRH-Ag) is the initial surge in sex steroids they cause. To address this, we employed a novel class of LHRH-antagonists (LHRH-Ant) that rapidly block the secretion of sex steroids without causing their initial surge that can be even more detrimental to thymopoiesis. Mice treated with LHRH-Ant showed a significantly faster increase in thymic cellularity compared with LHRH-Ag treated mice (Figure 1B). Given the negative regulation of DLL4 by the AR, we hypothesized that DLL4 expression would conversely increase after SSA in vivo. Indeed, we found a significant increase in DLL4 expression after SSA and also an increase in genes downstream of DLL4, such as Ptcra, Hes1 and Cd25 (Figure 1C). We next evaluated if treatment with the LHRH-Ant would provide a faster immune recovery after injury to the immune system. We found that mice treated with LHRH-Ant showed a faster thymic regeneration after total body irradiation (TBI) compared to the control irradiated mice (Figure 1D) and enhanced viral clearance (Figure 1E). Finally, we also found that LHRH-Ant enhanced thymic and peripheral reconstitution up to 3 months after allo-HSCT (Figure 1F). In conclusion, we found that down-regulation of DLL4 may represent one of the mechanisms underlying the effects of sex steroids on thymic function. We demonstrate that SSA with a novel LHRH-Ant increases DLL4 expression and enhances thymic and peripheral T cell recovery and function after immune injury. These findings suggest that the employment of a LHRH-Ant, which is already in clinical use for prostate cancer patients, represents a novel therapeutic strategy to enhance immune recovery and function in immunocompromised patients. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points Image-guided intrathymic injection of cells or drugs permits implementation of clinically relevant strategies to improve thymic function. Intrathymic injection of hematopoietic stem cells generates long-lasting antigen-specific T-cell immunity.