Role of endothelial cells in graft-versus-host disease.

To date, the only curative treatment for high-risk or refractory hematologic malignancies non-responsive to standard chemotherapy is allogeneic hematopoietic transplantation (allo-HCT). Acute graft-versus-host disease (GVHD) is a donor T cell-mediated immunological disorder that is frequently fatal and the leading cause of non-relapse mortality (NRM) in patients post allo-HCT. The pathogenesis of acute GVHD involves recognition of minor and/or major HLA mismatched host antigens by donor T cells followed by expansion, migration and finally end-organ damage due to combination of inflammatory cytokine secretion and direct cytotoxic effects. The endothelium is a thin layer of endothelial cells (EC) that line the innermost portion of the blood vessels and a key regulator in vascular homeostasis and inflammatory responses. Endothelial cells are activated by a wide range of inflammatory mediators including bacterial products, contents released from dying/apoptotic cells and cytokines and respond by secreting cytokines/chemokines that facilitate the recruitment of innate and adaptive immune cells to the site of inflammation. Endothelial cells can also be damaged prior to transplant as well as by alloreactive donor T cells. Prolonged EC activation results in dysfunction that plays a role in multiple post-transplant complications including but not limited to veno-occlusive disease (VOD), transplant associated thrombotic microangiopathy (TA-TMA), and idiopathic pneumonia syndrome. In this mini review, we summarize the biology of endothelial cells, factors regulating EC activation and the role of ECs in inflammation and GVHD pathogenesis.

Modulating endothelial cells with EGFL7 to diminish aGVHD after allogeneic bone marrow transplantation in mice.

Acute graft-versus-host disease (aGVHD) is the second most common cause of death after allogeneic hematopoietic stem cell transplantation (allo-HSCT), underscoring the need for novel therapies. Based on previous work that endothelial cell dysfunction is present in aGVHD and that epidermal growth factor-like domain 7 (EGFL7) plays a significant role in decreasing inflammation by repressing endothelial cell activation and T-cell migration, we hypothesized that increasing EGFL7 levels after allo-HSCT will diminish the severity of aGVHD. Here, we show that treatment with recombinant EGFL7 (rEGFL7) in 2 different murine models of aGVHD decreases aGVHD severity and improves survival in recipient mice after allogeneic transplantation with respect to controls without affecting graft-versus-leukemia effect. Furthermore, we showed that rEGFL7 treatment results in higher thymocytes, T, B, and dendritic cell counts in recipient mice after allo-HSCT. This study constitutes a proof of concept of the ability of rEGFL7 therapy to reduce GHVD severity and mortality after allo-HSCT.

Corrigendum: Role of endothelial cells in graft-versus-host disease.

[This corrects the article DOI: 10.3389/fimmu.2022.1033490.].

Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on MIR300 Antiproliferative and PP2A-Activating Functions.

Persistence of drug-resistant quiescent leukemic stem cells (LSC) and impaired natural killer (NK) cell immune response account for relapse of chronic myelogenous leukemia (CML). Inactivation of protein phosphatase 2A (PP2A) is essential for CML-quiescent LSC survival and NK cell antitumor activity. Here we show that MIR300 has antiproliferative and PP2A-activating functions that are dose dependently differentially induced by CCND2/CDK6 and SET inhibition, respectively. MIR300 is upregulated in CML LSCs and NK cells by bone marrow microenvironment (BMM) signals to induce quiescence and impair immune response, respectively. Conversely, BCR-ABL1 downregulates MIR300 in CML progenitors to prevent growth arrest and PP2A-mediated apoptosis. Quiescent LSCs escape apoptosis by upregulating TUG1 long noncoding RNA that uncouples and limits MIR300 function to cytostasis. Genetic and pharmacologic MIR300 modulation and/or PP2A-activating drug treatment restore NK cell activity, inhibit BMM-induced growth arrest, and selectively trigger LSC apoptosis in vitro and in patient-derived xenografts; hence, the importance of MIR300 and PP2A activity for CML development and therapy.

Studying Peripheral T Cell Homeostasis in Mice: A Concise Technical Review.

For several years, it was believed that the thymus was entirely responsible for maintaining T cell homeostasis. Today, it is well-known that homeostatic peripheral mechanisms are essential in order to maintain T cell numbers and diversity constant in the periphery. Naïve and memory T cells require continual access to self-peptide MHC class I and II molecules and/or cytokines to survive in the periphery. Under normal conditions, homeostatic resources are low, and lymphocytes undergo very slow proliferation and survive. Following T cell depletion, the bioavailability of homeostatic resources is significantly increased, and T cell proliferation is dramatically augmented. The development of lymphopenic mouse models has helped our current understanding of factors involved in the regulation of peripheral T cell homeostasis. In this minireview, we will give a brief overview about basic techniques used to study peripheral T cell homeostasis in mice.

IL-7 Is the Limiting Homeostatic Factor that Constrains Homeostatic Proliferation of CD8+ T Cells after Allogeneic Stem Cell Transplantation and Graft-versus-Host Disease.

Immune reconstitution after allogeneic hematopoietic stem cell transplantation relies primarily on homeostatic proliferation (HP) of mature T lymphocytes, but this process is typically impaired during graft-versus-host disease (GVHD). We previously showed that low IL-7 levels combined with lack of dendritic cell (DC) regeneration constrain CD4+ T cell HP during GVHD. However, it is not clear whether these alterations to the peripheral CD4+ T cell niche also contribute to impair CD8+ T cell regeneration during GVHD. We found that IL-7 therapy was sufficient for restoring CD8+ T cell HP in GVHD hosts while forcing DC regeneration with Flt3-L had only a modest effect on CD8+ T cell HP in IL-7 treated mice. Using bone marrow chimeras, we showed that HP of naïve CD8+ T cells is primarily regulated by MHC class I on radio-resistant stromal cells, yet optimal recovery of CD8+ T cell counts still requires expression of MHC class I on both radio-resistant and radio-sensitive hematopoietic cells. Thus, IL-7 level is the primary limiting factor that constrains naïve CD8+ T cell HP during GVHD, and accessibility of MHC class I on stromal cells explains how IL-7 therapy, as a single agent, can induce robust CD8 + T cell HP in the absence of DCs.

Double-Negative T Cell Levels Correlate with Chronic Graft-versus-Host Disease Severity.

Chronic graft-versus-host disease (cGVHD) is a major complication, affecting 50% to 80% of long-term survivors of allogeneic hematopoietic stem cell transplantation. Current cGVHD therapies are neither specific nor curative, and patients are typically maintained for several months to years under immunosuppressive regimens that are associated with important side effects and increased susceptibility to life-threatening infections. As a result, continued investigation into the pathology of the disease and the search for novel diagnostic and therapeutic strategies to treat cGVHD remains a high priority. We report that the cellular dynamics of various immune cell subsets are related to cGVHD onset and severity in a cohort of allogeneic hematopoietic stem cell transplantation recipients. We document a decrease in the proportion of CD45RO+ CD4-CD8- (double-negative [DN]) T cells at the onset of cGVHD, a time at which serum levels of B cell activating factor and B cells are increased. We also find that DN T cell levels are correlated with cGVHD severity. Our present findings are in line with the view that activated DN T cells exhibit their immunoregulatory potential by eliminating B cells in vivo. Taken together, these findings suggest that maintaining elevated DN T cell numbers before the onset of cGVHD may prevent pathological B cell responses.

Restoring T Cell Homeostasis After Allogeneic Stem Cell Transplantation; Principal Limitations and Future Challenges.

For several leukemia patients, allogeneic stem cell transplantation (allogeneic-SCT) is the unique therapeutic modality that could potentially cure their disease. Despite significant progress made in clinical management of allogeneic-SCT, acute graft-versus-host disease (aGVHD) and infectious complications remain the second and third cause of death after disease recurrence. Clinical options to restore immunocompetence after allogeneic-SCT are very limited as studies have raised awareness about the safety with regards to graft-versus-host disease (GVHD). Preclinical works are now focusing on strategies to improve thymic functions and to restore the peripheral niche that have been damaged by alloreactive T cells. In this mini review, we will provide a brief overview about the adverse effects of GVHD on the thymus and the peripheral niche and the resulting negative outcome on peripheral T cell homeostasis. Finally, we will discuss the potential relevance of coordinating our studies on thymic rejuvenation and improvement of the peripheral lymphoid niche to achieve optimal T cell regeneration in GVHD patients.

Homeostatic cytokines in immune reconstitution and graft-versus-host disease.

For numerous patients, allogeneic stem cell transplantation (SCT) is the only therapeutic option that could potentially cure their disease. Despite significant progress made in clinical management of allogeneic SCT, acute graft-versus-host disease (aGVHD) remains the second cause of death after disease recurrence. aGVHD is highly immunosuppressive and the adverse effect of allogeneic SCT on T cell regeneration is typically more important than the levels of immunosuppression normally seen after autologous SCT. In these patients, immune reconstitution often takes several years to occur and restoring immunocompetence after allogeneic SCT represents an important challenge, principally because clinical options are limited and current methods used to accelerate immune reconstitution are associated with increased GVHD. Interleukin-7 and IL-15 are both under clinical investigation and demonstrate the greatest potential on peripheral T cells regeneration in mice and humans. However, awareness has been raised about the use of IL-7 and IL-15 after allogeneic SCT with regards to potential adverse effects on aGVHD. In this review, we will discuss about recent progress made in lymphocyte regeneration, the critical role played by IL-7 and IL-15 in T cell homeostasis and how these cytokines could be used to improve immune reconstitution after allogeneic SCT.

Stromal-Derived Factor-1α and Interleukin-7 Treatment Improves Homeostatic Proliferation of Naïve CD4(+) T Cells after Allogeneic Stem Cell Transplantation.

Graft-versus-host disease (GVHD) impairs immune reconstitution after allogeneic stem cell transplantation (allo-SCT) and effective therapies aimed at restoring T cell counts in GVHD patients have yet to be developed. During GVHD, CD4(+) T cell reconstitution is particularly affected and current models hold that GVHD insult to the peripheral lymphoid niche is responsible for this effect. Here, we show that naïve CD4(+) T cell homeostatic proliferation (HP) is lost during GVHD because of low systemic IL-7 and impaired dendritic cell (DC) regeneration. We assessed factors involved in DC differentiation and found that although fms-like tyrosine kinase 3 ligand (Flt3-L) levels were normal, stromal-derived factor-1α (SDF-1α) was diminished in the blood of GVHD mice. Unlike Flt3-L treatment, the administration of SDF-1α specifically increased CD8α(+) DC numbers and did not worsen GVHD. Importantly, CD4(+) T cell HP was enhanced only when IL-7 and SDF-1α or Flt3L were coadministered, confirming the crucial role of DCs and IL-7 in restoring CD4(+) T cell regeneration during GVHD. Altogether, our results indicate that CD8α(+) DCs are part of the peripheral niche that controls CD4(+) T cell HP and that their depletion, combined with low systemic IL-7, explains how GVHD constrains naïve CD4(+) T cell reconstitution after allo-SCT.

Deficiency in the metabolite receptor SUCNR1 (GPR91) leads to outer retinal lesions.

Age-related macular degeneration (AMD) is a prominent cause of blindness in the Western world. To date, its molecular pathogenesis as well as the sequence of events leading to retinal degeneration remain largely ill-defined. While the invasion of choroidal neovessels in the retina is the primary mechanism that precipitates loss of sight, an earlier dry form precedes it. Here we provide the first evidence for the protective role of the Retinal Pigment Epithelium (RPE)-resident metabolite receptor, succinate receptor 1 (SUCNR1; G-Protein coupled Receptor-91 (GPR91), in preventing dry AMD-like lesions of the outer retina. Genetic analysis of 925 patients with geographic atrophy and 1199 AMD-free peers revealed an increased risk of developing geographic atrophy associated with intronic variants in theSUCNR1 gene. In mice, outer retinal expression of SUCNR1 is observed in the RPE as well as microglial cells and decreases progressively with age. Accordingly, Sucnr1-/- mice show signs of premature sub-retinal dystrophy with accumulation of oxidized-LDL, abnormal thickening of Bruch's membrane and a buildup of subretinal microglia. The accumulation of microglia in Sucnr1-deficient mice is likely triggered by the inefficient clearance of oxidized lipids by the RPE as bone marrow transfer of wild-type microglia into Sucnr1-/- mice did not salvage the patho-phenotype and systemic lipolysis was equivalent between wild-type and control mice. Our findings suggest that deficiency in SUCNR1 is a possible contributing factor to the pathogenesis of dry AMD and thus broaden our understanding of this clinically unmet need.

TCR triggering modulates the responsiveness and homeostatic proliferation of CD4+ thymic emigrants to IL-7 therapy.

Interleukin-7 (IL-7) is currently used in clinical trials to augment T-cell counts. Paradoxically, elevated systemic IL-7 found in lymphopenic humans is typically insufficient for CD4(+) T-cell regeneration, and thymopoiesis becomes critical in this process. Here we show that the proliferative effect of IL-7 is more pronounced on CD4(+)CD8(-) thymocytes compared with peripheral CD4(+) T cells. These cells express miR181a at higher levels and respond to lower concentrations of IL-7. As single-positive CD4(+) thymocytes (CD4(+)(SPT)) exit the thymus, they rapidly diminish their proliferation to IL-7 therapy, and this is mediated, at least in part, by major histocompatibility complex class II distribution outside the thymus. Interestingly, increasing T-cell receptor (TCR) stimulation augments IL-7 responsiveness and proliferation of peripheral CD4(+) T cells, whereas failure to stimulate TCR abrogates proliferation induced by IL-7. Finally, we demonstrated that IL-7 enhances the proliferation of CD4(+) T cells that undergo "slow proliferation" in lymphopenic hosts. To date, our results indicate that TCR signaling is a major controlling factor for CD4 responsiveness and proliferation to IL-7 therapy.

Val-boroPro accelerates T cell priming via modulation of dendritic cell trafficking resulting in complete regression of established murine tumors.

Although tumors naturally prime adaptive immune responses, tolerance may limit the capacity to control progression and can compromise effectiveness of immune-based therapies for cancer. Post-proline cleaving enzymes (PPCE) modulate protein function through N-terminal dipeptide cleavage and inhibition of these enzymes has been shown to have anti-tumor activity. We investigated the mechanism by which Val-boroPro, a boronic dipeptide that inhibits post-proline cleaving enzymes, mediates tumor regression and tested whether this agent could serve as a novel immune adjuvant to dendritic cell vaccines in two different murine syngeneic murine tumors. In mice challenged with MB49, which expresses the HY antigen complex, T cell responses primed by the tumor with and without Val-boroPro were measured using interferon gamma ELISPOT. Antibody depletion and gene-deficient mice were used to establish the immune cell subsets required for tumor regression. We demonstrate that Val-boroPro mediates tumor eradication by accelerating the expansion of tumor-specific T cells. Interestingly, T cells primed by tumor during Val-boroPro treatment demonstrate increased capacity to reject tumors following adoptive transfer without further treatment of the recipient. Val-boroPro -mediated tumor regression requires dendritic cells and is associated with enhanced trafficking of dendritic cells to tumor draining lymph nodes. Finally, dendritic cell vaccination combined with Val-boroPro treatment results in complete regression of established tumors. Our findings demonstrate that Val-boroPro has antitumor activity and a novel mechanism of action that involves more robust DC trafficking with earlier priming of T cells. Finally, we show that Val-boroPro has potent adjuvant properties resulting in an effective therapeutic vaccine.

Neuronal ER stress impedes myeloid-cell-induced vascular regeneration through IRE1α degradation of netrin-1.

In stroke and proliferative retinopathy, despite hypoxia driven angiogenesis, delayed revascularization of ischemic tissue aggravates the loss of neuronal function. What hinders vascular regrowth in the ischemic central nervous system remains largely unknown. Using the ischemic retina as a model of neurovascular interaction in the CNS, we provide evidence that the failure of reparative angiogenesis is temporally and spatially associated with endoplasmic reticulum (ER) stress. The canonical ER stress pathways of protein kinase RNA-like ER kinase (PERK) and inositol-requiring enzyme-1α (IRE1α) are activated within hypoxic/ischemic retinal ganglion neurons, initiating a cascade that results in angiostatic signals. Our findings demonstrate that the endoribonuclease IRE1α degrades the classical guidance cue netrin-1. This neuron-derived cue triggers a critical reparative-angiogenic switch in neural macrophage/microglial cells. Degradation of netrin-1, by persistent neuronal ER stress, thereby hinders vascular regeneration. These data identify a neuronal-immune mechanism that directly regulates reparative angiogenesis.

Graft-versus-host disease impairs vaccine responses through decreased CD4+ and CD8+ T cell proliferation and increased perforin-mediated CD8+ T cell apoptosis.

Tumor-targeted vaccines represent a strategy to enhance the graft-versus-leukemia effect after allogeneic blood and marrow transplantation (BMT). We have previously shown that graft-versus-host disease (GVHD) can negatively impact quantitative responses to vaccines. Using a minor histocompatibility Ag-mismatched BMT (B6 → B6 × C3H.SW) followed by adoptive transfer of HY-specific T cells and HY-expressing dendritic cells, we assessed whether GVHD induced by donor lymphocyte infusion (DLI) affects the persistence, proliferation, and survival of vaccine-responding, nonalloantigen reactive T cells. Both CD8(+) and CD4(+) HY-specific T cells undergo less vaccine-driven proliferation in allogeneic recipients with GVHD. Although vaccine-responding CD8(+) T cells show decreased IFN-γ and CD107a production, CD4(+) T cells exhibit increased programmed death 1 and T cell Ig mucin-like domain 3 expression. In addition, the degree of apoptosis in vaccine-responding CD8(+) T cells was higher in the presence of GVHD, but there was no difference in CD4(+) T cell apoptosis. Using Fas ligand-deficient or TRAIL-deficient DLI had no impact on apoptosis of HY-specific T cells. However, perforin-deficient alloreactive DLI induced significantly less apoptosis of vaccine-responding CD8(+) T cells and resulted in enhanced tumor protection. Thus, diminished vaccine responses during GVHD result from impaired proliferation of CD8(+) and CD4(+) T cells responding to vaccination, with an additional contribution from perforin-mediated CD8(+) T cell apoptosis. These results provide important insights toward optimizing vaccine responses after allogeneic BMT.

IL-7-dependent B lymphocytes are essential for the anti-polysaccharide response and protective immunity to Streptococcus pneumoniae.

Young children are impaired in their response to T cell-independent (TI) Ags, such as pneumococcal polysaccharide (PPS). B lymphopoeisis early in life is IL-7 independent, whereas in adults it is IL-7 dependent. Therefore, we hypothesized that IL-7-driven B lymphopoiesis plays a critical role in promoting Ab responses to TI Ags. Young but not adult mice are impaired in responses to PPS vaccination and to 4-hydroxy-3-nitrophenyl-acetyl-Ficoll, a widely studied model TI Ag, and B1b cells generate Ab responses to these Ags. In this paper, we show that, despite having B1b, B1a, and MZ B cells-all of which are involved in TI responses-young wild-type or adult mice deficient either in IL-7 or in IL-7Ralpha are severely impaired in anti-PPS responses and do not survive Streptococcus pneumoniae challenge, indicating IL-7-dependent B cells are required for TI immunity. Consistent with this, PPS immunization induced a robust TI response in young IL-7 transgenic mice that was comparable to adult wild-type responses. Moreover, immunized young or adult IL-7 transgenic mice were completely resistant to S. pneumoniae challenge. Our data indicate that activating the IL-7 signaling pathway could restore impaired TI responses in the young.

Pharmacologic modulation of niche accessibility via tyrosine kinase inhibition enhances marrow and thymic engraftment after hematopoietic stem cell transplantation.

Essential survival signals within hematopoietic stem cell (HSC) and thymic niches are mediated by receptor tyrosine kinases, which can be reversibly inhibited using clinically available drugs. We studied whether sunitinib, a multityrosine kinase inhibitor that inhibits KIT, enhances engraftment after bone marrow transplantation (BMT) in mice. Sunitinib diminished hematopoietic progenitor cell numbers, and sunitinib enhanced marrow, peripheral myeloid, and lymphoid engraftment after BMT in Rag1(-/-) mice. Sunitinib augmented HSC engraftment because recipients displayed increased myeloid and lymphoid engraftment and because sunitinib-treated recipients of purified HSCs showed enhanced engraftment of secondary hosts. However, sunitinib preferentially augmented T-cell engraftment with lesser effects on myeloid and HSC engraftment. Consistent with this, sunitinib preferentially depleted the early thymic progenitor subset in the thymus. Sunitinib did not increase engraftment in mice with deficient KIT signaling, and the pattern of more potent effects on T cell compared with HSC engraftment observed in sunitinib-treated hosts was also observed after BMT into KIT(W/Wv) mice. These results implicate KIT as a critical modulator of thymic niches. We conclude that transient, pharmacologic inhibition of KIT enhances accessibility of marrow and thymic niches, and provides a novel, noncytotoxic approach to accomplish engraftment after stem cell transplantation.

In vivo role of Flt3 ligand and dendritic cells in NK cell homeostasis.

IL-15 is required for NK cell development and homeostasis in vivo. Because IL-15 is presented in trans via its high-affinity IL-15Ralpha-chain to cells expressing the IL-15Rbetagamma complex, we postulated that certain IL-15-bearing cells must be required for NK cell homeostasis. Using IL-15(WT/WT) and IL-15(-/-) mice, bone marrow chimeras with normal cellularity, and a selective depletion of CD11c(hi) dendritic cells (DCs), we demonstrate that ablation of the resting CD11c(hi) DC population results in a highly significant decrease in the absolute number of mature NK cells. In contrast, administration of Flt3 ligand increases the CD11c(hi) DC population, which, when expressing IL-15, significantly expands mature NK cells via enhanced survival and proliferation. In summary, a CD11c(hi) DC population expressing IL-15 is required to maintain NK cell homeostasis under conditions of normal cellularity and also is required to mediate Flt3 ligand-induced NK cell expansion in vivo.

Harnessing the physiology of lymphopenia to support adoptive immunotherapy in lymphoreplete hosts.

Lymphopenia enhances the effectiveness of adoptive immunotherapy by facilitating expansion of transferred T cells but also limits the T-cell repertoire available to mediate immune responses and, in humans, is associated with chronic immune dysfunction. Previous studies concluded that lymphopenia augments adoptive immunotherapy by diminishing Tregs and increasing homeostatic cytokines. We sought to determine whether targeted therapies that replicate the physiology of lymphopenia in lymphoreplete hosts could provide a similarly supportive milieu. Pmel-1 T cells were transferred to B16-bearing lymphopenic versus lymphoreplete mice receiving alphaCD25 and/or recombinant human interleukin-7. Although CD25-based Treg depletion was inefficient because of peripheral expansion of CD4+CD25-FOXP3+ cells, outcomes were better in alphaCD25-treated lymphoreplete hosts than in lymphopenic hosts, and adoptive immunotherapy was most effective in lymphoreplete hosts receiving alphaCD25 plus recombinant human interleukin-7. Lymphopenic hosts supported increased proliferation of adoptively transferred antigen-specific T cells, but cells transferred to lymphoreplete recipients receiving targeted therapies showed superior function. Further, determinant spreading was substantial in lymphoreplete hosts but absent in lymphopenic hosts. These results demonstrate that targeted therapies delivered to mimic the "physiology of lymphopenia" enhance the efficacy of adoptive immunotherapy in lymphoreplete hosts and provide a potentially superior alternative to the induction of lymphopenia.

Interleukin 7 signaling in dendritic cells regulates the homeostatic proliferation and niche size of CD4+ T cells.

Interleukin 7 (IL-7) and T cell antigen receptor signals have been proposed to be the main drivers of homeostatic T cell proliferation. However, it is not known why CD4(+) T cells undergo less-efficient homeostatic proliferation than CD8(+) T cells do. Here we show that systemic IL-7 concentrations increased during lymphopenia because of diminished use of IL-7 but that IL-7 signaling on IL-7 receptor-alpha-positive (IL-7Ralpha(+)) dendritic cells (DCs) in lymphopenic settings paradoxically diminished the homeostatic proliferation of CD4(+) T cells. This effect was mediated at least in part by IL-7-mediated downregulation of the expression of major histocompatibility complex class II on IL-7Ralpha(+) DCs. Our results indicate that IL-7Ralpha(+) DCs are regulators of the peripheral CD4(+) T cell niche and that IL-7 signals in DCs prevent uncontrolled CD4(+) T cell population expansion in vivo.