P63 targeted deletion under the FOXN1 promoter disrupts pre-and post-natal thymus development, function and maintenance as well as induces severe hair loss.

Progressive immune deficiency of aging is characterized by severe thymic atrophy, contracted T cell repertoire, and poor immune function. p63 is critical for the proliferative potential of embryonic and adult stem cells, as well as thymic epithelial cells (TECs). Because p63 null mice experience rapid post-natal lethality due to epidermal and limb morphogenesis defects, studies to define a role for p63 expression in TEC biology focused on embryonic thymus development and in vitro experiments. Since post-natal thymic stromal development and function differs from that of the embryo, we assessed the impact of lineage-restricted p63 loss on pre- and post-natal murine TEC function by generating mice with a loss of p63 function targeted to TEC, termed p63TECko mice. In adult p63TECko mice, severe thymic hypoplasia was observed with a lack in a discernable segregation into medullary and cortical compartments and peripheral T cell lymphopenia. This profound thymic defect was seen in both neonatal as well as embryonic p63TECko mice. In addition to TECs, p63 also plays in important role in the development of stratified epithelium of the skin; lack of p63 results in defects in skin epidermal stratification and differentiation. Interestingly, all adult p63TECko mice lacked hair follicles despite having normal p63 expression in the skin. Together our results show a critical role of TEC p63 in thymic development and maintenance and show that p63 expression is critical for hair follicle formation.

Inhibition of inositol kinase B controls acute and chronic graft-versus-host disease.

T-cell activation releases inositol 1,4,5-trisphosphate (IP3), inducing cytoplasmic calcium (Ca2+) influx. In turn, inositol 1,4,5-trisphosphate 3-kinase B (Itpkb) phosphorylates IP3 to negatively regulate and thereby tightly control Ca2+ fluxes that are essential for mature T-cell activation and differentiation and protection from cell death. Itpkb pathway inhibition increases intracellular Ca2+, induces apoptosis of activated T cells, and can control T-cell-mediated autoimmunity. In this study, we employed genetic and pharmacological approaches to inhibit Itpkb signaling as a means of controlling graft-versus-host disease (GVHD). Murine-induced, Itpkb-deleted (Itpkb-/-) T cells attenuated acute GVHD in 2 models without eliminating A20-luciferase B-cell lymphoma graft-versus-leukemia (GVL). A highly potent, selective inhibitor, GNF362, ameliorated acute GVHD without impairing GVL against 2 acute myeloid leukemia lines (MLL-AF9-eGFP and C1498-luciferase). Compared with FK506, GNF362 more selectively deleted donor alloreactive vs nominal antigen-responsive T cells. Consistent with these data and as compared with FK506, GNF362 had favorable acute GVHD and GVL properties against MLL-AF9-eGFP cells. In chronic GVHD preclinical models that have a pathophysiology distinct from acute GVHD, Itpkb-/- donor T cells reduced active chronic GVHD in a multiorgan system model of bronchiolitis obliterans (BO), driven by germinal center reactions and resulting in target organ fibrosis. GNF362 treatment reduced active chronic GVHD in both BO and scleroderma models. Thus, intact Itpkb signaling is essential to drive acute GVHD pathogenesis and sustain active chronic GVHD, pointing toward a novel clinical application to prevent acute or treat chronic GVHD.

Donor and host B7-H4 expression negatively regulates acute graft-versus-host disease lethality.

B7-H4 is a negative regulatory B7 family member. We investigated the role of host and donor B7-H4 in regulating acute graft-versus-host disease (GVHD). Allogeneic donor T cells infused into B7-H4-/- versus WT recipients markedly accelerated GVHD-induced lethality. Chimera studies pointed toward B7-H4 expression on host hematopoietic cells as more critical than parenchymal cells in controlling GVHD. Rapid mortality in B7-H4-/- recipients was associated with increased donor T cell expansion, gut T cell homing and loss of intestinal epithelial integrity, increased T effector function (proliferation, proinflammatory cytokines, cytolytic molecules), and reduced apoptosis. Higher metabolic demands of rapidly proliferating donor T cells in B7-H4-/- versus WT recipients required multiple metabolic pathways, increased extracellular acidification rates (ECARs) and oxygen consumption rates (OCRs), and increased expression of fuel substrate transporters. During GVHD, B7-H4 expression was upregulated on allogeneic WT donor T cells. B7-H4-/- donor T cells given to WT recipients increased GVHD mortality and had function and biological properties similar to WT T cells from allogeneic B7-H4-/- recipients. Graft-versus-leukemia responses were intact regardless as to whether B7-H4-/- mice were used as hosts or donors. Taken together, these data provide new insights into the negative regulatory processes that control GVHD and provide support for developing therapeutic strategies directed toward the B7-H4 pathway.

Despite high levels of expression in thymic epithelial cells, miR-181a1 and miR-181b1 are not required for thymic development.

MicroRNAs (miRNAs) have been shown to be key modulators of post-transcriptional gene silencing in many cellular processes. In previous studies designed to understand the role of miRNAs in thymic development, we globally deleted miRNA exclusively in thymic epithelial cells (TECs), which are critical in thymic selection. This resulted in the loss of stromal cells that instruct T cell lineage commitment and affect thymocyte positive selection, required for mature T cell development. Since murine miR-181 is expressed in the thymus and miR-181 deficiency disrupts thymocyte development, we first quantified and thereby demonstrated that miR181a1 and miR181b1 are expressed in purified TECs. By generating mice with TEC targeted loss of miR-181a1 and miR-181b1 expression, we observed that neither TEC cellularity nor thymocyte number nor differentiation was adversely affected. Thus, disrupted thymopoiesis in miR-181 deficient mice was not due to miR-181 loss of expression in TECs. Importantly, in mice with restricted TEC deficiency of miR-181a1 and miR-181b1, there were similar numbers of mature T cells in the periphery in regards to frequencies, differentiation, and function as compared to controls. Moreover miR-181a1 and miR-181b1 were not required for maintenance of thymus integrity over time, as thymic involution was not accelerated in gene-targeted mice. Taken together our data indicate that miR-181a1 and miR-181b1 are dispensable for TEC differentiation, their control of thymocyte development and mature T cell export to and homeostasis within the periphery.

MicroRNA-155 Modulates Acute Graft-versus-Host Disease by Impacting T Cell Expansion, Migration, and Effector Function.

MicroRNA-155 (miR-155) is a small noncoding RNA critical for the regulation of inflammation as well as innate and adaptive immune responses. MiR-155 has been shown to be dysregulated in both donor and recipient immune cells during acute graft-versus-host disease (aGVHD). We previously reported that miR-155 is upregulated in donor T cells of mice and humans with aGVHD and that mice receiving miR-155-deficient (miR155-/-) splenocytes had markedly reduced aGVHD. However, molecular mechanisms by which miR-155 modulates T cell function in aGVHD have not been fully investigated. We identify that miR-155 expression in both donor CD8+ T cells and conventional CD4+ CD25- T cells is pivotal for aGVHD pathogenesis. Using murine aGVHD transplant experiments, we show that miR-155 strongly impacts alloreactive T cell expansion through multiple distinct mechanisms, modulating proliferation in CD8+ donor T cells and promoting exhaustion in donor CD4+ T cells in both the spleen and colon. Additionally, miR-155 drives a proinflammatory Th1 phenotype in donor T cells in these two sites, and miR-155-/- donor T cells are polarized toward an IL-4-producing Th2 phenotype. We further demonstrate that miR-155 expression in donor T cells regulates CCR5 and CXCR4 chemokine-dependent migration. Notably, we show that miR-155 expression is crucial for donor T cell infiltration into multiple target organs. These findings provide further understanding of the role of miR-155 in modulating aGVHD through T cell expansion, effector cytokine production, and migration.

Programmed death ligand-1 expression on donor T cells drives graft-versus-host disease lethality.

Programmed death ligand-1 (PD-L1) interaction with PD-1 induces T cell exhaustion and is a therapeutic target to enhance immune responses against cancer and chronic infections. In murine bone marrow transplant models, PD-L1 expression on host target tissues reduces the incidence of graft-versus-host disease (GVHD). PD-L1 is also expressed on T cells; however, it is unclear whether PD-L1 on this population influences immune function. Here, we examined the effects of PD-L1 modulation of T cell function in GVHD. In patients with severe GVHD, PD-L1 expression was increased on donor T cells. Compared with mice that received WT T cells, GVHD was reduced in animals that received T cells from Pdl1-/- donors. PD-L1-deficient T cells had reduced expression of gut homing receptors, diminished production of inflammatory cytokines, and enhanced rates of apoptosis. Moreover, multiple bioenergetic pathways, including aerobic glycolysis, oxidative phosphorylation, and fatty acid metabolism, were also reduced in T cells lacking PD-L1. Finally, the reduction of acute GVHD lethality in mice that received Pdl1-/- donor cells did not affect graft-versus-leukemia responses. These data demonstrate that PD-L1 selectively enhances T cell-mediated immune responses, suggesting a context-dependent function of the PD-1/PD-L1 axis, and suggest selective inhibition of PD-L1 on donor T cells as a potential strategy to prevent or ameliorate GVHD.

Targeted Rho-associated kinase 2 inhibition suppresses murine and human chronic GVHD through a Stat3-dependent mechanism.

Chronic graft-versus-host disease (cGVHD) remains a major complication following allogeneic bone marrow transplantation (BMT). The discovery of novel therapeutics is dependent on assessment in preclinical murine models of cGVHD. Rho-associated kinase 2 (ROCK2) recently was shown to be implicated in regulation of interleukin-21 (IL-21) and IL-17 secretion in mice and humans. Here, we report that the selective ROCK2 inhibitor KD025 effectively ameliorates cGVHD in multiple models: a full major histocompatibility complex (MHC) mismatch model of multiorgan system cGVHD with bronchiolitis obliterans syndrome and a minor MHC mismatch model of sclerodermatous GVHD. Treatment with KD025 resulted in normalization of pathogenic pulmonary function, which correlates with a marked reduction of antibody and collagen deposition in the lungs of treated mice to levels comparable to non-cGVHD controls. Spleens of mice treated with KD025 had decreased frequency of T follicular helper cells and increased frequency of T follicular regulatory cells, accompanied by a reduction in signal transducer and activator of transcription 3 (STAT3) and concurrent increase in STAT5 phosphorylation. The critical role of STAT3 in this cGVHD model was confirmed by data showing that mice transplanted with inducible STAT3-deficient T cells had pulmonary function comparable to the healthy negative controls. The therapeutic potential of targeted ROCK2 inhibition in the clinic was solidified further by human data demonstrating the KD025 inhibits the secretion of IL-21, IL-17, and interferon γ along with decreasing phosphorylated STAT3 and reduced protein expression of interferon regulatory factor 4 and B-cell lymphoma 6 (BCL6) in human peripheral blood mononuclear cells purified from active cGVHD patients. Together these data highlight the potential of targeted ROCK2 inhibition for clinical cGVHD therapy.

Targeting Syk-activated B cells in murine and human chronic graft-versus-host disease.

Novel therapies for chronic graft-versus-host disease (cGVHD) are needed. Aberrant B-cell activation has been demonstrated in mice and humans with cGVHD. Having previously found that human cGVHD B cells are activated and primed for survival, we sought to further evaluate the role of the spleen tyrosine kinase (Syk) in cGVHD in multiple murine models and human peripheral blood cells. In a murine model of multiorgan system, nonsclerodermatous disease with bronchiolitis obliterans where cGVHD is dependent on antibody and germinal center (GC) B cells, we found that activation of Syk was necessary in donor B cells, but not T cells, for disease progression. Bone marrow-specific Syk deletion in vivo was effective in treating established cGVHD, as was a small-molecule inhibitor of Syk, fostamatinib, which normalized GC formation and decreased activated CD80/86(+) dendritic cells. In multiple distinct models of sclerodermatous cGVHD, clinical and pathological disease manifestations were not eliminated when mice were therapeutically treated with fostamatinib, though both clinical and immunologic effects could be observed in one of these scleroderma models. We further demonstrated that Syk inhibition was effective at inducing apoptosis of human cGVHD B cells. Together, these data demonstrate a therapeutic potential of targeting B-cell Syk signaling in cGVHD.

B7-H3 expression in donor T cells and host cells negatively regulates acute graft-versus-host disease lethality.

Members of the B7 family have been shown to be important for regulating immune responses by providing either positive or negative costimulatory signals. The function of B7-H3 has been controversial. We show that B7-H3 is upregulated in graft-versus-host disease (GVHD) target organs, including the colon, liver, and lung. Infusion of allogeneic donor T cells into B7-H3(-/-) vs wild-type (WT) recipients resulted in increased GVHD lethality associated with increased T-cell proliferation, colonic inflammatory cytokines, and destruction of epithelial barriers. Allogeneic B7-H3(-/-) vs WT donor T cells also had increased T-cell proliferation and GVHD lethality associated with increased proliferation and cytokine secretion in the spleen, intraepithelial lymphocyte inflammatory cytokines, and intestinal permeability. Both resting and activated regulatory T cells (Tregs) lack B7-H3 messenger RNA. Consistent with these data, GVHD was augmented in recipients of B7-H3(-/-) Treg-depleted grafts. In two delayed lymphocyte infusion (DLI) models, T cells lacking B7-H3 are capable of providing graft-versus-leukemia (GVL) effects. We conclude that B7-H3 is responsible for providing a negative costimulatory signal. Our studies provide support for developing and testing new therapies directed toward the B7-H3 pathway, including approaches to augment host B7-H3 early after bone marrow transplantation to prevent GVHD and to develop potent antagonistic antibodies later after transplant to facilitate DLI-mediated GVL without GVHD complications.

The IL-33/ST2 axis augments effector T-cell responses during acute GVHD.

Interleukin (IL)-33 binding to the receptor suppression of tumorigenicity 2 (ST2) produces pro-inflammatory and anti-inflammatory effects. Increased levels of soluble ST2 (sST2) are a biomarker for steroid-refractory graft-versus-host disease (GVHD) and mortality. However, whether sST2 has a role as an immune modulator or only as a biomarker during GVHD was unclear. We show increased IL-33 production by nonhematopoietic cells in the gastrointestinal (GI) tract in mice post-conditioning and patients during GVHD. Exogenous IL-33 administration during the peak inflammatory response worsened GVHD. Conversely, GVHD lethality and tumor necrosis factor-α production was significantly reduced in il33(-/-) recipients. ST2 was upregulated on murine and human alloreactive T cells and sST2 increased as experimental GVHD progressed. Concordantly, st2(-/-) vs wild-type (WT) donor T cells had a marked reduction in GVHD lethality and GI histopathology. Alloantigen-induced IL-18 receptor upregulation was lower in st2(-/-) T cells, and linked to reduced interferon-γ production by st2(-/-) vs WT T cells during GVHD. Blockade of IL-33/ST2 interactions during allogeneic-hematopoietic cell transplantation by exogenous ST2-Fc infusions had a marked reduction in GVHD lethality, indicating a role of ST2 as a decoy receptor modulating GVHD. Together, these studies point to the IL-33/ST2 axis as a novel and potent target for GVHD therapy.

Therapeutic activity of multiple common γ-chain cytokine inhibition in acute and chronic GVHD.

The common γ chain (CD132) is a subunit of the interleukin (IL) receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Because levels of several of these cytokines were shown to be increased in the serum of patients developing acute and chronic graft-versus-host disease (GVHD), we reasoned that inhibition of CD132 could have a profound effect on GVHD. We observed that anti-CD132 monoclonal antibody (mAb) reduced acute GVHD potently with respect to survival, production of tumor necrosis factor, interferon-γ, and IL-6, and GVHD histopathology. Anti-CD132 mAb afforded protection from GVHD partly via inhibition of granzyme B production in CD8 T cells, whereas exposure of CD8 T cells to IL-2, IL-7, IL-15, and IL-21 increased granzyme B production. Also, T cells exposed to anti-CD132 mAb displayed a more naive phenotype in microarray-based analyses and showed reduced Janus kinase 3 (JAK3) phosphorylation upon activation. Consistent with a role of JAK3 in GVHD, Jak3(-/-) T cells caused less severe GVHD. Additionally, anti-CD132 mAb treatment of established chronic GVHD reversed liver and lung fibrosis, and pulmonary dysfunction characteristic of bronchiolitis obliterans. We conclude that acute GVHD and chronic GVHD, caused by T cells activated by common γ-chain cytokines, each represent therapeutic targets for anti-CD132 mAb immunomodulation.

Increased T follicular helper cells and germinal center B cells are required for cGVHD and bronchiolitis obliterans.

Chronic graft-versus-host disease (cGVHD) is a leading cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Having shown that germinal center (GC) formation and immunoglobulin deposition are required for multiorgan system cGVHD and associated bronchiolitis obliterans syndrome (BOS) in a murine model, we hypothesized that T follicular helper (Tfh) cells are necessary for cGVHD by supporting GC formation and maintenance. We show that increased frequency of Tfh cells correlated with increased GC B cells, cGVHD, and BOS. Although administering a highly depletionary anti-CD20 monoclonal antibody (mAb) to mice with established cGVHD resulted in peripheral B-cell depletion, B cells remained in the lung, and BOS was not reversed. BOS could be treated by eliminating production of interleukin-21 (IL-21) by donor T cells or IL-21 receptor (IL-21R) signaling of donor B cells. Development of BOS was dependent upon T cells expressing the chemokine receptor CXCR5 to facilitate T-cell trafficking to secondary lymphoid organ follicles. Blocking mAbs for IL-21/IL-21R, inducible T-cell costimulator (ICOS)/ICOS ligand, and CD40L/CD40 hindered GC formation and cGVHD. These data provide novel insights into cGVHD pathogenesis, indicate a role for Tfh cells in these processes, and suggest a new line of therapy using mAbs targeting Tfh cells to reverse cGVHD.

Host programmed death ligand 1 is dominant over programmed death ligand 2 expression in regulating graft-versus-host disease lethality.

Programmed death 1 (PD-1) and its ligands, PD-L1 and PD-L2, play an important role in the maintenance of peripheral tolerance. We explored the role of PD-1 ligands in regulating graft-versus-host disease (GVHD). Both PD-L1 and PD-L2 expression were upregulated in the spleen, liver, colon, and ileum of GVHD mice. Whereas PD-L2 expression was limited to hematopoietic cells, hematopoietic and endothelial cells expressed PD-L1. PD-1/PD-L1, but not PD-1/PD-L2, blockade markedly accelerated GVHD-induced lethality. Chimera studies suggest that PD-L1 expression on host parenchymal cells is more critical than hematopoietic cells in regulating acute GVHD. Rapid mortality onset in PD-L1-deficient hosts was associated with increased gut T-cell homing and loss of intestinal epithelial integrity, along with increased donor T-cell proliferation, activation, Th1 cytokine production, and reduced apoptosis. Bioenergetics profile analysis of proliferating alloreactive donor T-cells demonstrated increased aerobic glycolysis and oxidative phosphorylation in PD-L1-deficient hosts. Donor T-cells exhibited a hyperpolarized mitochondrial membrane potential, increased superoxide production, and increased expression of a glucose transporter in PD-L1-deficient hosts. Taken together, these data provide new insight into the differential roles of host PD-L1 and PD-L2 and their associated cellular and metabolic mechanisms controlling acute GVHD.

Inhibiting retinoic acid signaling ameliorates graft-versus-host disease by modifying T-cell differentiation and intestinal migration.

Graft-versus-host disease (GVHD) is a critical complication after allogeneic bone marrow transplantation. During GVHD, donor T cells are activated by host antigen-presenting cells and differentiate into T-effector cells (Teffs) that migrate to GVHD target organs. However, local environmental factors influencing Teff differentiation and migration are largely unknown. Vitamin A metabolism within the intestine produces retinoic acid, which contributes to intestinal homeostasis and tolerance induction. Here, we show that the expression and function of vitamin A-metabolizing enzymes were increased in the intestine and mesenteric lymph nodes in mice with active GVHD. Moreover, transgenic donor T cells expressing a retinoic acid receptor (RAR) response element luciferase reporter responded to increased vitamin A metabolites in GVHD-affected organs. Increasing RAR signaling accelerated GVHD lethality, whereas donor T cells expressing a dominant-negative RARα (dnRARα) showed markedly diminished lethality. The dnRARα transgenic T cells showed reduced Th1 differentiation and α4ß7 and CCR9 expression associated with poor intestinal migration, low GVHD pathology, and reduced intestinal permeability, primarily via CD4(+) T cells. The inhibition of RAR signaling augmented donor-induced Treg generation and expansion in vivo, while preserving graft-versus-leukemia effects. Together, these results suggested that reagents blunting donor T-cell RAR signaling may possess therapeutic anti-GVHD properties.

Contrasting acute graft-versus-host disease effects of Tim-3/galectin-9 pathway blockade dependent upon the presence of donor regulatory T cells.

T-cell immunoglobulin mucin-3 (Tim-3) is expressed on pathogenic T cells, and its ligand galectin-9 (gal-9) is up-regulated in inflamed tissues. When Tim-3(+) T cells encounter high gal-9 levels, they are deleted. Tim-3 is up-regulated on activated T cells during GVHD. Inhibition of Tim-3/gal-9 binding by infusion of a Tim-3-Ig fusion protein or Tim-3(-/-) donor T cells increased T-cell proliferation and GVHD lethality. When the Tim-3/gal-9 pathway engagement was augmented using gal-9 transgenic recipients, GVHD lethality was slowed. Together, these data indicate a potential for modulating this pathway to reduce disease by increasing Tim-3 or gal-9 engagement. Paradoxically, when Tim-3/gal-9 was inhibited in the absence of donor T-regulatory cells (Tregs), GVHD was inhibited. GVHD reduction was associated with decreased colonic inflammatory cytokines as well as epithelial barrier destruction. CD25-depleted Tim-3(-/-) donor T cells underwent increased activation-induced cell death because of increased IFN-γ production. To our knowledge, these studies are the first to show that although the absence of Tim-3/gal-9 pathway interactions augments systemic GVHD, concurrent donor Treg depletion paradoxically and surprisingly inhibits GVHD. Thus, although donor Tregs typically inhibit GVHD, under some conditions, such Tregs actually may contribute to GVHD by reducing activation-induced T-cell death.

FTY720 markedly increases alloengraftment but does not eliminate host anti-donor T cells that cause graft rejection on its withdrawal.

The immunomodulator FTY720 (FTY) is beneficial in models of graft-versus-host disease, solid organ transplantation, and autoimmunity and has been approved for use in patients with multiple sclerosis. FTY modifies the homing and migration of many cell types. We report that FTY has profound positive and negative effects on allogeneic bone marrow (BM) engraftment in sublethally irradiated recipients. FTY increased donor hematopoietic progenitors in the BM, resulting in high donor engraftment in the B cell, myeloid cell, and natural killer cell, but not T cell, lineages. Donor T cell progenitors within the thymus of FTY-treated recipients were dramatically reduced, resulting in a lack of donor T cell reconstitution. In addition to preventing the ingress of donor (and host) T cell progenitors, FTY prevented the egress of fully functional host CD4+CD8- and CD4-CD8+ thymocytes that on cessation of FTY administration were able to exit from the thymus and contribute to a rapid and complete rejection of a well-established donor BM graft. When used in combination with anti-CD40L mAbs to block the CD40L:CD40 costimulatory pathway, FTY markedly enhanced anti-CD40L mAb-mediated alloengraftment promotion. In contrast to FTY alone, the combination of anti-CD40L mAb and FTY resulted in a surprisingly stable, multilineage, long-term donor chimerism. These data illustrate FTY's profound migration modulating effects and suggest a use in combinatorial therapy in achieving stable alloengraftment under nonmyeloablative conditions.

Regulation of acute graft-versus-host disease by microRNA-155.

Acute graft-versus-host disease (aGVHD) remains a major complication of allogeneic hematopoietic stem cell transplant (alloHSCT), underscoring the need to further elucidate its mechanisms and develop novel treatments. Based on recent observations that microRNA-155 (miR-155) is up-regulated during T-cell activation, we hypothesized that miR-155 is involved in the modulation of aGVHD. Here we show that miR-155 expression was up-regulated in T cells from mice developing aGVHD after alloHSCT. Mice receiving miR-155-deficient donor lymphocytes had markedly reduced lethal aGVHD, whereas lethal aGVHD developed rapidly in mice recipients of miR-155 overexpressing T cells. Blocking miR-155 expression using a synthetic anti-miR-155 after alloHSCT decreased aGVHD severity and prolonged survival in mice. Finally, miR-155 up-regulation was shown in specimens from patients with pathologic evidence of intestinal aGVHD. Altogether, our data indicate a role for miR-155 in the regulation of GVHD and point to miR-155 as a novel target for therapeutic intervention in this disease.