Alarmins and Their Receptors as Modulators and Indicators of Alloimmune Responses.

Cell damage and death releases alarmins, self-derived immunomodulatory molecules that recruit and activate the immune system. Unfortunately, numerous processes critical to the transplantation of allogeneic materials result in the destruction of donor and recipient cells and may trigger alarmin release. Alarmins, often described as damage-associated molecular patterns, together with exogenous pathogen-associated molecular patterns, are potent orchestrators of immune responses; however, the precise role that alarmins play in alloimmune responses remains relatively undefined. We examined evolving concepts regarding how alarmins affect solid organ and allogeneic hematopoietic cell transplantation outcomes and the mechanisms by which self molecules are released. We describe how, once released, alarmins may act alone or in conjunction with nonself materials to contribute to cytokine networks controlling alloimmune responses and their intensity. It is becoming recognized that this class of molecules has pleotropic functions, and certain alarmins can promote both inflammatory and regulatory responses in transplant models. Emerging evidence indicates that alarmins and their receptors may be promising transplantation biomarkers. Developing the therapeutic ability to support alarmin regulatory mechanisms and the predictive value of alarmin pathway biomarkers for early intervention may provide opportunities to benefit graft recipients.

The Knife's Edge of Tolerance: Inducing Stable Multilineage Mixed Chimerism but With a Significant Risk of CMV Reactivation and Disease in Rhesus Macaques.

Although stable mixed-hematopoietic chimerism induces robust immune tolerance to solid organ allografts in mice, the translation of this strategy to large animal models and to patients has been challenging. We have previously shown that in MHC-matched nonhuman primates (NHPs), a busulfan plus combined belatacept and anti-CD154-based regimen could induce long-lived myeloid chimerism, but without T cell chimerism. In that setting, donor chimerism was eventually rejected, and tolerance to skin allografts was not achieved. Here, we describe an adaptation of this strategy, with the addition of low-dose total body irradiation to our conditioning regimen. This strategy has successfully induced multilineage hematopoietic chimerism in MHC-matched transplants that was stable for as long as 24 months posttransplant, the entire length of analysis. High-level T cell chimerism was achieved and associated with significant donor-specific prolongation of skin graft acceptance. However, we also observed significant infectious toxicities, prominently including cytomegalovirus (CMV) reactivation and end-organ disease in the setting of functional defects in anti-CMV T cell immunity. These results underscore the significant benefits that multilineage chimerism-induction approaches may represent to transplant patients as well as the inherent risks, and they emphasize the precision with which a clinically successful regimen will need to be formulated and then validated in NHP models.

In Vitro Induction of Human Regulatory T Cells Using Conditions of Low Tryptophan Plus Kynurenines.

Thymic regulatory T cells (tTregs) and induced regulatory T cells (iTregs) suppress murine acute graft-versus-host disease (GVHD). Previously, we demonstrated that the plasmacytoid dendritic cell indoleamine 2,3-dioxygenase (IDO) fosters the in vitro development of human iTregs via tryptophan depletion and kynurenine (Kyn) metabolites. We now show that stimulation of naïve CD4+ T cells in low tryptophan (low Trp) plus Kyn supports human iTreg generation. In vitro, low Trp + Kyn iTregs and tTregs potently suppress T effector cell proliferation equivalently but are phenotypically distinct. Compared with tTregs or T effector cells, bioenergetics profiling reveals that low Trp + Kyn iTregs have increased basal glycolysis and oxidative phosphorylation and use glutaminolysis as an energy source. Low Trp + Kyn iTreg viability was reliant on interleukin (IL)-2 in vitro. Although in vivo IL-2 administration increased low Trp + Kyn iTreg persistence on adoptive transfer into immunodeficient mice given peripheral blood mononuclear cells to induce GVHD, IL-2-supported iTregs did not improve recipient survival. We conclude that low Trp + Kyn create suppressive iTregs that have high metabolic needs that will need to be addressed before clinical translation.

Strategies to inhibit alloantibody production in alloprimed murine recipients of hematopoietic stem cell grafts.

Alloantibody, not primed T cells, is the major barrier to bone marrow (BM) engraftment in allosensitized mice. We have shown that a single intravenous injection of donor splenocytes, to mimic a blood transfusion, results in high, sustained levels of serum alloantibody sufficient to eliminate donor BM within 3 h, resulting in uniform mortality in lethally irradiated allogeneic recipients. Current studies focused preventing and treating allopriming. Blockade of B cell survival signals with mTACI-Ig pre- and postpriming was ineffective, as was the B cell but not plasma cell depleting anti-CD20 mAb. Germinal center formation inhibition by lymphotoxin-beta receptor-Ig (LßR-Ig) diminished allosensitization, although conditional Prmd1 (Blimp-1) deletion in CD19+ cells was highly effective. By combining anti-CD20 mAb to reduce B cells and LTßR-Ig to diminish the frequency of B cells that could form germinal centers pre- and postpriming, allosensitization was precluded, permitting long-term survival in T- and NK-depleted, irradiated allogeneic recipients, whereas combined therapy postpriming alone was ineffective. As evidence of the critical role of B cells, the proteosomal inhibitor, bortezomib, given unencapsulated or encapsulated, proved ineffective in influencing allosensitization. These data extend our understanding of allopriming and provide a potential therapy for patients at risk for allosensitization and BM graft rejection.

Superiority of rapamycin over tacrolimus in preserving nonhuman primate Treg half-life and phenotype after adoptive transfer.

Many critical issues remain concerning how best to deploy adoptive regulatory T cell (Treg) immunotherapy to the clinic. These include a determination of their pharmacokinetic characteristics, their optimal dose, their phenotypic stability and the best therapies with which to pair Tregs. By performing a CFSE-labeled autologous Treg pulse experiment, we determined that the accessible peripheral blood Treg pool in rhesus macaques is quite large (75 ± 11 × 10(6) Tregs/kg). Pharmacokinetic analysis revealed that Tregs have two phases of elimination: an α phase, with a T1/2 in the peripheral blood of 32.4 ± 11.3 h and a ß phase with a T1/2 of 120.4 ± 19.7 h. In addition to their short initial half-life, Tregs underwent rapid phenotypic shifts after infusion, with significant loss of both CD25 and FoxP3 by day +6. While tacrolimus stabilized CD25 expression, it did not improve T1/2 , nor mitigate the loss of FoxP3. In contrast, rapamycin significantly stabilized both CD25 and FoxP3, and supported an increased half-life, with an α phase of 67.7 ± 6.9 h and a ß phase of 252.1 ± 54.9 h. These results suggest that rapamycin may be a necessary addition to Treg immunotherapy, and that tacrolimus may be deleterious to Treg integrity posttransfer.

Nonhuman primate transplant models finally evolve: detailed immunogenetic analysis creates new models and strengthens the old.

Nonhuman primate (NHP) models play a critical role in the translation of novel therapies for transplantation to the clinic. However, although MHC disparity significantly affects the outcome of transplantation, until recently, experiments using NHP models were performed without the ability to rigorously control the degree of MHC disparity in transplant cohorts. In this review, we discuss several key technical breakthroughs in the field, which have finally enabled detailed immunogenetic data to be incorporated into NHP transplantation studies. These advances have created a new gold-standard for NHP transplantation research, which incorporates detailed information regarding the degree of relatedness and the degree of MHC haplotype disparity between transplant pairs and the precise MHC alleles that both donors and recipients express. The adoption of this new standard promises to increase the rigor of NHP transplantation studies and to ensure that these experiments are optimally translatable to patient care.

Regulatory T cells exhibit decreased proliferation but enhanced suppression after pulsing with sirolimus.

Although regulatory T cells (Tregs) suppress allo-immunity, difficulties in their large-scale production and in maintaining their suppressive function after expansion have thus far limited their clinical applicability. Here we have used our nonhuman primate model to demonstrate that significant ex vivo Treg expansion with potent suppressive capacity can be achieved and that Treg suppressive capacity can be further enhanced by their exposure to a short pulse of sirolimus. Both unpulsed and sirolimus-pulsed Tregs (SPTs) are capable of inhibiting proliferation of multiple T cell subpopulations, including CD4(+) and CD8(+) T cells, as well as antigen-experienced CD28(+) CD95(+) memory and CD28(-) CD95(+) effector subpopulations. We further show that Tregs can be combined in vitro with CTLA4-Ig (belatacept) to lead to enhanced inhibition of allo-proliferation. SPTs undergo less proliferation in a mixed lymphocyte reaction (MLR) when compared with unpulsed Tregs, suggesting that Treg-mediated suppression may be inversely related to their proliferative capacity. SPTs also display increased expression of CD25 and CTLA4, implicating signaling through these molecules in their enhanced function. Our results suggest that the creation of SPTs may provide a novel avenue to enhance Treg-based suppression of allo-immunity, in a manner amenable to large-scale ex vivo expansion and combinatorial therapy with novel, costimulation blockade-based immunosuppression strategies.

A low frequency of pancreatic islet insulin-expressing cells derived from cord blood stem cell allografts in humans.

AIMS/HYPOTHESIS: We sought to establish if stem cells contained in cord blood cell allografts have the capacity to differentiate into insulin-expressing beta cells in humans. METHODS: We studied pancreases obtained at autopsy from individuals (n = 11) who had prior opposite-sex cord blood transplants to reconstitute haematopoiesis. Pancreatic tissue sections were stained first by XY-fluorescence in situ hybridisation and then insulin immunohistochemistry. Pancreases obtained at autopsy from participants without cord blood cell infusions served as controls (n = 11). RESULTS: In the men with prior transplant of female cord blood, there were 3.4 ± 0.3% XX-positive insulin-expressing islet cells compared with 0.32 ± 0.05% (p < 0.01) in male controls. In women with prior transplant of male cord blood cells we detected 1.03 ± 0.20% XY insulin-expressing islet cells compared with 0.03 ± 0.03 in female controls (p < 0. 001). CONCLUSIONS/INTERPRETATION: Cord blood stem cells have the capacity to differentiate into insulin-expressing cells in non-diabetic humans. It remains to be established whether these cells have the properties of beta cells.

Generation and large-scale expansion of human inducible regulatory T cells that suppress graft-versus-host disease.

Adoptive transfer of thymus-derived natural regulatory T cells (nTregs) effectively suppresses disease in murine models of autoimmunity and graft-versus-host disease (GVHD). TGFß induces Foxp3 expression and suppressive function in stimulated murine CD4+25- T cells, and these induced Treg (iTregs), like nTreg, suppress auto- and allo-reactivity in vivo. However, while TGFß induces Foxp3 expression in stimulated human T cells, the expanded cells lack suppressor cell function. Here we show that Rapamycin (Rapa) enhances TGFß-dependent Foxp3 expression and induces a potent suppressor function in naive (CD4+ 25-45RA+) T cells. Rapa/TGFß iTregs are anergic, express CD25 at levels higher than expanded nTregs and few cells secrete IL-2, IFNγ or IL-17 even after PMA and Ionomycin stimulation in vitro. Unlike other published methods of inducing Treg function, Rapa/TGFß induces suppressive function even in the presence of memory CD4+ T cells. A single apheresis unit of blood yields an average ~240 × 109 (range ~ 70-560 × 109) iTregs from CD4+25- T cells in ≤ 2 weeks of culture. Most importantly, Rapa/TGFß iTregs suppress disease in a xenogeneic model of GVHD. This study opens the door for iTreg cellular therapy for human diseases.

p27kip1 functions as an anergy factor inhibiting interleukin 2 transcription and clonal expansion of alloreactive human and mouse helper T lymphocytes.

Although recent in vitro studies have begun to decipher the molecular events that characterize the anergic state, their in vivo biologic relevance and potential clinical importance remain unclear. Here, using anergic human T-cell clones and tolerant alloreactive mouse T cells that do not induce graft-versus-host disease, we show that p27kip1 cyclin-dependent kinase inhibitor is an essential regulator responsible for the blockade of clonal expansion of anergic T cells in vitro and in vivo. Moreover, in anergic cells, p27kip1 associates with the c-Jun co-activator JAB1, resulting in defective transactivation of AP-1 and interleukin 2 transcription. Therefore, pharmacological agents that upregulate the expression of or prevent the degradation of p27kip1 during antigen recognition should be part of new therapeutic strategies to induce antigen-specific T-cell unresponsiveness.

CD4(+)CD25(+) immune regulatory cells are required for induction of tolerance to alloantigen via costimulatory blockade.

Immune regulatory CD4(+)CD25(+) cells play a vital role in the induction and maintenance of self-tolerance and are essential for T cell homeostasis and the prevention of autoimmunity. Induction of tolerance to allogeneic donor grafts is a clinically desirable goal in bone marrow and solid organ transplantation. To determine whether CD4(+)CD25(+) cells regulate T cell responses to alloantigen and are critical for tolerance induction, murine CD4(+) T cells were tolerized to alloantigen via ex vivo CD40 ligand (CD40L)/CD40 or CD28/cytotoxic T lymphocyte-associated antigen 4/B7 blockade resulting in secondary mixed leukocyte reaction hyporesponsiveness and tolerance to alloantigen in vivo. CD4(+)CD25(+) T cells were found to be potent regulators of alloresponses. Depletion of CD4(+)CD25(+) T cells from the CD4(+) responder population completely abrogated ex vivo tolerance induction to alloantigen as measured by intact responses to alloantigen restimulation in vitro and in vivo. Addback of CD4(+)CD25(+) T cells to CD4(+)CD25(-) cultures restored tolerance induction. These data are the first to indicate that CD4(+)CD25(+) cells are essential for the induction of tolerance to alloantigen and have important implications for tolerance-inducing strategies targeted at T cell costimulatory pathways.

CD47 (integrin-associated protein) engagement of dendritic cell and macrophage counterreceptors is required to prevent the clearance of donor lymphohematopoietic cells.

Integrin-associated protein (CD47) is a broadly expressed protein that costimulates T cells, facilitates leukocyte migration, and inhibits macrophage scavenger function. To determine the role of CD47 in regulating alloresponses, CD47(+/+) or CD47(-/-) T cells were infused into irradiated or nonconditioned major histocompatibility complex disparate recipients. Graft-versus-host disease lethality was markedly reduced with CD47(-/-) T cells. Donor CD47(-/-) T cells failed to engraft in immunodeficient allogeneic recipients. CD47(-/-) marrow was unable to reconstitute heavily irradiated allogeneic or congenic immune-deficient CD47(+/+) recipients. These data suggested that CD47(-/-) T cells and marrow cells were cleared by the innate immune system. To address this hypothesis, dye-labeled CD47(-/-) and CD47(+/+) lymphocytes or marrow cells were infused in vivo and clearance was followed. Dye-labeled CD47(-/-) cells were engulfed by splenic dendritic cells and macrophages resulting in the clearance of virtually all CD47(-/-) lymphohematopoietic cells within 1 day after infusion. Host phagocyte-depleted CD47(+/+) recipients partially accepted allogeneic CD47(-/-) T cells. Thus, dendritic cells and macrophages clear lymphohematopoietic cells that have downregulated CD47 density. CD47 expression may be a critical indicator for determining whether lymphohematopoietic cells will survive or be cleared.

L-selectin is dispensable for T regulatory cell function postallogeneic bone marrow transplantation.

In murine models, the adoptive transfer of CD4(+) /CD25(+) regulatory T cells (T(regs) ) inhibited graft-versus-host disease (GvHD). Previous work has indicated a critical role for the adhesion molecule L-selectin (CD62L) in the function of T(regs) in preventing GvHD. Here we examined the capacity of naive wild-type (WT), CD62L(-/-) and ex vivo expanded CD62L(Lo) T(regs) to inhibit acute GvHD. Surprisingly, we found that CD62L(-/-) T(regs) were potent suppressors of GvHD, whereas CD62L(Lo) T(regs) were unable to inhibit disease despite being functionally competent to suppress allo T cell responses in vitro. Concomitant with improved outcomes, WT and CD62L(-/-) T(regs) significantly reduced liver pathology and systemic pro-inflammatory cytokine production, although CD62L(-/-) T(regs) were less effective in reducing lung pathology. While accumulation of CD62L(-/-) T(regs) in GvHD target organs was equivalent to WT T(regs) , CD62L(-/-) T(regs) did not migrate as well as WT T(regs) to peripheral lymph nodes (PLNs) over the first 2 weeks posttransplantation. This work demonstrated that CD62L was dispensable for T(reg) -mediated protection from GvHD.

Ex vivo inhibition of NF-kappaB signaling in alloreactive T-cells prevents graft-versus-host disease.

The ex vivo induction of alloantigen-specific hyporesponsiveness by costimulatory pathway blockade or exposure to immunoregulatory cytokines has been shown to inhibit proliferation, IL-2 production, and the graft-versus-host disease (GVHD) capacity of adoptively transferred T-cells. We hypothesized that inhibition of the intracellular NF-kappaB pathway in alloreactive T-cells, which is critical for T-cell activation events including IL-2 transcription, could lead to alloantigen hyporesponsiveness and loss of GVHD capacity. We demonstrate that treatment of mixed lymphocyte reaction (MLR) cultures with PS1145, a potent inhibitor of NF-kappaB activation, can induce T-cell hyporesponsiveness to alloantigen in primary and secondary responses while preserving in vitro responses to potent mitogenic stimulation. GVHD lethality in recipients of ex vivo PS1145-treated cells was profoundly inhibited. Parking of control or PS1145-treated MLR cells in syngeneic Rag(-/-) recipients resulted in intact contact hypersensitivity (CHS) responses. However, GVHD lethality capacity also was restored, suggesting that lymphopenic expansion uncoupled alloantigen hyporesponsiveness. These results indicate that the NF-kappaB pathway is a critical regulator of alloresponses and provide a novel small molecule inhibitor based approach that is effective in preventing early posttransplant GVHD lethality but that also permits donor T-cell responses to recover after a period of lymphopenic expansion.

Expanded nonhuman primate tregs exhibit a unique gene expression signature and potently downregulate alloimmune responses.

We have established two complementary strategies for purifying naturally occurring regulatory T cells (Tregs) from rhesus macaques in quantities that would be sufficient for use as an in vivo cellular therapeutic. The first strategy identified Tregs based on their being CD4+/CD25(bright). The second incorporated CD127, and purified Tregs based on their expression of CD4 and CD25 and their low expression of CD127. Using these purification strategies, we were able to purify as many as 1x10(6) Tregs from 120 cc of peripheral blood. Cultures of these cells with anti-CD3, anti-CD28 and IL-2 over 21 days yielded as much as a 450-fold expansion, ultimately producing as many as 4.7x10(8) Tregs. Expanded Treg cultures potently inhibited alloimmune proliferation as measured by a carboxyfluorescein succinimidyl ester- mixed lymphocyte reaction (CFSE-MLR) assay even at a 1:100 ratio with responder T cells. Furthermore, both responder-specific and third-party Tregs downregulated alloproliferation similarly. Both freshly isolated and cultured Tregs had gene expression signatures distinguishable from concurrently isolated bulk CD4+ T-cell populations, as measured by singleplex reverse transcriptase-polymerase chain reaction (RT-PCR) and gene array. Moreover, an overlapping yet distinct gene expression signature seen in freshly isolated compared to expanded Tregs identifies a subset of Treg genes likely to be functionally significant.

Donor and recipient plasma follistatin levels are associated with acute GvHD in Blood and Marrow Transplant Clinical Trials Network 0402.

Follistatin is an angiogenic factor elevated in the circulation after allogeneic hematopoietic cell transplantation (HCT). Elevations in follistatin plasma concentrations are associated with the onset of and poor survival after acute GvHD (aGvHD). Using data from the Blood and Marrow Transplant Clinical Trials Network 0402 study (n=247), we sought to further quantify the longitudinal associations between plasma follistatin levels in transplant recipients, as well as baseline HCT donor follistatin levels, and allogeneic HCT outcomes. Higher recipient baseline follistatin levels were predictive of development of aGvHD (P=0.04). High donor follistatin levels were also associated with the incidence of aGvHD (P<0.01). Elevated follistatin levels on day 28 were associated with the onset of grade II-IV aGvHD before day 28, higher 1-year non-relapse mortality (NRM) and lower overall survival. In multivariate analyses, individuals with follistatin levels >1088 pg/mL at day 28 had a 4-fold increased risk for NRM (relative risk (RR)=4.3, 95% confidence interval (CI) 1.9-9.9, P<0.01) and a nearly three-fold increased overall risk for mortality (RR=2.8, 95% CI 1.5-5.2, P<0.01). Given the multiple roles of follistatin in tissue inflammation and repair, and the confirmation that this biomarker is predictive of important HCT outcomes, the pathobiology of these relationships need further study.

CD4(+) T cells tolerized ex vivo to host alloantigen by anti-CD40 ligand (CD40L:CD154) antibody lose their graft-versus-host disease lethality capacity but retain nominal antigen responses.

A major goal of the transplant field is to tolerize donor T cells to prevent graft-versus-host disease (GVHD) (1). We describe an ex vivo approach in which the blockade of CD40 ligand (CD40L:CD154):CD40 interactions, a pathway required for optimal T cell expansion, induces donor CD4(+) T cells to become tolerant to host alloantigens (2). High doses of tolerized cells did not cause GVHD lethality in vivo. T cells had intact responses to antigens not present during tolerization. Tolerance was long lived and not readily reversible in vivo. These data have significant implications for the use of tolerization approaches to prevent human GVHD.

The critical early proinflammatory events associated with idiopathic pneumonia syndrome in irradiated murine allogeneic recipients are due to donor T cell infusion and potentiated by cyclophosphamide.

We have hypothesized that lung damage occurring in the peri-bone marrow transplant (BMT) period is critical for the subsequent generation of idiopathic pneumonia syndrome (IPS), a major complication following human BMT. The proinflammatory events induced by a common pre-BMT conditioning regimen, cyclophosphamide (Cytoxan(R)) (Cy) and total body irradiation, were analyzed in a murine BMT model. Electron microscopy indicated that Cy exacerbated irradiation-induced epithelial cell injury as early as day 3 after BMT. Allogenicity was an important contributing factor to lung injury as measured by lung wet and dry weights and decreased specific lung compliance. The most significant pulmonary dysfunction was seen in mice receiving both allogeneic T cells and Cy conditioning. IPS was associated with an influx of T cells, macrophages, and neutrophils early post-BMT. Hydroxyproline levels were not increased, indicating that the injury was not fibrotic early post-BMT. As early as 2 h after chemoradiation, host macrophages increased in number in the lung parenchyma. Continued increases in macrophages occurred if splenic T cells were administered with the donor graft. The expression of costimulatory B7 molecules correlated with macrophage numbers. Frequencies of cells expressing mRNA for the inflammatory proteins TNF-alpha, IL-1beta, and TGFbeta were increased. Cy accelerated the upregulation of TGFbeta and increase in host macrophages. The exacerbation of macrophage activation and severity of IPS was dependent on allogeneic T cells, implicating immune-mediated mechanisms as critical to the outcome of IPS. This demonstration of early injury after BMT indicates the need for very early therapeutic intervention before lung damage becomes profound and irreversible.

Differential effects of the absence of interferon-gamma and IL-4 in acute graft-versus-host disease after allogeneic bone marrow transplantation in mice.

Graft-versus-host disease (GVHD), in which immunocompetent donor cells attack the host, remains a major cause of morbidity after allogeneic bone marrow transplantation (BMT). To understand the role of cytokines in the pathobiology of GVHD, we used cytokine knockout (KO) mice as a source of donor T cells. Two different MHC-disparate strain combinations were examined: BALB/c (H2(d)) donors into lethally irradiated C57BL/6 (H2(b)) recipients or C57BL/6 (H2(b)) donors into B10.BR (H2(k)) recipients. Donor cells were from mice in which either the interferon-gamma (IFN-gamma) or the IL-4 gene was selectively disrupted to understand the role of these cytokines in acute GVHD. In both strain combinations the same pattern was noted with regard to GVHD onset and morbidity. All mice exhibited the classic signs of acute GVHD: weight loss with skin, gut, and liver pathology resulting in morbidity and mortality. Surprisingly, donor cells obtained from mice lacking IFN-gamma gave rise to accelerated morbidity from GVHD when compared with cells from wild-type control donors. Similar results were obtained using normal donors when neutralizing antibodies to IFN-gamma were administered immediately after the BMT. These results suggest that IFN-gamma plays a role in protection from acute GVHD. In marked contrast, cells obtained from IL-4 KO mice resulted in protection from GVHD compared with control donors. Splenocytes from IFN KO mice stimulated with a mitogen proliferated to a significantly greater extent and produced more IL-2 compared with splenocytes obtained from IL-4 KO or control mice. Additionally, there was increased IL-2 production in the spleens of mice undergoing GVHD using IFN-gamma KO donors. These results therefore indicate, with regard to the TH1/ TH2 cytokine paradigm, the absence of a TH1-type cytokine can be deleterious in acute GVHD, whereas absence of a TH2 cytokine can be protective.