Rejection Of Solid Organ Allografts Research Articles

The self-peptide repertoire plays a critical role in transplant tolerance induction.

While direct allorecognition underpins both solid organ allograft rejection and tolerance induction, the specific molecular targets of most directly alloreactive CD8+ T cells have not been defined. In this study, we used a combination of genetically engineered major histocompatibility complex class I (MHC I) constructs, mice with a hepatocyte-specific mutation in the class I antigen-presentation pathway, and immunopeptidomic analysis to provide definitive evidence for the contribution of the peptide cargo of allogeneic MHC I molecules to transplant tolerance induction. We established a systematic approach for the discovery of directly recognized pMHC epitopes and identified 17 strongly immunogenic H-2Kb-associated peptides recognized by CD8+ T cells from B10.BR (H-2k) mice, 13 of which were also recognized by BALB/c (H-2d) mice. As few as 5 different tetramers used together were able to identify a high proportion of alloreactive T cells within a polyclonal population, suggesting that there are immunodominant allogeneic MHC-peptide complexes that can account for a large component of the alloresponse.

Use of Post-transplant Cyclophosphamide Treatment to Build a Tolerance Platform to Prevent Liquid and Solid Organ Allograft Rejection.

Corneal transplantation (CT) is the most frequent type of solid organ transplant (SOT) performed worldwide. Unfortunately, immunological rejection is the primary cause of graft failure for CT and therefore advances in immune regulation to induce tolerance remains an unmet medical need. Recently, our work and others in pre-clinical studies found that cyclophosphamide (Cy) administered after (“post-transplant,” PTCy) hematopoietic stem cell transplantation (HSCT), i.e., liquid transplants is effective for graft vs. host disease prophylaxis and enhances overall survival. Importantly, within the past 10 years, PTCy has been widely adopted for clinical HSCT and the results at many centers have been extremely encouraging. The present studies found that Cy can be effectively employed to prolong the survival of SOT, specifically mouse corneal allografts. The results demonstrated that the timing of PTCy administration is critical for these CT and distinct from the kinetics employed following allogeneic HSCT. PTCy was observed to interfere with neovascularization, a process critically associated with immune rejection of corneal tissue that ensues following the loss of ocular “immune privilege.” PTCy has the potential to delete or directly suppress allo-reactive T cells and treatment here was shown to diminish T cell rejection responses. These PTCy doses were observed to spare significant levels of CD4+ FoxP3+ (Tregs) which were found to be functional and could readily receive stimulating signals leading to their in vivo expansion via TNFRSF25 and CD25 agonists. In total, we posit future studies can take advantage of Cy based platforms to generate combinatorial strategies for long-term tolerance induction.

Metabolic reprogramming augments potency of human pSTAT3-inhibited iTregs to suppress alloreactivity.

Immunosuppressive donor Tregs can prevent graft-versus-host disease (GVHD) or solid-organ allograft rejection. We previously demonstrated that inhibiting STAT3 phosphorylation (pSTAT3) augments FOXP3 expression, stabilizing induced Tregs (iTregs). Here we report that human pSTAT3-inhibited iTregs prevent human skin graft rejection and xenogeneic GVHD yet spare donor antileukemia immunity. pSTAT3-inhibited iTregs express increased levels of skin-homing cutaneous lymphocyte-associated antigen, immunosuppressive GARP and PD-1, and IL-9 that supports tolerizing mast cells. Further, pSTAT3-inhibited iTregs significantly reduced alloreactive conventional T cells, Th1, and Th17 cells implicated in GVHD and tissue rejection and impaired infiltration by pathogenic Th2 cells. Mechanistically, pSTAT3 inhibition of iTregs provoked a shift in metabolism from oxidative phosphorylation (OxPhos) to glycolysis and reduced electron transport chain activity. Strikingly, cotreatment with coenzyme Q10 restored OxPhos in pSTAT3-inhibited iTregs and augmented their suppressive potency. These findings support the rationale for clinically testing the safety and efficacy of metabolically tuned, human pSTAT3-inhibited iTregs to control alloreactive T cells.

Natural Killer Cells: Critical Effectors During Antibody-mediated Rejection of Solid Organ Allografts.

Antibody-mediated rejection (AMR) is an important cause of graft loss and continues to present a formidable obstacle to successful transplantation. Unresolved problems continue to be the absence of effective strategies to ablate the donor-specific antibody (DSA) response as well as to attenuate the antibody-mediated graft tissue injury. While the properties of DSA that cause greater graft tissue injury and the characteristic microvascular pathology of the graft injury are well documented, the mechanisms underlying the injury mediated by the antibodies remains unclear. Recent transcriptome interrogation of kidney and heart biopsies procured during ongoing AMR has indicated the expression of genes associated with natural killer (NK) cell activation that is absent during T cell-mediated rejection. The expression of NK cell transcripts during AMR correlates with the presence of CD56+ cells in the microcirculation inflammation observed during AMR. Several mouse models have recently demonstrated the role of NK cells in antibody-mediated chronic vasculopathy in heart allografts and the requirement for NK cell activation during acute AMR of kidney allografts. In the latter model, NK cell activation within kidney allografts is regulated by the activation of myeloid cells producing myeloperoxidase. Overall, the studies to date indicate that AMR constitutes a complex series of DSA-induced interactions with components of the innate immune response. The innate immune participants and their expressed effector functions resulting in the rejection are beginning to be identified. The identification of these components should uncover novel targets that can be used to attenuate acute graft tissue injury in the presence of DSA.

Tocilizumab in transplantation.

Tocilizumab, a monoclonal antibody directed against the IL-6 receptor, might block detrimental effects of IL-6 on transplantation. IL-6 plays a considerable role in cytokine storm after stem cell transplantation as well as graft versus host disease, and it has also been shown to be involved in solid organ allograft rejection; therefore, tocilizumab is expected to promote graft survival. Nonetheless, due to the small number of studies and disparate methods of drug administration and outcome evaluation, for which types of transplantation, at which stages, and to what extent tocilizumab could be applied remains to be defined. The Pubmed,SCOPUS and Google Scholar search engines were used to collect data. The keywords were determined by Pubmed MeSH. No time limitation was set and all types of articles were allowed. RESULTS: According to the potential ofTocilozumab in controlling both cellular and humoral immunity it could be considered as a promising agent in tolerance induction; however, blocking IL-6 signaling might result in augmented infection rate in recipients. The need for providing effective and safe immunosuppressive agents to protect transplanted cells and organs against allo-reactivity urges the collection and discussion of all available findings about inhibition ofdeterminingimmune components including cytokines;herein, we have summarized the clinical consequences of blocking IL-6 by tocilizumab in stem cell and solid organ transplantations.

Update on C1 Esterase Inhibitor in Human Solid Organ Transplantation.

Complement plays important roles in both ischemia-reperfusion injury (IRI) and antibody-mediated rejection (AMR) of solid organ allografts. One approach to possibly improve outcomes after transplantation is the use of C1 inhibitor (C1-INH), which blocks the first step in both the classical and lectin pathways of complement activation and also inhibits the contact, coagulation, and kinin systems. C1-INH can also directly block leukocyte-endothelial cell adhesion. C1-INH contrasts with eculizumab and other distal inhibitors, which do not affect C4b or C3b deposition or noncomplement pathways. Authors of reports on trials in kidney transplant recipients have suggested that C1-INH treatment may reduce IRI and delayed graft function, based on decreased requirements for dialysis in the first month after transplantation. This effect was particularly marked with grafts with Kidney Disease Profile Index ≥ 85. Other clinical studies and models suggest that C1-INH may decrease sensitization and donor-specific antibody production and might improve outcomes in AMR, including in patients who are refractory to other modalities. However, the studies have been small and often only single-center. This article reviews clinical data and ongoing trials with C1-INH in transplant recipients, compares the results with those of other complement inhibitors, and summarizes potentially productive directions for future research.

Skin-restricted commensal colonization accelerates skin graft rejection.

Solid organ transplantation can treat end-stage organ failure, but the half-life of transplanted organs colonized with commensals is much shorter than that of sterile organs. Whether organ colonization plays a role in this shorter half-life is not known. We have previously shown that an intact whole-body microbiota can accelerate the kinetics of solid organ allograft rejection in untreated colonized mice when compared to germ-free (GF) or to antibiotic-pre-treated colonized mice, by enhancing the capacity of antigen presenting cells (APCs) to activate graft-reactive T cells. However, the contribution of intestinal versus skin microbiota to these effects was unknown. Here, we demonstrate that colonizing the skin of GF mice with a single commensal, Staphylococcus epidermidis (S. epi), while preventing intestinal colonization with oral vancomycin, was sufficient to accelerate skin graft rejection. Notably, unlike the mechanism by which whole-body microbiota accelerates skin graft rejection, cutaneous S. epi did not enhance the priming of alloreactive T cells in the skin-draining lymph nodes (LNs). Rather, cutaneous S. epi augmented the ability of skin APCs to drive the differentiation of alloreactive T cells. This study reveals that the extra-intestinal donor microbiota can affect transplant outcome and may contribute to the shorter half-life of colonized organs.

Natural killer cells as modulators of alloimmune responses.

Natural killer (NK) cells are effector cells of the innate immune system that can lyse target cells without prior sensitization and are important in host defense to virally infected and transformed cells. Although the concept of 'missing-self' would suggest NK cells could target foreign allografts, the prevailing dogma has been that NK cells are not active participants in the rejection of solid organ allografts. This review summarizes recent studies that challenge this conclusion and instead suggest NK cells are important in outcomes posttransplant. NK cells expressing specific cell surface receptors may promote graft damage and rejection. However, recent studies suggest some NK cell subsets have tolerogenic or immunoregulatory potential and promote graft stability, suggesting a dichotomous role for NK cells after transplant. Furthermore, NK cells respond to cells infected with cytomegalovirus and Epstein-Barr virus, and studies suggest some NK cells have immune memory. Our understanding of the role of NK cells posttransplant has evolved from 'no role' to the current idea that NK cells may have 'complex interactions' that impact graft outcomes. Additional studies, using cutting edge techniques to comprehensively analyze the phenotypic and functional subsets of NK cells in transplant recipients, are clearly necessary.

A randomized control trial to establish the feasibility and safety of rapamycin treatment in an older human cohort: Immunological, physical performance, and cognitive effects

Inhibition of the mechanistic target of rapamycin (mTOR) pathway by rapamycin (RAPA), an FDA-approved immunosuppressive drug used as a clinical therapy to prevent solid organ allograft rejection, enhances longevity in mice. Importantly, RAPA was efficacious even when initiated in relatively old animals, suggesting that mTOR inhibition could potentially slow the progression of aging-associated pathologies in older humans (Harrison et al., 2009; Miller et al., 2011). However, the safety and tolerability of RAPA in older human subjects have not yet been demonstrated. Towards this end, we undertook a placebo-controlled pilot study in 25 generally healthy older adults (aged 70–95 years); subjects were randomized to receive either 1 mg RAPA or placebo daily. Although three subjects withdrew, 11 RAPA and 14 controls completed at least 8 weeks of treatment and were included in the analysis. We monitored for changes that would indicate detrimental effects of RAPA treatment on metabolism, including both standard clinical laboratory assays (CBC, CMP, HbA1c) and oral glucose tolerance tests (OGTTs). We also monitored parameters typically associated with aging that could potentially be modified by RAPA; these included cognitive function which was assessed by three different tools: Executive Interview-25 (EXIT25); Saint Louis University Mental Status Exam (SLUMS); and Texas Assessment of Processing Speed (TAPS). In addition, physical performance was measured by handgrip strength and 40-foot timed walks. Lastly, changes in general parameters of healthy immune aging, including serum pro-inflammatory cytokine levels and blood cell subsets, were assessed. Five subjects reported potential adverse side effects; in the RAPA group, these were limited to facial rash (1 subject), stomatitis (1 subject) and gastrointestinal issues (2 subjects) whereas placebo treated subjects only reported stomatitis (1 subject). Although no other adverse events were reported, statistically significant decrements in several erythrocyte parameters including hemoglobin (HgB) and hematocrit (Hct) as well as in red blood cell count (RBC), red blood cell distribution width (RDW), mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) were observed in the RAPA-treatment group. None of these changes manifested clinically significant effects during the short duration of this study. Similarly, no changes were noted in any other clinical laboratory, cognitive, physical performance, or self-perceived health status measure over the study period. Immune parameters were largely unchanged as well, possibly due to the advanced ages of the cohort (70–93 years; mean age 80.5). RAPA-associated increases in a myeloid cell subset and in TREGS were detected, but changes in most other PBMC cell subsets were not statistically significant. Importantly, the OGTTs revealed no RAPA-induced change in blood glucose concentration, insulin secretion, and insulin sensitivity. Thus, based on the results of our pilot study, it appears that short-term RAPA treatment can be used safely in older persons who are otherwise healthy; a trial with a larger sample size and longer treatment duration is warranted.

Utility of IL-2 Complexes in Promoting the Survival of Murine Orthotopic Forelimb Vascularized Composite Allografts.

Vascularized composite allografts (VCA) are novel, life-enhancing forms of transplantation (Tx). However, host immune responses to the various VCA components, especially those involving skin, are complex and make selection of appropriate therapy challenging. Although the interplay between Foxp3+ T regulatory (Treg) cells and CD4 and CD8 effector T cells is of central importance in determining the acceptance or rejection of solid organ allografts, there is little information available concerning the contribution of Treg cells to VCA survival. In addition, the effects of therapeutic expansion in vivo of host Treg cell populations on VCA survival are unknown. We established a fully major histocompatibility complex-disparate (BALB/c- > C57BL/6) murine orthotopic forelimb Tx model to explore the benefits of pre- and post-Tx IL-2/anti-IL-2 monoclonal antibody complex (IL-2C) administration to expand the host Treg cell population and thereby attempt to promote Treg cell-dependent VCA survival. Both strategies expanded the Treg cell population in vivo and prolonged VCA survival (P < 0.001), but IL-2C administration pre-Tx led to significantly longer survival compared with IL-2C administration post-Tx (P < 0.01). In addition, compared with post-Tx therapy, pre-Tx therapy resulted in an increased ratio of Treg cells to CD8+ T cells (P < 0.001), reduced proliferation of CD4 and CD8 effector T cells, and reduced production of IFN-γ. Optimal effects were seen when combined with rapamycin therapy, whereas the combination of IL-2C therapy plus calcineurin inhibitor was counterproductive. Our studies involving different IL-2C-mediated Treg cell expansion strategies demonstrate that pre-Tx IL-2C therapy may be a useful component for developing strategies to promote VCA survival.

Augmentation of Recipient Adaptive Alloimmunity by Donor Passenger Lymphocytes within the Transplant.

SummaryChronic rejection of solid organ allografts remains the major cause of transplant failure. Donor-derived tissue-resident lymphocytes are transferred to the recipient during transplantation, but their impact on alloimmunity is unknown. Using mouse cardiac transplant models, we show that graft-versus-host recognition by passenger donor CD4 T cells markedly augments recipient cellular and humoral alloimmunity, resulting in more severe allograft vasculopathy and early graft failure. This augmentation is enhanced when donors were pre-sensitized to the recipient, is dependent upon avoidance of host NK cell recognition, and is partly due to provision of cognate help for allo-specific B cells from donor CD4 T cells recognizing B cell MHC class II in a peptide-degenerate manner. Passenger donor lymphocytes may therefore influence recipient alloimmune responses and represent a therapeutic target in solid organ transplantation.

CD8+ T Cells Mediate Antibody-Independent Platelet Clearance Following Transfusion

Background: Alloimmunization to major histocompatibility complex (MHC) antigens is the leading immunological barrier to transplantation and platelet transfusion therapy. In the context of platelet transfusion medicine, alloimmunization to MHC antigens can substantially diminish the therapeutic efficacy of subsequent blood transfusions. Presently, MHC alloantibodies are recognized as the primary mediators of immune-mediated platelet clearance. However, individuals previously exposed to MHC alloantigens can display significant platelet clearance in the absence of detectable anti-platelet antibodies. Given the ability of cellular immunity to mediate rejection of solid organ allografts across MHC differences, we hypothesized that cellular immunity likewise facilitates the clearance of platelets in MHC alloimmunized recipients.Methods: FVB (H-2Kq) MHC-immunized or non-immunized C57BL/6 (H-2Kb) recipients were transfused with filter leukoreduced platelet rich plasma (PRP) isolated from C57BL/6 donors expressing green fluorescent protein (GFP) under a H-2Kb promoter (B6 GFP) or PRP obtained from a FVB X B6 GFP F1 cross, which generates GFP+ H-2Kq+ platelets. Following transfusion, mice were bled at 10 minutes, 1 hour, 2 hours or 24 hours to calculate the percentage of GFP+ transfused platelets remaining using flow cytometric analysis. To test the hypothesis that platelet clearance can occur in an antibody-independent process, FVB H-2Kq MHC-immunized or non-immunized μMT recipients were similarly transfused with B6 GFP or FVB X B6 GFP PRP. CD8+ T cells in immunized μMT recipients were depleted by i.p. injection of anti-CD8 (clone 2.43) antibody for 3 consecutive days prior to transfusion. NK cells were similarly depleted by injection of anti-NK1.1 (clone PK-136) antibody, 1 day prior to transfusion. CD4, CD8 T cell and NK cell depletion was evaluated in the peripheral blood prior to platelet transfusion, and compared to non-treated and isotype control treated recipients.Results: While GFP+ platelets were readily detected following transfusion into non-immunized or MHC matched recipients, transfusion of FVB-B6 platelets into H-2Kq MHC-immunized C57BL/6 recipients resulted in rapid clearance within the first hour following transfusion. In contrast, though little platelet clearance was detected in H-2Kq MHC-immunized μMT recipients within an hour following transfusion, very few transfused platelets were observed after 24 hours. Moreover, despite platelet clearance, anti-MHC antibodies were not detectable in μMT recipients. To determine the cellular component responsible for clearance in alloimmunized μMT recipients, CD8+ T cells or NK cells were depleted prior to transfusion. NK cell depletion failed to impact platelet clearance in alloimmunized μMT recipients; however, depletion of CD8+ T cells prior to platelet transfusion abrogated platelet clearance in immunized μMT recipients.Conclusion: Immunized recipients rapidly clear platelets in an alloantigen specific fashion following platelet transfusion in a murine model. Though platelet clearance occurred at a faster rate in intact immunized C57BL/6 recipients, B cell deficient MHC-immunized μMT recipients possessed the capacity to induce significant clearance at later time points. Evaluation of the cellular populations responsible for antibody-independent clearance in μMT recipients strongly suggest that CD8+ T cells play a key role in antibody-independent immune-mediated platelet clearance. These results suggest that, in addition to antibodies, CD8+ T cells can play a role in the development of platelet refractoriness and may contribute to platelet non-responsiveness in patients with little to no detectable anti-platelet alloantibodies. DisclosuresNo relevant conflicts of interest to declare.

Management Strategies for Posterior Reversible Encephalopathy Syndrome (PRES) in Patients Receiving Calcineurin-Inhibitor or Sirolimus Therapy for Hematologic Disorders and Allogeneic Transplantation

▪Background: Posterior reversible encephalopathy syndrome (PRES) is a potentially life-threatening complication of therapy with calcineurin inhibitors (CNI). Medications in this class include tacrolimus (Tac) and cyclosporine A (CSA). Sirolimus (Siro), an inhibitor of the mammalian target of rapamycin (mTOR) complex, may also be associated with PRES. These therapies are generally used in the management of patients with severe aplastic anemia (SAA), to prevent or treat graft-versus-host disease following allogeneic hematopoietic cell transplantation (alloHCT), or to prevent rejection of solid organ allografts. PRES is associated with hypertension and symptomatic features include headaches, seizures, and occasionally, intracranial hemorrhage or ischemic strokes. Classical findings on magnetic resonance imaging (MRI) studies include leukoencephalopathy with vasogenic edema in the occipital cerebrum. Diffusion restriction is observed on diffusion-weighted imaging. Management of CNI/Siro-associated PRES has not been well-studied.Methods: We performed a single-institution retrospective review of all patients identified to have possible PRES on MRI between October 2000 and February 2014. Patients were identified by searching a database of all neuroimaging studies performed at our institution during this time period for the terms: PRES, posterior reversible encephalopathy, or reversible posterior leukoencephalopathy. Radiography studies were reevaluated for this study to uniformly collect data regarding imaging features. Medical chart review was conducted to determine patient features at diagnosis, management decisions with focus on medication changes, and outcomes.Results: Our review identified 191 patients who underwent 236 MRI studies that demonstrated findings suggesting a possible diagnosis of PRES. On medical record review, pertinent clinical data were available for 167 patients, of whom 93 (56%) were deemed to have a clinical diagnosis consistent with PRES. Amongst this cohort, 27 patients (29%) were diagnosed with PRES while taking a CNI or Siro – 22 (81%) were receiving Tac, 4 (15%) were receiving CSA, and 1 patient (3.7%) was receiving Siro at diagnosis. The median age at PRES diagnosis was 52 (range 7 - 68), with no apparent association between age and the occurrence of CNI/Siro-associated PRES. Indications for CNI/Siro therapy included: alloHCT (19%), SAA (3.7%), systemic lupus erythematosus (7.4%), and solid organ transplantation (70%). Symptomatic features and physical signs at diagnosis of CNI/Siro-associated PRES included: hypertension (74%), seizure (52%), headache (44%), intracranial hemorrhage (22%), and cerebrovascular accident (15%). Presentation with these symptoms was not significantly different between CNI/Siro-associated PRES and other forms of PRES (p = 0.36, 0.48, 0.88, 0.97, and 0.44, respectively). The median time from initiation of CNI/Siro therapy to onset of PRES was 22 days (range 1 - 1924). Only 3 patients (11%) discontinued CNI/Siro therapy at PRES diagnosis and did not resume, whereas 12 (44%) continued the same medication, 10 (37%) were changed to alternative CNI (Tac-to-CSA or CSA-to-Tac) therapy, and 2 (7.4%) were changed from CNI to Siro. Two patients experienced recurrent PRES following these initial management decisions. All other patients continued therapy without recurrence.Conclusions: CNI/Siro therapy, mostly in the setting of allogeneic hematopoietic cell or solid organ transplantation, was associated with roughly one-third of PRES cases at our institution. Management of CNI/Siro-associated PRES has not been well studied, and the low incidence of this complication is prohibitive to the implementation of prospective studies. In this retrospective review, we evaluated the management strategies and outcomes of patients who developed PRES while receiving CNI/Siro therapy. The vast majority of patients (77%) tolerated continuation of the same CNI or changing to alternative CNI therapy. An additional 11% tolerated conversion from CNI to Siro. The recurrence rate with continuation of CNI/Siro therapy was low at 2/24 (8.3%) and no patients experienced fatal complications of PRES, suggesting this management strategy is safe. Additional analyses are underway to determine whether clinical or radiographic features may help predict the recurrence of PRES in patients who continue CNI/Siro therapy. DisclosuresOff Label Use: Off-label use of tacrolimus, cyclosporine, and sirolimus for immune prophylaxis in hematopoietic cell transplant patients is discussed..

Current Clinical Applications of Extracorporeal Photochemotherapy

From the 1980s, extracorporeal photochemotherapy (ECP) has been shown to be effective in a variety of pathological conditions such as erythrodermic cutaneous T-cell lymphoma, autoimmune diseases, solid organ allograft rejection and graft versus host disease. To date, ECP represents a non-aggressive immune modulatory therapy with a low spectrum of toxicity. ECP reduces the alloreactivity promoting the immune tolerance to self. At the same time, it allows the maintenance of immune response integrity of both naive and memory T-cells. However, the molecular mechanisms of action by which ECP exerts its therapeutic activity are still under investigation. Here, we review molecular mechanisms and clinical applications involved in ECP. The outcome of ECP is difficult due to the lack of reliable predictor factors for the selection of patients and their adequate follow-up. Since the study of such predictors is important, we also describe some biological markers that enable us to investigate the clinical management of the patients considered for the use of ECP.

BK virus (BKV) nephropathy, collecting duct cell proliferation and malignancy in a renal allograft: Case history and review of the literature

Several lines of evidence indicate that viral infections, particularly with cytomegalovirus (CMV), play a role in the pathogenesis of solid organ allograft rejection. A diagnostic feature of acute rejection is infiltration of allograft parenchyma by lymphocytes, a process by induction of adhesion molecules on vascular endothelial cells and their ligands on leucocytes. Data derived from biopsies of CMV-infected transplant recipients, as well as from experimental models of transplantation, indicate that CMB infection can result in an upregulation of such adhesion molecules, thereby facilitating the inflammatory process. Infection with CMV is also associated with an increased expression of MHC class II on multiple cell types. Since recognition of nonself MHC antigens is the major determination of allograft rejection, an upregulation of these molecules could contribute to graft failure. Infection with CMV has als been implicated in the induction of smooth muscle proliferation and intimal thickening, both hallmarks of transplant atherosclerosis, which constitutes the most common cause of heart allograft failure. CMV can be classified into four, possibly five, different genotypes based on restriction length polymorphism of the envelope glycoprotein B gene; these genotypes may exhibit varied geographic and demographic frequency distributions and also differ in their pathogenicity and cell tropism. Further studies are needed to evaluate these issues and in particular the genetic contribution of the recipient to CMV modulation of rejection.

NK cells after transplantation: friend or foe

Natural killer (NK) cells are effector cells of the innate immune system that can lyse target cells without prior sensitization and have an important role in host defense to pathogens and transformed cells. A balance between negative and positive signals transmitted via germ line-encoded inhibitory and activating receptors controls the function of NK cells. Although the concept of "missing-self" would suggest that NK cells could target foreign allografts, the prevailing dogma has been that NK cells are not active participants in the mechanisms that culminate in the rejection of solid organ allografts. Recent studies, however, challenge this conclusion and instead implicate NK cells in contributing to both graft rejection and tolerance to an allograft. In this review, we highlight recent studies with the goal of understanding the complex NK cell interactions that impact alloimmunity.

Perforin and human diseases.

Natural killer (NK) cells and cytotoxic T lymphocytes (CTL) use a highly toxic pore-forming protein perforin (PFN) to destroy cells infected with intracellular pathogens and cells with pre-cancerous transformations. However, mutations of PFN and defects in its expression can cause an abnormal function of the immune system and difficulties in elimination of altered cells. As discussed in this chapter, deficiency of PFN due to the mutations of its gene, PFN1, can be associated with malignancies and severe immune disorders such as familial hemophagocytic lymphohistiocytosis (FHL) and macrophage activation syndrome. On the other hand, overactivity of PFN can turn the immune system against autologous cells resulting in other diseases such as systemic lupus erythematosus, polymyositis, rheumatoid arthritis and cutaneous inflammation. PFN also has a crucial role in the cellular rejection of solid organ allografts and destruction of pancreatic β-cells resulting in type 1 diabetes. These facts highlight the importance of understanding the biochemical characteristics of PFN.

Roles of Toll-Like Receptors in Allogeneic Islet Transplantation

Toll-like receptors (TLRs) are involved in the rejection of solid organ allografts. However, the roles of TLRs in islets are still controversial. We investigated the roles of TLRs in donor islets together with those in recipients in allogeneic islet transplantation. To assess the roles of TLRs in either donor islets or recipients, allogeneic islet transplantation was performed using myeloid differentiation factor 88 (MyD88)-knockout (KO), TLR4-KO, or Toll/interleukin-1 receptor domain-containing adaptor-inducing interferon-β (TRIF)-KO mice. Both polyriboinosinic polyribocytidylic acid and lipopolysaccharide (LPS) stimulation induced the mRNA expression of regulated and normal T cell expressed and secreted, interferon-γ-inducible protein-10, monocyte chemotactic protein-1, interleukin-8, and inducible nitric oxide synthase in murine islets, whereas the induction was attenuated in TRIF-KO, interferon-β promoter stimulator-1-KO, and TLR4-KO mice. When islets from MyD88-KO, TLR4-KO, or TRIF-KO C57BL/6 mice were transplanted to BALB/c recipients, graft survival was not better than that of wild-type (WT) islets. However, the survival of the MyD88-KO islet allograft was significantly prolonged when combined with anti-CD40L. In parallel, LPS stimulation in donor islets interfered with anti-CD40L blockade-mediated long-term survival of islet allografts in TLR4-KO recipients. LPS stimulation increased the perigraft infiltration of both T cells and macrophages. Then again, when islets from WT BALB/c mice were transplanted to MyD88-KO, TRIF-KO, or WT C57BL/6 mice, there was no difference in graft survival, although some of the MyD88-KO recipients obtained long-term graft survival. However, anti-CD40L prolonged graft survival significantly in MyD88-KO recipients. The absence of MyD88 in either donors or recipients decreased the perigraft infiltration of inflammatory cells when combined with anti-CD40L. TLRs in both donor islets and recipients are involved in islet allograft rejection.

Cell-Based Therapies in the Prevention of Solid Organ Transplant Rejection

Problem statement: Organ transplantation is a life-saving and increasi ngly common procedure, as it often serves as the only treatment available for end-stage organ disease. Although th e constant development of new and more effective immunosuppressive drugs has revolutionized the prevention and treatment of acute graft rejection, these drugs have significant toxicity, greatly incr ease patient susceptibility to neoplasms and infection a nd exert little impact on chronic rejection. Approach: The literature was reviewed to illuminate the mech anisms by which the anti-donor immune response is initiated and how cellular therapies im pact this response. Results: Data show that Donor Specific Transfusion, Apoptotic Cell therapies and Dendritic Cell therapies all function as a source o f alloantigen to suppress the anti-donor T cell respo nse. Conclusion: Cellular therapies hold promise in the prevention of solid organ allograft rejection, but require optimization and study in large animal mode ls before clinical implementation.

Janus Kinase-2 Inhibition Induces Durable Tolerance to Alloantigen by Dendritic Cell-Stimulated Human T Cells, Yet Preserves Immunity to Recall Antigen

Abstract 1903Janus kinase 2 (JAK2) conveys signals from receptor binding by several inflammatory cytokines, including IL-6, IL-12, and IL-23, via phosphorylation of signal transducer and activator of transcription 3 (STAT3). These cytokines promote the development and expansion of T helper 1 (Th1) cells, which use IL-12, and Th17 cells, which use IL-6 and IL-23. Th1 and Th17 cells can in turn induce alloreactive end organ damage in GvHD. JAK2 is therefore a principle gatekeeper of alloreactivity and inflammation, and it represents an attractive target to control GvHD. We demonstrate that TG101348, a synthetic small molecule, highly selective JAK2 inhibitor, when present during initial and secondary encounters between human T cells and allogeneic monocyte-derived dendritic cells (moDCs), induces durable, profound, and specific T cell tolerance on reexposure to the same alloantigens. TG101348 ablates IL-6/JAK2-mediated phosphorylation of STAT3 in T cells (24% v 0%, P<0.05, n=5) but has no off-target effects on IL-2 or IL-15/JAK3/pSTAT5-dependent signaling, which would interfere with Tregs and other effector T cell responses. JAK2 inhibition preserves Treg numbers and thereby enhances the ratio of CD4+ Tregs to CD8+CD25+ effector T cells in favor of Tregs (Treg:Effector 1:2 vs 1:1, P<0.05, n=5). JAK2 inhibition also reduces the production of IL-6 (255 vs 157 pg/ml, P<0.05, n=5) and TNF-alpha (50 vs 15 pg/ml, P<0.05, n=4) in allogeneic MLRs, impairing the activation of central and effector memory T cells as well as the expansion of responder Th1 (24% vs 14%, P<0.05, n=4) and Th17 (2% vs 1%, P<0.05, n=4). T cells, stimulated by moDCs in the presence of TG101348, demonstrate a marked reduction in proliferative capacity upon reexposure to fresh allogeneic moDCs from the original stimulating party, even in the absence of the JAK2 inhibitor during the secondary MLR (P<0.05, n=4). Responses to stimulation de novo by influenza matrix peptide (fluMP), a pathogenic nominal antigen, remain intact (P=NS, n=4). Conversely, TG101348 also inhibits secondary MLR responses by T cells, which have already been sensitized to alloantigens in a primary MLR without the JAK2 inhibitor (P<0.05, n=4). The durable tolerance against full HLA disparities in each of these settings provides preclinical data in vitro that JAK2 represents a relevant biologic target for preventing or treating graft-versus-host disease (GvHD), or even solid organ allograft rejection, without broader immune impairment. Disclosures:No relevant conflicts of interest to declare.