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Introduction: Thymus resident B cells were described more than 40 years ago. In early human life, these cells are found predominantly in the medulla and overwhelmingly display an unswitched IgM+ phenotype. The reactivity of thymic IgM B cells, however, is still unclear. Methods: Here, we generated 120 IgM-producing B cell clones from 3 separate thymus specimens obtained from infant, adolescent, and adult donors. Using flow cytometry and a unique high-dimensional ELISA platform, we investigated the clones' reactivity to apoptotic cells as well as to common chemical adducts exposed on modified amino acids and other macromolecules. Results: Regardless of the age, approximately 30-40% of thymic IgM B cells reacted to apoptotic cells. Further, 30-40% displayed reactivity to at least one adduct, including malondialdehyde, Homocysteine, and NEDD 8. Four distinct reactivity patterns were identified through this profiling. Notably, a significant association was observed between reactivity to apoptotic cells, and to one or more adducts, suggesting that the same determinants were recognized in both assays. Additionally, thymic IgM B cells reactive to adducts were more likely to recognize intra-nuclear or intra-cytoplasmic structures in Hep-2 cells as revealed by immunofluorescence staining. Conclusion/Discussion: Collectively, our findings suggest that thymic IgM B cells actively uptake apoptotic bodies and cellular debris in the medulla by binding specific chemical adducts. This mechanism could underpin their antigenpresenting function and further support their role in T-cell negative selection. [ABSTRACT FROM AUTHOR]

Background. The relationship between circulating effector memory T and B cells long after transplantation and their susceptibility to immunosuppression are unknown. To investigate the impact of antirejection therapy on T cell-B cell coordinated immune responses, we assessed IFN-γ-producing memory cells and natural antibodies (nAbs) that potentially bind to autoantigens on the graft. Methods. Plasma levels of IgG nAbs to malondialdehyde (MDA) were measured in 145 kidney transplant recipients at 5–7 years after transplantation. In 54 of these patients, the number of donor-reactive IFN-γ-producing cells was determined. 35/145 patients experienced rejection, 18 of which occurred within 1 year after transplantation. Results. The number of donor-reactive IFN-γ-producing cells and the levels of nAbs were comparable between rejectors and nonrejectors. The nAbs levels were positively correlated with the number of donor-reactive IFN-γ-producing cells (rs = 0.39, p = 0.004). The positive correlation was only observed in rejectors (rs = 0.53, p = 0.003 ; nonrejectors: rs = 0.24, p = 0.23). Moreover, we observed that intravenous immune globulin treatment affected the level of nAbs and this effect was found in patients who experienced a late ca-ABMR compared to nonrejectors (p = 0.008). Conclusion. The positive correlation found between alloreactive T cells and nAbs in rejectors suggests an intricate role for both components of the immune response in the rejection process. Treatment with intravenous immune globulin impacted nAbs. [ABSTRACT FROM AUTHOR]

Aims: Allograft rejection following heart transplantation (HTx) is a serious complication even in the era of modern immunosuppressive regimens and causes up to a third of early deaths after HTx. Allograft rejection is mediated by a cascade of immune mechanisms leading to acute cellular rejection (ACR) and/or antibody‐mediated rejection (AMR). The gold standard for monitoring allograft rejection is invasive endomyocardial biopsy that exposes patients to complications. Little is known about the potential of circulating miRNAs as biomarkers to detect cardiac allograft rejection. We here present a systematic analysis of circulating miRNAs as biomarkers and predictors for allograft rejection after HTx using next‐generation small RNA sequencing. Methods and results: We used next‐generation small RNA sequencing to investigate circulating miRNAs among HTx recipients (10 healthy controls, 10 heart failure patients, 13 ACR, and 10 AMR). MiRNA profiling was performed at different time points before, during, and after resolution of the rejection episode. We found three miRNAs with significantly increased serum levels in patients with biopsy‐proven cardiac rejection when compared with patients without rejection: hsa‐miR‐139‐5p, hsa‐miR‐151a‐5p, and hsa‐miR‐186‐5p. We identified miRNAs that may serve as potential predictors for the subsequent development of ACR: hsa‐miR‐29c‐3p (ACR) and hsa‐miR‐486‐5p (AMR). Overall, hsa‐miR‐486‐5p was most strongly associated with acute rejection episodes. Conclusions: Monitoring cardiac allograft rejection using circulating miRNAs might represent an alternative strategy to invasive endomyocardial biopsy. [ABSTRACT FROM AUTHOR]

Touched by a plasmacytoid dendritic cell: Plasmacytoid dendritic cells (pDCs) are crucial to generating strong antiviral immune responses, yet it is unclear how these cells recognize viral infections. Yun et al. tested the cytokine production of human pDCs in response to free cytomegalovirus (CMV) or CMV-infected cells in vitro. They found that pDCs generated robust type I and III interferon responses to CMV-infected cells but not free CMV. These pDC interferon responses were long-lasting and facilitated strong antiviral NK cell responses. Elevated interferons and pDC activation were confirmed in patients with active CMV infections. Similar pDC responses were seen with influenza-infected cells. Together, these data suggest a more general phenomenon whereby antiviral immune responses are supported by the direct recognition of virally infected cells by pDCs. Plasmacytoid dendritic cells (pDCs) can rapidly produce interferons and other soluble factors in response to extracellular viruses or virus mimics such as CpG-containing DNA. pDCs can also recognize live cells infected with certain RNA viruses, but the relevance and functional consequences of such recognition remain unclear. We studied the response of primary DCs to the prototypical persistent DNA virus, human cytomegalovirus (CMV). Human pDCs produced high amounts of type I interferon (IFN-I) when incubated with live CMV-infected fibroblasts but not with free CMV; the response involved integrin-mediated adhesion, transfer of DNA-containing virions to pDCs, and the recognition of DNA through TLR9. Compared with transient polyfunctional responses to CpG or free influenza virus, pDC response to CMV-infected cells was long-lasting, dominated by the production of IFN-I and IFN-III, and lacked diversification into functionally distinct populations. Similarly, pDC activation by influenza-infected lung epithelial cells was highly efficient, prolonged, and dominated by interferon production. Prolonged pDC activation by CMV-infected cells facilitated the activation of natural killer cells critical for CMV control. Last, patients with CMV viremia harbored phenotypically activated pDCs and increased circulating IFN-I and IFN-III. Thus, recognition of live infected cells is a mechanism of virus detection by pDCs that elicits a unique antiviral immune response. [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the activation of autoreactive T and B cells, autoantibody production, and immune complex deposition in various organs. Previous evidence showed abnormal accumulation of B cells in the thymus of lupus-prone mice, but the role of this population in the progression of the disease remains mostly undefined. Here we analyzed the spatial distribution, function, and properties of this thymic B cell population in the BWF1 murine model of SLE. We found that in diseased animals, thymic B cells proliferate, and cluster in structures that resemble ectopic germinal centers. Moreover, we detected antibody-secreting cells in the thymus of diseased-BWF1 mice that produce anti-dsDNA IgG autoantibodies. We also found that thymic B cells from diseased-BWF1 mice induced the differentiation of thymocytes to follicular helper T cells (TFH). These data suggest that the accumulation of B cells in the thymus of BWF1 mice results in the formation of germinal center-like structures and the expansion of a TFH population, which may, in turn, activate and differentiate B cells into autoreactive plasma cells. Therefore, the thymus emerges as an important niche that supports the maintenance of the pathogenic humoral response in the development of murine SLE. [ABSTRACT FROM AUTHOR]

Abstract: Innate B cells and natural antibodies (Nabs) have been extensively studied in normal physiological conditions as well as in several diseases. However, their significance in the context of ABO‐compatible solid organ transplantation is only emerging. This review summarizes recent studies exploring these often neglected innate immune elements in situations related to sensitization and clinical graft rejection. A focus is placed on class‐switched IgG Nabs that develop amidst inflammation, rather than IgM Nabs abundant at the steady state, as new evidence point to their implication in serum reactivity to HLA and kidney graft failure. The involvement of innate B cells in the pathophysiology of CAV is also presented. Lastly, we discuss key questions that need answering to understand whether and how innate B‐cell immunity contributes to the outcome of solid organ transplantation. [ABSTRACT FROM AUTHOR]

Abstract: CD4+CD25+FOXP3+ regulatory T cells (Treg) successfully control graft-versus-host-disease (GVHD) in animal models. In humans, incomplete reconstitution of Treg after allogeneic hematopoietic stem cell transplantation (HSCT) has been associated with chronic GVHD (cGVHD). Recent studies have demonstrated that interleukin (IL)-2 infusions expand Treg in vivo. However, the effectiveness of this therapy depends on the number of cells capable of responding to IL-2. We examined the effect of low-dose IL-2 infusions on Treg populations after HSCT in patients who also received infusions of donor CD4+ lymphocytes. Utilizing FOXP3 as a Treg marker, we found that patients who received CD4+DLI concomitantly with IL-2 had greater expansion of Treg compared to patients who received IL-2 (P = .03) or CD4+DLI alone (P = .001). FOXP3 expression correlated with absolute CD4+CD25+ cell counts. Moreover, expanded CD4+CD25+ T cells displayed normal suppressive function and treatment with CD4+DLI and IL-2 was not associated with GVHD. This study suggests that administration of low-dose IL-2 combined with adoptive CD4+ cellular therapy may provide a mechanism to expand Treg in vivo. [Copyright &y& Elsevier]

Abstract: Naturally occurring CD4+CD25+ regulatory T cells (Treg) are differentiated T lymphocytes actively involved in the control of peripheral immunity. Over the past few years, a number of animal studies have demonstrated the critical role of these cells in the outcome of allogeneic hematopoietic stem cell transplantation (HCT). In these models, Treg can exert a potent suppressive effect on immune effector cells reactive to host antigens and prevent graft versus host disease (GVHD) while preserving the graft-versus-leukemia effect (GVL). The present review summarizes current knowledge on the role of Treg populations in humans following allogeneic HCT. Recent investigations focusing on Treg in transplant patients have generated conflicting results mostly due to the use of different parameters to assess Treg. Nonetheless, these studies suggested that an imbalance between Treg and effector cells during immune reconstitution can substantially impair regulatory mechanisms and contributes to the development of GVHD. Building on these studies, a number of therapeutic strategies are being developed to positively modulate Treg pools in vivo and prevent or even correct GVHD. Conversely, clinical interventions can also be envisaged to decrease Treg activity in vivo and enhance the GVL effect. These potential strategies are discussed herein. Coming years will undoubtedly yield additional knowledge on how to use Treg subsets in vivo and successfully control and modulate immune responses in patients post-HCT. [Copyright &y& Elsevier]

Background Antibody-mediated rejection (AMR) has been associated with increased death and cardiac allograft vasculopathy (CAV). Early studies suggested that late AMR was rarely associated with graft dysfunction, whereas recent reports have demonstrated an association with increased mortality. We investigated the timing of AMR and its association with graft dysfunction, death, and CAV. Methods This retrospective cohort study identified all adult orthotopic heart transplant (OHT) recipients ( N = 689) at Columbia University Medical Center from 2004 to 2013. There were 68 primary cases of AMR, which were stratified by early (< 1 year post-OHT) or late (> 1 year post-OHT) AMR. Kaplan-Meier survival analysis and modeling was performed with multivariable logistic regression and Cox proportional hazards regression. Results From January 1, 2004, through October 1, 2015, early AMR (median 23 days post-OHT) occurred in 43 patients and late AMR (median 1,084 days post-OHT) occurred in 25. Graft dysfunction was less common with early compared with late AMR (25.6% vs 56%, p = 0.01). Patients with late AMR had decreased post-AMR survival compared with early AMR (1 year: 80% vs 93%, 5 years: 51% vs 73%, p < 0.05). When stratified by graft dysfunction, only those with late AMR and graft dysfunction had worse survival (30 days: 79%, 1 year: 64%, 5 years: 36%; p < 0.006). The association remained irrespective of age, sex, donor-specific antibodies, left ventricular assist device use, reason for OHT, and recovery of graft function. Similarly, those with late AMR and graft dysfunction had accelerated development of de novo CAV (50% at 1 year; hazard ratio, 5.42; p = 0.009), whereas all other groups were all similar to the general transplant population. Conclusions Late AMR is frequently associated with graft dysfunction. When graft dysfunction is present in late AMR, there is an early and sustained increased risk of death and rapid development of de novo CAV despite aggressive treatment. [ABSTRACT FROM AUTHOR]

Antibody mediated rejection (AMR) is an increasingly studied cause of graft failure after heart transplantation. AMR diagnosis previously required the detection of circulating donor specific antibodies (DSA); however, the most recent criteria only require pathological findings. This classification defined a subset of patients with AMR, yet without known antibodies. Here, we sought to evaluate differences in the transcriptome profile associated with different types of AMR. RNA sequencing was used on endomyocardial biopsies to analyze and compare transcriptomic profiles associated with different subtypes of AMR defined by immunopathological and histopathological findings, as well as the presence or absence of DSA. Gene expression profiles were characterized for each diagnostic group. The most divergent gene expression profiles were observed between patients with or without DSA. AMR subtypes associated with DSA showed expression of signature genes involved in monocyte activation and response to interferon. There was also substantial difference between the transcriptomic profiles of AMR defined by histopathological and immunopathological findings, the latter being associated with expression of mucin genes. In contrast, there was no differential RNA expression between patients with pAMR1i without DSA and those without AMR. Likewise, no differential expression was observed between patients with pAMR1h with DSA and pAMR2. Overall, our studies reveal different expression profiles in endomyocardial biopsies in relation to some key criteria used to diagnose AMR. These findings support the view that the diagnosis of AMR encompasses several phenotypes that may rely on distinct mechanisms of injury. [ABSTRACT FROM AUTHOR]

Primary graft dysfunction (PGD) is the leading cause of early mortality after heart transplant. Pre-transplant predictors of PGD remain elusive and its etiology remains unclear. Microvesicles were isolated from 88 pre-transplant serum samples and underwent proteomic evaluation using TMT mass spectrometry. Monte Carlo cross validation (MCCV) was used to predict the occurrence of severe PGD after transplant using recipient pre-transplant clinical characteristics and serum microvesicle proteomic data. Putative biological functions and pathways were assessed using gene set enrichment analysis (GSEA) within the MCCV prediction methodology. Using our MCCV prediction methodology, decreased levels of plasma kallikrein (KLKB1), a critical regulator of the kinin-kallikrein system, was the most predictive factor identified for PGD (AUROC 0.6444 [0.6293, 0.6655]; odds 0.1959 [0.0592, 0.3663]. Furthermore, a predictive panel combining KLKB1 with inotrope therapy achieved peak performance (AUROC 0.7181 [0.7020, 0.7372]) across and within (AUROCs of 0.66–0.78) each cohort. A classifier utilizing KLKB1 and inotrope therapy outperforms existing composite scores by more than 50 percent. The diagnostic utility of the classifier was validated on 65 consecutive transplant patients, resulting in an AUROC of 0.71 and a negative predictive value of 0.92–0.96. Differential expression analysis revealed a enrichment in inflammatory and immune pathways prior to PGD. Pre-transplant level of KLKB1 is a robust predictor of post-transplant PGD. The combination with pre-transplant inotrope therapy enhances the prediction of PGD compared to pre-transplant KLKB1 levels alone and the resulting classifier equation validates within a prospective validation cohort. Inflammation and immune pathway enrichment characterize the pre-transplant proteomic signature predictive of PGD. [ABSTRACT FROM AUTHOR]

Background Pre-transplant sensitization is a limiting factor in solid-organ transplantation. In heart transplants, ventricular assist device (VAD) implantation has been associated with sensitization to human leukocyte antigens (HLA). The effect of VAD on non-HLA antibodies is unclear. We have previously shown that polyreactive natural antibodies (Nabs) contribute to pre-sensitization in kidney allograft recipients. Here we assessed generation of Nabs after VAD implantation in pre-transplant sera and examined their contribution to cardiac allograft outcome. Methods IgM and IgG Nabs were tested in pre-transplant serum samples collected from 206 orthotopic heart transplant recipients, including 128 patients with VAD (VAD patients) and 78 patients without VAD (no-VAD patients). Nabs were assessed by testing serum reactivity to apoptotic cells by flow cytometry and to the generic oxidized epitope, malondialdehyde, by enzyme-linked immunosorbent assay. Results No difference was observed in serum levels of IgM Nabs between VAD and no-VAD patients. However, serum IgG Nabs levels were significantly increased in VAD compared with no-VAD patients. This increase was likely due to the presence of the VAD, as revealed by lower serum IgG Nabs levels before implantation. Elevated pre-transplant IgG Nabs level was associated with development of primary graft dysfunction (PGD). Conclusions Our study demonstrates that VAD support elicits IgG Nabs reactive to apoptotic cells and oxidized epitopes. These findings further support broad and non-specific B-cell activation by VAD, resulting in IgG sensitization. Moreover, the association of serum IgG Nabs levels with development of PGD suggests a possible role for these antibodies in the inflammatory reaction accompanying this complication. [ABSTRACT FROM AUTHOR]

Background Donor-specific anti-HLA antibodies (DSA) are common after heart transplantation and are associated with rejection, cardiac allograft vasculopathy, and mortality. A noninvasive diagnostic test for pathologic antibody-mediated rejection (pAMR) does not exist. Methods From January 1, 2010, through August 31, 2013, 221 consecutive adult patients underwent heart transplantation and were followed through October 1, 2015. The primary objective was to determine whether the presence of DSA could detect AMR at the time of pathologic diagnosis. Secondary analyses included association of DSA (stratified by major histocompatibility complex class and de novo status) during AMR with new graft dysfunction, graft loss (mortality or retransplantation), and development of cardiac allograft vasculopathy. Results During the study period, 69 patients (31.2%) had DSA (24% had de novo DSA), and there were 74 episodes of pAMR in 38 patients. Sensitivity of DSA at any mean fluorescence intensity to detect concurrent pAMR was only 54.3%. The presence of any DSA during pAMR increased the odds of graft dysfunction (odds ratio = 5.37; 95% confidence interval [CI], 1.34–21.47; p = 0.018), adjusting for age, sex, and timing of AMR. Circulating class II DSA after transplantation increased risk of future pAMR (hazard ratio = 2.97; 95% CI, 1.31–6.73; p = 0.009). Patients who developed de novo class II DSA had 151% increased risk of graft loss (contingent on 30-day survival) compared with patients who did not have DSA (95% CI, 1.11–5.69; p = 0.027). Conclusions DSA were inadequate to diagnose pAMR. Class II DSA provided prognostic information regarding future pAMR, graft dysfunction with pAMR, and graft loss. [ABSTRACT FROM AUTHOR]

Background and Objectives: Chronic rejection leads to kidney allograft failure and develops in many kidney transplant recipients. One cause of chronic rejection, chronic antibody mediated rejection (CAMR), is attributed to alloantibodies. Maintenance immunosuppression including prednisone, mycophenolate mofetil (MMF) and calcineurin inhibitors may limit alloantibody production in some patients, but many maintain or develop alloantibody production, leading to CAMR. Therefore, no efficacious therapy to treat CAMR is presently available to prevent the progression of CAMR to kidney allograft failure. Design, Setting, Participants, and Measurements: We performed a retrospective review of 31 subjects with CAMR, of which 14 received Rituximab and 17 subjects did not. Response to Rituximab was defined as decline or stabilization of serum creatinine for at least one year. Data reviewed included demographic, clinical, allograft, post-transplant, and pathological variables. Pathological variables in the diagnostic allograft biopsy were scored according to Banff criteria. Results: The median survival time (MST) for allografts in the control group was 439 days, and for the Rituximab treated group was 685 days. The Rituximab group was dichotomous with 8 subjects showing a medial survival time of 1180 days, and 6 subjects having a median survival time of 431 days. The MST for the responders was statistically significant from the non-responders and controls. No pathological parameter distinguished any subset of subjects. Conclusions: These data show that Rituximab followed by standard maintenance immunosuppression shows a therapeutic effect in the treatment of CAMR, which is confined to a subset of treated subjects, not identifiable a priori. [ABSTRACT FROM AUTHOR]

It is well established that HLA antibodies play an important role in antibody-mediated rejection (AMR) and there is a growing body of literature demonstrating that non-HLA antibodies may also contribute to AMR. To study the source of non-donor specific HLA antibodies and self-reactive antibodies in patients with kidney graft loss, Porcheray et al. (Am J Transplant 2012; 12: 2088–2097) expanded immortalized polyreactive B-cell clones from a patient with kidney graft rejection and showed that some of these natural antibodies are cross-reactive to HLA and self-antigens. Gao et al. (Am J Transplant 2014; 14:1581–1591) also showed that the presence of IgG natural antibodies, with activity to apoptotic cells, correlates with late kidney allograft loss even after excluding patients with high reactivity to HLA. We have further characterized the HLA reactivity of 4 polyreactive B-cell clones isolated by Porcheray, Gao et al. using solid phase assays (Immucor, Inc.). We report here that while these clones do not react with HLA on the Immucor single antigen beads, they react to other HLA products, with some variation between different solid phase assays. Their reactivity appears to be enhanced upon denaturation, as judged by the monoclonal antibody 210.4, which reacts with denatured antigen, suggesting that they primarily react to cryptic epitopes. B. Ray: Employee; Company/Organization; Immucor. [ABSTRACT FROM AUTHOR]