Germinal center humoral autoimmunity independently mediates progression of allograft vasculopathy.

The development of humoral autoimmunity following organ transplantation is increasingly recognised, but of uncertain significance. We examine whether autoimmunity contributes independently to allograft rejection. In a MHC class II-mismatched murine model of chronic humoral rejection, we report that effector antinuclear autoantibody responses were initiated upon graft-versus-host allorecognition of recipient B cells by donor CD4 T-cells transferred within heart allografts. Consequently, grafts were rejected more rapidly, and with markedly augmented autoantibody responses, upon transplantation of hearts from donors previously primed against recipient. Nevertheless, rejection was dependent upon recipient T follicular helper (TFH) cell differentiation and provision of cognate (peptide-specific) help for maintenance as long-lived GC reactions, which diversified to encompass responses against vimentin autoantigen. Heart grafts transplanted into stable donor/recipient mixed haematopoietic chimeras, or from parental strain donors into F1 recipients (neither of which can trigger host adaptive alloimmune responses), nevertheless provoked GC autoimmunity and were rejected chronically, with rejection similarly dependent upon host TFH cell differentiation. Thus, autoantibody responses contribute independently of host adaptive alloimmunity to graft rejection, but require host TFH cell differentiation to maintain long-lived GC responses. The demonstration that one population of helper CD4 T-cells initiates humoral autoimmunity, but that a second population of TFH cells is required for its maintenance as a GC reaction, has important implications for how autoimmune-related phenomena manifest.

Data regarding transplant induced germinal center humoral autoimmunity.

This data is related to the research article entitled "Germinal center humoral autoimmunity independently mediates progression of allograft vasculopathy" (Harper et al., 2016) [2]. The data presented here focuses on the humoral autoimmune response triggered by transferred allogeneic CD4 T cells and includes details on: (a) the recipient splenic germinal center (GC) response; (b) augmentation of humoral autoimmunity and accelerated heart allograft rejection following transplantation from donors primed against recipient; (c) flow cytometric analysis of donor and recipient CD4 T cells for signature markers of T follicular helper cell differentiation; (d) in vitro donor endothelial cell migration in response to column purified autoantibody from recipient sera; (e) analysis of development of humoral responses in recipients following adoptive transfer of donor CD4 T cells and; (f) the development of humoral autoimmunity in mixed haematopoietic chimeric mice.

Germinal Center Alloantibody Responses Mediate Progression of Chronic Allograft Injury.

Different profiles of alloantibody responses are observed in the clinic, with those that persist, often despite targeted treatment, associated with poorer long-term transplant outcomes. Although such responses would suggest an underlying germinal center (GC) response, the relationship to cellular events within the allospecific B cell population is unclear. Here we examine the contribution of germinal center (GC) humoral alloimmunity to chronic antibody mediated rejection (AMR). A murine model of chronic AMR was developed in which T cell deficient (Tcrbd-/-) C57BL/6 recipients were challenged with MHC-mismatched BALB/c heart allografts and T cell help provided by reconstituting with 103 "TCR75" CD4 T cells that recognize self-restricted allopeptide derived from the H-2Kd MHC class I alloantigen. Reconstituted recipients developed Ig-switched anti-Kd alloantibody responses that were slow to develop, but long-lived, with confocal immunofluorescence and flow cytometric characterization of responding H-2Kd-allospecific B cells confirming persistent splenic GC activity. This was associated with T follicular helper (TFH) cell differentiation of the transferred TCR75 CD4 T cells. Heart grafts developed progressive allograft vasculopathy, and were rejected chronically (MST 50 days), with explanted allografts displaying features of humoral vascular rejection. Critically, late alloantibody responses were abolished, and heart grafts survived indefinitely, in recipients reconstituted with Sh2d1a-/- TCR75 CD4 T cells that were genetically incapable of providing TFH cell function. The GC response was associated with affinity maturation of the anti-Kd alloantibody response, and its contribution to progression of allograft vasculopathy related principally to secretion of alloantibody, rather than to enhanced alloreactive T cell priming, because grafts survived long-term when B cells could present alloantigen, but not secrete alloantibody. Similarly, sera sampled at late time points from chronically-rejecting recipients induced more vigorous donor endothelial responses in vitro than sera sampled earlier after transplantation. In summary, our results suggest that chronic AMR and progression of allograft vasculopathy is dependent upon allospecific GC activity, with critical help provided by TFH cells. Clinical strategies that target the TFH cell subset may hold therapeutic potential. This work is composed of two parts, of which this is Part II. Please read also Part I: Alsughayyir et al., 2019.

Relative Frequencies of Alloantigen-Specific Helper CD4 T Cells and B Cells Determine Mode of Antibody-Mediated Allograft Rejection.

Humoral alloimmunity is now recognized as a major determinant of transplant outcome. MHC glycoprotein is considered a typical T-dependent antigen, but the nature of the T cell alloresponse that underpins alloantibody generation remains poorly understood. Here, we examine how the relative frequencies of alloantigen-specific B cells and helper CD4 T cells influence the humoral alloimmune response and how this relates to antibody-mediated rejection (AMR). An MHC-mismatched murine model of cardiac AMR was developed, in which T cell help for alloantibody responses in T cell deficient (Tcrbd-/-) C57BL/6 recipients against donor H-2Kd MHC class I alloantigen was provided by adoptively transferred "TCR75" CD4 T cells that recognize processed H-2Kd allopeptide via the indirect-pathway. Transfer of large numbers (5 × 105) of TCR75 CD4 T cells was associated with rapid development of robust class-switched anti-H-2Kd humoral alloimmunity and BALB/c heart grafts were rejected promptly (MST 9 days). Grafts were not rejected in T and B cell deficient Rag2-/- recipients that were reconstituted with TCR75 CD4 T cells or in control (non-reconstituted) Tcrbd-/- recipients, suggesting that the transferred TCR75 CD4 T cells were mediating graft rejection principally by providing help for effector alloantibody responses. In support, acutely rejecting BALB/c heart grafts exhibited hallmark features of acute AMR, with widespread complement C4d deposition, whereas cellular rejection was not evident. In addition, passive transfer of immune serum from rejecting mice to Rag2-/- recipients resulted in eventual BALB/c heart allograft rejection (MST 20 days). Despite being long-lived, the alloantibody responses observed at rejection of the BALB/c heart grafts were predominantly generated by extrafollicular foci: splenic germinal center (GC) activity had not yet developed; IgG secreting cells were confined to the splenic red pulp and bridging channels; and, most convincingly, rapid graft rejection still occurred when recipients were reconstituted with similar numbers of Sh2d1a-/- TCR75 CD4 T cells that are genetically incapable of providing T follicular helper cell function for generating GC alloimmunity. Similarly, alloantibody responses generated in Tcrbd-/- recipients reconstituted with smaller number of wild-type TCR75 CD4 T cells (103), although long-lasting, did not have a discernible extrafollicular component, and grafts were rejected much more slowly (MST 50 days). By modeling antibody responses to Hen Egg Lysozyme protein, we confirm that a high ratio of antigen-specific helper T cells to B cells favors development of the extrafollicular response, whereas GC activity is favored by a relatively high ratio of B cells. In summary, a relative abundance of helper CD4 T cells favors development of strong extrafollicular alloantibody responses that mediate acute humoral rejection, without requirement for GC activity. This work is composed of two parts, of which this is Part I. Please read also Part II: Chhabra et al., 2019.

Identifying inflamed carotid plaques using in vivo USPIO-enhanced MR imaging to label plaque macrophages.

BACKGROUND: Inflammation within atherosclerotic lesions contributes to plaque instability and vulnerability to rupture. We set out to evaluate the use of a macrophage labeling agent to identify carotid plaque inflammation by in vivo magnetic resonance imaging (MRI). METHODS AND RESULTS: Thirty patients with symptomatic severe carotid stenosis scheduled for carotid endarterectomy underwent multi-sequence MRI of the carotid bifurcation before and after injection of ultrasmall superparamagnetic particles of iron oxide (USPIOs). USPIO particles accumulated in macrophages in 24 of 30 plaques (80%). Areas of signal intensity reduction, corresponding to USPIO/macrophage-positive histological sections, were visualized in 24 of 27 (89%) patients, with an average reduction in signal intensity induced by the USPIO particles of 24% (range, 3.1% to 60.8%). CONCLUSIONS: USPIO-enhanced MRI can identify plaque inflammation in vivo by accumulation of USPIO within macrophages in carotid plaques.

In vivo detection of macrophages in human carotid atheroma: temporal dependence of ultrasmall superparamagnetic particles of iron oxide-enhanced MRI.

UNLABELLED: Background- It has been suggested that inflammatory cells within vulnerable plaques may be visualized by superparamagnetic iron oxide particle-enhanced MRI. The purpose of this study was to determine the time course for macrophage visualization with in vivo contrast-enhanced MRI using an ultrasmall superparamagnetic iron oxide (USPIO) agent in symptomatic human carotid disease. METHODS: Eight patients scheduled for carotid endarterectomy underwent multisequence MRI of the carotid bifurcation before and 24, 36, 48, and 72 hours after Sinerem (2.6 mg/kg) infusion. RESULTS: USPIO particles accumulated in macrophages in 7 of 8 patients given Sinerem. Areas of signal intensity reduction, corresponding to USPIO/macrophage-positive histological sections, were visualized in all 7 of these patients, optimally between 24 and 36 hours, decreasing after 48 hours, but still evident up to 96 hours after infusion. CONCLUSIONS: USPIO-enhanced MRI of carotid atheroma can be used to identify macrophages in vivo. The temporal change in the resultant signal intensity reduction on MRI suggests an optimal time window for the detection of macrophages on postinfusion imaging.

ACE2 gene expression is up-regulated in the human failing heart.

BACKGROUND: ACE2 is a novel homologue of angiotensin converting enzyme (ACE). ACE2 is highly expressed in human heart and animal data suggest that ACE2 is an essential regulator of cardiac function in vivo. Since overactivity of the renin-angiotensin system contributes to the progression of heart failure, this investigation assessed changes in gene expression of ACE2, ACE, AT1 receptor and renin in the human failing heart. METHODS: The sensitive technique of quantitative reverse transcriptase polymerase chain reaction was used to determine the level of mRNA expression of ACE and ACE2 in human ventricular myocardium from donors with non-diseased hearts (n = 9), idiopathic dilated cardiomyopathy (IDC, n = 11) and ischemic cardiomyopathy (ICM, n = 12). Following logarithmic transformation of the data, a one-way analysis of variance was performed for each target gene followed by a Dunnett's test to compare the two disease groups IDC and ICM versus control. RESULTS: As anticipated, ACE mRNA was found to be significantly increased in the failing heart with a 3.1 and 2.4-fold up-regulation found in IDC and ICM relative to non-diseased myocardium. Expression of ACE2 mRNA was also significantly up-regulated in IDC (2.4-fold increase) and ICM (1.8-fold increase) versus non-diseased myocardium. No change in angiotensin AT1 receptor mRNA expression was found in failing myocardium and renin mRNA was not detected. CONCLUSIONS: These data suggest that ACE2 is up-regulated in human IDC and ICM and are consistent with the hypothesis that differential regulation of this enzyme may have important functional consequences in heart failure. This strengthens the hypothesis that ACE2 may be a relevant target for the treatment of heart failure and will hopefully spur further studies to clarify the functional effects in human myocardium of ACE2 derived peptides.

Use of 18FDG-pet to discriminate between infection and rejection in lung transplant recipients.

18F-fluorodeoxyglucose (18FDG) uptake measured by positron emission tomography (PET) allows assessment of neutrophil activity in vivo and is increased in patients with airway inflammation or infection. Because infection but not rejection elicits a highly neutrophilic response, we assessed the ability of this non-invasive technique to differentiate these two events in lung transplant recipients. 18FDG-PET was measured in 15 patients classified by clinical, radiologic, and pathologic criteria. 18FDG-PET signal was increased with proven infection but not when no infection was identified (mean [standard error of mean]: 8.00 [1.81] and 3.16 [0.61], respectively [P = 0.021]. Rejection alone did not increase the signal. These data confirm that neutrophil activation is not a feature of acute rejection and indicate that a high 18FDG-PET signal is indicative of infection but not rejection in lung transplant recipients. This non-invasive and repeatable test could reduce the number of transbronchial biopsies required during episodes of breathlessness after lung transplantation.

Posttransplant lymphoproliferative disorder associated with primate gamma-herpesvirus in cynomolgus monkeys used in pig-to-primate renal xenotransplantation and primate renal allotransplantation.

BACKGROUND: A series of immunosuppressed cynomolgus monkeys were used in porcine-to-primate and primate-to-primate renal transplantation. In a number of animals nodal and extranodal lymphomas as well as areas of lymphoid hyperplasia in multiple organs (posttransplant lymphoproliferative disorder, PTLD) were recorded. METHODS: PTLD was characterized with respect to manifestation sites, histopathology, immunophenotype, and association with primate Epstein Barr-like Virus by in situ hybridization and quantitative polymerase chain reaction. RESULTS: PTLD was observed in 10 of 245 xenotransplanted and 9 of 231 allotransplanted monkeys; its detection in xenotransplanted animals was significantly earlier after transplantation than that in allo-transplanted animals (median, 40 and 104 days, respectively; P<0.001). In the xenotransplanted animals, four cases showed a B-cell lymphoma and six cases were nonneoplastic (lymphoid hyperplasia). All nine PTLD cases from allotransplanted animals were diagnosed as lymphoma. There was no clear relationship between the use of a particular drug or drug combination in maintenance immunosuppression and the occurrence of PTLD. Fourteen of 19 animals (six of the cases from xenotransplants, eight from the allotransplant series) were positive by in situ hybridization with oligonucleotide probes detecting primate gamma-herpesvirus. CONCLUSION: These data indicate that PTLD in the xeno- and allotransplanted cynomolgus monkeys are associated with primate gamma-herpesvirus-induced B-cell proliferation.

Histopathology of cardiac xenograft rejection in the pig-to-baboon model.

BACKGROUND: The use of pig organs transgenic for human decay accelerating factor (hDAF) has largely overcome the problems of hyperacute rejection. With improved immunosuppressive protocols, life supporting grafts are showing greater survival times bringing the possibility of clinical xenotransplantation closer. Examination of the histopathology of the rejection process provides insight into the underlying mechanism and may suggest ways in which new immunosuppressive strategies should be directed. METHODS: 44 baboons (Papio anubis) underwent heart transplants of which 39 were from transgenic donors. The transplanted organs were examined histologically and stained for evidence of immunoglobulin and complement deposition as well as cellular infiltrates. RESULTS: In the transgenic animals survival times were 2 to 99 days (mean 23.5) and the heterotopic group and 1 to 39 days (mean 11.7) in the orthotopic group. There were 3 cases of hyperacute rejection between the 2 groups. Rejected organs showed areas of old and recent myocardial infarction associated with vascular thrombosis. There was widespread deposition within vessels of immunoglobulins IgM and IgG together with complement fractions C3 and C5b to 9 in those organs that were rejected. The amount of complement positive in the longer surviving organs was less than those rejecting early. Cellular infiltate was predominantly macrophage with some later appearing T or natural killer cells. CONCLUSIONS: The histopathological changes support the importance of immunoglobulin and complement in delayed xenograft or acute vascular rejection. With time there is an increase in cellular infiltrate predominantly macrophages and these findings suggest an increasingly important role for the cells and the rejection process. The presence of areas of infarction and underlying vascular thrombosis is in keeping with endothelial activation and the establishment of procoagulant phenotype which may be due to immunoglobulin, complement, secreted cytokines and direct cellular effects.

Neointimal smooth muscle cells in human cardiac allograft coronary artery vasculopathy are of donor origin.

BACKGROUND: Transplant coronary artery vasculopathy (CAV) is a fibro-proliferative process that leads to lumen occlusion and cardiac failure. Current theories suggest that the process evolves over time in response to inflammation and proliferation of donor derived medial smooth muscle cells (SMC). Animal models of cardiac transplantation have suggested that the neointima is formed by recipient derived circulating progenitor cells. The aim of this investigation is to determine the origin of the neointimal SMC within epicardial coronary arteries from human cardiac allografts by using sex mis-matched recipients and donors, and a Y specific chromosome probe. METHODS: Coronary arteries from 14 patients previously assessed histologically to have CAV were analyzed-eight male recipients of female donor organs, 2 female-to-female, and 4 male-to-male transplants. A double immunocytochemistry and in-situ hybridization technique using a Y chromosome DNA probe and either antibodies to smooth muscle actin or Ham-56 a macrophage marker were employed. RESULTS: No Y chromosome bodies could be identified in the female-to-female allografts. In the 4 male donor and male recipient cases, cells positive for the Y chromosome probe were identified. In sex mis-matched transplants, female to male, inflammatory cells marked with Ham-56 were also positive for Y chromosome probe. Flattened cells positive for Y chromosome were observed just beneath the endothelial surface. When double stained, these were identified as infiltrating macrophages. No double staining smooth muscle cells and Y chromosome positive cells could be identified within the neointima. CONCLUSIONS: This study confirms the source of SMC of the neointima of CAV lesions from epicardial coronary arteries to be of donor origin. In contrast to animal models, circulating progenitor cells do not appear to play a role within the neointima of human transplant CAV.

Deposition of C4d and C3d in cardiac transplants: a factor in the development of coronary artery vasculopathy.

BACKGROUND: Coronary artery vasculopathy (CAV) is the major life-limiting factor in cardiac transplantation, after 1 year. Antibody-mediated rejection (AMR) has been associated with development of both acute and chronic rejection. We analyzed endomyocardial biopsies for pathologic markers of AMR (C4d and C3d), from the first 2 years post-transplantation, to determine complement deposition in relation to the development of CAV. METHODS: A retrospective, matched-pair study was used. Group 1 subjects (n = 26) were CAV-negative at 8 years, and Group 2 (n = 26) had angiographically detectable CAV at 4 years. Biopsies from six time-points were studied (total = 282). Immunohistochemistry was performed for C4d, C3d and CD68. Biopsies were graded for rejection using ISHLT criteria. RESULTS: Although CAV was not significantly associated with C4d deposition, it was associated with C3d deposition (p = 0.043). Only 4% of C4d and 5% of C3d biopsies were completely negative. Group 1 had 6 AMR-positive biopsies, with Group 2 having 8. There was no significant relationship between acute cellular rejection or AMR events and CAV. CONCLUSIONS: This study demonstrates that complement deposition is a frequent occurrence in the first 2 years post-transplantation. Although acute rejection is a known risk factor for CAV, in this study the relationship was found not to be significant. No relationship was found with the development of CAV and histologic features of AMR, when assessed by C4d deposition alone. However, an association between C3d deposition and the development of CAV was determined in this study group, suggesting that complement activation may play a role in the pathogenesis of CAV.