Conversion from calcineurin inhibitors to belatacept-based immunosuppressive therapy skews terminal proliferation of non-classical monocytes and lowers lymphocyte counts.

Belatacept, a modified form of CTLA-Ig that blocks CD28-mediated co-stimulation of T cells, is an immune-suppressant that can be used as an alternative to calcineurin inhibitors (CNIs). In kidney transplant recipients, belatacept has been associated with improved renal function and reduced cardiovascular toxicity. Monocytes as well as T-lymphocytes play causal roles in the pathophysiology of atherosclerotic disease. We hypothesized that the beneficial impact of the use of belatacept over CNIs on cardiovascular risk could be partly explained by the impact of belatacept therapy on these circulating leukocytes. Hence, we phenotyped circulating leukocytes in transplanted patients with a stable renal function that were randomized between either continuation of CNI or conversion to belatacept in two international studies in which we participated. In 41 patients, we found that belatacept-treated patients consistently showed lower numbers of B-lymphocytes, T-lymphocytes as well as CD14-negative monocytes (CD14NM), especially in non-diabetic patients. Our observation that this decrease was associated to plasma concentrations of TNFα is consistent with a model where CD14NM-production of TNFα is diminished by belatacept-treatment, due to effects on the antigen-presenting cell compartment.

Presence of CD163+ macrophages in DCD kidneys with high DGF reduces the risk for acute cellular rejection in 6 months after kidney transplantation.

Acute cellular rejection (ACR) occurs in 10% of renal allograft recipients and is characterized by leukocyte infiltration as observed in needle biopsies. ACR onset is subject to several risk factors, including delayed graft function (DGF). As the impact of DGF on the etiology of ACR remains unclear, this study analyzed the association between presence of leukocyte subsets and ACR onset, in DCD kidney biopsies with extensive DGF following transplantation. Immunohistochemical analysis of protocol biopsies taken 10 days after kidney transplantation revealed that patients with high levels of renal CD163+ macrophages have a decreased risk (OR = 0.021, P = 0.008) for ACR in the first 6 months after transplantation. In pre-transplant biopsies of a comparable DCD cohort, with >80% DGF, presence of donor CD163+ macrophages showed no effect on ACR risk. Therefore, leukocyte infiltrate present during the inflammatory response at the time of DGF may contain anti-inflammatory macrophages that exert a protective effect against ACR development.

Is complement the main accomplice in IgA nephropathy? From initial observations to potential complement-targeted therapies.

IgA Nephropathy (IgAN) is the main cause of primary glomerulonephritis, globally. This disease is associated with a wide range of clinical presentations, variable prognosis and a spectrum of histological findings. More than fifty years after its first description, this heterogeneity continues to complicate efforts to understand the pathogenesis. Nevertheless, involvement of the complement system in IgAN was identified early on. Dysfunction of the immunoglobulin A (IgA) system, the principal offender in this disease, including modification of isoforms and glycoforms of IgA1, the nature of immune complexes and autoantibodies to galactose deficient IgA1 might all be responsible for complement activation in IgAN. However, the specific mechanisms engaging complement are still under examination. Research in this domain should allow for identification of patients that may benefit from complement-targeted therapy, in the foreseeable future.

Effect of seminal plasma on dendritic cell differentiation in vitro depends on the serum source in the culture medium.

Dendritic cells (DCs) are key in shaping immune responses and are recruited to the human cervix after coitus by seminal plasma (SP). SP has been shown to skew the differentiation of monocyte-derived DCs towards an anti-inflammatory profile when cultured in medium containing fetal calf serum (FCS). Here, we confirmed that SP skewed DCs cultured in fetal bovine serum (FBS) towards a tolerogenic profile. To create a setting more similar to the in vivo situations in humans, we tested the immune regulatory effect of SP on DCs in cell cultures containing human serum (HS). SP-DCs cultured in HS did show increased CD14 and decreased CD1a, indicating an inhibited maturation phenotype. Gene expression of TGF-ß and IL-10 and IL-10 protein expression were elevated in LPS-activated SP-DCs, whereas IL-12p70 protein levels were decreased compared to LPS-activated control DCs. In contrast to FBS culture conditions, in the presence of HS co-cultures of SP-DCs with allogeneic peripheral blood mononuclear cells (PBMCs) did not result in decreased T cell proliferation and inflammatory cytokine production. Thus, under HS culture conditions SP can skew the differentiation of monocyte-derived DCs phenotypically towards alternatively activated DCs, but this immune regulatory phenotype is functionally less pronounced compared to SP-treated DCs cultured in FBS containing medium. These findings highlight the importance of the source of the serum that is used in SP treated cell cultures in vitro.

Diagnosis and treatment of C3 glomerulopathy in a center of expertise.

C3 glomerulopathy is a rare renal disease that has been distinguished as a renal disease for about 10 years. It is caused by an excessive activation of the alternative complement pathway in the circulation, which leads to deposition of complement factor C3 in glomeruli. It is diagnosed based on clinical presentation, histological patterns in a kidney biopsy and tests of the complement pathways. It can closely resemble immune complexmediated glomerulonephritis and postinfectious glomerulonephritis. Renal failure develops in up to half of all patients within 10 years after presentation. A curative treatment is not available. Treatment relies on renoprotective measures, occasional immunosuppressive medication and experimental novel complement inhibitors. Because the disease is rare, its care and cure are concentrated in centers of expertise. Here we provide an overview of the state-ofthe-art diagnosis and treatment of C3 glomerulopathy in a center of expertise in the Netherlands.

Urinary metabolites predict prolonged duration of delayed graft function in DCD kidney transplant recipients.

Extending kidney donor criteria, including donation after circulatory death (DCD), has resulted in increased rates of delayed graft function (DGF) and primary nonfunction. Here, we used Nuclear Magnetic Resonance (NMR) spectroscopy to analyze the urinary metabolome of DCD transplant recipients at multiple time points (days 10, 42, 180, and 360 after transplantation). The aim was to identify markers that predict prolonged duration of functional DGF (fDGF). Forty-seven metabolites were quantified and their levels were evaluated in relation to fDGF. Samples obtained at day 10 had a different profile than samples obtained at the other time points. Furthermore, at day 10 there was a statistically significant increase in eight metabolites and a decrease in six metabolites in the group with fDGF (N = 53) vis-à-vis the group without fDGF (N = 22). In those with prolonged fDGF (≥21 days) (N = 17) urine lactate was significantly higher and pyroglutamate lower than in those with limited fDGF (<21 days) (N = 36). In order to further distinguish prolonged fDGF from limited fDGF, the ratios of all metabolites were analyzed. In a logistic regression analysis, the sum of branched-chain amino acids (BCAAs) over pyroglutamate and lactate over fumarate, predicted prolonged fDGF with an AUC of 0.85. In conclusion, kidney transplant recipients with fDGF can be identified based on their altered urinary metabolome. Furthermore, two ratios of urinary metabolites, lactate/fumarate and BCAAs/pyroglutamate, adequately predict prolonged duration of fDGF.

IgG is elevated in obese white adipose tissue but does not induce glucose intolerance via Fcγ-receptor or complement.

BACKGROUND/OBJECTIVES: In obesity, B cells accumulate in white adipose tissue (WAT) and produce IgG, which may contribute to the development of glucose intolerance. IgG signals by binding to Fcγ receptors (FcγR) and by activating the complement system. The aim of our study was to investigate whether activation of FcγR and/or complement C3 mediates the development of high-fat diet-induced glucose intolerance. METHODS: We studied mice lacking all four FcγRs (FcγRI/II/III/IV-/-), only the inhibitory FcγRIIb (FcγRIIb-/-), only the central component of the complement system C3 (C3-/-), and mice lacking both FcγRs and C3 (FcγRI/II/III/IV/C3-/-). All mouse models and wild-type controls were fed a high-fat diet (HFD) for 15 weeks to induce obesity. Glucose metabolism was assessed and adipose tissue was characterized for inflammation and adipocyte functionality. RESULTS: In obese WAT of wild-type mice, B cells (+142%, P<0.01) and IgG (+128% P<0.01) were increased compared to lean WAT. Macrophages of FcγRI/II/III/IV-/-mice released lower levels of cytokines compared to wild-type mice upon IgG stimulation. Only C3-/- mice showed reduced HFD-induced weight gain as compared to controls (-18%, P<0.01). Surprisingly, FcγRI/II/III/IV-/- mice had deteriorated glucose tolerance (AUC +125%, P<0.001) despite reduced leukocyte number (-30%, P<0.05) in gonadal WAT (gWAT), whereas glucose tolerance and leukocytes within gWAT in the other models were unaffected compared to controls. Although IgG in gWAT was increased (+44 to +174%, P<0.05) in all mouse models lacking FcγRIIb, only FcγRI/II/III/IV/C3-/- mice exhibited appreciable alterations in immune cells in gWAT, for example, increased macrophages (+36%, P<0.001). CONCLUSIONS: Lack of FcγRs reduces the activity of macrophages upon IgG stimulation, but neither FcγR nor C3 deficiency protects against HFD-induced glucose intolerance or reduces adipose tissue inflammation. This indicates that if obesity-induced IgG contributes to the development of glucose intolerance, this is not mediated by FcγR or complement activation.

Production of complement components by cells of the immune system.

The complement system is an important part of the innate immune defence. It contributes not only to local inflammation, removal and killing of pathogens, but it also assists in shaping of the adaptive immune response. Besides a role in inflammation, complement is also involved in physiological processes such as waste disposal and developmental programmes. The complement system comprises several soluble and membrane-bound proteins. The bulk of the soluble proteins is produced mainly by the liver. While several complement proteins are produced by a wide variety of cell types, other complement proteins are produced by only a few related cell types. As these data suggest that local production by specific cell types may have specific functions, more detailed studies have been employed recently analysing the local and even intracellular role of these complement proteins. Here we review the current knowledge about extrahepatic production and/or secretion of complement components. More specifically, we address what is known about complement synthesis by cells of the human immune system.

Microarray Gene Expression Analysis to Evaluate Cell Type Specific Expression of Targets Relevant for Immunotherapy of Hematological Malignancies.

Cellular immunotherapy has proven to be effective in the treatment of hematological cancers by donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation and more recently by targeted therapy with chimeric antigen or T-cell receptor-engineered T cells. However, dependent on the tissue distribution of the antigens that are targeted, anti-tumor responses can be accompanied by undesired side effects. Therefore, detailed tissue distribution analysis is essential to estimate potential efficacy and toxicity of candidate targets for immunotherapy of hematological malignancies. We performed microarray gene expression analysis of hematological malignancies of different origins, healthy hematopoietic cells and various non-hematopoietic cell types from organs that are often targeted in detrimental immune responses after allogeneic stem cell transplantation leading to graft-versus-host disease. Non-hematopoietic cells were also cultured in the presence of IFN-γ to analyze gene expression under inflammatory circumstances. Gene expression was investigated by Illumina HT12.0 microarrays and quality control analysis was performed to confirm the cell-type origin and exclude contamination of non-hematopoietic cell samples with peripheral blood cells. Microarray data were validated by quantitative RT-PCR showing strong correlations between both platforms. Detailed gene expression profiles were generated for various minor histocompatibility antigens and B-cell surface antigens to illustrate the value of the microarray dataset to estimate efficacy and toxicity of candidate targets for immunotherapy. In conclusion, our microarray database provides a relevant platform to analyze and select candidate antigens with hematopoietic (lineage)-restricted expression as potential targets for immunotherapy of hematological cancers.

Beneficial Immune Effects of Myeloid-Related Proteins in Kidney Transplant Rejection.

Acute rejection is a risk factor for inferior long-term kidney transplant survival. Although T cell immunity is considered the main effector in clinical acute rejection, the role of myeloid cells is less clear. Expression of S100 calcium-binding protein A8 (S100A8) and S100A9 was evaluated in 303 biopsies before and after transplantation from 190 patients. In two independent cohorts of patients with acute rejection (n = 98 and n = 11; mostly cellular rejections), high expression of S100 calcium-binding protein A8 (S100A8) and A9 (S100A9) was related to improved graft outcome. Mechanisms of action of the S100 molecules were investigated. In the graft and peripheral blood cells, S100A8 and S100A9 expression correlated with myeloid-derived suppressor markers. In line with this finding, recombinant S100A8 and S100A9 proteins inhibited maturation and the allogeneic T cell stimulatory capacity of dendritic cells. S100A9 enhanced the production of reactive oxygen species by macrophages, which suppressed T cell activity at low concentrations in the form of hydrogen peroxide. Intragraft S100A8 and S100A9 expression linked to reduced expression of T cell immunity and tissue injury markers and higher expression of immune regulatory molecules. This study sheds new light on the importance of myeloid cell subsets in directing the outcome of T cell-mediated acute rejection.

Functional assessment of rat complement pathway activities and quantification of soluble C5b-9 in an experimental model of renal ischemia/reperfusion injury.

There is a growing interest in the monitoring of complement activation, not only in clinical settings but also in experimental models. However, for rodents only a limited number of tools are available to assess complement activity and activation. Here we describe three ELISAs for the measurement of rat classical (CP), MB-lectin (LP) and alternative (AP) pathway activities in serum and plasma. Moreover, we optimised a soluble C5b-9 (sC5b-9) ELISA for the detection of low level complement activation in rat. We determined the conditions for correct sample handling and showed that the assays had low inter- and intra-assay variation. We applied these assays to monitor complement activation in an experimental rat model of renal ischemia/reperfusion injury. We did not observe major complement consumption following reperfusion in CP or LP, and only minor AP consumption at 24h post reperfusion. However, MBL depletion prior to ischemia/reperfusion using a monoclonal antibody, transiently and specifically inhibited 75% of LP activity and ameliorated the AP consumption at 24h. To further assess complement activation during renal IRI, we monitored serum sC5b-9 and found that it was only significantly increased 72h post-reperfusion, but not when rats were pre-treated with anti-MBL or after sham surgery. In conclusion the described assays enable sensitive, reproducible and comprehensive assessment of complement activation in experimental rat models.

Diagnosis of early pancreas graft failure via antibody-mediated rejection: single-center experience with 256 pancreas transplantations.

Early pancreas graft loss is usually attributed to technical failure while the possibility of antibody-mediated rejection (AMR) is generally overlooked. To investigate the role of AMR in early pancreas graft loss, we retrospectively assessed 256 patients with simultaneous pancreas-kidney transplantation (SPK) between 1985 and 2010 at our institute. We included 33 SPK patients who lost their pancreas graft <1 year after transplantation. AMR was diagnosed based on donor-specific antibodies, C4d and histology in 7 cases, 8 cases were suspicious for AMR and 18 pancreas graft losses were not due to AMR. Acute AMR occurred >1 month after transplantation in 6/7 cases, whereas all other causes typically led to loss <1 month after transplantation. Thrombotic lesions occurred equally among the 33 cases. In 12/18 concurrent kidney specimens, the diagnostic results paralleled those of the pancreas graft. All patients with acute AMR of the pancreas graft lost their renal grafts <1 year after transplantation. In the setting of a thrombotic event, histopathological analysis of early pancreas graft loss is advisable to rule out the possibility of AMR, particularly because a diagnosis of acute AMR has important consequences for renal graft outcomes.

Randomized trial of short-course high-dose erythropoietin in donation after cardiac death kidney transplant recipients.

Eryhropoiesis-stimulating agents have demonstrated tissue-protective effects in experimental models of ischemia-reperfusion injury. PROTECT was a 12-month, randomized, double-blind, placebo-controlled, single center study with high-dose recombinant human erythropoietin-ß (Epoetin) in 92 donation after cardiac death (DCD) kidney transplant recipients. Patients were randomized to receive an intravenous bolus of Epoetin (3.3 × 10(4) international unit (IU); n = 45) or placebo (saline 0.9% solution; n = 47) on 3 consecutive days, starting 3-4 h before the transplantation and 24 h and 48 h after reperfusion. The immunosuppressive regimen included an anti-CD25 antibody, steroids, mycophenolate mofetil and delayed introduction of cyclosporine. Primary end point was a composite of the incidence of primary nonfunction and delayed graft function, either defined by spontaneous functional recovery or need for dialysis in the first week. Secondary objectives included duration of delayed function, renal function and proteinuria up to 1 year and thrombotic adverse events. Results showed no differences in the incidence or duration of delayed graft function and/or primary nonfunction (Epoetin 77.8 vs. placebo 78.7%, p = 1.00). Epoetin treatment significantly increased the risk of thrombotic events at 1 month and 1 year (Epoetin 24.4% vs. placebo 6.4%, p = 0.02).

Mannan-binding lectin mediates renal ischemia/reperfusion injury independent of complement activation.

Ischemia/reperfusion injury (IRI) remains a major problem in renal transplantation. Clinical studies have identified that high serum levels of Mannan-binding lectin (MBL), the initiator of the lectin pathway of complement activation, are associated with inferior renal allograft survival. Using a rat model, we identified an entirely novel role for MBL in mediating renal IRI. Therapeutic inhibition of MBL was protective against kidney dysfunction, tubular damage, neutrophil and macrophage accumulation, and expression of proinflammatory cytokines and chemokines. Following reperfusion, exposure of tubular epithelial cells to circulation-derived MBL resulted in internalization of MBL followed by the rapid induction of tubular epithelial cell death. Interestingly, this MBL-mediated tubular injury was completely independent of complement activation since attenuation of complement activation was not protective against renal IRI. Our identification that MBL-mediated cell death precedes complement activation strongly suggests that exposure of epithelial cells to MBL immediately following reperfusion is the primary culprit of tubular injury. In addition, also human tubular epithelial cells in vitro were shown to be susceptible to the cytotoxic effect of human MBL. Taken together, these data reveal a crucial role for MBL in the early pathophysiology of renal IRI and identify MBL as a novel therapeutic target in kidney transplantation.

Pancreas allograft biopsies with positive c4d staining and anti-donor antibodies related to worse outcome for patients.

C4d+ antibody-mediated rejection following pancreas transplantation has not been well characterized. Therefore, we assessed the outcomes of 27 pancreas transplantation patients (28 biopsies), with both C4d staining and donor-specific antibodies (DSA) determined, from a cohort of 257 patients. The median follow-up was 50 (interquartile range [IQR] 8-118) months. Patients were categorized into 3 groups: group 1, patients with minimal or no C4d staining and no DSA (n = 13); group 2, patients with either DSA present but no C4d, diffuse C4d+ and no DSA or focal C4d+ and DSA (n = 6); group 3, patients with diffuse C4d+ staining and DSA (n = 9). Active septal inflammation, acinar inflammation and acinar cell injury/necrosis were significantly more abundant in group 3 than in group 2 (respective p-values: 0.009; 0.033; 0.025) and in group 1 (respective p-values: 0.034; 0.009; 0.002). The overall uncensored pancreas graft survival rate for groups 1, 2 and 3 were 53.3%, 66.7% and 34.6%, respectively (p = 0.044). In conclusion, recipients of pancreas transplants with no C4d or DSA had excellent long-term graft survival in comparison with patients with both C4d+ and DSA present. Hence, C4d should be used as an additional marker in combination with DSA in the evaluation of pancreas transplant biopsies.

Calcineurin inhibitors affect B cell antibody responses indirectly by interfering with T cell help.

In general, humoral immune responses depend critically upon T cell help. In transplantation, prevention or treatment of humoral rejection therefore require drugs that ideally inhibit both B cell and T helper cell activity. Here, we studied the effects of commonly used immunosuppressive drugs [tacrolimus, cyclosporin, mycophenolic acid (MPA) and rapamycin] on T cell helper activity and on T cell-dependent B cell responses. T cells were activated polyclonally in the presence of immunosuppressive drugs in order to analyse the effect of these drugs on T cell proliferation, co-stimulatory ligand expression and cytokines. The impact of immunosuppressive drugs on T cell-dependent immunoglobulin production by B cells was addressed in T-B cell co-cultures. All drugs affected T cell proliferation and attenuated T cell co-stimulatory ligand (CD154 and CD278) expression when T cells were activated polyclonally. Tacrolimus, cyclosporin and rapamycin also attenuated B cell stimulatory cytokine mRNA levels in T cells. As a consequence, a decrease in immunoglobulin levels was observed in autologous T-B cell co-cultures, where T cell help is essential for immunoglobulin production. In contrast, when pre-activated T cells were used to stimulate autologous B cells, calcineurin inhibitors failed to inhibit B cell immunoglobulin production, whereas MPA and rapamycin did show inhibition. From these studies, it is evident that calcineurin inhibitors affect the humoral immune response by interfering with T helper signals, but not by targeting B cells directly. Furthermore, our studies support the necessity of intervening in T cell helper function to attenuate humoral responses.

Accumulation of autoreactive effector T cells and allo-specific regulatory T cells in the pancreas allograft of a type 1 diabetic recipient.

AIMS/HYPOTHESIS: Simultaneous kidney-pancreas transplantation is an established treatment for patients with type 1 diabetes and end-stage renal failure, even though restored beta cell function may become affected by recurrent islet autoimmunity or graft rejection. We characterised infiltrating lymphocytes isolated from a pancreatic graft with normal endocrine function in a kidney-pancreas recipient with type 1 diabetes. METHODS: The pancreas graft was removed due to recurrent graft pancreatitis of unknown cause. Pancreas-infiltrating lymphocytes and peripheral blood mononuclear cells (PBMC) were isolated and characterised phenotypically and functionally. RESULTS: Compared with PBMC, pancreas-infiltrating lymphocytes exhibited a distinct activation/memory phenotype and T cell receptor profile that were indicative of selective infiltration of the pancreas. Islet autoreactive CD8(+) T cells could be detected in the pancreas and were increased in frequency compared with PBMC. Additionally, an augmentation of CD8(+) CD28(-) regulatory T cells was observed in the pancreas; these induced expression of the inhibitory receptor immunoglobulin-like transcript-3 on antigen-presenting cells in a donor HLA class I-specific manner. CONCLUSIONS/INTERPRETATION: These data demonstrate the simultaneous presence of regulatory and effector T cells in the pancreas allograft of a recipient with type 1 diabetes. They also indicate that circulating islet autoreactive T cells may reflect immunological processes in pancreatic tissue, even though their frequency in the periphery may lead to underestimation of their presence in the pancreas. Additional specificities were also present in the pancreas that were undetectable in the circulation.

IgA1 glycosylation in IgA nephropathy: as sweet as it can be.

Abnormally O-glycosylated IgA1 is likely to be involved in the pathogenesis of IgA nephropathy (IgAN). Buck et al. show that the enzyme activity and gene expression of specific glycosyltransferases, in purified B cells isolated from peripheral blood and bone marrow, is not reduced in IgAN patients. As only a small fraction of IgA in IgAN patients is abnormally glycosylated, it is probable that a more detailed molecular analysis at the single cell level is required to unravel the cause of this abnormality.

Secretory immunoglobulin A (IgA) responses in IgA nephropathy patients after mucosal immunization, as part of a polymeric IgA response.

Secretory immunoglobulin A (SIgA), although generated at mucosal surfaces, is also found in low concentrations in the circulation. Recently, SIgA was demonstrated in mesangial deposits of patients with immunoglobulin A nephropathy (IgAN), suggesting a role in the pathogenesis. This finding is in line with the belief that high molecular weight (HMW) immunoglobulin A (IgA) is deposited in the kidney. However, there is little information on the size distribution of antigen-specific IgA in circulation upon mucosal challenge. In this study we measured antigen-specific IgA, including SIgA, in serum following challenge of IgAN patients and controls via intranasal vaccination with a neoantigen, cholera toxin subunit B (CTB). We size-fractionated serum and nasal washes to study the size distribution of total IgA, SIgA and CTB-specific IgA. Finally, we compared the size distribution of antigen-specific IgA after mucosal immunization with the distribution upon systemic immunization. A significant induction of antigen-specific SIgA was detectable in serum of both patients with IgAN and controls after mucosal immunization with CTB. Independent of the route of immunization, in both groups the antigen-specific IgA response was predominantly in the polymeric IgA fractions. This is in contrast to total IgA levels in serum that are predominantly monomeric. We conclude that mucosal challenge results in antigen-specific SIgA in the circulation, and that the antigen-specific IgA response in both IgAN patients and in controls is of predominantly HMW in nature. No differences between IgAN patients and controls were detected, suggesting that the size distribution of antigen-specific IgA in the circulation is not disturbed specifically in IgAN patients.