Donor-specific Antibody Production Research Articles

Antibody-mediated rejection in heart transplantation

The role of antibody-mediated rejection in predicting survival among heart recipients has been studied in clinical transplantology for over 20 years. This condition is a significant risk factor for heart failure and graft vasculopathy. Antibody-mediated rejection results from activation of the humoral immune system and production of donorspecific antibodies that cause myocardial injury through the complement system. The presence of donor-specific antibodies is associated with lower allograft survival. Treatment of antibody-mediated rejection should take into account the rejection category and the presence or absence of graft dysfunction. The main principle of treatment is to suppress humoral immunity at different levels. World clinical practice has made significant inroads into the study of this issue. However, further research is required to identify and develop optimal treatment regimens for patients with humoral rejection in cardiac transplantation.

Blockade of IL-6/IL-6R Signaling Attenuates Acute Antibody-Mediated Rejection in a Mouse Cardiac Transplantation Model.

Acute antibody-mediated rejection (AAMR) is an important cause of cardiac allograft dysfunction, and more effective strategies need to be explored to improve allograft prognosis. Interleukin (IL)-6/IL-6R signaling plays a key role in the activation of immune cells including B cells, T cells and macrophages, which participate in the progression of AAMR. In this study, we investigated the effect of IL-6/IL-6R signaling blockade on the prevention of AAMR in a mouse model. We established a mouse model of AAMR for cardiac transplantation via presensitization of skin grafts and addition of cyclosporin A, and sequentially analyzed its features. Tocilizumab, anti-IL-6R antibody, and recipient IL-6 knockout were used to block IL-6/IL-6R signaling. We demonstrated that blockade of IL-6/IL-6R signaling significantly attenuated allograft injury and improved survival. Further mechanistic research revealed that signaling blockade decreased B cells in circulation, spleens, and allografts, thus inhibiting donor-specific antibody production and complement activation. Moreover, macrophage, T cell, and pro-inflammatory cytokine infiltration in allografts was also reduced. Collectively, we provided a highly practical mouse model of AAMR and demonstrated that blockade of IL-6/IL-6R signaling markedly alleviated AAMR, which is expected to provide a superior option for the treatment of AAMR in clinic.

The Role of Hyperleptinaemia and Low Values of Interleukin 10 in De Novo Donor-Specific Antibody Production After Kidney Transplantation

The Role of Hyperleptinaemia and Low Values of Interleukin 10 in De Novo Donor-Specific Antibody Production After Kidney Transplantation

Alloreactive Immune Response Associated to Human Mesenchymal Stromal Cells Treatment: A Systematic Review.

The well-known immunomodulatory and regenerative properties of mesenchymal stromal cells (MSCs) are the reason why they are being used for the treatment of many diseases. Because they are considered hypoimmunogenic, MSCs treatments are performed without considering histocompatibility barriers and without anticipating possible immune rejections. However, recent preclinical studies describe the generation of alloantibodies and the immune rejection of MSCs. This has led to an increasing number of clinical trials evaluating the immunological profile of patients after treatment with MSCs. The objective of this systematic review was to evaluate the generation of donor specific antibodies (DSA) after allogeneic MSC (allo-MSC) therapy and the impact on safety or tolerability. Data from 555 patients were included in the systematic review, 356 were treated with allo-MSC and the rest were treated with placebo or control drugs. A mean of 11.51% of allo-MSC-treated patients developed DSA. Specifically, 14.95% of these patients developed DSA and 6.33% of them developed cPRA. Neither the production of DSA after treatment nor the presence of DSA at baseline (presensitization) were correlated with safety and/or tolerability of the treatment. The number of doses administrated and human leucocyte antigen (HLA) mismatches between donor and recipient did not affect the production of DSA. The safety of allo-MSC therapy has been proved in all the studies and the generation of alloantibodies might not have clinical relevance. However, there are very few studies in the area. More studies with adequate designs are needed to confirm these results.

Development of De Novo Donor-specific HLA Antibodies and AMR in Renal Transplant Patients Depends on CYP3A5 Genotype.

Background.The single-nucleotide polymorphism CYP3A5 rs776746 is related to a reduction in the metabolizing activity of the CYP3A5 enzyme. People carrying at least one copy of the wild-type allele, defined as CYP3A5 expressers, exhibit higher clearance and lower trough concentrations of tacrolimus than homozygous nonexpressers, and this difference may affect alloimmunization and allograft function.Methods.We retrospectively studied 400 kidney transplant recipients treated with a tacrolimus-based immunosuppression regimen to detect CYP3A5 genotype, de novo formation of HLA antibodies and donor-specific antibodies (DSAs), and clinical outcome up to 5 y after transplant.Results.We found that 69 (17%) of the 400 patients were CYP3A5 expressers. During the first 3 y after transplant, CYP3A5 expressers tended to have lower tacrolimus trough levels than nonexpressers, although their tacrolimus dosage was as much as 80% higher. De novo DSAs were found more frequently in CYP3A5 expressers than in nonexpressers (13/69 [19%] versus 33/331 [10%], P = 0.02). De novo DSA-free survival rates (P = 0.02) were significantly lower for expressers than for nonexpressers. CYP3A5 genotype had no effect on allograft failure, but CYP3A5 expressers exhibited a significantly higher frequency of antibody-mediated rejection. CYP3A5 expresser status was an independent risk factor for the development of de novo DSAs (relative risk, 2.34, P = 0.01).Conclusions.Early detection of CYP3A5 expressers, enabling genotype-based dose adjustment of tacrolimus immediately after renal transplant, may be a useful strategy for reducing the risk of de novo DSA production and antibody-mediated rejection.

IRF4 ablation in B cells abrogates allogeneic B cell responses and prevents chronic transplant rejection

B cells contribute to chronic transplant rejection by producing donor-specific antibodies and promoting T cell response, but how these processes are regulated at the transcriptional level remains unclear. Herein, we investigate the role of transcription factor interferon regulatory factor 4 (IRF4) in controlling B cell response during chronic transplant rejection. We generated the Irf4gfp reporter mice to determine IRF4 expression in B cell lineage. We then used mice with B cell-specific IRF4 deletion to define the role of IRF4 in B cell response after NP-KLH immunization or allogeneic heart transplantation. In particular, graft survival and histology, as well as B and T cell responses, were evaluated after transplantation. IRF4 is dynamically expressed at different stages of B cell development and is absent in germinal center (GC) B cells. However, IRF4 ablation in the B cell lineage primarily eliminates GC B cells in both naïve and NP-KLH immunized mice. In the transplantation setting, IRF4 functions intrinsically in B cells and governs allogeneic B cell responses at multiple levels, including GC B cell generation, plasma cell differentiation, donor-specific antibody production, and support of T cell response. B cell-specific IRF4 deletion combined with transient CTLA4-Ig treatment abrogates acute and chronic cardiac allograft rejection in naïve recipient mice but not in donor skin-sensitized recipients. B cells require IRF4 to mediate chronic transplant rejection. IRF4 ablation in B cells abrogates allogeneic B cell responses and may also inhibit the ability of B cells to prime allogenic T cells. Targeting IRF4 in B cells represents a potential therapeutic strategy for eliminating chronic transplant rejection.

Clinical Significance of Shared T Cell Epitope Analysis in Early De Novo Donor-Specific Anti-HLA Antibody Production After Kidney Transplantation and Comparison With Shared B cell Epitope Analysis

In pre-sensitizing events, immunological memory is mainly created via indirect allorecognition where CD4+ T cells recognize foreign peptides in the context of self-HLA class II (pHLA) presented on antigen-presenting cells. This recognition makes it possible for naive CD4+ T-helper cells to differentiate into memory cells, resulting in the creation of further antibody memory. These responses contribute to effective secretion of donor-specific anti-HLA antibodies (DSA) after second encounters with the same peptide. Preformed donor-reactive CD4+ memory T cells may induce early immune responses after transplantation; however, the tools to evaluate them are limited. This study evaluated shared T cell epitopes (TEs) between the pre-sensitizing and donor HLA using an in silico assay, an alternative to estimate donor-reactive CD4+ memory T cells before transplantation. In 578 living donor kidney transplants without preformed DSA, 69 patients had anti-HLA antibodies before transplantation. Of them, 40 had shared TEs and were estimated to have donor-reactive CD4+ memory T cells. De novo DSA formation in the early phase was significantly higher in the shared TE-positive group than in the anti-HLA antibody- and shared TE-negative groups (p=0.001 and p=0.02, respectively). In conclusion, evaluation of shared TEs for estimating preformed donor-reactive CD4+ memory T cells may help predict the risk of early de novo DSA formation after kidney transplantation.

Role of Memory B Cells in Antibody Mediated Rejection: A Proof of Concept

<h3>Introduction</h3> Antibody mediated rejection (AMR) diagnosis, monitoring and treatment is exclusively based on circulating donor specific antibodies (DSA). However, DSA often fails to predict and diagnose AMR, since it does not represent the whole HLA-specific memory B cell (mBc) repertoire. Central and peripheral donor (HLA)-sp B-cell responses may offer a new diagnostic and therapeutic target. <h3>Case Report</h3> 44 years old female affected from an advanced Chagasic cardiomyopathy refractory to therapy was listed for heart transplant with a PRA of 67% in 03/2019. She was transplanted in 11/2019, with a negative CDC-XM. Induction was based on basiliximab and tacrolimus-based triple therapy with Mycophenolate mofetil and steroids. Despite a negative CDC-XM, 5 C3q-fixing DSA were detected (A3, DR1, DR7, DPB1, DQ2 and DQ5). Post-op was uneventful and biventricular function was preserved at day 7. The 14-day endomyocardial biopsy showed acute cellular rejection G2R and p(AMR)1 i+ with persistent DSAs. High doses of esteroids, IVIG and plasmapheresis were started. The SAB assay on day 25 showed persistent detection of all DSA, although with lower MFI levels. To characterize B-cell immune response, we used a novel HLA-specific B-cell FluorSpot assay. Bone marrow aspirate and peripheral blood demonstrated high frequencies of donor(HLA)-sp IgG-producing plasma cells and circulating donor-specific mBc, respectively, against different donor(HLA)-Ag specificities <b>(figure 1)</b>. Given patient's favorable evolution, with two main immune compartments showing anti-donor B-cell activation requiring a combined immunosuppressive therapy, no additional treatment was started. At 12 months of follow-up the patient remains clinically stable with negative ACR/pAMR EMB. <h3>Summary</h3> Our data highlights the need of a more accurate evaluation of the anti-donor humoral immune response leading to DSA production in order to refine current immune-risk stratification and ultimately help decision-making regarding the type of rescue immunosuppressive therapy.

Graft-derived extracellular vesicles transported across subcapsular sinus macrophages elicit B cell alloimmunity after transplantation.

Despite the role of donor-specific antibodies (DSAs) in recognizing major histocompatibility complex (MHC) antigens and mediating transplant rejection, how and where recipient B cells in lymphoid tissues encounter donor MHC antigens remains unclear. Contrary to the dogma, we demonstrated here that migration of donor leukocytes out of skin or heart allografts is not necessary for B or T cell allosensitization in mice. We found that mouse skin and cardiac allografts and human skin grafts release cell-free donor MHC antigens via extracellular vesicles (EVs) that are captured by subcapsular sinus (SCS) macrophages in lymph nodes or analog macrophages in the spleen. Donor EVs were transported across the SCS macrophages, and donor MHC molecules on the EVs were recognized by alloreactive B cells. This triggered B cell activation and DSA production, which were both prevented by SCS macrophage depletion. These results reveal an unexpected role for graft-derived EVs and open venues to interfere with EV biogenesis, trafficking, or function to restrain priming or reactivation of alloreactive B cells.

A CD31-Derived Peptide Prevents the Development of Antibody-Mediated Lesions in a Rat Model of Aortic Allograft

BackgroundAntibody-mediated rejection (AMR) is a major cause of graft loss. The development of donor-specific antibodies (DSAs) directed against the allogeneic HLA molecules expressed by the graft also leads to accelerated arteriosclerosis. CD31 is a protein expressed on endothelial and immune cells, ensuring homeostasis at this interface. When strong immune stimulation occurs, the regulatory function of CD31 is lost owing to cleavage of its extracellular portion. P8RI, a synthetic peptide that binds to the ectodomain of CD31, is able to restore the CD31 immunomodulatory function. In this study, we hypothesized that CD31 could represent an attractive molecular target in AMR and investigated whether P8RI could prevent the development of vascular antibody-mediated lesions. Materials and methodsA rat model of orthotopic aortic allograft was used, and P8RI was administered for 28 days. Circulating DSAs were quantified to assess the alloimmune humoral response, and histologic and immunohistochemical analyses of aortic allografts were performed to estimate antibody-mediated lesions in the allograft. ResultsAorta-allografted rats receiving P8RI developed fewer DSAs than control animals (mean fluorescence intensity 344 vs 741). The density of nuclei in the media (3.4 x 10-5 vs 2.2 x 10-5 nuclei/px2) and media surface area (2.33 x 106 vs 2.02 x 106 px2) were higher in animals treated with P8RI than in control animals. ConclusionsThese data support a therapeutic potential for molecules able to restore the CD31 signaling to fight AMR. P8RI, an agonist synthetic peptide targeting CD31, might prevent DSA production and have a beneficial effect in limiting arterial antibody-mediated lesions. CD31 agonists may become therapeutic tools to prevent and treat solid organ transplant arteriosclerosis.

Optimal blood levels of (extended-release) tacrolimus in living donor kidney transplantation to prevent de novo donor-specific antibody production: A retrospective cohort study

Chronic antibody-mediated rejection, caused by de novo donor-specific antibody (dnDSA) production, results in poor graft survival. To prevent dnDSA production, optimal blood levels of immunosuppressive drugs in living donor kidney transplant recipients were determined. A total of 772 recipients underwent living donor kidney transplantation between January 2008 and December 2017. Graft survival and risk factors for dnDSA production were investigated in 647 recipients. Optimal blood levels of tacrolimus (TAC) and extended-release TAC (TACER) were measured in recipients receiving steroids and mycophenolate mofetil, combined with TAC (n = 53) or TACER (n = 135). Receiver operating characteristic (ROC) curve analysis and comparisons between dnDSA-negative and dnDSA-positive recipients were carried out. The Kaplan-Meier method revealed significantly poor graft survival in dnDSA-positive recipients (P < 0.001). Cox regression models indicated calcineurin inhibitor withdrawal as a significant risk for dnDSA production (P < 0.001; hazard ratio 6.637; 95% confidence interval 2.667–6.517). Average trough levels of TAC and TACER in dnDSA-negative recipients were significantly higher than those in dnDSA-positive recipients (4.88 vs 3.69 ng TAC/ml, P = 0.023, and 4.60 vs 3.85 ng TACER/ml, P = 0.001). ROC curve analysis indicated 4.325 and 3.990 ng/ml as the best trough levels under TAC- and TACER-based regimens, respectively, to prevent dnDSA production (areas under the curve: 0.788 and 0.813, respectively). Maintenance of the trough levels of TAC > 4.325 ng/ml and TACER > 3.990 ng/ml may prevent dnDSA production.

New concepts in chronic antibody-mediated kidney allograft rejection: prevention and treatment.

Chronic antibody-mediated rejection (AMR) is a cardinal cause of transplant failure, with currently no proven effective prevention or treatment. The present review will focus on new therapeutic concepts currently under clinical evaluation. One interesting treatment approach may be interference with interleukin-6 (IL-6) signaling to modulate B-cell immunity and donor-specific antibody (DSA) production. Currently, a large phase III randomized controlled trial is underway to clarify the safety and efficacy of clazakizumab, a high-affinity anti-IL-6 antibody, in chronic AMR. A prevention/treatment strategy may be costimulation blockade using belatacept to interfere with germinal center responses and DSA formation. In a recent uncontrolled study, belatacept conversion was shown to stabilize renal function and dampen AMR activity. Moreover, preliminary clinical results suggest efficacy of CD38 antibodies to deplete plasma and natural killer cells to treat AMR, with anecdotal reports demonstrating at least transient resolution of active rejection. There are promising concepts on the horizon for the prevention and treatment of chronic AMR. The design of adequately powered placebo-controlled trials to clarify the safety and efficacy of such new therapies, however, remains a big challenge, and will rely on the definition of precise surrogate endpoints predicting long-term allograft survival. Mapping the natural history of AMR would greatly help the understanding of who would derive benefits from treatment.

Preceding T-Cell-Mediated Rejection Is Associated with the Development of Chronic Active Antibody-Mediated Rejection by de Novo Donor-Specific Antibody

Aim: Chronic active antibody-mediated rejection (CAABMR) is an important cause of late-stage renal allograft loss. Early inflammatory events such as acute rejection and infection after transplantation are considered to be the risk factors of de novo donor-specific antibody (dnDSA) production. In this study, we investigated the relationship between pre­disposing T-cell-mediated rejection and dnDSA-positive CAABMR. Methods: We recruited 365 patients who underwent ABO-compatible renal transplantation at our hospital. Among them, 16 patients diagnosed as having dnDSA-positive CAABMR were designated as a CAABMR group, and 38 randomly selected patients were designated as a control group. All biopsies from 1 month after transplantation were included in the study. The presence or absence of borderline changes (BLCs), acute T-cell-mediated rejection (ATMR), microvascular inflammation (MVI), and C4d positive on peritubular capillaries (C4d-P) was examined. Results: In the CAABMR group, BLC/ATMR was found in 12 cases (75%), and the mean duration until appearance of BLC/ATMR was 282.7 ± 328.7 days. C4d-P was found in 11 cases (68.8%), and the mean duration until its appearance was 1,432 ± 1,307 days. MVI was found in all cases, and the mean duration until its appearance was 1,333 ± 1,126 days. The mean duration until diagnosis of CAABMR was 2,268 ± 1,191 days. In the control group, BLC/ATMR was found in 13 cases (34.2%), and the mean duration until the appearance of BLC/ATMR was 173.1 ± 170.4 days. C4d-P was found in 2 cases (5.3%), and the durations until its appearance were 748 and 1,881 days. No cases of MVI were found in the control group. The frequency of BLC/ATMR was significantly higher in the CAABMR group (p < 0.01). Conclusion: Preceding BLC/ATMR is associated with the development of CAABMR with dnDSA.

Galectin-9 is required for endometrial regenerative cells to induce long-term cardiac allograft survival in mice

BackgroundEndometrial regenerative cells (ERCs), a novel type of mesenchymal-like stem cells, were identified as an attractive candidate for immunoregulation and induction of cardiac allograft tolerance. However, the underlying mechanisms of ERCs in immune regulation still remain largely unclear. The present study is designed to determine whether the expression of Galectin-9 (Gal-9), a soluble tandem-repeat member of the galectin family, is crucial for ERC-based immunomodulation.MethodsIn this study, we measured Gal-9 expression on ERCs and then co-cultured Gal-9-ERCs, ERCs, and ERCs+lactose (Gal-9 blocker) with activated C57BL/6-derived splenocytes. Furthermore, we performed mouse heart transplantation between BALB/c (H-2d) donor and C57BL/6 (H-2b) recipient. ERCs were administrated 24 h after the surgery, either alone or in combination with rapamycin.ResultsOur data demonstrate that ERCs express Gal-9, and this expression is increased by IFN-γ stimulation in a dose-dependent manner. Moreover, both in vitro and in vivo results show that Gal-9-ERC-mediated therapy significantly suppressed Th1 and Th17 cell response, inhibited CD8+ T cell proliferation, abrogated B cell activation, decreased donor-specific antibody production, and enhanced the Treg population. The therapeutic effect of ERCs was further verified by their roles in prolonging cardiac allograft survival and alleviating graft pathological changes.ConclusionsTaken together, these data indicate that Gal-9 is required for ERC-mediated immunomodulation and prevention of allograft rejection.

Analysis of T and B Cell Epitopes to Predict the Risk of de novo Donor-Specific Antibody (DSA) Production After Kidney Transplantation: A Two-Center Retrospective Cohort Study.

Risk prediction of de novo donor specific antibody (DSA) would be very important for long term graft outcome after organ transplantation. The purpose of this study was to elucidate the association of eplet mismatches and predicted indirectly recognizable HLA epitopes (PIRCHE) scores with de novo DSA production. Our retrospective cohort study enrolled 691 living donor kidney transplantations. HLA-A, B, DRB and DQB eplet mismatches and PIRCHE scores (4 digit of HLA-A, B, DR, and DQ) were determined by HLA matchmaker (ver 2.1) and PIRCHE-II Matching Service, respectively. Weak correlation between eplet mismatches and PIRCHE scores was identified, although both measurements were associated with classical HLA mismatches. Class II (DRB+DQB) eplet mismatches were significantly correlated with the incidence of de novo class II (DR/DQ) DSA production [8/235 (3.4%) in eplet mismatch ≤ 13 vs. 92/456 (20.2%) in eplet mismatch ≥ 14, p < 0.001]. PIRCHE scores were also significantly correlated with de novo class II DSA production [26/318 (8.2%) in PIRCHE ≤ 175 vs. 74/373 (19.8%) in PIRCHE ≥ 176, p < 0.001]. Patients with low levels of both class II eplet mismatches and PIRCHE scores developed de novo class II DSA only in 4/179 (2.2%). Analysis of T cell and B cell epitopes can provide a beneficial information on the design of individualized immunosuppression regimens for prevention of de novo DSA production after kidney transplantation.

P1758TOCILIZUMAB TREATMENT IN DSA POSITIVE ANTIBODY MEDIATED REJECTION AMONG KIDNEY TRANSPLANT RECIPIENTS

Abstract Background and Aims Pretransplant or de novo donor specific antibodies (DSA) may lead to active or chronic active antibody mediated rejection (a and cABMR) as leading cause of graft loss after kidney transplantation. There is no treatment protocol approved for ABMR. Anti Il-6 tocilizumab (TCZ), inhibitor of DSA production, is a potential approach to stabilize kidney allograft function, however, evidence based results are not available. In our retrospective case series analysis, we assessed the changes in DSA and eGFR during and at the end of treatment with TCZ. Method In our single center case series analysis, 10 kidney transplant patients with biopsy proven ABMR (aABMR 6, cABMR 4, age 43±10.5ys, 6 males, time since transplantation 18(2-119)months, 7 first transplant, serum creatinine 224±80 umol/L at baseline) were studied between January 2017 – June 2019. Total plasma exchange (PE) (5x) was followed by TCZ (8 mg/kg, 1x monthly for 6 months) in case of a and cABMR. Intravenous immunoglobulin (IVIG, 1gr/kg) was added in case of aABMR. Routine laboratory parameters and DSA were reported retrospectively. Results 6 aABMR patients completed the treatment protocol. Class I DSA decreased significantly (MFI 4457(635-14084) vs. 877(595-5678); p=0.007), but Class II DSA remained the same during the treatment (MFI 4725 (586-17615) vs. 8097 (671-14636) p=NS). eGFR of aABMR patients and 1 cABMR patient were stabilized. 3 cABMR patients returned to dialysis. Reversible elevation of liver transaminases were detected in three patients. There was no any serious adverse event recorded. Conclusion The opportunely timed TCZ seems to be effective in reducing Class I DSA and stabilize graft function in patients with aABMR. Further studies are needed to prove the long-term efficacy and the exact role of TCZ in cABMR among kidney transplant patients.

Mechanisms of organ transplant injury mediated by B cells and antibodies: Implications for antibody-mediated rejection.

Recent adjustments to the histological diagnosis and the introduction of molecular classification are providing renewed support for the paradigm that antibody-mediated rejection (ABMR) is an important clinical problem for which there is an urgent need for better therapies. Acute ABMR is observed when the graft is exposed to rapid increases in high-titer donor-specific antibodies (DSA) that are most often generated as anamnestic responses in sensitized recipients or de novo responses in nonsensitized patients who are nonadherent. Chronic ABMR is associated with slower increases in DSA, which may be high or low titer and transient or persistent. These DSA elicit cycles of injury and repair that manifest as multilamination of the peritubular capillary basement membrane or arteriopathy manifesting as intimal fibrosis. Mitigating the problem of AMBR requires the anamnestic and de novo DSA responses to be prevented and established DSA responses to be reversed. To this end, a better understanding of the immunobiology of DSA production is necessary and also the development of assays capable of detecting early humoral immune responses.Recent advances in understanding the immunobiology of B cells and areas requiring further investigation that might lead to new therapies or better diagnosis are discussed in this review.

Vascular Signaling in Allogenic Solid Organ Transplantation - The Role of Endothelial Cells.

Graft rejection remains the major obstacle after vascularized solid organ transplantation. Endothelial cells, which form the interface between the transplanted graft and the host’s immunity, are the first target for host immune cells. During acute cellular rejection endothelial cells are directly attacked by HLA I and II-recognizing NK cells, macrophages, and T cells, and activation of the complement system leads to endothelial cell lysis. The established forms of immunosuppressive therapy provide effective treatment options, but the treatment of chronic rejection of solid organs remains challenging. Chronic rejection is mainly based on production of donor-specific antibodies that induce endothelial cell activation—a condition which phenotypically resembles chronic inflammation. Activated endothelial cells produce chemokines, and expression of adhesion molecules increases. Due to this pro-inflammatory microenvironment, leukocytes are recruited and transmigrate from the bloodstream across the endothelial monolayer into the vessel wall. This mononuclear infiltrate is a hallmark of transplant vasculopathy. Furthermore, expression profiles of different cytokines serve as clinical markers for the patient’s outcome. Besides their effects on immune cells, activated endothelial cells support the migration and proliferation of vascular smooth muscle cells. In turn, muscle cell recruitment leads to neointima formation followed by reduction in organ perfusion and eventually results in tissue injury. Activation of endothelial cells involves antibody ligation to the surface of endothelial cells. Subsequently, intracellular signaling pathways are initiated. These signaling cascades may serve as targets to prevent or treat adverse effects in antibody-activated endothelial cells. Preventive or therapeutic strategies for chronic rejection can be investigated in sophisticated mouse models of transplant vasculopathy, mimicking interactions between immune cells and endothelium.

DNA-PKcs Inhibition Extends Allogeneic Skin Graft Survival and Suppresses EGR1 and NFκB-mediated IL6 Production

Abstract Organ transplantation is the only option for patients in end-stage organ failure. Therefore; it is imperative that immunological mechanisms that foster graft rejection be elucidated to develop more effective immunosuppressants for transplant patients. In recent studies, the double-strand DNA break repair kinase, DNA-dependent protein kinase catalytic subunit(DNA-PKcs) has been implicated as a signaling molecule necessary for both B and T cell activity highlighting its potential role in graft rejection. In this study, we performed a phospho-proteomic screen to identify immunological targets of DNA-PKcs and a murine allogeneic skin graft study using DNA-PKcs inhibitors to investigate its effects on graft rejection. We identified a mechanism whereby DNA-PKcs is required for the protein stability of EGR1, a transcription factor known to promote NFκB expression. In primary splenocytes, DNA-PKcs inhibition abrogated EGR1 expression causing a decrease in NFκB-p65 levels and a reduction in the levels of the pro-inflammatory cytokine IL6 which can promote maturation of plasma cells. DNA-PKcs inhibition significantly reduced skin graft necrosis and extended graft survival compared to controls. DNA-PKcs inhibition reduced the infiltration of CD3+ lymphocytes into the grafts and the level of circulating CD19+ CD138+ lymphocytes. Additionally, DNA-PKcs inhibition reduced the production of donor-specific IgG and IgM antibodies. These results highlight a novel function for DNA-PKcs in the immune system and support further investigations into the use DNA-PKcs inhibitors in transplant therapy.

Therapeutic Effects and Functional Mechanism of Intravenous Immunoglobulin in Preclinical Rat Renal Transplant Model of Antibody-Mediated Rejection

BackgroundIntravenous immunoglobulin (Ig) therapy is used based on empirical findings for treatment of antibody-mediated rejection (AMR) in cases of renal transplantation, although its therapeutic efficacy has not been proven and the functional mechanism of an administered Ig remains elusive. In this study, the therapeutic effects of an Ig were examined in a preclinical rat renal transplant model of AMR to investigate this mechanism. MethodsTo establish an AMR renal graft model, skin graft specimens were obtained from Brown-Norway (BN) rats and transplanted to Lewis rats to produce donor-specific antibodies (DSAs), after which kidney transplantation from the Brown-Norway to Lewis rats was performed. AMR model rats were administered the Ig at a dose of 2 g/kg or saline as a control. Survival period, renal graft histopathology, complement factors, and DSA levels were assessed. ResultsThe survival period of the group administered the Ig was significantly prolonged. Histopathological examinations of renal grafts also showed significant suppression of glomerulitis and peritubular capillaritis in the Ig group, and real-time polymerase chain reaction analysis results demonstrated significantly lower levels of expression of the complement factors C1q and C3. When the Ig was given to rats that underwent skin grafting but not renal transplantation, DSA was decreased after 6 hours and remained lower than the baseline level for at least 7 days. ConclusionIg administration suppressed DSA production. The present results showed that suppression of the complement system contributed to effective Ig treatment for AMR.