Circulating exosomes induced by respiratory viral infections in lung transplant recipients activate cellular stress,innate immune pathways and epithelial to mesenchymal transition

BackgroundChronic lung transplant rejection occurs in over 50% of lung transplant recipients and mechanism of chronic rejection is unknown. Evaluation of potential mechanism of exosomes from lung transplant recipients diagnosed with respiratory viral infection (RVI) in inducing chronic lung allograft dysfunction (CLAD).MethodExosomes were isolated from lung transplant recipients followed by DNA and RNA isolation from exosomes. Cell signaling mechanisms were studied by co-culturing exosomes with human epithelial cells. Mice were immunized with exosomes and lung homogenates were studied for immune signaling proteins.ResultsExosomes from lung transplant recipients with RVI carry nucleic acids which are capable of inducing innate immune signaling, endoplasmic reticulum stress, and epithelial mesenchymal transition.ConclusionTherefore, we propose that RVI can lead to induction of exosomes that initiate the process leading to CLAD in mice models. These novel findings identified the molecular mechanisms by which RVI increases the risk of CLAD.     

Influence of immune activation on the risk of allograft rejection in human immunodeficiency virus-infected kidney transplant recipients

BackgroundHIV infection is associated with high rates of acute rejection following kidney transplantation. The underlying mechanisms for such predisposition are incompletely understood. Pathological immune activation is a hallmark of chronic HIV infection that persists despite effective antiretroviral therapy. We hypothesized that the baseline levels of T cell activation in HIV⁺ candidates would correlate with their risk of acute rejection following kidney transplantation.MethodsSingle-center retrospective cohort analysis of HIV⁺ adult kidney transplants performed between October 2006 and September 2013. The frequency of CD3⁺ HLA-DR⁺ cells measured by flow cytometry served as a surrogate marker of immune activation. Patients were categorized into tertiles of activation, and the rates of biopsy-proven acute rejection were compared across groups.Results(1) Compared to matched HIV⁻ controls, the baseline number of CD3⁺ HLA-DR⁺ cells was higher in HIV⁺ kidney transplant candidates. (2) Abnormally high levels of activation did not decrease with transplant-associated immunosuppression. (3) Patients categorized within the lower and middle CD3⁺ HLA-DR⁺ tertiles had higher probability of rejection during the first 3 years post-transplant compared to those in the higher activation tertile (36.9% vs. 0%; log-rank P = 0.04).ConclusionsPathological immune activation in HIV⁺ transplant candidates does not explain their increased susceptibility to allograft rejection. Paradoxically, those with the highest levels of immune activation seem to be less prone to rejection.     

Kinetics of cellular and humoral immunity in a successful case of positive crossmatch kidney transplantation: a case report

A positive crossmatch remains one of the major barriers to successful kidney transplantation. Highly sensitized patients are at greater risk of hyperacute rejection and subsequent graft loss after transplantation. Although recent advances in desensitization therapy allow kidney transplantation in these patients, the success rate is quite low. Herein, we have reported a successful case of positive crossmatch living donor kidney transplantation using a desensitization protocol with an immune monitoring assay.A 42-year-old woman with end-stage renal disease due to IgA nephropathy had been on hemodialysis for 36 months. She showed positive T-cell and B-cell cytotoxic crossmatches with her husband owing to pretransplantation blood transfusions. We performed a preconditioning regimen comprising a single dose of rituximab (375 mg/m²) combined with double-filtration plasmapheresis (DFPP) followed by low doses of intravenous immunoglobulin (DFPP/IVIG treatment). Tacrolimus (target trough level, 5-10 ng/mL) and mycophenolate mofetil (1500 mg/body) were started 2 weeks before the DFPP/IVIG treatment. After 6 DFPP/IVIG sessions, the crossmatch became negative. An induction quadruple immunosuppression protocol included tacrolimus, mycophenolate mofetil, basiliximab, and methylprednisolone. After the transplantation, the patient's immune status was evaluated regularly by mixed lymphocyte reactions (MLR) using an intracellular carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeling technique (CFSE-MLR assay) and immunosuppressant therapy was adjusted accordingly. During the observation period, neither antibody-mediated rejection nor acute cellular rejection was encountered in this patient.     

Increased Delta Neutrophil Index Is Associated With Poor Prognosis in Cadaver Donor Kidney Transplantation

ObjectiveDelta neutrophil index (DNI), representing an elevated fraction of circulating immature granulocyte in acute infection, has been reported as a useful, predictable marker for mortality in patients with sepsis. We have hypothesized that an increased recipient DNI is associated with poor prognosis in cadaver donor kidney transplantation.MethodsWe investigated patients undergoing kidney transplantation from cadaver donors from March 2013 to January 2018. Rejection was diagnosed by kidney biopsy with Banff classification and excluded subclinical rejection.ResultsIn a total of 73 patients undergoing cadaver kidney transplantation, 25 (34.2%) patients were diagnosed with rejection based on the Banff classification. Among them, 11 patients were diagnosed with early rejection. The recipients' postoperative DNI (%) was different between patients with early rejection and patients without rejection (0.18 vs 1.21, P < .001). In the univariate logistic regression analysis, cold ischemic time, donor preoperative last creatinine level, postoperative DNI level, and perioperative infection were predictive of early rejection. However, in a multivariate adjusted logistic regression test, only a high level of DNI (odds ratio 12.307, 95% confidence interval [CI] 1.22–129.82) was associated with early rejection. The C-statistic was 0.777 (95% CI 0.604–0.951, P = .004) for DNI. In multivariate Cox regression analysis, the donor's last creatinine level (hazard ratio 2.25, 95% CI 1.26–4.13) and preoperative DNI (hazard ratio 14.02 95% CI 2.62–75.26) were predictors of renal survival.ConclusionsIncreased DNI in cadaver donor kidney transplantation recipients might be one of the predictive values of early kidney rejection and prognosis.     

Hyperacute rejection in ABO-incompatible kidney transplantation: Significance of isoagglutinin subclass

IntroductionABO-incompatible transplantation has expanded the limited donor pool for kidney transplantation. Despite the successful desensitization protocols and immunosuppression, undesirable cases of hyperacute rejection occurs.ObjectiveFlow cytometry was used to measure isoagglutinin titer and its IgG subclasses in assessment of the cause of hyperacute rejection in ABO-incompatible kidney transplantation.Materials and methodsThe recipient was admitted for kidney transplantation due to end-stage renal disease. Pre-transplantation work-up for ABO-incompatible kidney transplantation included blood group typing, HLA DNA typing and HLA antibody analyses. HLA crossmatch analysis was conducted using donor lymphocytes and anti-HLA antibody assay using Luminex panel reactive antibody test (One Lambda, Inc., Canoga Park, CA). Desensitization protocol was composed of therapeutic plasma exchange sessions and rituximab.ResultsDespite negative HLA crossmatch results, a case of hyperacute rejection occurred after living donor kidney transplantation. Rejection resulted in immediate removal of graft, and the patient later received a second kidney transplantation. Retrospective evaluation of isoagglutinin titer and its subclasses using flow cytometry identified the cause of rejection to increased IgG1 subclass. Desensitization protocol for ABO-incompatible kidney transplantation now implements further caution for blood group O recipients.DiscussionHyperacute rejection resulting from increased IgG1 isoagglutinin subclass has not been previously confirmed using flow cytometry. Unfortunate outcome of this rejection case provides insight to how we should approach and ensure successful ABO-incompatible kidney transplantation.     

Infections after organ transplantation and immune response

Organ transplantation has provided another chance of survival for end-stage organ failure patients. Yet, transplant rejection is still a main challenging factor. Immunosuppressive drugs have been used to avoid rejection and suppress the immune response against allografts. Thus, immunosuppressants increase the risk of infection in immunocompromised organ transplant recipients. The infection risk reflects the relationship between the nature and severity of immunosuppression and infectious diseases. Furthermore, immunosuppressants show an immunological impact on the genetics of innate and adaptive immune responses. This effect usually reactivates the post-transplant infection in the donor and recipient tissues since T-cell activation has a substantial role in allograft rejection. Meanwhile, different infections have been found to activate the T-cells into CD₄⁺ helper T-cell subset and CD₈⁺ cytotoxic T-lymphocyte that affect the infection and the allograft. Therefore, the best management and preventive strategies of immunosuppression, antimicrobial prophylaxis, and intensive medical care are required for successful organ transplantation. This review addresses the activation of immune responses against different infections in immunocompromised individuals after organ transplantation.     

Elevated intragraft expression of innate immunity and cell death-related markers is a risk factor for adverse graft outcome

BackgroundMolecules of the innate immune response are increasingly recognized as important mediators in allograft injury during and after kidney transplantation. We therefore aimed to establish the relationship between the expression of these genes at implantation, during an acute rejection (AR) and on graft outcome.MethodsA total of 19 genes, including Toll like receptors (TLRs), complement components and regulators, and apoptosis-related genes were analyzed at the mRNA level by qPCR in 123 biopsies with acute rejection and paired pre-transplantation tissue (n = 75).ResultsBefore transplantation, relative mRNA expression of BAX:BCL2 ratio (apoptosis marker) and several complement genes was significantly higher in tissue samples from deceased donors compared to living donors. During AR, TLRs and complement genes showed an increased expression compared to pre-transplant conditions, whereas complement regulators were decreased. A relatively high TLR4 expression level and BAX:BCL2 ratio during AR in the deceased donor group was associated with adverse graft outcome, independently of clinical risk factors.ConclusionsComplement- and apoptosis-related gene expression is elevated in deceased donor transplants before transplantation. High BAX:BCL2 ratio and TLR4 expression during AR may reflect enhanced intragraft cell death and immunogenic danger signals, and pose a risk factor for adverse graft outcome.     

Antibody-mediated rejection owing to donor-specific HLA-DQA1 antibodies after renal transplantation: A case report

BackgroundDonor-specific HLA antibodies are important risk factors in antibody-mediated rejection and graft loss after renal transplantation and are associated with higher rejection rates and lower graft survival. Most de novo donor specific antibodies (dnDSA) after renal transplantation are directed toward donor HLA-DQ antigens. An HLA-DQ antigen is a heterodimer consisting of an alpha and beta chain. Traditionally, HLA-DQA1 typing has not been part of pretransplant evaluation. Therefore, DQ alpha proteins are not usually considered in the interpretation of HLA-DQ antibody reactions.MethodsThe renal transplant recipient had a 0% panel reactive antibody pretransplant. Two years after transplantation, he developed symptoms of abdominal distension and bilateral lower extremity edema. Histopathological findings on renal puncture biopsy showed a combination of T-cell-mediated acute rejection type IIA and antibody-mediated rejection with a trend toward chronicity in the transplanted kidney. DSAs were investigated by HLA-I (HLA-A/B) and HLA-II (HLA-DRB1/DQA1/DQB1) single antigen bead (SAB) assay. HLA typing was performed to explain the antibody reactivity patterns by PCR-SSO and Sequencing-based typing (SBT). HLAMatchmaker analysis was performed to identify eplets that explain antibody reactivity patterns.ResultsHLA-II SAB analysis of the patient's serum at the time of rejection showed positive reactions with all DQB1*03:03-carrying beads with high mean fluorescence intensity (MFI). However, DQB1*03:03 was not a dnDSA antigen. High-resolution HLA typing revealed that HLA-DQA1*05:01 and DQA1*03:02 were mismatched donor antigens. HLA Matchmaker analysis demonstrated reactivity toward 130R and 116 V eplet on DQA1 and DQB1.ConclusionsAntibodies specific to DQα chains after renal transplantation were highlighted.     

Recipient Myd88 Deficiency Promotes Spontaneous Resolution of Kidney Allograft Rejection

The myeloid differentiation protein 88 (MyD88) adapter protein is an important mediator of kidney allograft rejection, yet the precise role of MyD88 signaling in directing the host immune response toward the development of kidney allograft rejection remains unclear. Using a stringent mouse model of allogeneic kidney transplantation, we demonstrated that acute allograft rejection occurred equally in MyD88-sufficient (wild-type [WT]) and MyD88^−/− recipients. However, MyD88 deficiency resulted in spontaneous diminution of graft infiltrating effector cells, including CD11b⁻Gr-1⁺ cells and activated CD8 T cells, as well as subsequent restoration of near-normal renal graft function, leading to long-term kidney allograft acceptance. Compared with T cells from WT recipients, T cells from MyD88^−/− recipients failed to mount a robust recall response upon donor antigen restimulation in mixed lymphocyte cultures ex vivo. Notably, exogenous IL-6 restored the proliferation rate of T cells, particularly CD8 T cells, from MyD88^−/− recipients to the proliferation rate of cells from WT recipients. Furthermore, MyD88^−/− T cells exhibited diminished expression of chemokine receptors, specifically CCR4 and CXCR3, and the impaired ability to accumulate in the kidney allografts despite an otherwise MyD88-sufficient environment. These results provide a mechanism linking the lack of intrinsic MyD88 signaling in T cells to the effective control of the rejection response that results in spontaneous resolution of acute rejection and long-term graft protection.     

Immutol regulates CD4+Tregs,CD8+Tregs and pDCs via IDO signaling pathway to induce immune tolerance in rat heart allograft transplant

Indoleamine 2,3-dioxygenase (IDO) can promote tryptophan metabolism to kynurenine and modulate regulatory T cells (Tregs), thereby maintains lower efficiency to induce tolerance. Our aim is to investigate the mechanism of tolerance induction by a IDO metabolite named Immutol.MethodsWe established rat heterotopic heart transplantation models and treated them with Immutol, cyclosporine A (CsA) and 1-methyl-DL-tryptophan (1-MT) in vivo. The drugs were administered via gavage to all but the control group one day before surgery. CsA was gavaged continually for 20 days and Immutol for 60 days; after withdrawal of the drugs, the recipients were observed for at least 10 months. Immune cells were analyzed by flow cytometry. The IDO signaling pathway was evaluated by Western blotting, RT-PCR and immunochemical staining. Enzyme-linked immunosorbent assays (ELISAs) were used to detect changes in cytokines.ResultsCsA or Immutol alone prolonged survival but did not induce tolerance after withdrawal. Immutol+CsA inhibited acute rejection, and the grafts survived more than 400 d, with tolerance detected in most rats (13/15). Increased protein IDO and kynurenine could regulate the accumulation of CD4⁺Tregs, CD8⁺Tregs and pDC to induce immune tolerance. I-MT specifically blocked IDO, weakened the expression of IDO and kynurenine, and produced grafts rejection. Additionally, Tregs could down-regulate immune responses through production of the immunosuppressive cytokines IL-10 and TGF-beta, thus induce immune tolerance. CD8⁺ Tregs produce IFN-γ, and tolerance is dependent on both IFN-γ and IDO.ConclusionImmutol combined with CsA can control acute rejection and induce tolerance in rats with cardiac allografts after withdrawal. Immutol may become a novel drug for future clinical use.     

Isolation ad infusion of donor CD34+ bone marrow cells in cadaver kidney transplantation

Background: Infusion of donor bone marrow cells induces tolerance in allograft models. CD34⁺ stem cells present in human bone marrow could be endowed with tolerogenic properties. Methods: CD34⁺ stem cells were isolated from bone marrow extracted from vertebral bodies of cadaveric donors. Donor CD34⁺ cells (0.6-3.7 x 10⁶/kg) were infused during surgery in 10 kidney transplant recipients receiving OKT3 as induction therapy. Chimerism was investigated using nested PCR for donor-specific HLA alleles. Results: The infusion of CD34⁺ stem cells was perfectly tolerated. Five patients remained free of acute rejection at follow-up, 47-325 days post-operatively. The five other patients underwent a single episode of corticosensitive acute rejection. Long-term chimerism was not induced in the seven patients investigated for the persistence of donor DNA. Conclusions: Infusion of donor CD34⁺ stem cells in kidney transplantation is safe. The clinical usefulness of the procedure remains to be established.     

High terminal creatinine donors should not preclude simultaneous kidney and pancreas transplantation

BackgroundSimultaneous pancreas and kidney transplantation (SPK) in the setting of end-stage renal disease offers unmatched outcomes in insulin dependent diabetic patients. Donor pool expansion through the transplantation of kidneys with acute kidney injury (AKI) is controversial.Methods59 SPK transplants were classified by presence of donor AKI, defined as donor terminal creatinine ≥ 1.5x the initial creatinine or donor terminal creatinine > 4.0 mg/dL. Endpoints included graft and patient survival, delayed graft function (DGF), serum creatinine, glomerular filtration rate (GFR), Hemoglobin A1c (HbA1c) and acute rejection.ResultsThe donor AKI group (n = 35) had significantly higher rates of DGF (38 v. 9%, p = 0.01). There was no difference in creatinine or GFR at 1, 3, 6 and 12 months. HbA1c was comparable at 3, 6 and 12 months. There was no significant difference in the percentage of patients that required anti-diabetic agents after transplant (14 v. 4%, p = 0.56).ConclusionsWe observed increased rates of DGF in SPK recipients with donor AKI. However, equivalent outcomes of pancreas and kidney function in both groups were observed.     

Bacterial products in donor airways prevent the induction of lung transplant tolerance

Although postoperative bacterial infections can trigger rejection of pulmonary allografts, the impact of bacterial colonization of donor grafts on alloimmune responses to transplanted lungs remains unknown. Here, we tested the hypothesis that bacterial products present within donor grafts at the time of implantation promote lung allograft rejection. Administration of the toll-like receptor 2 (TLR2) agonist Pam₃Cys₄ to Balb/c wild-type grafts triggered acute cellular rejection after transplantation into B6 wild-type recipients that received perioperative costimulatory blockade. Pam₃Cys₄-triggered rejection was associated with an expansion of CD8⁺ T lymphocytes and CD11c⁺CD11b^hiMHC (major histocompatibility complex) class II⁺ antigen-presenting cells within the transplanted lungs. Rejection was prevented when lungs were transplanted into TLR2-deficient recipients but not when MyD88-deficient donors were used. Adoptive transfer of B6 wild-type monocytes, but not T cells, following transplantation into B6 TLR2-deficient recipients restored the ability of Pam₃Cys₄ to trigger acute cellular rejection. Thus, we have demonstrated that activation of TLR2 by a bacterial lipopeptide within the donor airways prevents the induction of lung allograft tolerance through a process mediated by recipient-derived monocytes. Our work suggests that donor lungs harboring bacteria may precipitate an inflammatory response that can facilitate allograft rejection.     

Pretransplant soluble CD30 serum concentration does not affect kidney graft outcomes 3 years after transplantation

IntroductionAn elevated serum concentration of soluble the form of CD30 (sCD30), an activation marker of mainly T_H2-type cytokines producing T lymphocytes, has been reported as a predictive factor for acute cellular rejection episodes and poor graft outcomes in kidney transplantation. This historic cohort study investigated the association of a pretransplant sCD30 serum concentrations with kidney graft function and graft survival 3 years posttransplantation in adult recipients of deceased donor kidney grafts, treated with monoclonal anti-CD25 antibodies as an induction treatment combined with a cyclosporine (CsA)-based maintenance triple therapy.Materials and MethodsThe pretransplant sera of 296 recipients were tested for sCD30 content using a microsphere flow-cytometry assay. The estimated glomerular filtration rate (eGFR) was determined by the 4-variable Modification of Diet in Renal Disease equation. The incidences of graft loss were calculated with the use of Kaplan-Meier survival analysis and compared using the log-rank test.ResultsAccording to the distribution of the pretransplant sCD30 levels concentration ≥2700 pg/mL was defined as high (n = 146) and concentration <2700 pg/mL as low (n = 150). Three years posttransplantation, the eGFR was not significantly different in the recipients in high and low sCD30 groups (65 ± 24 vs 67 ± 21 mL/min/1.73 m²; P = .43); there was no association between the eGFR 3 years after transplantation and the pretransplant sCD30 levels (r² = 0.002; P = .49). Graft survival 3 years after transplantation was also not different in the recipients in high and low sCD30 groups (P = .52).ConclusionIn our adult deceased-donor kidney graft recipients, the pretransplant sCD30 serum concentration was not a predictive factor of immunologic risk associated with the kidney graft function 3 years posttransplantation; neither did it affect graft survival 3 years after transplantation. The immunosuppression with anti-CD25 antibodies as an induction treatment combined with the CsA-based maintenance triple therapy could possibly be decisive for our findings.     

Pancreatic islets engineered with a FasL protein induce systemic tolerance at the induction phase that evolves into long‐term graft‐localized immune privilege

We have previously shown that pancreatic islets engineered to transiently display a modified form of FasL protein (SA‐FasL) on their surface survive indefinitely in allogeneic recipients without a need for chronic immunosuppression. Mechanisms that confer long‐term protection to allograft are yet to be elucidated. We herein demonstrated that immune protection evolves in two distinct phases; induction and maintenance. SA‐FasL‐engineered allogeneic islets survived indefinitely and conferred protection to a second set of donor‐matched, but not third‐party, unmanipulated islet grafts simultaneously transplanted under the contralateral kidney capsule. Protection at the induction phase involved a reduction in the frequency of proliferating alloreactive T cells in the graft‐draining lymph nodes, and required phagocytes and TGF‐β. At the maintenance phase, immune protection evolved into graft site‐restricted immune privilege as the destruction of long‐surviving SA‐FasL‐islet grafts by streptozotocin followed by the transplantation of a second set of unmanipulated islet grafts into the same site from the donor, but not third party, resulted in indefinite survival. The induced immune privilege required both CD4⁺CD25⁺Foxp3⁺ Treg cells and persistent presence of donor antigens. Engineering cell and tissue surfaces with SA‐FasL protein provides a practical, efficient, and safe means of localized immunomodulation with important implications for autoimmunity and transplantation.     

Host immune suppression after small bowel/liver transplantation in rats

Simultaneous liver grafting in the Lewis (RT1¹)-to-DA (RT1^a) rat strain combination protects small intestinal grafts from rejection. The present study examined host immune responses after combined small bowel/liver transplantation (SBL) in this model. Orthotopic liver transplantation and heterotopic small intestinal transplantation were performed simultaneously and compared with isolated small bowel allografts (SBA) and isolated small bowel isografts (SBI). All rats were sacrificed on postoperative day (POD) 7 or 14 for immunological and histological studies. The mean time to rejection of the SBA was 6.6±0.3 days. Incontrast, there was no clinical or histological evidence of intestinal rejection in SBL recipients during the 14 days of follow-up. The SBL recipients showed clinical and histological evidence of graft-versushost disease (GVHD). Lmphocyte proliferation and IL-2 production in response to donor antigens were suppressed after SBL transplantation compared with the SBA or the SBI controls (P<0.05). Cell-mediated cytotoxicity and lymphocytotoxic antibody production against donor cells were also significantly inhibited in the SBL recipients compared with the SBA control group (P<0.05). We conclude that SBL transplantation in the Lewis-toDA rat strain combination: (1) suppresses host alloimmune responses, (2) prevents early intestinal rejection, and (3) favors the development of GVHD.     

Soluble CD83 inhibits acute rejection by up regulating TGF-β and IDO secretion in rat liver transplantation

BackgroundAllogeneic transplantation immune tolerance is currently a hot research issue and soluble CD83(sCD83) is a novel immunomodulator with great potential in inducing transplantation tolerance.ObjectiveTo investigate the mechanism of the immune tolerance effect of sCD83 on rat liver transplantation.MethodA rat liver transplantation model was established to study the effects of sCD83 on the expression levels of IL-2, IL-10, and TGF-β in peripheral blood and the mRNA expressions of foxp3, TGF-β, and Indoleamine 2,3-dioxygenase (IDO) in liver. The expression changes of costimulatory molecules CD80, CD86, and MHC-II on the surface of DC cells and the expressions of IDO ⁺ DC cell, TGF-β ⁺ CD4 ⁺ T cell, and CD4 ⁺ CD25 ⁺ Foxp3 ⁺ T cell were analyzed and compared.ResultssCD83 alleviated the rejection activity index (RAI) of rat liver transplantation in the early stage, increased the expressions of TGF-β, IL-10 in peripheral blood and the mRNAs of IDO, TGF-β and foxp3 in the transplanted liver, and down-regulated the expressions of MHC-II, CD86, and CD80 in DC cells, resulting in significant increased numbers of tolerogenic TGF-β ⁺ CD4 ⁺ T cells, Treg cells, and IDO ⁺ DC cells with low expression.ConclusionsCD83 inhibited acute rejection after liver transplantation in an allogeneic rat, and the mechanism was associated with the effect that sCD83 increased the expression of TGF-β, activated IDO immunosuppressive pathway, and increased tolerogenic DC cells and Treg cells.