Acute Lung Allograft Rejection Research Articles

Resolution of Acute Lung Allograft Rejection Detected by Probe-based Confocal Laser Endomicroscopy.

Resolution of Acute Lung Allograft Rejection Detected by Probe-based Confocal Laser Endomicroscopy.

Donor IL-17 receptor A regulates LPS-potentiated acute and chronic murine lung allograft rejection.

Chronic lung allograft dysfunction (CLAD) is a major complication after lung transplantation that results from a complex interplay of innate inflammatory and alloimmune factors, culminating in parenchymal and/or obliterative airway fibrosis. Excessive IL-17A signaling and chronic inflammation have been recognized as key factors in these pathological processes. Herein, we developed a model of repeated airway inflammation in mouse minor alloantigen-mismatched single-lung transplantation. Repeated intratracheal LPS instillations augmented pulmonary IL-17A expression. LPS also increased acute rejection, airway epithelial damage, and obliterative airway fibrosis, similar to human explanted lung allografts with antecedent episodes of airway infection. We then investigated the role of donor and recipient IL-17 receptor A (IL-17RA) in this context. Donor IL-17RA deficiency significantly attenuated acute rejection and CLAD features, whereas recipient IL-17RA deficiency only slightly reduced airway obliteration in LPS allografts. IL-17RA immunofluorescence positive staining was greater in human CLAD lungs compared with control human lung specimens, with localization to fibroblasts and myofibroblasts, which was also seen in mouse LPS allografts. Taken together, repeated airway inflammation after lung transplantation caused local airway epithelial damage, with persistent elevation of IL-17A and IL-17RA expression and particular involvement of IL-17RA on donor structural cells in development of fibrosis.

Cross-Regulation of F-Box Protein FBXL2 with T-bet and TNF-α during Acute and Chronic Lung Allograft Rejection.

Chronic lung allograft dysfunction is the major barrier to long-term survival in lung transplant recipients. Evidence supports type 1 alloimmunity as the predominant response in acute/chronic lung rejection, but the immunoregulatory mechanisms remain incompletely understood. We studied the combinatorial F-box E3 ligase system: F-box protein 3 (FBXO3; proinflammatory) and F-box and leucine-rich repeat protein 2 (FBXL2; anti-inflammatory and regulates TNFR-associated factor [TRAF] protein). Using the mouse orthotopic lung transplant model, we evaluated allografts from BALB/c → C57BL/6 (acute rejection; day 10) and found significant induction of FBXO3 and diminished FBXL2 protein along with elevated T-bet, IFN-γ, and TRAF proteins 1-5 compared with isografts. In the acute model, treatment with costimulation blockade (MR1/CTLA4-Ig) resulted in attenuated FBXO3, preserved FBXL2, and substantially reduced T-bet, IFN-γ, and TRAFs 1-5, consistent with a key role for type 1 alloimmunity. Immunohistochemistry revealed significant changes in the FBXO3/FBXL2 balance in airway epithelia and infiltrating mononuclear cells during rejection compared with isografts or costimulation blockade-treated allografts. In the chronic lung rejection model, DBA/2J/C57BL/6F1 > DBA/2J (day 28), we observed persistently elevated FBXO3/FBXL2 balance and T-bet/IFN-γ protein and similar findings from lung transplant recipient lungs with chronic lung allograft dysfunction versus controls. We hypothesized that FBXL2 regulated T-bet and found FBXL2 was sufficient to polyubiquitinate T-bet and coimmunoprecipitated with T-bet on pulldown experiments and vice versa in Jurkat cells. Transfection with FBXL2 diminished T-bet protein in a dose-dependent manner in mouse lung epithelial cells. In testing type 1 cytokines, TNF-α was found to negatively regulate FBXL2 protein and mRNA levels. Together, our findings show the combinatorial E3 ligase FBXO3/FBXL2 system plays a role in the regulation of T-bet through FBXL2, with negative cross-regulation of TNF-α on FBXL2 during lung allograft rejection.

Fibrosis of Chronic Lung Allograft Dysfunction After Orthotopic Lung Transplantation is Prevented in the Absence of Secondary Lymphoid Organs in a Mouse Model

<h3>Purpose</h3> It was reported that acute lung allograft rejection occurred without secondary lymphoid organs (SLOs) in a mouse allogenic lung transplantation model. The objective of this study was to investigate the role of SLOs in chronic lung allograft dysfunction (CLAD) after allogenic mouse lung transplantation using splenectomized alymphoplastic (aly/aly) mice as SLOs-deficient recipient. <h3>Methods</h3> Left lung of BALB/c mouse was transplanted into C57BL/6J (B6) or aly/aly mouse with B6 background. Low-dose cyclosporine (10 mg/kg, 3 times/week) and methylprednisolone (1.6 mg/kg, 3 times/week) were given in all the recipient mice until sacrifice. The recipient mice were sacrificed at day 15 (D15, n=4, each group) or day 30 (D30, n=5, each group) to evaluate histology. <h3>Results</h3> Allograft in B6 mice showed A2-3 rejection on D15 and A2-3 rejection with severe peri-airway and peri-vascular fibrosis on D30. On the contrary, allograft in aly/aly mouse showed A1-2 rejection on D15 and D30 without fibrosis (Figure 1). Quantification of peri-airway fibrosis thickness demonstrated significantly less fibrotic change in aly/aly mice than B6 mice on D30 (p = 0.012) (Figure 2). The A grade rejection score was significantly low on D30 in aly/aly mice (median A1, range A1-A2) than B6 mice (median A3, range A2-A3) (p = 0.015). <h3>Conclusion</h3> Fibrotic change with cellular rejection did not occur in the absence of SLOs in a mouse model of CLAD induced by inadequate immunosuppression, which may provide a platform for novel strategy to prevent CLAD.

Blocking hyaluronan synthesis alleviates acute lung allograft rejection.

Lung allograft rejection results in the accumulation of low–molecular weight hyaluronic acid (LMW-HA), which further propagates inflammation and tissue injury. We have previously shown that therapeutic lymphangiogenesis in a murine model of lung allograft rejection reduced tissue LMW-HA and was associated with improved transplant outcomes. Herein, we investigated the use of 4-Methylumbelliferone (4MU), a known inhibitor of HA synthesis, to alleviate acute allograft rejection in a murine model of lung transplantation. We found that treating mice with 4MU from days 20 to 30 after transplant was sufficient to significantly improve outcomes, characterized by a reduction in T cell–mediated lung inflammation and LMW-HA content and in improved pathology scores. In vitro, 4MU directly attenuated activation, proliferation, and differentiation of naive CD4+ T cells into Th1 cells. As 4MU has already been demonstrated to be safe for human use, we believe examining 4MU for the treatment of acute lung allograft rejection may be of clinical significance.

IL-17 Receptor on Donor Cells Regulates Acute and Chronic Lung Allograft Rejection Potentiated by Repeated Endotoxin Inhalations

<h3>Purpose</h3> Chronic lung allograft dysfunction is a major complication after lung transplantation (LTx). Excessive IL17 receptor A (IL17RA) signaling has been recognized as a key factor; however, its precise mechanism is unclear. In a mouse minor alloantigen mismatched LTx model (C57BL/10 [B10, H-2^b] → C57BL/6 [B6. H-2^b]), we found that repeated airway endotoxin (lipopolysaccharide, LPS) challenge augments airway epithelial damage, acute and chronic rejection with airway obliteration, together with increased IL17A transcription in allografts but not isografts. We hypothesized that both donor and recipient cell responses to IL17A are important in mediating these processes. We therefore investigated the role of IL17RA on donor and recipient cells in the context of chronic rejection. <h3>Methods</h3> B10→B6 LTx and B6→B10 LTx were performed with or without intratracheal LPS administration (5 ug LPS/50 ul PBS, Day 3, 7, 10, 14, 17, 21). B6 IL17RA^−/− mice were used as donors or recipients. Grafts were assessed with micro CT scans on days 3 and 27; histological scoring and quantitative PCR were performed 28 days after LTx. <h3>Results</h3> Without LPS, neither donor nor recipient IL17RA deficiency modified histological scores or lung graft volume or density by CT scan. LPS-treated IL17RA^−/− recipients showed a similar degree of lung radiographic change and histological acute rejection compared to IL17RA^+/+ recipients; however airway obliteration was attenuated (Figure A). In contrast, recipients of IL17RA^−/− donor lungs showed reduced gain of graft density on CT, and diminished acute rejection and peri-airway fibrosis (Figure B-C). IL17RA^−/− donor grafts had fewer CXCL1 (a neutrophil chemoattractant) transcripts (Figure D). <h3>Conclusion</h3> IL17RA signaling on donor cells regulates acute and chronic rejection in LTx recipients experiencing airway inflammation.

A post-preservation vascular flush removes significant populations of donor leukocytes prior to lung transplantation

BackgroundDonor leukocytes are intrinsically involved in acute lung allograft rejection, via self-presentation of donor antigens to recipient leukocytes. Therapeutic modalities to remove donor leukocytes are currently unavailable. We evaluated if a vascular flush immediately following preservation can be used for this purpose. MethodsA post-preservation flush was performed with STEEN solution in n = 6 porcine lungs following static cold storage. The first 500 ml effluent from the left atrium was collected and an inflammatory profile performed. ResultsA total of 1.17 billion (±2.8 × 108) viable leukocytes were identified within the effluent. T cells were the dominant cell population, representing 82% of the total mobilised leukocytes, of which <0.01% were regulatory T cells. IL-18 was the most abundant cytokine, with a mean concentration of 84,216 pg (±153,552 pg). In addition, there was a mean concentration of 8819 ng (±4415) cell-free mitochondrial DNA. ConclusionThere is an immediate transfer of donor leukocytes, cytokines and damage-associated molecular patterns following reperfusion. Such a pro-inflammatory donor load may enhance alloantigen presentation and drive recipient alloimmune responses. A post-preservation flush may therefore be an effective method for reducing the immune burden of the donor lung prior to transplantation.

Distinct molecular and immunological properties of circulating exosomes isolated from pediatric lung transplant recipients with bronchiolitis obliterans syndrome - a retrospective study.

Long-term success following human lung transplantation is poor due to chronic rejection. We demonstrated circulating exosomes of lung origin during acute and chronic lung allograft rejection. We analyzed plasma from pediatric lung transplant recipients (LTxRs) enrolled in the CTOT-C-03 to determine whether circulating exosomes are released into circulation during bronchiolitis obliterans syndrome (BOS). Plasma exosomes were isolated, and human leukocyte antigens (HLA) were detected. Exosomes were analyzed for lung self-antigens (SAgs), co-stimulatory molecules transcription factors, major histocompatibility complex class II (MHC-II), adhesion molecules, and 20S proteasome. Mice were immunized with exosomes from BOS or stable to determine their immunogenicity. Circulating exosomes from BOS LTxRs contained increased levels of SAgs, donor HLA class I, MHC-II, transcription factors, co-stimulatory molecules, and 20S proteasome compared with stable. Serial analysis of exosomes containing SAgs demonstrated that exosomes are detectable in the circulation before BOS. Mice immunized with exosomes from BOS, or stable, demonstrated that exosomes from BOS are distinct in inducing both humoral and cellular immune responses to SAgs. Circulating exosomes from BOS LTxRs elicit distinct humoral and cellular response. In addition, detection of SAgs on circulatory exosomes 12months before diagnosis of BOS suggest that exosomes could serve as biomarker.

IL-17 Receptor on Donor Cells Regulates Acute and Chronic Lung Allograft Rejection Potentiated by Repeated Endotoxin Inhalations

Chronic lung allograft dysfunction is a major complication after lung transplantation (LTx). Excessive IL17 receptor A (IL17RA) signaling has been recognized as a key factor; however, its precise mechanism is unclear. In a mouse minor alloantigen mismatched LTx model (C57BL/10 [B10, H-2b] → C57BL/6 [B6. H-2b]), we found that repeated airway endotoxin (lipopolysaccharide, LPS) challenge augments airway epithelial damage, acute and chronic rejection with airway obliteration, together with increased IL17A transcription in allografts but not isografts. We hypothesized that both donor and recipient cell responses to IL17A are important in mediating these processes. We therefore investigated the role of IL17RA on donor and recipient cells in the context of chronic rejection. B10→B6 LTX and B6→B10 LTx were performed with or without intratracheal LPS administration (5ug LPS/ 50ul PBS, Day 3, 7, 10, 14, 17, 21). B6 IL17RA-/- mice were used as donors or recipients. Grafts were assessed with micro CT scans on days 3 and 27; histological scoring and quantitative PCR were performed 28 days after LTx. Without LPS, neither donor nor recipient IL17RA deficiency modified histological scores or lung graft volume or density by CT scan. LPS-treated IL17RA-/- recipients showed a similar degree of lung radiographic change and histological acute rejection compared to IL17RA+/+ recipients; however airway obliteration was attenuated (Figure A). In contrast, recipients of IL17RA-/- donor lungs showed reduced gain of graft density on CT, and diminished acute rejection and periairway fibrosis (Figure B-C). IL17RA-/- donor grafts had fewer CXCL1 transcripts (Figure D). IL17RA signaling on donor cells regulates acute and chronic rejection in LTx recipients experiencing airway inflammation.

HDAC6-specific inhibitor suppresses Th17 cell function via the HIF-1α pathway in acute lung allograft rejection in mice.

Background: Previous animal experiments and clinical studies indicated the critical role of Th17 cells in lung transplant rejection. Therefore, the downregulation of Th17 cell function in lung transplant recipients is of great interest. Methods: We established an orthotopic mouse lung transplantation model to investigate the role of histone deacetylase 6-specific inhibitor (HDAC6i), Tubastatin A, in the suppression of Th17 cells and attenuation of pathologic lesions in lung allografts. Moreover, mechanism studies were conducted in vitro. Results: Tubastatin A downregulated Th17 cell function in acute lung allograft rejection, prolonged the survival of lung allografts, and attenuated acute rejection by suppressing Th17 cell accumulation. Consistently, exogenous IL-17A supplementation eliminated the protective effect of Tubastatin A. Also, hypoxia-inducible factor-1α (HIF-1α) was overexpressed in a lung transplantation mouse model. HIF-1α deficiency suppressed Th17 cell function and attenuated lung allograft rejection by downregulating retinoic acid-related orphan receptor γt (ROR γt) expression. We showed that HDAC6i downregulated HIF-1α transcriptional activity under Th17-skewing conditions in vitro and promoted HIF-1α protein degradation in lung allografts. HDAC6i did not affect the suppression of HIF-1α-/- naïve CD4+ T cell differentiation into Th17 cell and attenuation of acute lung allograft rejection in HIF-1α-deficient recipient mice. Conclusion: These findings suggest that Tubastatin A downregulates Th17 cell function and suppresses acute lung allograft rejection, at least partially, via the HIF-1α/ RORγt pathway.

Vendor-specific microbiome controls both acute and chronic murine lung allograft rejection by altering CD4+ Foxp3+ regulatory T cell levels.

Despite standardized postoperative care, some lung transplant patients suffer multiple episodes of acute and chronic rejection while others avoid graft problems for reasons that are poorly understood. Using an established model of C57BL/10 to C57BL/6 minor antigen mismatched single lung transplantation, we now demonstrate that the recipient microbiota contributes to variability in the alloimmune response. Specifically, mice from the Envigo facility in Frederick, Maryland contain nearly double the number of CD4+ Foxp3+ regulatory T cells (Tregs ) than mice from the Jackson facility in Bar Harbor, Maine or the Envigo facility in Indianapolis, Indiana (18 vs 9 vs 7%). Lung graft recipients from the Maryland facility thus do not develop acute or chronic rejection. Treatment with broad-spectrum antibiotics decreases Tregs and increases both acute and chronic graft rejection in otherwise tolerant strains of mice. Constitutive depletion of regulatory T cells, using Foxp3-driven expression of diphtheria toxin receptor, leads to the development of chronic rejection and supports the role of Tregs in both acute and chronic alloimmunity. Taken together, our data demonstrate that the microbiota of certain individuals may contribute to tolerance through Treg -dependent mechanisms and challenges the practice of indiscriminate broad-spectrum antibiotic use in the perioperative period.

FCGR3A and FCGR2A Genotypes Differentially Impact Allograft Rejection and Patients' Survival After Lung Transplant.

Fc gamma receptors (FcγRs) play a major role in the regulation of humoral immune responses. Single-nucleotide polymorphisms (SNPs) of FCGR2A and FCGR3A can impact the expression level, IgG affinity and function of the CD32 and CD16 FcγRs in response to their engagement by the Fc fragment of IgG. The CD16 isoform encoded by FCGR3A [158V/V] controls the intensity of antibody-dependent cytotoxic alloimmune responses of natural killer cells (NK) and has been identified as a susceptibility marker predisposing patients to cardiac allograft vasculopathy after heart transplant. This study aimed to investigate whether FCGR2A and FCGR3A polymorphisms can also be associated with the clinical outcome of lung transplant recipients (LTRs). The SNPs of FCGR2A ([131R/H], rs1801274) and FCGR3A ([158V/F], rs396991) were identified in 158 LTRs and 184 Controls (CTL). The corresponding distribution of genotypic and allelic combinations was analyzed for potential links with the development of circulating donor-specific anti-HLA alloantibodies (DSA) detected at months 1 and 3 after lung transplant (LTx), the occurrence of acute rejection (AR) and chronic lung allograft dysfunction (CLAD), and the overall survival of LTRs. The FCGR3A [158V/V] genotype was identified as an independent susceptibility factor associated with higher rates of AR during the first trimester after LTx (HR 4.8, p < 0.0001, 95% CI 2.37–9.61), but it could not be associated with the level of CD16- mediated NK cell activation in response to the LTR's DSA, whatever the MFI intensity and C1q binding profiles of the DSA evaluated. The FCGR2A [131R/R] genotype was associated with lower CLAD-free survival of LTRs, independently of the presence of DSA at 3 months (HR 1.8, p = 0.024, 95% CI 1.08–3.03). Our data indicate that FCGR SNPs differentially affect the clinical outcome of LTRs and may be of use to stratify patients at higher risk of experiencing graft rejection. Furthermore, these data suggest that in the LTx setting, specific mechanisms of humoral alloreactivity, which cannot be solely explained by the complement and CD16-mediated pathogenic effects of DSA, may be involved in the development of acute and chronic lung allograft rejection.

Microbiome Dependent Regulatory T Cell Generation Correlates with Acute and Chronic Lung Allograft Rejection in a Murine Model

Purpose Despite the growing experience in lung transplantation a significant number of patients rapidly succumb to chronic rejection in the form of bronchiolitis obliterans (OB). The reasons for such discrepancies are unknown but have been suspected to relate to individual variability in gut and lung microbiome. Methods To study the role of the microbiome in controlling lung immunology we transplanted C57BL/10 lungs to C57BL/6 recipients from either Jackson Lab, Maine or Envigo facilities in Maryland vs Indiana. Histological grading of acute and chronic rejection, in the form of OB was performed on days 7 and 21 respectively with flow cytometric analysis of the lungs in parallel. Results Recipients from the Maine facility rapidly rejected lung grafts with high ISHLT rejection grades (2.8±0.37) by day 7 while Maryland recipients were rejection-free (0.25±0.25). Delayed histologic evaluation on day 21 post-engraftment revealed evidence of OB in 40% of Indiana recipients while 0% in Maine or Maryland mice(Fig.1A). Flow cytometric analysis revealed a high prevalence of CD4+Foxp3+ Tregs in Maryland recipients (29.56±2.32%) but lower in Maine and Indiana mice while T cell IL-17 levels were also different between them. Systemic different TGF-β levels were also observed. Treatment of Maryland recipients with broad-spectrum antibiotics affected TGF-β levels, decreased the Treg proportion (14.24±1.47%, p Conclusion Our data demonstrate that mice from different origins mount different lung allograft-specific microbiome-related immune responses. Furthermore it suggests that microbiome-dependent TGF beta levels may contribute to the generation of protective Tregs and ameliorate both acute and chronic rejection. Such data puts into question the notion that TGF-β contributes to lung fibrosis and opens up therapeutic avenues of microbiome manipulation to augment the TGF-β -Treg axis.

Stem cell secretome attenuates acute rejection in rat lung allotransplant.

Stem cells secrete significant amounts of bioactive factors in their secretome that can be immunosuppressive. We studied the effect of the secretome obtained from bone marrow-derived mesenchymal stem cells (BMSC-sec) in combination with cyclosporine A following acute rejection of lung allografts in the rat. Lung allotransplants were performed from male Brown Norway donor rats to recipient male Fisher 344 rats. Rat BMSC-sec was introduced intratracheally in the recipient every day after the transplant until the day the animal was sacrificed. Group A (n = 5) received control medium and cyclosporine A (2.5 mg/kg body weight intraperitoneally) for 5 days post-transplant and group B (n = 5) received BMSC-sec and cyclosporine A. Blood gas analysis was performed to assess graft function at day 5 only from the graft, and the tissue was sampled for measurement of the wet/dry ratio and histological grading of rejection. All control animals (group A) showed severe signs of rejection. At day 5 grafts in group B showed improved gas exchange (i.e. mean PaO2 mmHg 237.9 ± 130 mmHg vs 24.9 ± 7.8 mmHg in group A). Histological examination according to the International Society of Heart and Lung Transplantation (ISHLT) revealed moderate to severe rejection in all animals in group A (III B) and a significant improvement in group B (I-IIA). The wet/dry ratio was also reduced in group B to 6.19 ± 0.6 compared to 9.36 ± 2 in group A. Furthermore, in vitro T-cell proliferation was reduced after treatment with BMSC-sec for CD 3 cells (69.55 ± 07 vs 73 ± 0.84), for CD 4 (24.95 ± 1.2 vs 27.75 ± 0.21) and for CD 8 cells (3.75 ± 0.2 vs 5.68 ± 0.02). The BMSC-sec is a promising novel cell-based therapeutic option for acute rejection in a rat lung allograft model.

Recipient Emphysema Differentially Affects Immunologic Responses During Acute Lung Allograft Rejection in a Pre-Clinical Model

Immune-modifying processes such as IPF and COPD result in a recipient environment that can be hostile to allografts by, as of yet, ill-defined mechanisms. In an era driving towards personalized medicine, a better understanding of the potentially differing immune mechanisms in different populations may identify novel therapeutic approaches to improve outcomes. Here we utilize a systems biology approach to determine the impact of recipient pre-transplant emphysema on post transplant graft survival.

Azithromycin Fails to Prevent Accelerated Airway Obliteration in T-bet-/- Mouse Lung Allograft Recipients

BackgroundCellular and molecular mechanisms of acute and chronic lung allograft rejection have yet to be clearly defined, and obliterative bronchiolitis (OB) remains the primary limitation to survival in lung transplant recipients (LTRs). We have previously shown that T-bet–deficient recipients of full major histocompatibility complex (MHC)-mismatched, orthotopic left lung transplants develop accelerated obliterative airway disease (OAD) in the setting of acute cellular rejection characterized by robust alloimmune CD8+ interleukin (IL)-17 and interferon (IFN)-γ responses that are attenuated with neutralization of IL-17. Azithromycin has been shown to be beneficial in some LTRs with bronchiolitis obliterans syndrome/OB. Here, we evaluated the effects of azithromycin on rejection pathology and T-cell effector responses in T-bet-/- recipients of lung transplants. MethodsOrthotopic left lung transplantation was performed in BALB/c → B6 wild type or BALB/c → B6 T-bet-/- strain combinations as previously described. Mice treated with azithromycin received 10 mg/kg or 50 mg/kg subcutaneously daily. Lung allograft histopathology was analyzed at day 10 or day 21 post-transplantation, and neutrophil staining for quantification was performed using anti-myeloperoxidase. Allograft mononuclear cells were isolated at day 10 for T-cell effector cytokine response assessment using flow cytometry. ResultsWe show that while azithromycin significantly decreases lung allograft neutrophilia and CXCL1 levels and attenuates allospecific CD8+ IL-17 responses early post-transplantation, OAD persists in T-bet–deficient mice. ConclusionsOur results indicate that lung allograft neutrophilia is not essential for the development of OAD in this model and suggest allospecific T-cell responses that remain despite marked attenuation of CD8+ IL-17 are sufficient for obliterative airway inflammation and fibrosis.

Bone marrow stem cells modified with human interleukin 10 attenuate acute rejection in rat lung allotransplantation.

The aim of this study was to investigate new therapeutic options to attenuate acute rejection in a rat lung allograft model. Cell-based gene therapies have recently been reported as a novel curative option in acute and chronic diseases for which conventional treatments are not available. We studied the effect of human interleukin 10 (hIL-10) on expressing bone marrow-derived mesenchymal stem cells (BMSCs) in combination with cyclosporine A (CsA) on acute rejection of lung allografts in the rat. Lung allotransplantation was performed from male Brown Norway donor to male Fisher (F344) rats. Rat BMSCs were transfected with hIL-10 in vitro and introduced in the graft prior to implantation. Group A (n = 5) received CsA intraperitoneally (2.5 mg/kg body weight) for 5 days post-transplant; Group B (n = 5) received BMSC and CsA and Group C (n = 5) received hIL-10-BMSC before implantation and CsA. Graft function was assessed by blood gas levels only from the graft on day 5; tissue was sampled for histological grading of rejection and measurement of the wet-to-dry ratio. All Group A control animals showed severe signs of rejection. On Day 5, all grafts in Group C showed improved gas exchange (mean arterial partial pressure of oxygen 222.2 ± 40.38 mmHg vs 92.36 ± 20.92 mmHg in Group B and 42.72 ± 18.07 mmHg in Group A). Histological examination revealed moderate-to-severe rejection in all animals in Group A [International Society for Heart and Lung Transplantation Level III B (ISHLT)] in contrast to low-to-moderate rejection in Group B (II-IIIA) and much improved histological grade in Group C (I-IIA). Moreover, the wet-to-dry ratio was also reduced in Group C (4.8 ± 1.19 compared with 4.78 ± 0.62 in Group B and 9.36 ± 0.90 in Group A). The hIL-10 BMSC represent a promising novel method for localized cell-based gene therapy for acute rejection in a rat lung allograft model.

Rapid detection of donor cell free DNA in lung transplant recipients with rejections using donor-recipient HLA mismatch

Fiberoptic bronchoscopy and transbronchial lung biopsy are currently the gold standard for detection of acute rejection following human lung transplantation (LTx). However, these surveillance procedures are expensive and invasive. Up to now, there are few new methods that have demonstrated clinical utility for detecting early stages of rejection following human lung transplantation. We optimized and technically validated a novel method to quantify donor-derived circulating cell free DNA (DcfDNA) that can be used as an early biomarker for lung allograft rejection. The method involves the initial development of a panel of probes in which each probe will specifically target a unique sequence of a human leukocyte antigen (HLA) allele. After transplantation, donor/recipient specific probes are chosen based on the mismatched HLA loci, followed by droplet digital PCR (ddPCR) used as a quantitative assay to accurately track the trace amount of DcfDNA in an ample excess of recipient DNA background. The average false positive rate noted was about 1 per 800,000 molecules. Serially 2-fold diluted cfDNA, representing donor fractions of cfDNA, were spiked into a constant level of cfDNA representing the recipient cfDNA. The fraction of spiked cfDNA was measured and quantitative linearity was observed across seven serially diluted cfDNA samples. We were able to measure the minor portion of cfDNA as low as 0.2% of total cfDNA. We subsequently applied the method to a pilot set of 18 LTx recipients grouped into biopsy-proven acute rejection, bronchiolitis obliterans syndrome (BOS) or stable groups. Serial plasma samples were used to identify the percentage of DcfDNA over total cfDNA. The level of DcfDNA was significantly elevated in patients diagnosed with acute rejection (10.30±2.80, n=18), compared to that from stable (1.71±0.50, n=24) or from BOS patients (2.52±0.62, n=20). In conclusion, we present results validating the application of digital PCR to quantify DcfDNA assay in primary clinical specimens, which demonstrate that DcfDNA can be used as an early non-invasive biomarker for acute lung allograft rejection.

Donor-derived exosomes: key in lung allograft rejection?

In a recent publication, Gunasekaran and colleagues conducted a case-control observational study in patients who underwent bilateral lung transplantation (LTx), determining whether exosomes are generated during allograft rejection and define their origin and antigenic phenotype (1). LTx is the sole effective treatment to save the life for patients with end-stage lung disorders (2). One of the major drawbacks for the clinical outcome of LTx is allograft rejection by acute rejection (AR) or bronchiolitis obliterans syndrome (BOS) (2). A link between autoimmunity and immune responses to tissue restricted self-antigens (SAgs) such as K-alpha-1-tubulin (K-α1T) and Collagen V (Col-V) with acute or chronic lung allograft rejection has been proposed (3,4). However, the mechanisms underlying the evolution of acute or chronic lung rejection are poorly understood.

Donor-Reactive Regulatory T Cell Frequency Increases During Acute Cellular Rejection of Lung Allografts.

Acute cellular rejection is a major cause of morbidity after lung transplantation. Because regulatory T (Treg) cells limit rejection of solid organs, we hypothesized that donor-reactive Treg increase after transplantation with development of partial tolerance and decrease relative to conventional CD4 (Tconv) and CD8 T cells during acute cellular rejection. To test these hypotheses, we prospectively collected 177 peripheral blood mononuclear cell specimens from 39 lung transplant recipients at the time of transplantation and during bronchoscopic assessments for acute cellular rejection. We quantified the proportion of Treg, CD4 Tconv, and CD8 T cells proliferating in response to donor-derived, stimulated B cells. We used generalized estimating equation-adjusted regression to compare donor-reactive T cell frequencies with acute cellular rejection pathology. An average of 16.5 ± 9.0% of pretransplantation peripheral blood mononuclear cell Treg cell were donor-reactive, compared with 3.8% ± 2.9% of CD4 Tconv and 3.4 ± 2.6% of CD8 T cells. These values were largely unchanged after transplantation. Donor-reactive CD4 Tconv and CD8 T cell frequencies both increased 1.5-fold (95% confidence interval [95% CI], 1.3-1.6; P < 0.001 and 95% CI, 1.2-1.6; P = 0.007, respectively) during grade A2 rejection compared with no rejection. Surprisingly, donor-reactive Treg frequencies increased by 1.7-fold (95% CI, 1.4-1.8; P < 0.001). Contrary to prediction, overall proportions of donor-reactive Treg cells are similar before and after transplantation and increase during grade A2 rejection. This suggests how A2 rejection can be self-limiting. The observed increases over high baseline proportions in donor-reactive Treg were insufficient to prevent acute lung allograft rejection.