B cell reconstitution following alemtuzumab induction under a belatacept‐based maintenance regimen

Lymphocyte depletion has been shown to control costimulation blockade‐resistant rejection but, in some settings, to exacerbate antibody‐mediated rejection (AMR). We have used alemtuzumab, which depletes T and B cells, combined with belatacept and rapamycin and previously reported control of both costimulation blockade‐resistant rejection and AMR. To evaluate this regimen's effect on B cell signatures, we investigated 40 patients undergoing this therapy. B cell counts and phenotypes were interrogated using flow cytometry, and serum was analyzed for total IgG, IgM, and donor‐specific alloantibody (DSA). Alemtuzumab induction produced pan‐lymphocyte depletion; B cells repopulated faster and more completely than T cells. Reconstituting B cells were predominantly naïve, and memory B cells were significantly reduced (P = .001) post repopulation. Two B cell populations with potential immunomodulatory effects—regulatory (CD38^hiCD24^hiIgM^hiCD20^hi) and transitional B cells (CD19⁺CD27^?IgD⁺CD38^hi)—were enriched posttransplant (P = .001). Total serum IgG decreased from baseline (P = .016) while IgM levels remained stable. Five patients developed DSAs within 36 months posttransplant, but none developed AMR. Baseline IgG levels in these patients were significantly higher than those in patients without DSAs. These findings suggest that belatacept and rapamycin together limit homeostatic B cell activation following B cell depletion and may lessen the risk of AMR. This regimen warrants prospective, comparative study. ClinicalTrials.gov NCT00565773.     

Pseudomonas aeruginosa Induced Airway Epithelial Injury Drives Fibroblast Activation: A Mechanism in Chronic Lung Allograft Dysfunction

Bacterial infections after lung transplantation cause airway epithelial injury and are associated with an increased risk of developing bronchiolitis obliterans syndrome. The damaged epithelium is a source of alarmins that activate the innate immune system, yet their ability to activate fibroblasts in the development of bronchiolitis obliterans syndrome has not been evaluated. Two epithelial alarmins were measured longitudinally in bronchoalveolar lavages from lung transplant recipients who developed bronchiolitis obliterans syndrome and were compared to stable controls. In addition, conditioned media from human airway epithelial cells infected with Pseudomonas aeruginosa was applied to lung fibroblasts and inflammatory responses were determined. Interleukin‐1 alpha (IL‐1α) was increased in bronchoalveolar lavage of lung transplant recipients growing P. aeruginosa (11.5 [5.4–21.8] vs. 2.8 [0.9–9.4] pg/mL, p < 0.01) and was significantly elevated within 3 months of developing bronchiolitis obliterans syndrome (8.3 [1.4–25.1] vs. 3.6 [0.6–17.1] pg/mL, p < 0.01), whereas high mobility group protein B1 remained unchanged. IL‐1α positively correlated with elevated bronchoalveolar lavage IL‐8 levels (r² = 0.6095, p < 0.0001) and neutrophil percentage (r² = 0.25, p = 0.01). Conditioned media from P. aeruginosa infected epithelial cells induced a potent pro‐inflammatory phenotype in fibroblasts via an IL‐1α/IL‐1R‐dependent signaling pathway. In conclusion, we propose that IL‐1α may be a novel therapeutic target to limit Pseudomonas associated allograft injury after lung transplantation.     

Cytotoxicity of Natural Killer Cells Activated Through NKG2D Contributes to the Development of Bronchiolitis Obliterans in a Murine Heterotopic Tracheal Transplant Model

Bronchiolitis obliterans after lung transplantation is a major cause of postoperative mortality in which T cell–mediated immunity is known to play an important role. However, the exact contribution of natural killer (NK) cells, which have functions similar to CD8⁺ T cells, has not been defined. Here, we assessed the role of NK cells in murine bronchiolitis obliterans through heterotopic tracheal transplantations and found a greater percentage of NK cells in allografts than in isografts. Depletion of NK cells using an anti‐NK1.1 antibody attenuated bronchiolitis obliterans in transplant recipients compared with controls. In terms of NK cell effector functions, an improvement in bronchiolitis obliterans was observed in perforin‐KO recipient mice compared to wild type (WT). Furthermore, we found upregulation of NKG2D‐ligand in allografts and demonstrated the significance of this using grafts expressing Rae‐1, a murine NKG2D‐ligand, which induced severe bronchiolitis obliterans in WT and Rag‐1 KO recipients. This effect was ameliorated by injection of anti‐NKG2D blocking antibody. Together, these results suggest that cytotoxicity resulting from activation of NK cells through NKG2D leads to the development of murine bronchiolitis obliterans.     

Absence of evidence that respiratory viral infections influence pediatric lung transplantation outcomes: Results of the CTOTC‐03 study

Based on reports in adult lung transplant recipients, we hypothesized that community‐acquired respiratory viral infections (CARVs) would be a risk factor for poor outcome after pediatric lung transplant. We followed 61 pediatric lung transplant recipients for 2+ years or until they met a composite primary endpoint including bronchiolitis obliterans syndrome/obliterative bronchiolitis, retransplant, or death. Blood, bronchoalveolar lavage, and nasopharyngeal specimens were obtained with standard of care visits. Nasopharyngeal specimens were obtained from recipients with respiratory viral symptoms. Respiratory specimens were interrogated for respiratory viruses by using multiplex polymerase chain reaction. Donor‐specific HLA antibodies, self‐antigens, and ELISPOT reactivity were also evaluated. Survival was 84% (1 year) and 68% (3 years). Bronchiolitis obliterans syndrome incidence was 20% (1 year) and 38% (3 years). The primary endpoint was met in 46% of patients. CARV was detected in 156 patient visits (74% enterovirus/rhinovirus). We did not find a relationship between CARV recovery from respiratory specimens and the primary endpoint (hazard ratio 0.64 [95% confidence interval: 0.25‐1.59], P = .335) or between CARV and the development of alloimmune or autoimmune humoral or cellular responses. These findings raise the possibility that the immunologic impact of CARV following pediatric lung transplant is different than that observed in adults.     

Calcineurin and mTOR inhibitors have opposing effects on regulatory T cells while reducing regulatory B cell populations in kidney transplant recipients

BackgroundRegulatory B (Breg) and T (Treg) cells represent a biomarker for tolerance in transplant patients. Despite the importance of Treg and Breg in transplantation and the suggested crosstalk between both suppressive cell populations, little is known on how they are influenced by long-term immunosuppressive treatment. The aim of the present study was to investigate the effect of different immunosuppressive drugs used in routine clinical practice on Treg and Breg cell numbers.MethodsThirty-six kidney transplant recipients with stable graft function were recruited and classified according to their concomitant therapy: 22 patients received calcineurin inhibitors (CNI) and 14 patients received mammalian target of rapamycin (mTOR) inhibitors. A group of 8 healthy untreated subjects was included as control. Absolute numbers of peripheral blood-derived IL10-producing B cells (CD19⁺IL10⁺), CD19⁺CD24^hiCD38^hi transitional B cells and Treg cells (CD4⁺CD25⁺FOXP3⁺) were quantified in all KT patients and controls by flow cytometry.ResultsCD19⁺CD24^hiCD38^hi transitional B cells increased over time and seem to be related with long-term therapeutic graft survival irrespective of the treatment regimen. CNI and mTOR inhibitors significantly reduced numbers of Breg cells when compared with healthy individuals, whereas mTOR inhibitors expanded Treg cells in comparison with CNI drugs.ConclusionsBridging the drug-mediated reduction of Breg cell numbers in vivo with the requirements of Breg cells for long-term transplant success remains an as yet unresolved task for therapeutic intervention. Further larger cohort studies that evaluate the effect of different treatment regimen on defined lymphocyte subpopulations are warranted.     

T Cells Promote Bronchial Epithelial Cell Secretion of Matrix Metalloproteinase‐9 via a C‐C Chemokine Receptor Type 2 Pathway: Implications for Chronic Lung Allograft Dysfunction

Chronic lung allograft dysfunction (CLAD) is the major limitation of long‐term survival after lung transplantation. CLAD manifests as bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS). Alloimmune reactions and epithelial‐to‐mesenchymal transition have been suggested in BOS. However, little is known regarding the role of allogenicity in epithelial cell differentiation. Primary human bronchial epithelial cells (BECs) were treated with activated T cells in the presence or absence of transforming growth factor (TGF)‐β. The expression of epithelial and mesenchymal markers was investigated. The secretion of inflammatory cytokines and matrix metalloproteinase (MMP)‐9 was measured in culture supernatants and in plasma from lung transplant recipients (LTRs): 49 stable, 29 with BOS, and 16 with RAS. We demonstrated that C‐C motif chemokine 2 secreted by T cells supports TGF‐β–induced MMP‐9 production by BECs after binding to C‐C chemokine receptor type 2. Longitudinal investigation in LTRs revealed a rise in plasma MMP‐9 before CLAD onset. Multivariate analysis showed that plasma MMP‐9 was independently associated with BOS (odds ratio [OR] = 6.19, p = 0.002) or RAS (OR = 3.9, p = 0.024) and predicted the occurrence of CLAD 12 months before the functional diagnosis. Thus, immune cells support airway remodeling through the production of MMP‐9. Plasma MMP‐9 is a potential predictive biomarker of CLAD.     

Multicenter evaluation of parametric response mapping as an indicator of bronchiolitis obliterans syndrome after hematopoietic stem cell transplantation

Parametric response mapping (PRM) is a novel computed tomography (CT) technology that has shown potential for assessment of bronchiolitis obliterans syndrome (BOS) after hematopoietic stem cell transplantation (HCT). The primary aim of this study was to evaluate whether variations in image acquisition under real‐world conditions affect the PRM measurements of clinically diagnosed BOS. CT scans were obtained retrospectively from 72 HCT recipients with BOS and graft‐versus‐host disease from Fred Hutchinson Cancer Research Center, Karolinska Institute, and the University of Michigan. Whole lung volumetric scans were performed at inspiration and expiration using site‐specific acquisition and reconstruction protocols. PRM and pulmonary function measurements were assessed. Patients with moderately severe BOS at diagnosis (median forced expiratory volume at 1 second [FEV1] 53.5% predicted) had similar characteristics between sites. Variations in site‐specific CT acquisition protocols had a negligible effect on the PRM‐derived small airways disease (SAD), that is, BOS measurements. PRM‐derived SAD was found to correlate with FEV1% predicted and FEV1/ forced vital capacity (R = ?0.236, P = .046; and R = ?0.689, P < .0001, respectively), which suggests that elevated levels in the PRM measurements are primarily affected by BOS airflow obstruction and not CT scan acquisition parameters. Based on these results, PRM may be applied broadly for post‐HCT diagnosis and monitoring of BOS.     

Immune responses following tocilizumab therapy to desensitize HLA-sensitized kidney transplant candidates

Kidney transplant candidates (KTCs) who are HLA highly sensitized (calculated panel-reactive alloantibodies >95%) have poor access to deceased kidney transplantation. In this single-center prospective study, 13 highly sensitized desensitization-naïve KTCs received IV tocilizumab (8 mg/kg) every 4 weeks. We evaluated tolerability as well as immune responses, that is, T cell, B cell, T follicular helper (Tfh) subsets, blood cytokines (IL-6, soluble IL-6 receptor-sIL-6R-, IL-21), blood chemokines (CXCL10, CXCL13), and anti-HLA alloantibodies. Tocilizumab treatment was well-tolerated except in one patient who presented spondylodiscitis, raising a note of caution. Regarding immune parameters, there were no significant changes of percentages of lymphocyte subsets, that is, CD3⁺, CD3⁺/CD4⁺, CD3⁺/CD8⁺ T cells, and NK cells. This was also the case for Tfh cell subsets, B cells, mature B cells, plasma cells, pre-germinal center (GC) B cells, and post-GC B cells, whereas we observed a significant increase in naïve B cells (p = .02) and a significant decrease in plasmablasts (p = .046) over the tocilizumab treatment course. CXCL10, CXCL13, IL-21, total IgG, IgA, and IgM levels did not significantly change during tocilizumab therapy; conversely, there was a significant increase in IL-6 levels (p = .03) and a huge increase in sIL-6R (p = .00004). There was a marginal effect on anti-HLA alloantibodies (class I and class II). To conclude in highly sensitized KTCs, tocilizumab as a monotherapy limited B cell maturation; however, it had almost no effect on anti-HLA alloantibodies.     

Dynamic changes of B‐cell compartments in kidney transplantation: lack of transitional B cells is associated with allograft rejection

B cells play an important role in the immune responses which affect the outcomes of kidney allografts. Dynamic changes of B‐cell compartments in clinical kidney transplantation are still poorly understood. B‐cell subsets were prospectively monitored using flow cytometry for 1 year in 98 kidney transplant recipients. Data were correlated with immunosuppression and clinical outcomes. An increase in the total population of B lymphocytes was observed during the first week after transplantation. The level of IgM^highCD38^highCD24^high transitional B cells reduced significantly up until the third month, with partial repopulation in the first year. Lower numbers of transitional B cells in the third month were associated with higher risk of graft rejection. IgM⁺IgD⁺CD27^? naive B cells did not change within follow‐up. IgM⁺CD27⁺ nonswitched memory B cells and IgM^?CD27⁺ switched memory B cells increased on post‐operative day 7. IgM^?CD38^highCD27^high plasmablasts showed similar kinetics during the first post‐transplant year, similar to transitional B cells. In conclusion, sensitized kidney transplant recipients as well as those with either acute or chronic rejection within the first post‐transplant year exhibited lower levels of transitional B cells. Therefore, these data further support the hypothesis that transitional B cells have a protective role in kidney transplantation.     

Decrease of immature B cell and interleukin-10 during early-post-transplant period in renal transplant recipients under tacrolimus based immunosuppression

B lymphocytes are known to play a role in kidney transplantation (KT) outcomes. Here, we evaluated the proportion of B cell subsets before and after KT. Twenty-one patients, who showed stable allograft function without acute rejection in the month following the KT, were included in this study. Peripheral blood samples were obtained from these patients before transplantation as well as 1 month after transplantation. Changes in the proportion of B cell subsets after transplantation were investigated using multi-color flow cytometry. The proportion of lymphocytes in the peripheral blood mononuclear cells (PBMCs) and of CD19⁺ B cells in the total leukocyte population did not change after KT. Similarly, the proportions of CD19⁺CD24⁺ lymphocytes, mature B cells (CD24^IntCD38^Inter/CD19⁺), and memory B cells (CD24⁺CD38^?/CD19⁺) did not change post-KT. However, the proportion of immature B cells (CD24⁺CD38⁺/CD19⁺ B cells) decreased significantly after transplantation (P < 0.01). The levels of IL-10, and IL-21, and expression of the B cell marker BLNK also decreased significantly after transplantation. Incubation of PBMCs with tacrolimus (0.1, 1, and 10 ng/mL) and mycophenolate mofetil (200 μg/mL) an immunosuppressant, resulted in significant reduction in the percentage of immature B cells. In contrast, the proportion of memory and mature B cells was not affected. Taken together, these results show that while the total B lymphocyte count and the proportion of memory/mature B cell subsets do not change after KT, the proportion of immature B cells and the associated cytokines that they secrete decrease significantly.     

Contrasting effects of B cell depletion on CD4+ and CD8+ memory T cell responses generated after transplantation

Alloreactive memory T cells play a key role in transplantation by accelerating allograft rejection and preventing tolerance induction. Some studies using µMT mice, which are constitutionally devoid of B cells, showed that B cells were required for the generation of memory T cells after allotransplantation. However, whether B cell depletion in normal adult mice has the same effect on memory responses by CD4⁺ and CD8⁺ T cells activated after transplantation has not been thoroughly investigated. In this study, we tested the effect of anti‐CD20 antibody‐mediated B cell depletion on CD4⁺ and CD8⁺ memory T cell alloresponses after skin transplantation in wild‐type mice. We found that B cell depletion prevented the development of memory alloresponses by CD4⁺ T cells but enhanced that of CD8⁺ memory T cells. Next, we tested the influence of B cell depletion on hematopoietic chimerism. In OT‐II CD4⁺ anti‐OVA TCR transgenic mice sensitized to ovalbumin antigen, B cell depletion also impaired allospecific memory T cell responses and thereby enhanced donor hematopoietic chimerism and T cell deletion after bone marrow transplantation. This study underscores the complexity of the relationships between B and T cells in the generation and reactivation of different memory T cell subsets after transplantation.     

In Vivo Mobilization and Functional Characterization of Nonhuman Primate Monocytic Myeloid‐Derived Suppressor Cells

Increasing evidence from small animal models shows that myeloid‐derived suppressor cells (MDSCs) can play a crucial role in inhibiting allograft rejection and promoting transplant tolerance. We identified CD3^?CD20^?HLA‐DR^?CD14⁺CD33⁺CD11b⁺ cells in peripheral blood of healthy rhesus macaques. These putative monocytic MDSCs constituted 2.1% ± 1.7% of lin^?HLA‐DR^? peripheral blood mononuclear cells. Administration of granulocyte‐macrophage colony‐stimulating factor (CSF) and granulocyte CSF increased their incidence to 5.3% ± 3.4%. The total number of MDSCs that could be flow sorted from a single whole rhesus leukapheresis product was 38 ± 13 × 10⁶ (n = 10 monkeys). Freshly isolated or cryopreserved MDSCs from mobilized monkeys incorporated in cultures of anti‐CD3– and anti‐CD28–stimulated autologous T cells markedly suppressed CD4⁺ and CD8⁺ T cell proliferation and cytokine secretion (interferon γ, IL‐17A). Moreover, these MDSCs enhanced CD4⁺CD25^hiFoxp3⁺ regulatory T cell (Treg) expansion while inhibiting proliferation of activated memory T cells and increasing Treg relative to effector and terminally differentiated memory T cells. Inhibition of arginase‐1, but not inducible nitric oxide synthase activity, partially reversed the inhibitory effect of the MDSCs on CD8⁺ T cell proliferation. Consequently, functional MDSCs can be isolated from nonhuman primates for prospective use as therapeutic cellular vaccines in transplantation.

     

Microvasculature partial endothelial mesenchymal transition in early posttransplant biopsy with acute tubular necrosis identifies poor recovery renal allografts

Acute tubular necrosis (ATN), a frequent histopathological feature in the early post–renal transplant biopsy, affects long‐term graft function. Appropriate markers to identify patients at risk of no or incomplete recovery after delayed graft function are lacking. In this study, we first included 41 renal transplant patients whose biopsy for cause during the first month after transplantation showed ATN lesions. Using partial microvasculature endothelial (fascin, vimentin) and tubular epithelial (vimentin) to mesenchymal transition markers, detected by immunohistochemistry, we found a significant association between partial endothelial to mesenchymal transition and poor graft function recovery (Spearman's rho = ?0.55, P = .0005). Transforming growth factor–β1 was strongly expressed in these phenotypic changed endothelial cells. Extent of ATN was also correlated with short‐ and long‐term graft dysfunction. However, the association of extensive ATN with long‐term graft dysfunction (24 months posttransplant) was observed only in patients with partial endothelial to mesenchymal transition marker expression in their grafts (Spearman's rho = ?0.64, P = .003), but not in those without. The association of partial endothelial to mesenchymal transition with worse renal graft outcome was confirmed on 34 other early biopsies with ATN from a second transplant center. Our results suggest that endothelial cell activation at the early phase of renal transplantation plays a detrimental role.     

Rejection of xenogeneic porcine islets in humanized mice is characterized by graft‐infiltrating Th17 cells and activated B cells

Xenogeneic porcine islet transplantation is a promising potential therapy for type 1 diabetes (T1D). Understanding human immune responses against porcine islets is crucial for the design of optimal immunomodulatory regimens for effective control of xenogeneic rejection of porcine islets in humans. Humanized mice are a valuable tool for studying human immune responses and therefore present an attractive alternative to human subject research. Here, by using a pig‐to‐humanized mouse model of xenogeneic islet transplantation, we described the human immune response to transplanted porcine islets, a process characterized by dense islet xenograft infiltration of human CD45⁺ cells comprising activated human B cells, CD4⁺CD44⁺IL‐17⁺ Th17 cells, and CD68⁺ macrophages. In addition, we tested an experimental immunomodulatory regimen in promoting long‐term islet xenograft survival, a triple therapy consisting of donor splenocytes treated with ethylcarbodiimide (ECDI‐SP), and peri‐transplant rituximab and rapamycin. We observed that the triple therapy effectively inhibited graft infiltration of T and B cells as well as macrophages, promoted transitional B cells both in the periphery and in the islet xenografts, and provided a superior islet xenograft protection. Our study therefore indicates an advantage of donor ECDI‐SP treatment in controlling human immune cells in promoting long‐term islet xenograft survival.     

Early Fundoplication Prevents Chronic Allograft Dysfunction in Patients with Gastroesophageal Reflux Disease

Background

Chronic allograft dysfunction limits the long-term success of lung transplantation. Increasing evidence suggests nonimmune mediated injury such as due to reflux contributes to the development of bronchiolitis obliterans syndrome. We have previously demonstrated that fundoplication can reverse bronchiolitis obliterans syndrome in some lung transplant recipients with reflux. We hypothesized that treatment of reflux with early fundoplication would prevent bronchiolitis obliterans syndrome and improve survival.

Methods

A retrospective analysis of 457 patients who underwent lung transplantation from April 1992 through July 2003 was conducted. Patients were stratified into four groups: no history of reflux, history of reflux, history of reflux and early (< 90 days) fundoplication and history of reflux and late fundoplication.

Results

Incidence of postoperative reflux was 76% (127 of 167 patients) in pH confirmed subgroups. In 14 patients with early fundoplication, actuarial survival was 100% at 1 and 3 years when compared with those with reflux and no intervention (92% ± 3.3, 76% ± 5.8; p < 0.02). Further, those who underwent early fundoplication had improved freedom from bronchiolitis obliterans syndrome at 1 and 3 years (100%, 100%) when compared with no fundoplication in patients with reflux (96% ± 2.5, 60% ± 7.5; p < 0.01).

Conclusions

Reflux is a frequent medical complication after lung transplantation. Although the number of patients undergoing early fundoplication is small, our results suggest early aggressive surgical treatment of reflux results in improved rates of bronchiolitis obliterans syndrome and survival. Further research into the mechanisms and treatment of nonalloimmune mediated lung allograft injury is needed to reduce rates of chronic lung failure.