Posttransplant oxygen inhalation improves the outcome of subcutaneous islet transplantation: A promising clinical alternative to the conventional intrahepatic site

Subcutaneous tissue is a promising site for islet transplantation, due to its large area and accessibility, which allows minimally invasive procedures for transplantation, graft monitoring, and removal of malignancies as needed. However, relative to the conventional intrahepatic transplantation site, the subcutaneous site requires a large number of islets to achieve engraftment success and diabetes reversal, due to hypoxia and low vascularity. We report that the efficiency of subcutaneous islet transplantation in a Lewis rat model is significantly improved by treating recipients with inhaled 50% oxygen, in conjunction with prevascularization of the graft bed by agarose–basic fibroblast growth factor. Administration of 50% oxygen increased oxygen tension in the subcutaneous site to 140 mm Hg, compared to 45 mm Hg under ambient air. In vitro, islets cultured under 140 mm Hg oxygen showed reduced central necrosis and increased insulin release, compared to those maintained in 45 mm Hg oxygen. Six hundred syngeneic islets subcutaneously transplanted into the prevascularized graft bed reversed diabetes when combined with postoperative 50% oxygen inhalation for 3 days, a number comparable to that required for intrahepatic transplantation; in the absence of oxygen treatment, diabetes was not reversed. Thus, we show oxygen inhalation to be a simple and promising approach to successfully establishing subcutaneous islet transplantation.     

Macrophage subpopulations and their impact on chronic allograft rejection versus graft acceptance in a mouse heart transplant model

Macrophages infiltrating the allografts are heterogeneous, consisting of proinflammatory (M1 cells) as well as antiinflammatory and fibrogenic phenotypes (M2 cells); they affect transplantation outcomes via diverse mechanisms. Here we found that macrophage polarization into M1 and M2 subsets was critically dependent on tumor necrosis factor receptor–associated factor 6 (TRAF6) and mammalian target of rapamycin (mTOR), respectively. In a heart transplant model we showed that macrophage‐specific deletion of TRAF6 (LysM^CreTraf6 ^fl/fl) or mTOR (LysM^CreMtor^fl/fl) did not affect acute allograft rejection. However, treatment of LysM^CreMtor^fl/fl recipients with CTLA4‐Ig induced long‐term allograft survival (>100 days) without histological signs of chronic rejection, whereas the similarly treated LysM^CreTraf6 ^fl/fl recipients developed severe transplant vasculopathy (chronic rejection). The presentation of chronic rejection in CTLA4‐Ig–treated LysM^CreTraf6 ^fl/fl mice was similar to that of CTLA4‐Ig–treated wild‐type B6 recipients. Mechanistically, we found that the graft‐infiltrating macrophages in LysM^CreMtor^fl/fl recipients expressed high levels of PD‐L1, and that PD‐L1 blockade readily induced rejection of otherwise survival grafts in the LysM^CreMtor^fl/fl recipients. Our findings demonstrate that targeting mTOR‐dependent M2 cells is critical for preventing chronic allograft rejection, and that graft survival under such conditions is dependent on the PD‐1/PD‐L1 coinhibitory pathway.     

Neutrophil derived CSF1 induces macrophage polarization and promotes transplantation tolerance

The colony‐stimulating factor 1 (CSF1) regulates the differentiation and function of tissue macrophages and determines the outcome of the immune response. The molecular mechanisms behind CSF1‐mediated macrophage development remain to be elucidated. Here we demonstrate that neutrophil‐derived CSF1 controls macrophage polarization and proliferation, which is necessary for the induction of tolerance. Inhibiting neutrophil production of CSF1 or preventing macrophage proliferation, using targeted nanoparticles loaded with the cell cycle inhibitor simvastatin, abrogates the induction of tolerance. These results provide new mechanistic insights into the developmental requirements of tolerogenic macrophages and identify CSF1 producing neutrophils as critical regulators of the immunological response.     

Heterogeneity of memory B cells

Potential solid organ transplant recipients broadly sensitized to HLA have long wait times, low transplant rates and poor outcomes. The new kidney allocation system has improved access to the most highly sensitized recipients; however, their long‐term outcomes are unknown. Emerging data suggest that memory B cell repertoire is broader than the plasma cell repertoire, therefore, despite refinements in anti‐HLA antibody detection technology, donor‐specific HLA‐ specific memory B cells may in fact be present in some, if not most, highly sensitized recipients with no detectable donor‐specific antibodies. In addition, new findings have underscored the heterogeneity in memory B cell generation, and in the signals that determine memory versus plasma cell fate during primary antigen encounter, as well as memory B cell differentiation upon antigen reencounter into plasma cells or reentry into germinal centers to subsequently emerge as higher affinity and class‐switched plasma cells. Thus, heterogeneity memory B cells generation may affect the efficacy of specific immunomodulation during the recall response. We propose that the ability to quantify donor‐specific B cell in transplant recipients is urgently required to provide insights into the mechanisms of sensitization and recall, and for the early detection of acute and chronic AMR.     

Subcutaneous administration of a neutralizing IL‐1β antibody prolongs limb allograft survival

Cytokine‐expression profiles revealed IL‐1ß highly upregulated in rejecting skin of limb allografts. We investigate the effect of intragraft treatment with a neutralizing IL‐1β antibody in limb transplantation. Following allogenic hind‐limb transplantation, Lewis rats were either left untreated 1 or treated with anti‐lymphocyte serum + tacrolimus (baseline) 2 ; baseline immunosuppression + anti‐IL‐1β (1 mg/kg once/week, 6‐8 subcutaneous injections) into the transplanted 3 or contralateral 4 limb. Endpoint was rejection grade III or day 100. Graft rejection was assessed by histology, immunohistochemistry, flow cytometry phenotyping of immune cells, and monitoring cytokine expression. Anti‐IL‐1β injections into the allograft or contralateral limb resulted in a significant delay of rejection onset (controls: 58.60 ± 0.60; group 3: 75.80 ± 10.87, P = .044; group 4: 73.00 ± 6.49, P = .008) and prolongation of graft survival (controls: 64.60 ± 0.87; group 3: 86.60 ± 5.33, P = .002; group 4: 93.20 ± 3.82, P = .002), compared to controls. Although the phenotype of the graft infiltrating immune cells did not differ between groups, significantly decreased skin protein levels of IL‐1β, IL‐4, IL‐13, IP‐10, MCP‐1, and MCP‐3 in long‐term‐survivors indicate an overall decrease of chemoattraction and infiltration of immune cells as the immunosuppressive mechanism of anti‐IL‐1β. Inhibition of IL‐1β with short‐term systemic immunosuppression prolongs limb allograft survival and represents a promising target for immunosuppression in extremity transplantation.     

Comparing Humoral and Cellular Immune Response Against HBV Vaccine in Kidney Transplant Patients

Host protection upon vaccination usually results from the complex interplay of humoral and cellular components of the immune system. Exploring hepatitis B surface antigen (HBsAg)‐specific T cell responses and their correlation with humoral responses under immunosuppression, we analyzed 51 renal transplant recipients, differing in HBV vaccine–specific antibody titers (non [NRs]‐, low [LRs]‐, and high responders [HRs]) and in 22 healthy controls (HCs) in a cross‐sectional study. HBsAg‐specific T cells were analyzed by flow cytometry according to expression of activation markers CD40L and/or CD69, and the cytokines IFNγ, IL‐2, TNFα, and IL‐17. No significant differences in responder rate and magnitude of HBsAg‐specific T cell responses were found between HCs and HRs. Interestingly, HBsAg‐specific Th‐cells were also observed in 50% of humoral NRs. Frequencies of HBsAg‐specific CD40L+ Th‐cells were significantly higher in HRs compared to LRs (p = 0.009) and in LRs in comparison to NRs (p = 0.043). All but NRs showed a predominance of multi‐potent HBsAg‐specific TNFα+IL‐2+ Th‐cells. As expected, HBsAg‐specific CD8⁺ T cells were rarely found. In conclusion, mounting of hepatitis B vaccine‐specific T cell responses is possible in kidney transplant recipients despite immunosuppression. Detection of HBV‐specific Th‐cells in a significant proportion of humoral NRs contributes to the current discussion on conferring immune protection by cellular memory in such patients.     

Pathogen Stimulation History Impacts Donor‐Specific CD8+ T Cell Susceptibility to Costimulation/Integrin Blockade–Based Therapy

Recent studies have shown that the quantity of donor‐reactive memory T cells is an important factor in determining the relative heterologous immunity barrier posed during transplantation. Here, we hypothesized that the quality of T cell memory also potently influences the response to costimulation blockade‐based immunosuppression. Using a murine skin graft model of CD8⁺ memory T cell–mediated costimulation blockade resistance, we elicited donor‐reactive memory T cells using three distinct types of pathogen infections. Strikingly, we observed differential efficacy of a costimulation and integrin blockade regimen based on the type of pathogen used to elicit the donor‐reactive memory T cell response. Intriguingly, the most immunosuppression‐sensitive memory T cell populations were composed primarily of central memory cells that possessed greater recall potential, exhibited a less differentiated phenotype, and contained more multi‐cytokine producers. These data, therefore, demonstrate that the memory T cell barrier is dependent on the specific type of pathogen infection via which the donor‐reactive memory T cells are elicited, and suggest that the immune stimulation history of a given transplant patient may profoundly influence the relative barrier posed by heterologous immunity during transplantation.     

CD4 T Cells but Not Th17 Cells Are Required for Mouse Lung Transplant Obliterative Bronchiolitis

Lung transplant survival is limited by obliterative bronchiolitis (OB), but the mechanisms of OB development are unknown. Previous studies in a mouse model of orthotopic lung transplantation suggested a requirement for IL‐17. We have used this orthotopic mouse model to investigate the source of IL‐17A and the requirement for T cells producing IL‐17A. The major sources of IL‐17A were CD4⁺ T cells and γδ T cells. Depletion of CD4⁺ T cells led to a significantly decreased frequency and number of IL‐17A⁺ lymphocytes and was sufficient to prevent acute rejection and OB. However, mice with STAT3‐deficient T cells, which are unable to differentiate into Th17 cells, rejected lung allografts and developed OB similar to control mice. The frequency of IL‐17A⁺ cells was not decreased in mice with STAT3‐deficient T cells due mainly to the presence of IL‐17A⁺ γδ T cells. Deficiency of γδ T cells also did not affect the development of airway fibrosis. Our data suggest that CD4⁺ T cells are required for OB development and expansion of IL‐17A responses in the lung, while Th17 and γδ T cells are not absolutely required and may compensate for each other.