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The role of endothelial cells in the pathophysiology of antibody-mediated rejection after renal transplantation has been widely investigated. We expand this scenario to the impact of epithelial cells on the microenvironment during rejection. Primary proximal tubular epithelial cells were stimulated via HLA class I, CD155 and CD166 based on their potential signal-transducing capacity to mediate back signaling after encounter with either T/NK cells or donor-specific antibodies. Upon crosslinking of these ligands with mAbs, PTEC secreted IL-6, CXCL1,8,10, CCL2, and sICAM-1. These proteins were also released by PTEC as consequence of a direct interaction with T/NK cells. Downmodulation of the receptor CD226 on effector cells confirmed the involvement of this receptor/ligand pair in back signaling. In vivo, CD155 and CD166 expression was detectable in proximal and distal tubuli of renal transplant biopsies, respectively. The composition of the protein microenvironment in these biopsies showed a substantial overlap with the PTEC response. Cluster and principal component analyses of the microenvironment separated unsuspicious from rejection biopsies and, furthermore, ABMR, TCMR, and borderline rejection. In conclusion, our results provide evidence that epithelial cells may contribute to the rejection process and pave the way to a better understanding of the pathomechanisms of kidney allograft rejection.

Purpose Organ-specific differences are discussed for ischemia/reperfusion injury (IRI) in cardiothoracic transplantation but rarely compared directly in a clinical setting. Therefore, we compared a cohort of combined heart/lung transplants (HLTx) with cohorts of isolated heart (HTx) or lung transplantations (LuTx), respectively, with regards to cytokines and endothelial markers in recipient blood and perfusates. Our aim was to determine whether the microenvironment of HLTx patients and perfusates would be rather related to the HTx or LuTx setting. Methods Blood plasma pre Tx and post Tx at T0, T24 and 3 wks as well as perfusion solutions (taken from storage bags at the end of cold ischemia) of 5 HLTx, 24 HTx and 21 LuTx patients were analyzed for cytokines, chemokines and soluble endothelial markers (60 proteins) using multiplex assays. Results Early after transplantation at T0 and T24, HLTx and HTx were clustered together and separated from LuTx recipients based on their significantly higher plasma levels of IL-6, CXCL8/IL-8, Ang-2, IGFBP-1, PAI-1 compared to LTx recipients. Endoglin, IL-18, sFASL, TNF-α, VEGF-A, -C, -D, HB-EGF, EGF, uPA and PAI-1 contributed to the discriminative pattern between the three groups with similar kinetics of a significant increase at T0 (all p Conclusion A direct comparison of combined heart/lung with isolated heart or lung transplantation revealed that the early systemic reperfusion response of HLTx recipients in blood is dominated by heart-associated endothelial markers like IGFBP-1, Ang-2, and PAI-1 which groups them together with HTx patients. In contrast, the ischemic damage pattern of HLTx perfusates was clustering closer to lung than to heart perfusates. These differences between recipient blood and perfusates strongly suggest that the ischemia response is dominated by lung whereas the systemic reperfusion response is guided by heart. These results underline the organ-specific impact on IRI with distinct heart- vs lung-associated signatures.

Purpose After lung transplantation (LuTx), the development of early donor HLA-specific antibodies (eDSA) has been shown to be associated with antibody-mediated rejection (AMR) and poor graft survival. Since 2013, patients with eDSA within the first month after LuTx in our center are treated with IgA/IgM enriched intravenous immunoglobulins (IgGAM), combined with anti-CD20 antibody (Rituximab). We addressed the hypothesis that naive vs. memory B cell subsets differ between eDSA-positive and negative patients already early after LuTx before onset of the treatment regimen. Methods In a pilot study of 31 out of 97 LuTx recipients in our immune monitoring cohort, B cell subsets were analysed pre, post (T0), 24h (T24) and 3 wks after LuTx. B cells were phenotyped using flow cytometry with CD19, CD27, IgD, CD24 mab. The kinetics of B cell subsets were compared between eDSA-positive (n=7) and -negative (n=24) patients. Results During the first 24h, relative B cell frequencies increased and returned to baseline at 3 wks without differences between eDSA-positive and negative LuTx patients. In eDSA-positive patients, higher frequencies of IgD+CD27-CD24hi naive and lower frequencies of IgD-CD27-CD24lo memory B cell subsets were observed constantly pre, at T0, T24 and 3wks. In contrast, a transient decrease in IgG+CD27+ switch memory B cells was detected at T0 in both groups, returning to baseline levels already at T24. Conclusion In the context of lung transplantation, the ratio between naive and memory B cells even before and directly after LuTx may be associated with the development of de novo DSA. Therefore, a refined B cell monitoring may be able to identify patients with a higher risk for eDSA development. The impact of the treatment regimen on these B cell subsets is currently further investigated in terms of differential effects on their depletion and reconstitution, respectively.

Purpose After lung transplantation (LuTx), a transient chimerism of donor cells exists in the blood of recipients due to the migration of lymphocytes from the transplanted lung into the periphery. We aimed to characterize the phenotype of donor CD8+ and CD4+ T and NK cells and to investigate whether they might represent tissue-resident memory (TRM) cells. Methods Lymphocyte dynamics in recipient blood were determined in 97 lung transplant patients directly (T0), 24 hours (T24) and 3 (wks) weeks after LuTx using flow cytometry with lineage-, tissue-, and donor HLA class I allele-specific mAb. The same makers were used to determine the phenotype of lymphocytes present in organ storage solution (perfusate, n=102), recipient explanted lung parenchyma (n=28) and donor trachea (n=17). Results In peripheral blood of all recipients, donor-derived CD4+ and CD8+ T and CD56dim NK cells were detected at T0, T24 and 3 wks after LuTx and had higher CD69 expression compared to recipient cells (p Conclusion Our results demonstrate that donor T and NK cells found in the blood of LuTx recipients are a distinct subset from circulating lymphocytes and TRM cells present in lung tissue, since they express CD69 but lack expression of other classical TRM markers. They display a higher functional capacity despite the onset of immunosuppression. Donor T cells might be clinically relevant for tolerance induction and long-term survival after transplantation due to their unique features.

Purpose After lung transplantation (LuTx), the development of early donor HLA-specific antibodies (eDSA) has been shown to be associated with antibody-mediated rejection (AMR) and poor graft survival. Since 2013, patients with eDSA within the first month after LuTx in our center are treated with IgA/IgM enriched intravenous immunoglobulins (IgGAM), combined with anti-CD20 antibody (Rituximab). We addressed the hypothesis that naive vs. memory B cell subsets differ between eDSA-positive and negative patients already early after LuTx before onset of the treatment regimen. Methods In a pilot study of 31 out of 97 LuTx recipients in our immune monitoring cohort, B cell subsets were analysed pre, post (T0), 24h (T24) and 3 wks after LuTx. B cells were phenotyped using flow cytometry with CD19, CD27, IgD, CD24 mab. The kinetics of B cell subsets were compared between eDSA-positive (n=7) and -negative (n=24) patients. Results During the first 24h, relative B cell frequencies increased and returned to baseline at 3 wks without differences between eDSA-positive and negative LuTx patients. In eDSA-positive patients, higher frequencies of IgD+CD27−CD24hi naive and lower frequencies of IgD−CD27−CD24lo memory B cell subsets were observed constantly pre, at T0, T24 and 3wks. In contrast, a transient decrease in IgG+CD27+ switch memory B cells was detected at T0 in both groups, returning to baseline levels already at T24. Conclusion In the context of lung transplantation, the ratio between naive and memory B cells even before and directly after LuTx may be associated with the development of de novo DSA. Therefore, a refined B cell monitoring may be able to identify patients with a higher risk for eDSA development. The impact of the treatment regimen on these B cell subsets is currently further investigated in terms of differential effects on their depletion and reconstitution, respectively.

Purpose In previous studies, we identified donor lymphocytes in peripheral blood of recipients directly after transplantation and persisting at least three weeks that were characterized by the tissue retention marker CD69. In order to determine their origin, we hypothesized that donor T and NK cells have a peculiar tissue-resident memory phenotype that is shared between cells derived from lung perfusates, trachea parenchyma and lymph nodes. Methods Donor lymphocytes in recipient blood were determined in 27 lung transplant patients at T0, T24, 3 weeks by staining of donor HLA class I molecules in combination with lineage- and tissue-specific markers using flow cytometry. The phenotype of T and NK cells in perfusates (n=30), donor trachea (n=5), and lymph node (n=10), recipient explanted parenchyma (n=15) was compared to circulating cells using the same markers. Results In peripheral blood of all lung transplant recipients, donor derived T and NK cells were detected at T0, T24 and 3 weeks and had higher CD69 expression compared to recipient cells (p=0.001 to 0.03) and were mostly CD25−. This phenotype was similar to T and NK cells in corresponding perfusates, with significantly increased CD69 expression compared to circulating PBMCs (all p Conclusion Our results suggest that donor T and NK cells found in the periphery of lung transplant recipients are derived from lung parenchyma and represent tissue-resident memory cells. This transient chimerism in recipient blood might be clinically relevant for tolerance induction after transplantation due to unique features of TRM T and NK cells.

Purpose Organ-specific differences are discussed for ischemia/reperfusion injury (IRI) in cardiothoracic transplantation (Tx) but rarely compared directly in a clinical setting. Therefore, we compared a cohort of combined heart/lung transplants (HLTx) with cohorts of isolated heart (HTx) or lung transplantations (LTx), respectively, with respect to cytokines and endothelial markers in recipient blood and perfusates. Despite the evident clinical differences, our aim was to determine whether the microenvironment of HLTx patients would be rather related to HTx or LTx patients. Methods Blood plasma pre Tx and post Tx at T0, T24 and 3 weeks as well as perfusion solutions of 5 HLTx, 24 HTx and 21 LTx patients were analysed for cytokines and soluble endothelial markers using multiplex assays. Results Early after transplantation at T0 and T24, HLTx and HTx recipients displayed significantly higher plasma levels of IL-6, CXCL8/IL-8, Ang-2, IGFBP-1, PAI-1 compared to LTx recipients that returned to baseline after three weeks. Identical kinetics with minimal changes were detected in the three groups for TNF, HB-EGF, EGF, PLGF, sFasL, TGF-α. Unsupervised cluster and principal component analyses clearly grouped HLTx and HTx patients together, separating LTx recipients apart with IGFBP-1, Ang-2, and PAI-1 as lead parameters (all p Conclusion A direct comparison of combined heart/lung with isolated heart or lung transplantation revealed that the systemic IRI response of HLTx recipients is, perhaps counterintuitively, dominated by heart-associated endothelial markers like IGFBP-1, Ang-2, and PAI-1 which groups them together with HTx patients. The difference between recipient blood and perfusates strongly suggests that the ischemia response is dominated by lung whereas the systemic reperfusion response is guided by heart. These results underline the organ-specific impact on IRI with distinct heart- vs lung-associated signatures.