C. Neudörfl

NK Cells of Kidney Transplant Recipients Display an Activated Phenotype that Is Influenced by Immunosuppression and Pathological Staging

To explore phenotype and function of NK cells in kidney transplant recipients, we investigated the peripheral NK cell repertoire, capacity to respond to various stimuli and impact of immunosuppressive drugs on NK cell activity in kidney transplant recipients. CD56dim NK cells of kidney transplanted patients displayed an activated phenotype characterized by significantly decreased surface expression of CD16 (p=0.0003), CD226 (p<0.0001), CD161 (p=0.0139) and simultaneously increased expression of activation markers like HLA-DR (p=0.0011) and CD25 (p=0.0015). Upon in vitro stimulation via Ca++-dependent signals, down-modulation of CD16 was associated with induction of interferon (IFN)-γ expression. CD16 modulation and secretion of NFAT-dependent cytokines such as IFN-γ, TNF-α, IL-10 and IL-31 were significantly suppressed by treatment of isolated NK cells with calcineurin inhibitors but not with mTOR inhibitors. In kidney transplant recipients, IFN-γ production was retained in response to HLA class I-negative target cells and to non-specific stimuli, respectively. However, secretion of other cytokines like IL-13, IL-17, IL-22 and IL-31 was significantly reduced compared to healthy donors. In contrast to suppression of cytokine expression at the transcriptional level, cytotoxin release, i.e. perforin, granzyme A/B, was not affected by immunosuppression in vitro and in vivo in patients as well as in healthy donors. Thus, immunosuppressive treatment affects NK cell function at the level of NFAT-dependent gene expression whereby calcineurin inhibitors primarily impair cytokine secretion while mTOR inhibitors have only marginal effects. Taken together, NK cells may serve as indicators for immunosuppression and may facilitate a personalized adjustment of immunosuppressive medication in kidney transplant recipients.

Ex Vivo Lung Perfusion using the portable OCS Maintains Endothelial Integrity in the Context of Reduced Severe PGD Rates

Purpose The INSPIRE trial revealed significant reduction of PGD grade 3, i.e. less ischemia reperfusion injury (IRI) using the Organ Care System (OCS) compared to controls for lung preservation. In order to investigate endothelial mechanisms initiated by cold vs. normothermic preservation, blood and perfusates of INSPIRE patients were assessed for proteins involved in endothelial integrity. We hypothesized that OCS preservation also supports endothelial integrity in parallel to an anti-inflammatory milieu. Methods Blood plasma pre, T0, T24 post Tx and perfusion solutions from 33 OCS and 26 SOC patients with control-preserved lungs were analysed for 100 cytokines, angiogenic factors, etc. by multiplex assays. Donor and recipient demographics, cold ischemic times and PGD scores were assessed and correlated with protein levels. Results Clinical evaluation (OCS/control) revealed mean recipient age: 50 vs. 49 years, diagnosis: idiopathic fibrosis (n=17/10), cystic fibrosis (n=7/8), idiopathic pulmonary hypertension (n=3/3) and emphysema (n=6/5), mean total cold ischemic times (CIT) 258±6 vs. 549±22 min (p<0.0001). In the OCS group, no cumulative PGD score > 2 was observed compared to 19% PGD3 in SOC (p=0.035). Less IRI in OCS patients was shown by significantly reduced IL-6, CXCL8, CXCL10, CCL2 plasma levels at T0. OCS plasma levels at T0 were also significantly lower for sCD31 (p=0.002), ICAM-1 (p=0.025), PAI-1 (p=0.03), leading to a higher PAI-1/uPA ratio of 82 in OCS compared to 67 in SOC. Lower VCAM-1, IGFBP-1, Ang-2, uPA, sHer2/neu, sVEGFR2 levels were detected in OCS compared to SOC recipients but did not reach statistical significance. Plasma levels of endoglin (CD105, p=0.01), PlGF (p=0.02) correlated with CIT. In contrast to the PGD correlation to IL-6 in SOC patients, none of these proteins showed a PGD correlation at T0 or T24. In contrast to plasma, significantly higher concentrations of these proteins were measured in OCS vs. SOC perfusates (p<0.01). Conclusion During normothermic lung preservation using the OCS system, reduced IRI is accompanied with protection of the endothelium, which can be detected by lower T0 plasma levels of endothelial activation markers. Thus, lung preservation using the OCS initiates an anti-inflammatory cascade and a tissue-protective milieu resulting in improved graft function. SFB738, B3. IFB-Tx OPEX_2.