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.

Pre-transplant allosensitization to human leukocyte antigens (HLA) increases the recipient waiting-list time and mortality in lung transplantation. Rather than waiting for crossmatch-negative donors, since 2013, recipients with preformed donor specific anti-HLA antibodies (pfDSA) have been managed with repeated IgA- and IgM-enriched intravenous immunoglobulin (IgGAM) infusions, usually in combination with plasmapheresis before IgGAM and a single dose of anti-CD20 antibody. This retrospective study presents our 9-year experience in patients transplanted with pfDSA. Records of patients transplanted between 02/2013 and 05/2022 were reviewed. Outcomes were compared between patients with pfDSA and those without any de-novo DSA. Median follow-up time was 50 months. Of the 1043 lung-transplanted patients, 758 (72.7%) did not develop any early DSA and 62 (5.9%) patients exhibited pfDSA. Among the 52 (84%) patients who completed treatment, pfDSA were cleared in 38 (73%) patients. In pfDSA vs. control patients and at 8-year follow-up, respectively, graft survival (%) was 75 vs. 65 (p=0.493) and freedom from chronic lung allograft dysfunction (CLAD, %) was 63 vs. 65 (p=0.525). In lung transplantation, crossing the preformed HLA-antibody barrier is safe using a treatment protocol based on IgGAM. Patients with pfDSA have a good 8-year graft survival and freedom from CLAD similar to control patients.

After pediatric liver transplantation (pLT), children undergo life-long immunosuppression since reliable biomarkers for the assessment of rejection probability are scarce. In the multicentre (n=7) prospective clinical cohort "ChilSFree" study, we aimed to characterize longitudinal dynamics of soluble and cellular immune mediators during the first year after pLT and identify early biomarkers associated with outcome. Using paired Luminex-based multiplex technique and flow cytometry, we characterized longitudinal dynamics of soluble immune mediators (SIM, n=50) and immune cells in the blood of 244 patients at 8 visits over one year: before, 7/14/21/28 days, 3/6/12 months after pLT. The unsupervised clustering of patients based on SIM profiles revealed 6 unique SIM signatures associated with clinical outcome. From 3 signatures linked to improved outcome, one was associated with one-year-long rejection-free survival and stable graft function and was characterized by low levels of pro-inflammatory (CXCL8/9/10/12, CCL7, SCGF-β, sICAM-1), high levels of regenerative (SCF, TNF-β), and pro-apoptotic (TRAIL) SIM (all, p100). Of note, this SIM signature appeared two weeks after pLT and remained stable over the entire year, pointing towards its potential as a novel early biomarker for minimizing or weaning immunosuppression. In the blood of these patients, a higher frequency of CD56^bright NK cells (p<0.01), a known hallmark also associated with operationally tolerant pLT patients, was detected. The concordance of the model for prediction of rejection based on identified SIM signatures was 0.715, and 0.795, in combination with living-related transplantation as co-variate, respectively. SIM blood signatures may enable the non-invasive and early assessment of rejection risks in the first year after pLT, paving the way to improved therapeutic options.

Steroid-free immunosuppression protocols gained popularity in pediatric liver transplantation (pLT) after the introduction of IL-2–receptor blockade for induction therapy. We analyzed the clinical and immunologic outcome data of the multicenter prospective observational ChilSFree study to compare the impact of steroid-free versus steroid-containing immunosuppressive therapy following pLT in a real-life scenario. Two hundred forty-six children [55.3% male, age at pLT median: 2.4 (range: 0.2–17.9) y] transplanted for biliary atresia (43%), metabolic liver disease (9%), acute liver failure (4%), hepatoblastoma (9%), and other chronic end-stage liver diseases (39%) underwent immune monitoring and clinical data documentation over the first year after pLT. Patient and graft survival at 1 year was 98.0% and 92.7%, respectively. Primary immunosuppression was basiliximab induction followed by tacrolimus (Tac) monotherapy (55%), Tac plus steroid tapering over 3 months (29%), or cyclosporine and steroid tapering (7%). One center used intraoperative steroids instead of basiliximab followed by Tac plus mycophenolate mofetil (7% of patients). N = 124 biopsy-proven T-cell–mediated rejections were documented in n = 82 (33.3%) patients. T-cell–mediated rejection occurred early (median: 41 d, range: 3–366 d) after pLT. Patients initially treated with Tac plus steroids experienced significantly fewer episodes of rejection than patients treated with Tac alone (chi-square p<0.01). The use of steroids was associated with earlier downregulation of proinflammatory cytokines interferon (IFN)-γ, Interleukin (IL)-6, CX motif chemokin ligand (CXCL)8, IL-7, and IL-12p70. Both primary immunosuppression with Tac plus steroids and living donor liver transplantation were independent predictors of rejection-free survival 1 year after pLT on logistic regression analysis. Adjunctive steroid therapy after pLT leads to earlier suppression of the post-pLT proinflammatory response and significantly reduced rejection rates during the first year after pLT (15.9%). Fifty-one percent of patients initially treated without steroids remain steroid-free over the first 12 months without rejection.

Background: The current gold standard to diagnose T-cell-mediated acute rejection (TCMR) requires liver histology. Using data from the ChilSFree study on immune response after paediatric liver transplantation (pLT), we aimed to assess whether soluble cytokines can serve as an alternative diagnostic tool in children suspected to have TCMR. Methods: A total of n = 53 blood samples obtained on the day of or up to 3 days before liver biopsy performed for suspected TCMR at median 18 days (range 7–427) after pLT in n = 50 children (38% female, age at pLT 1.8 (0.5–17.5) years) were analysed for circulating cytokine levels using Luminex-based Multiplex technology. Diagnostic accuracy of cytokine concentrations was assessed using a multivariable model based on elastic net regression and gradient boosting machine analysis. Results: TCMR was present in 68% of biopsies. There was strong evidence that patients with TCMR had increased levels of soluble CXCL8, CXCL9, CXCL10, IL-16, IL-18, HGF, CCL4, MIF, SCGF-β, and HGF before biopsy. There was some evidence for increased levels of sCD25, ICAM-1, IL-6, IL-3, and CCL11. Diagnostic value of both single cytokine levels and a combination of cytokines and clinical markers was poor, with AUROCs not exceeding 0.7. Conclusion: Patients with TCMR showed raised levels of cytokines and chemokines reflective of T-cell activation and chemotaxis. Despite giving insight into the mechanisms of TCMR, the diagnostic value of soluble cytokines for the confirmation of TCMR in a clinical scenario of suspected TCMR is poor.

Natural killer (NK) cell–targeting immunotherapies are emerging, yet the differentiation and functional states of tumor-infiltrating NK cells remain poorly understood. Using matched single-nucleus RNA and ATAC sequencing of samples from patients with non–small cell lung cancer (NSCLC), we resolved the transcriptional and epigenetic landscape of intratumoral NK cells. We identified two tumor-associated NK (taNK) cell subsets marked by expression of ITGAE (CD103) and ITGA1 (CD49a) that display features of tissue residency and dysfunction while preserving cytotoxic function. Trajectory and regulon analyses revealed an inflammation-driven transition from early granzyme K ( GZMK ) ⁺ NK cells toward an ENTPD1 ⁺ (CD39 ⁺ ) effector state characterized by interferon-stimulated gene (ISG) programs. Functional profiling established CD39 ⁺ taNK cells as the dominant cytotoxic NK cell population with superior killing capacity that was further potentiated by NKG2A blockade. This study offers mechanistic insights into NK cell differentiation in NSCLC and establishes CD39 ⁺ taNK cells as a targetable effector population for immunotherapy.