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Donor-reactive memory cells represent a barrier to long-term kidney graft survival. A better understanding of regulatory mechanisms that counterbalance alloreactive memory responses may help to identify patients with operational tolerance. This prospective study investigated the equilibrium between memory T cell subsets and regulatory T or B cells (Tregs, Bregs) in peripheral blood of kidney transplant recipients with operational tolerance (N=8), chronic rejection (N=8), and different immunosuppressive treatment regimens (N=81). Patients on hemodialysis and healthy individuals served as controls (N=50). In addition, the expression of Treg- and Breg-associated molecule genes was analyzed. Patients with chronic rejection showed a disrupted memory T cell composition with a significantly higher frequency of circulating CD8(+) terminally differentiated effector memory (TEMRA) T cells than patients with operational tolerance, patients on hemodialysis, or healthy controls (P<0.001). Low frequency of CD8(+) TEMRA and high frequency of Tregs and transitional Bregs were found in operationally tolerant patients. Consequently, operationally tolerant patients showed, as compared to all other transplant recipients with different immunosuppressive regiments, the lowest ratios between CD8(+) TEMRA T cells and Tregs or Bregs (for both P<0.001). Moreover, a specific peripheral blood transcription pattern was found in operationally tolerant patients with an increased expression of Breg- and Treg-associated genes CD22 and FoxP3 and a decreased Fc gamma RIIA/Fc gamma RIIB transcript ratio (for all P<0.001). In conclusion, monitoring the balance between circulating CD8(+) TEMRA T cells and regulatory cell subsets and their transcripts may help to distinguish transplant recipients with operational tolerance from recipients at risk of graft loss., European Union (EU); Horizon 2020; Research and Innovation Programme; Physician Scientist Program of the Heidelberg Faculty of Medicine; Olympia-Morata Program of the Heidelberg Faculty of Medicine

Donor-derived cell-free DNA (dd-cfDNA) identifies allograft injury and discriminates active rejection from no rejection. In this prospective study, 106 kidney transplant recipients with 108 clinically indicated biopsies were enrolled at Heidelberg University Hospital between November 2020 and December 2022 to validate the clinical value of dd-cfDNA in a cohort of German patients. dd-cfDNA was quantified at biopsy and correlated to histopathology. Additionally, dd-cfDNA was determined on days 7, 30, and 90 post-biopsy and analyzed for potential use to monitor response to anti-rejection treatment. dd-cfDNA levels were with a median (IQR) % of 2.00 (0.48-3.20) highest in patients with ABMR, followed by 0.92 (0.19-11.25) in patients with TCMR, 0.44 (0.20-1.10) in patients with borderline changes and 0.20 (0.11-0.53) in patients with no signs of rejection. The AUC for dd-cfDNA to discriminate any type of rejection including borderline changes from no rejection was at 0.72 (95% CI 0.62-0.83). In patients receiving anti-rejection treatment, dd-cfDNA levels significantly decreased during the 7, 30, and 90 days follow-up compared to levels at the time of biopsy (p = 0.006, p = 0.002, and p < 0.001, respectively). In conclusion, dd-cfDNA significantly discriminates active rejection from no rejection. Decreasing dd-cfDNA following anti-rejection treatment may indicate response to therapy., European Union (EU); Horizon 2020; Research and Innovation Programme; LB is funded by the Olympia Morata Program of Heidelberg University. CSp is funded by the Physician Scientist Program of the Heidelberg Faculty of Medicine. For the publication fee we acknowledge financial support by Deutsche Forschungsgemeinschaft within the funding program “Open Access Publikationskosten” as well as by Heidelberg University.

Seroconversion after COVID-19 vaccination is impaired in kidney transplant recipients. Emerging variants of concern such as the B.1.617.2 (delta) and the B.1.1.529 (omicron) variants pose an increasing threat to these patients. In this observational cohort study, we measured anti-S1 IgG, surrogate neutralizing, and anti-receptor-binding domain antibodies three weeks after a third mRNA vaccine dose in 49 kidney transplant recipients and compared results to 25 age-matched healthy controls. In addition, vaccine-induced neutralization of SARS-CoV-2 wild-type, the B.1.617.2 (delta), and the B.1.1.529 (omicron) variants was assessed using a live-virus assay. After a third vaccine dose, anti-S1 IgG, surrogate neutralizing, and anti-receptor-binding domain antibodies were significantly lower in kidney transplant recipients compared to healthy controls. Only 29/49 (59%) sera of kidney transplant recipients contained neutralizing antibodies against the SARS-CoV-2 wild-type or the B.1.617.2 (delta) variant and neutralization titers were significantly reduced compared to healthy controls (p < 0.001). Vaccine-induced cross-neutralization of the B.1.1.529 (omicron) variants was detectable in 15/35 (43%) kidney transplant recipients with seropositivity for anti-S1 IgG, surrogate neutralizing, and/or anti-RBD antibodies. Neutralization of the B.1.1.529 (omicron) variants was significantly reduced compared to neutralization of SARS-CoV-2 wild-type or the B.1.617.2 (delta) variant for both, kidney transplant recipients and healthy controls (p < .001 for all)., Dietmar Hopp Stiftung; Heidelberg Faculty of Medicine Rahel Goitein-Straus Program; Heidelberg Faculty of Medicine Physician Scientist Program; German Federal Research Network Applied Surveillance and Testing (BFAST); Network University Medicine; Helmholtz Association Initiative and Networking Fund Project Virological and Immunological Determinants of COVID-19 Pathogenesis

Background: to characterize humoral response after standard anti-SARS-CoV-2 vaccination in Rituximab-treated patients and to determine the optimal time point after last Rituximab treatment for appropriate immunization. Methods: sixty-four patients who received Rituximab within the last seven years prior to the first anti-SARS-CoV-2 vaccination were recruited in a prospective observational study. Anti-S1 IgG, SARS-CoV-2 specific neutralization, and various SARS-CoV-2 target antibodies were determined. A live virus assay was used to assess neutralizing antibody activity against B.1.617.2 (delta). In Rituximab-treated patients, CD19+ peripheral B-cells were quantified using flow cytometry. Results: after second vaccination, all antibodies were significantly reduced compared to healthy controls. Neutralizing antibody activity against B.1.617.2 (delta) was detectable with a median (IQR) ID50 of 0 (0–1:20) compared to 1:320 (1:160–1:320) in healthy controls (for all p < 0.001). Longer time period since last Rituximab administration correlated with higher anti-SARS-CoV-2 antibody levels and a stronger neutralization of B.1.617.2 (delta). With one exception, only patients with a CD19+ cell proportion ? 1% had detectable neutralizing antibodies. Conclusion: our data indicate that a reconstitution of the B-cell population to >1% seems crucial in developing neutralizing antibodies against SARS-CoV-2. We suggest that anti-SARS-CoV-2 vaccination should be administered at least 8–12 months after the last Rituximab treatment for sufficient humoral responses., Dietmar Hopp Stiftung; Heidelberg Faculty of Medicine Rahel Goitein-Strauss Program; Heidelberg Faculty of Medicine Physician Scientist Program; German Federal Research Network Applied Surveillance and Testing (BFAST); Network University Medicine; Virological and Immunological Determinants of COVID-19 Pathogenesis; Helmholtz Association Initiative and Networking Fund Project

Background and objectives: antibody response after severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) vaccination is impaired in kidney transplant recipients. Emerging variants, such as B.1.617.2 ( d), are of particular concern because of their higher transmissibility and partial immune escape. Little is known about protection against these variants in immunocompromised patients. Design, setting, participants, and measurements: in this prospective two-center study, antispike 1 IgG and surrogate neutralizing antibodies were measured in 173 kidney transplant recipients and 166 healthy controls with different vaccination schedules. In addition, different SARS-CoV-2 epitope antibodies from 135 vaccinated kidney transplant recipients were compared with antibodies in 25 matched healthy controls after second vaccination. In 36 kidney transplant recipients with seroconversion, neutralization against B.1.1.7 ( a), B.1.351 ( b), and B.1.617.2 ( d) was determined on VeroE6 cells and compared with neutralization in 25 healthy controls. Results: kidney transplant recipients had significantly lower seroconversion rates compared with healthy controls. After the second vaccination, antispike 1, antireceptor-binding domain, and surrogate neutralizing antibodies were detectable in 30%, 27%, and 24% of kidney transplant recipients, respectively. This compares with 100%, 96%, and 100% in healthy controls, respectively (P,0.001). Neutralization against B.1.1.7 was detectable in all kidney transplant recipients with seroconversion, with a median serum dilution that reduces infection of cells by 50% of 80 (interquartile range, 80-320). In contrast, only 23 of 36 (64%) and 24 of 36 (67%) kidney transplant recipients showed neutralization against B.1.351 and B.1.617.2, respectively, with median serum dilutions that reduce infection of cells by 50% of 20 (interquartile range, 0-40) and 20 (interquartile range, 0-40), respectively. Neutralization against different variants was signif, Dietmar Hopp Stiftung; German Federal Research Network Applied Surveillance and Testing; Network University Medicine; fightCOVID@DKFZ Initiative; Helmholtz Association Initiative and Networking Fund Project Virological and Immunological Determinants of COVID-19 Pathogenesis; Lessons to Get Prepared for Future Pandemics; Heidelberg Faculty of Medicine Rahel Goitein-Strauss Program; Heidelberg Faculty of Medicine Physician Scientist Program

Due to the current organ shortage, living donor kidney transplantation is increasingly performed across HLA (human leukocyte antigen) or ABO antibody barriers. There is still uncertainty about the risk of antibody-mediated rejection (AMR) episodes, which may limit long-term graft survival. From March 2007 to December 2016, 58 sensitized living donor kidney transplant candidates were identified and 38 patients eventually included in the study: 36 patients (95%) had pre-transplant and pre-desensitization Luminex-detected donor-specific HLA antibodies (DSA), and 17/36 patients (47%) in addition had a positive crossmatch result. Two patients had no detectable DSA but a positive CDC B-cell crossmatch result. Patients were treated with pre- and post-transplant apheresis and powerful immunosuppression including the anti-CD20 antibody rituximab (N = 36) in combination with thymoglobulin (N = 20) or anti-IL2 receptor antibody (N = 18). The results of the 38 successfully desensitized and transplanted patients were retrospectively compared to the results of 76 matched standard-risk recipients. Desensitized patients showed patient and graft survival rates similar to that of standard-risk recipients (P = 0.55 and P = 0.16, respectively). There was a trend toward reduced death-censored graft survival in desensitized patients (P = 0.053) which, however, disappeared when the 34 patients who were transplanted after introduction of sensitive Luminex testing were analyzed (P = 0.43). The incidence of rejection episodes without borderline changes were in desensitized patients with 21% similar to the 18% in standard-risk patients (P = 0.74). Thirty-six patients had pre-transplant HLA class I and/or II DSA that were reduced by 85 and 81%, respectively, during pre-transplant desensitization (P < 0.001 for both). On day 360 after transplantation, 20 of 36 (56%) patients had lost their DSA. The overall AMR rate was 6% in these patients, but as high as 60% in 5 (14%) patients with persistent, Dietmar Hopp Stiftung

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released from the cardiovascular cells into the circulation. Circulating miRNAs are highly stable and can be quantified. The quantitative expression of specific miRNAs can be linked to the pathology, and some miRNAs show high tissue and disease specificity. Finding novel biomarkers for cardiovascular diseases is of importance for medical research. Quite recently, digital polymerase chain reaction (dPCR) has been invented. dPCR, combined with fluorescent hydrolysis probes, enables specific direct absolute quantification. dPCR exhibits superior technical qualities, including a low variability, high linearity, and high sensitivity compared to the quantitative polymerase chain reaction (qPCR). Thus, dPCR is a more accurate and reproducible method for directly quantifying miRNAs, particularly for the use in large multi-center cardiovascular clinical trials. In this publication, we describe how to effectively perform digital PCR in order to assess the absolute copy number in serum samples.

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released from the cardiovascular cells into the circulation. Circulating miRNAs are highly stable and can be quantified. The quantitative expression of specific miRNAs can be linked to the pathology, and some miRNAs show high tissue and disease specificity. Finding novel biomarkers for cardiovascular diseases is of importance for medical research. Quite recently, digital polymerase chain reaction (dPCR) has been invented. dPCR, combined with fluorescent hydrolysis probes, enables specific direct absolute quantification. dPCR exhibits superior technical qualities, including a low variability, high linearity, and high sensitivity compared to the quantitative polymerase chain reaction (qPCR). Thus, dPCR is a more accurate and reproducible method for directly quantifying miRNAs, particularly for the use in large multi-center cardiovascular clinical trials. In this publication, we describe how to effectively perform digital PCR in order to assess the absolute copy number in serum samples.

Background Data on humoral immune response to standard COVID-19 vaccination are scarce in adolescent patients and lacking for children below 12 years of age with chronic kidney disease including kidney transplant recipients. Methods We therefore investigated in this retrospective two-center study (DRKS00024668; registered 23.03.2021) the humoral immune response to a standard two-dose mRNA vaccine regimen in 123 CKD patients aged 5-30 years. A live-virus assay was used to assess the serum neutralizing activity against the SARS-CoV-2 omicron (BA.1) variant. Results Children aged 5-11 years had a comparable rate and degree of immune response to adolescents despite lower vaccine doses (10 mu g vs. 30 mu g BNT162b2). Treatment with two (odds ratio 9.24) or three or more (odds ratio 17.07) immunosuppressants was an independent risk factor for nonresponse. The immune response differed significantly among three patient cohorts: 48 of 77 (62.3%) kidney transplant recipients, 21 of 26 (80.8%) patients on immunosuppressive therapy, and 19 of 20 (95.0%) patients with chronic kidney disease without immunosuppressive therapy responded. In the kidney transplant recipients, immunosuppressive regimens comprising mycophenolate mofetil, an eGFR of< 60 mL/min/1.73 m(2), and female sex were independent risk factors for nonresponse. Two of 18 (11.1%) and 8 of 16 (50.0%) patients with an anti-S1-RBD IgG of 100-1411 and > 1411 BAU/mL, respectively, showed a neutralization activity against the omicron variant. Conclusion A standard mRNA vaccine regimen in immunosuppressed children and adolescents with kidney disease elicits an attenuated humoral immune response with effective live virus neutralization against the omicron variant in approximately 10% of the patients, underlying the need for omicron-adapted vaccination.