Effects of Reduced-Dose Anti-Human T-Lymphocyte Globulin on Overall and Donor-Specific T-Cell Repertoire Reconstitution in Sensitized Kidney Transplant Recipients.

Background: Pre-sensitized kidney transplant recipients have a higher risk for rejection following kidney transplantation and therefore receive lymphodepletional induction therapy with anti-human T-lymphocyte globulin (ATLG) whereas non-sensitized patients are induced in many centers with basiliximab. The time course of lymphocyte reconstitution with regard to the overall and donor-reactive T-cell receptor (TCR) specificity remains elusive. Methods/Design: Five kidney transplant recipients receiving a 1.5-mg/kg ATLG induction therapy over 7 days and five patients with 2 × 20 mg basiliximab induction therapy were longitudinally monitored. Peripheral mononuclear cells were sampled pre-transplant and within 1, 3, and 12 months after transplantation, and their overall and donor-reactive TCRs were determined by next-generation sequencing of the TCR beta CDR3 region. Overall TCR repertoire diversity, turnover, and donor specificity were assessed at all timepoints. Results: We observed an increase in the donor-reactive TCR repertoire after transplantation in patients, independent of lymphocyte counts or induction therapy. Donor-reactive CD4 T-cell frequency in the ATLG group increased from 1.14% + -0.63 to 2.03% + -1.09 and from 0.93% + -0.63 to 1.82% + -1.17 in the basiliximab group in the first month. Diversity measurements of the entire T-cell repertoire and repertoire turnover showed no statistical difference between the two induction therapies. The difference in mean clonality between groups was 0.03 and 0.07 pre-transplant in the CD4 and CD8 fractions, respectively, and was not different over time (CD4: F(1.45, 11.6) = 0.64 p = 0.496; CD8: F(3, 24) = 0.60 p = 0.620). The mean difference in R20, a metric for immune dominance, between groups was -0.006 in CD4 and 0.001 in CD8 T-cells and not statistically different between the groups and subsequent timepoints (CD4: F(3, 24) = 0.85 p = 0.479; CD8: F(1.19, 9.52) = 0.79 p = 0.418). Conclusion: Reduced-dose ATLG induction therapy led to an initial lymphodepletion followed by an increase in the percentage of donor-reactive T-cells after transplantation similar to basiliximab induction therapy. Furthermore, reduced-dose ATLG did not change the overall TCR repertoire in terms of a narrowed or skewed TCR repertoire after immune reconstitution, comparable to non-depletional induction therapy.

ImmunoDataAnalyzer: a bioinformatics pipeline for processing barcoded and UMI tagged immunological NGS data.

BACKGROUND: Next-generation sequencing (NGS) is nowadays the most used high-throughput technology for DNA sequencing. Among others NGS enables the in-depth analysis of immune repertoires. Research in the field of T cell receptor (TCR) and immunoglobulin (IG) repertoires aids in understanding immunological diseases. A main objective is the analysis of the V(D)J recombination defining the structure and specificity of the immune repertoire. Accurate processing, evaluation and visualization of immune repertoire NGS data is important for better understanding immune responses and immunological behavior. RESULTS: ImmunoDataAnalyzer (IMDA) is a pipeline we have developed for automatizing the analysis of immunological NGS data. IMDA unites the functionality from carefully selected immune repertoire analysis software tools and covers the whole spectrum from initial quality control up to the comparison of multiple immune repertoires. It provides methods for automated pre-processing of barcoded and UMI tagged immune repertoire NGS data, facilitates the assembly of clonotypes and calculates key figures for describing the immune repertoire. These include commonly used clonality and diversity measures, as well as indicators for V(D)J gene segment usage and between sample similarity. IMDA reports all relevant information in a compact summary containing visualizations, calculations, and sample details, all of which serve for a more detailed overview. IMDA further generates an output file including key figures for all samples, designed to serve as input for machine learning frameworks to find models for differentiating between specific traits of samples. CONCLUSIONS: IMDA constructs TCR and IG repertoire data from raw NGS reads and facilitates descriptive data analysis and comparison of immune repertoires. The IMDA workflow focus on quality control and ease of use for non-computer scientists. The provided output directly facilitates the interpretation of input data and includes information about clonality, diversity, clonotype overlap as well as similarity, and V(D)J gene segment usage. IMDA further supports the detection of sample swaps and cross-sample contamination that potentially occurred during sample preparation. In summary, IMDA reduces the effort usually required for immune repertoire data analysis by providing an automated workflow for processing raw NGS data into immune repertoires and subsequent analysis. The implementation is open-source and available on https://bioinformatics.fh-hagenberg.at/immunoanalyzer/ .

Prospective Tracking of Donor-Reactive T-Cell Clones in the Circulation and Rejecting Human Kidney Allografts.

Background: Antigen recognition of allo-peptides and HLA molecules leads to the activation of donor-reactive T-cells following transplantation, potentially causing T-cell-mediated rejection (TCMR). Sequencing of the T-cell receptor (TCR) repertoire can be used to track the donor-reactive repertoire in blood and tissue of patients after kidney transplantation. Methods/Design: In this prospective cohort study, 117 non-sensitized kidney transplant recipients with anti-CD25 induction were included. Peripheral mononuclear cells (PBMCs) were sampled pre-transplant and at the time of protocol or indication biopsies together with graft tissue. Next-generation sequencing (NGS) of the CDR3 region of the TCRbeta chain was performed after donor stimulation in mixed lymphocyte reactions to define the donor-reactive TCR repertoire. Blood and tissue of six patients experiencing a TCMR and six patients without rejection on protocol biopsies were interrogated for these TCRs. To elucidate common features of T-cell clonotypes, a network analysis of the TCR repertoires was performed. Results: After transplantation, the frequency of circulating donor-reactive CD4 T-cells increased significantly from 0.86 ± 0.40% to 2.06 ± 0.40% of all CD4 cells (p < 0.001, mean dif.: -1.197, CI: -1.802, -0.593). The number of circulating donor-reactive CD4 clonotypes increased from 0.72 ± 0.33% to 1.89 ± 0.33% (p < 0.001, mean dif.: -1.168, CI: -1.724, -0.612). No difference in the percentage of donor-reactive T-cells in the circulation at transplant biopsy was found between subjects experiencing a TCMR and the control group [p = 0.64 (CD4+), p = 0.52 (CD8+)]. Graft-infiltrating T-cells showed an up to six-fold increase of donor-reactive T-cell clonotypes compared to the blood at the same time (3.7 vs. 0.6% and 2.4 vs. 1.5%), but the infiltrating TCR repertoire was not reflected by the composition of the circulating TCR repertoire despite some overlap. Network analysis showed a distinct segregation of the donor-reactive repertoire with higher modularity than the overall TCR repertoire in the blood. These findings indicate an unchoreographed process of diverse T-cell clones directed against numerous non-self antigens found in the allograft. Conclusion: Donor-reactive T-cells are enriched in the kidney allograft during a TCMR episode, and dominant tissue clones are also found in the blood. Trial Registration: Clinicaltrials.gov: NCT: 03422224 (https://clinicaltrials.gov/ct2/show/NCT03422224).

Steroid pretreatment of deceased donors and liver allograft function - Ten years follow-up of a blinded randomized placebo controlled trial.

BACKGROUND: Within the last decade numerous attempts have been reported in order to expand the donor pool and alleviate organ shortage in the setting of liver transplantation. Aim of this blinded randomized controlled trial was to evaluate the effect of donor steroid pretreatment on outcomes after liver transplantation. METHODS: We performed an international, multi-center double-blinded randomized placebo controlled trial. Donors received 1000 mg methylprednisone or placebo before organ procurement. Primary endpoint were patient and graft survival. Secondary end points were rate of BPAR and liver functions trajectories after transplantation. Follow up was 10 years. RESULTS: There was no effect of steroid pretreatment vs. placebo on overall patient survival (50% vs. 46%, p = n.s.) as well as graft survival (47% vs. 51%, p= n.s.). Further donor steroid pretreatment did not alter the rate of biopsy proven acute rejections (34% steroid group vs. 36% placebo, p = n.s.). Evaluating short term and long term graft function, steroid pretreatment had minor effect on immediate liver function trajectories within the first 2 weeks after transplantation. This was not seen in long-term follow up. CONCLUSION: In conclusion we found no evidence that donor steroid pretreatment translates in improved outcomes after liver transplantation.

Next generation sequencing based assessment of the alloreactive T cell receptor repertoire in kidney transplant patients during rejection: a prospective cohort study.

BACKGROUND: Kidney transplantation is the optimal treatment in end stage renal disease but the allograft survival is still hampered by immune reactions against the allograft. This process is driven by the recognition of allogenic antigens presented to T-cells and their unique T-cell receptor (TCR) via the major histocompatibility complex (MHC), which triggers a complex immune response potentially leading to graft injury. Although the immune system and kidney transplantation have been studied extensively, the subtlety of alloreactive immune responses has impeded sensitive detection at an early stage. Next generation sequencing of the TCR enables us to monitor alloreactive T-cell populations and might thus allow the detection of early rejection events. METHODS/DESIGN: This is a prospective cohort study designed to sequentially evaluate the alloreactive T cell repertoire after kidney transplantation. The TCR repertoire of patients who developed biopsy confirmed acute T cell mediated rejection (TCMR) will be compared to patients without rejection. To track the alloreactive subsets we will perform a mixed lymphocyte reaction between kidney donor and recipient before transplantation and define the alloreactive TCR repertoire by next generation sequencing of the complementary determining region 3 (CDR3) of the T cell receptor beta chain. After initial clonotype assembly from sequencing reads, TCR repertoire diversity and clonal expansion of T cells of kidney transplant recipients in periphery and kidney biopsy will be analyzed for changes after transplantation, during, prior or after a rejection. The goal of this study is to describe changes of overall T cell repertoire diversity, clonality in kidney transplant recipients, define and track alloreactive T cells in the posttransplant course and decipher patterns of expanded alloreactive T cells in acute cellular rejection to find an alternative monitoring to invasive and delayed diagnostic procedures. DISCUSSION: Changes of the T cell repertoire and tracking of alloreactive T cell clones after combined bone marrow and kidney transplant has proven to be of potential use to monitor the donor directed alloresponse. The dynamics of the donor specific T cells in regular kidney transplant recipients in rejection still rests elusive and can give further insights in human alloresponse. TRIAL REGISTRATION: Clinicaltrials.gov: NCT03422224 , registered February 5th 2018.

Contribution of non-HLA incompatibility between donor and recipient to kidney allograft survival: genome-wide analysis in a prospective cohort.

BACKGROUND: The introduction of HLA matching of donors and recipients was a breakthrough in kidney transplantation. However, half of all transplanted kidneys still fail within 15 years after transplantation. Epidemiological data suggest a fundamental role of non-HLA alloimmunity. METHODS: We genotyped 477 pairs of deceased donors and first kidney transplant recipients with stable graft function at three months that were transplanted between Dec 1, 2005, and April 30, 2015. Genome-wide genetic mismatches in non-synonymous single nucleotide polymorphisms (nsSNPs) were calculated to identify incompatibilities in transmembrane and secreted proteins. We estimated the association between nsSNP mismatch and graft loss in a Cox proportional hazard model, adjusting for HLA mismatch and clinical covariates. Customised peptide arrays were generated to screen for antibodies against genotype-derived mismatched epitopes in 25 patients with biopsy-confirmed chronic antibody-mediated rejection. FINDINGS: 59 268 nsSNPs affecting a transmembrane or secreted protein were analysed. The median number of nsSNP mismatches in immune-accessible transmembrane and secreted proteins between donors and recipients was 1892 (IQR 1850-1936). The degree of nsSNP mismatch was independently associated with graft loss in a multivariable model adjusted for HLA eplet mismatch (HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR). Each increase by a unit of one IQR had an HR of 1·68 (95% CI 1·17-2·41, p=0·005). 5-year death censored graft survival was 98% in the quartile with the lowest mismatch, 91% in the second quartile, 89% in the third quartile, and 82% in the highest quartile (p=0·003, log-rank test). Customised peptide arrays verified a donor-specific alloimmune response to genetically predicted mismatched epitopes. INTERPRETATION: Genetic mismatch of non-HLA haplotypes coding for transmembrane or secreted proteins is associated with an increased risk of functional graft loss independently of HLA incompatibility. As in HLA alloimmunity, donor-specific alloantibodies can be identified against genotype derived non-HLA epitopes. FUNDING: Austrian Science Fund, WWTF (Vienna Science and Technology Fund), and Ministry of Health of the Czech Republic.

Steroid pretreatment of organ donors does not impact on early rejection and long-term kidney allograft survival: Results from a multicenter randomized, controlled trial.

Steroid pretreatment of deceased donors reduces inflammation in allografts and is recommended by organ procurement guidelines. The impact on long-term graft outcome, however, remains elusive. In this multicenter randomized controlled trial, 306 deceased donors providing organs for 455 renal transplant recipients were randomized to 1000 mg of methylprednisolone or placebo prior to organ procurement (ISRCTN78828338). The incidence of biopsy-confirmed rejection (Banff>1) at 3 months was 23 (10%) in the steroid group and 26 (12%) in the placebo group (P = .468). Five-year functional graft survival was 84% and 82% for the steroid group and placebo group, respectively (P-value = .941). The hazard ratio of functional graft loss was 0.90 (95% confidence interval 0.57-1.42, P = .638) for steroid vs placebo in a multivariate Cox model. We did not observe effect modification by any of the predictors of graft survival and treatment modality. A robust sandwich estimate was used to account for paired grafts of some donors. The mean estimated GFR at 5 years was 47 mL/min per 1.73 m2 in the steroid group and 48 mL/min per 1.73 m2 in the placebo group (P = .756). We conclude that steroid pretreatment does not impact on long-term graft survival. In a donor population with higher risk of delayed graft function, however, repetitive and higher doses of steroid treatment may result in different findings.

miR-182-5p Inhibition Ameliorates Ischemic Acute Kidney Injury.

Acute kidney injury (AKI) remains a major clinical event with high mortality rates. We previously identified renal miR-182 as the main driver of post-transplantation AKI. Therefore, we tested the causal inference of miR-182 by inhibiting its renal expression in vivo. In 45 rats AKI was induced by right nephrectomy and contralateral clamping of the renal pedicle for 40 minutes. Systemically administered antisense oligonucleotide (ASO) inhibited miR-182 in the kidneys up to 96 hours. The maximum creatinine elevation was on day 2 after injury (mg/dL; median and interquartile range): ASO 2.5mg/kg: 1.9 (1.3; 3.2), ASO 25mg/kg: 2.8 (0.7; 5.0), mismatch oligonucleotide (MM) 25mg/kg: 5.7 (5,0; 5.8), saline: 4.4 (3.5; 5.8) (P = 0.016, analysis of variance). Blinded semiquantitative histologic evaluation of renal biopsies showed better preserved morphology in both ASO groups than saline- and MM-treated kidneys (median and interquartile range of overall injury scores): ASO both concentrations 1 (1, 1), saline 3 (3, 3) and MM 3 (3, 3) (P< 0.001, analysis of variance). ASO facilitated cell proliferation, metabolism, and angiogenesis on a genome-wide level. ASO when applied in normothermic kidney machine perfusion reduced renal miR-182 expression by more than two magnitudes. In summary, we showed that in vivo inhibition of miR-182 by ASO improved kidney function and morphology after AKI. This technique may be applicable to reduce the high rate of AKI in the human renal transplantation setting.

microRNA and Kidney Transplantation.

The kidney serves as the main clearance organ of our body, filtrating and excreting metabolic waste products. Various intrinsic and extrinsic conditions can lead to kidney injury, roughly 0.1% of the population suffer from end stage renal disease. Renal transplantation reinstitutes an almost normal quality of life; again it is cost effective and thus the preferred treatment of terminal renal failure. miRNAs play pivotal roles in immune responses and inflammation, which makes them particularly interesting in the field of transplantation and in understanding the molecular pathways of allograft pathologies such as delayed function or cellular and antibody mediated rejection. As kidney biopsy is part of the routine disease monitoring, the identification of miRNA pattern is feasible in different stages of the injury. Furthermore miRNAs are easy to detect not only in tissue samples but also in body fluids such as blood and urine. Their regulatory capacity of biological processes together with their stability makes them excellent candidates for noninvasive monitoring of kidney pathology. There is an accumulating knowledge about diseases-specific miRNA signatures in distinct kidney injuries. In the following chapter we present the current understanding of miRNAs regulation of intragraft processes after kidney transplantation.

Genome-wide studies to identify risk factors for kidney disease with a focus on patients with diabetes.

Chronic kidney disease (CKD) affects 10-13% of the general population and diabetic nephropathy (DN) is the leading cause of end-stage renal disease (ESRD). In addition to known demographic, biochemical and lifestyle risk factors, genetics is also contributing to CKD risk. In recent years, genome-wide association studies (GWAS) have provided a hypothesis-free approach to identify common genetic variants that could account for the genetic risk component of common diseases such as CKD. The identification of these variants might reveal the biological processes underlying renal impairment and could aid in improving risk estimates for CKD. This review aims to describe the methods as well as strengths and limitations of GWAS in CKD and to summarize the findings of recent GWAS in DN. Several loci and SNPs have been found to be associated with distinct CKD traits such as eGFR and albuminuria. For diabetic kidney disease, several loci were identified in different populations. Subsequent functional studies provided insights into the mechanism of action of some of these variants, such as UMOD or CERS2. However, overall, the results were ambiguous, and a few of the variants were not consistently replicated. In addition, the slow progression from albuminuria to ESRD could limit the power of longitudinal studies. The typically small effect size associated with genetic variants as well as the small portion of the variability of the phenotype explained by these variants limits the utility of genetic variants in improving risk prediction. Nevertheless, identifying these variants could give a deeper understanding of the molecular pathways underlying CKD, which in turn, could potentially lead to the development of new diagnostic and therapeutic tools.

Absence of pneumococcal PcsB is associated with overexpression of LysM domain-containing proteins.

The streptococcal protein required for cell separation B (PcsB) is predicted to play an important role in peptidoglycan metabolism, based on sequence motifs and altered phenotypes of gene deletion mutant cells exhibiting defects in cell separation. However, no enzymic activity has been demonstrated for PcsB so far. By generating gene deletion mutant strains in four different genetic backgrounds we could demonstrate that pcsB is not essential for cell survival in Streptococcus pneumoniae, but is essential for proper cell division. Deletion mutant cells displayed cluster formation due to aberrant cell division, reduced growth and antibiotic sensitivity that were fully reverted by transformation with a plasmid carrying pcsB. Immunofluorescence staining revealed that PcsB was localized to the cell poles, similarly to PBP3 and LytB, enzymes with demonstrated peptidoglycan-degrading activity required for daughter cell separation. Similarly to other studies with PcsB homologues, we could not detect peptidoglycan-lytic activity with recombinant or native pneumococcal PcsB in vitro. In addition to defects in septum placement and separation, the absence of PcsB induced an increased release of several proteins, such as enolase, MalX and the SP0107 LysM domain protein. Interestingly, genes encoding both LysM domain-containing proteins that are present in the pneumococcal genome (SP0107 and SP2063) and predicted to be involved in cell wall metabolism were found to be highly overexpressed (14-33-fold increase) in ΔpcsB cells in two different genetic backgrounds. Otherwise, we detected very few changes in the global gene expression profile of cells lacking PcsB. Thus our data suggest that LysM domain proteins partially compensate for the lack of PcsB function and allow the survival and slow growth of the pneumococcus.

The pneumococcal eukaryotic-type serine/threonine protein kinase StkP co-localizes with the cell division apparatus and interacts with FtsZ in vitro.

The importance of serine/threonine phosphorylation in signalling and regulation of gene expression in prokaryotes has been widely recognized. Driven by our interest in StkP (the pneumococcal serine/threonine kinase homologue) for vaccine development, we studied its cellular localization. We found that the C-terminally located PASTA (penicillin-binding protein and serine/threonine kinase associated) domains, but not the N-terminal kinase domain of StkP, were located on the surface of live pneumococcal cells grown in vitro and were also accessible to antibodies during pneumococcal infection in mice and man. Most importantly, we discovered, by immunofluorescence microscopy, that StkP co-localized with the cell division apparatus. StkP and FtsZ, the prokaryotic tubulin homologue, co-localized at mid-cell in most cells. Formation and constriction of the ring-like structure of StkP followed the dynamic changes of FtsZ in dividing cells. This pattern resembles that of the 'late' divisome protein penicillin-binding protein 2X. The lack of StkP in gene deletion mutants did not disturb FtsZ ring formation, further suggesting that StkP joins the divisome after the FtsZ ring is assembled. We also present evidence that StkP binds and phosphorylates recombinant FtsZ in vitro; however, we could not detect changes in the phosphorylation of FtsZ in a stkP deletion strain relative to wild-type cells. Based on its cell-division-dependent localization and interaction with FtsZ, we propose that StkP plays a currently undefined role in cell division of pneumococcus.