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Background: Donor-derived cell-free DNA (ddcfDNA) is a promising minimally invasive biomarker for acute rejection (AR) in kidney transplant recipients. To assess the diagnostic value of ddcfDNA as a marker for AR, ddcfDNA was quantified at multiple time points after kidney transplantation with a novel high-throughput droplet digital PCR indel method that allowed for the absolute quantification of ddcfDNA., Methods: In this study, ddcfDNA in plasma samples from 223 consecutive kidney transplant recipients was analyzed pretransplantation; at 3, 7, and 180 d after transplantation; and at time of for-cause biopsies obtained within the first 180 d after transplantation., Results: Median (interquartile range) ddcfDNA concentration was significantly higher on day 3 (58.3 [17.7-258.3] copies/mL) and day 7 (25.0 [10.4-70.8] copies/mL) than on day 180 after transplantation (4.2 [0.0-8.3] copies/mL; P < 0.001 and P < 0.001, respectively). At time of biopsy-proven AR (BPAR), between day 11 and day 180 after transplantation, ddcfDNA concentration was significantly higher (50.0 [25.0-108.3] copies/mL) than those when biopsies showed non-AR (0.0 [0.0-15.6] copies/mL; P < 0.05). ddcfDNA concentration within the first 10 d after transplantation showed no significant difference between recipients with BPAR and those with non-AR in their biopsy or between recipients with BPAR and ddcfDNA measured at day 3 and day 7., Conclusions: Unfortunately, ddcfDNA concentration is not a good biomarker to detect AR within the first 10 d after transplantation; however, BPAR occurring after 10 d after transplantation can be detected in kidney transplant recipients by ddcfDNA using a novel and unique high-throughput droplet digital PCR indel method., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

The effect of immunosuppressive drugs on the generation of T follicular helper (Tfh) cells, specialized in supporting B-cell differentiation, is largely unknown. We examined whether the calcineurin inhibitor tacrolimus (TAC) and the mammalian target of rapamycin (mtor) inhibitor sirolimus (SRL) inhibit Tfh cell differentiation, and affect subsequent B-cell functions. Isolated naive T cells were polarized into Tfh-like cells in the presence of TAC or SRL. To demonstrate their functionality, we co-cultured these cells with isolated B cells in the presence of alloantigen and studied the activation and differentiation of these B cells. Tfh-like cells were defined as CD4 + CXCR5 + T cells, expressing immunoinhibitory programmed death protein 1 (pd1) and inducible T-cell costimulator (icos). We found that TAC and SRL significantly inhibited Tfh-like cell differentiation. Therapeutic concentrations of TAC and SRL reduced the percentage of pd1 + and icos + Tfh cells compared to controls. In addition, T cells grown in the presence of TAC or SRL expressed less IL-21 and provided less B-cell help. TAC and SRL both inhibited Tfh-dependent alloantigen-activated B-cell proliferation and differentiation into plasma cells and transitional B cells. In conclusion, TAC and SRL inhibited the differentiation of naive T cells into functional Tfh-like cells, a finding that can be extrapolated to immunosuppressive regimens in transplant patients., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Natural occurring regulatory T cells (nTregs) have the potential to offer a targeted approach of immunosuppression and are the cell type of interest for inducing tolerance in kidney transplantation. End-stage renal disease (ESRD) profoundly affects the composition and function of circulating T cells but little is known with respect to how nTreg potential is affected. To address this, nTregs of patients with ESRD (on dialysis or not) and healthy individuals were isolated, expanded using allogeneic mature monocyte-derived dendritic cells followed by anti-CD3/anti-CD28-coated beads and the different nTregs were extensively characterized by the demethylation status of the Treg-specific demethylated region within FOXP3 and expression of typical nTreg markers. Additionally, the suppressive capacity as well as cytokine producing cells were analyzed for allogeneic mature monocyte-derived dendritic cell-expanded nTregs. Compared to age- and gender-matched healthy individuals, similar frequencies of nTregs were present within the circulation of patients with ESRD either on dialysis or not. The isolated nTregs could be equally well or even better expanded using allogeneic mature monocyte-derived dendritic cells and extensive characterization did not reveal significant differences. The demethylation status of the Treg-specific demethylated region was maintained or even further promoted as was the expression of markers characteristic for nTregs. Moreover, suppressive capacity and the cytokine profile of allogeneic mature monocyte-derived dendritic cell-expanded nTregs was similar to that of healthy individuals. Thus, circulating nTregs of patients with ESRD can effectively be expanded to stable allo antigen-specific nTregs with potential clinical applicability., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: Interleukin-17 (IL-17) is regarded as a major effector cytokine with pro-inflammatory actions. It has pleiotropic and environment-specific functions by promoting adaptive cytotoxic T-lymphocyte responses during inflammation. Therefore, it is tempting to speculate that IL-17 plays a major role in inflammatory responses in transplant recipients. We questioned whether IL-17 is expressed in the transplanted heart during acute rejection (AR), or during immunologic quiescence, and which graft-infiltrating lymphocytes produce IL-17. In addition, we analyzed donor-specific IL-17-producing cells in peripheral blood cells in comparable periods after transplantation., Methods: Endomyocardial biopsies from heart transplant recipients with early or late AR or in an immunologic quiescence period were analyzed for the presence of IL-17 mRNA. In addition, the capacity of graft-infiltrating lymphocytes (GILs) to produce IL-17 was analyzed. Moreover, we determined the frequency of donor-reactive IL-17-producing peripheral blood mononuclear cells (PBMCs) using an Elispot assay., Results: Twenty-one percent (14 of 67) of the biopsies assessed were positive for IL-17 mRNA. Thirteen of 41 biopsies were observed in the early period (≤3 months) after transplantation. One (of 26) of the late biopsies expressed IL-17 (p = 0.006). Specifically, IL-17 was expressed during early AR (57%, or 8 of 14), whereas biopsies from late AR (0 of 5) did not express IL-17 mRNA (p = 0.02). During AR, IL-17 is derived from IL-17-producing CD4(+)CD161(+), and not CD8(+), GILs. In contrast to the graft findings, we detected circulating donor-reactive IL-17-producing cells mostly during immunologic quiescence., Conclusions: Particularly early after heart transplantation, IL-17-producing CD4(+) T cells home to the graft, which contributes to the AR process., (Copyright © 2015 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

FOXP3(+) regulatory T cells (Treg) play a role in controlling alloreactivity. It has been shown that short (GT)n dinucleotide repeats (≤(GT)15; S) in the promoter region of the FOXP3 gene enhance the promoter activity when compared to long (GT)n repeats (≥(GT)16; L). The present study retrospectively investigated the influence of this (GT)n FOXP3 gene polymorphism on renal allograft survival. A total of 599 consecutive first-time kidney transplant patients (median follow-up time 7.7 years) were subdivided according to their FOXP3 genotype into the S-genotype group (SG) and the L-genotype group (LG). The SG was superior to the LG in both general graft survival censored for death (logrank test, p=0.013) and graft survival following acute rejection (p=0.021). Multivariate analysis defined the (GT)n FOXP3 dinucleotide repeat polymorphism as an independent factor and confirmed an advantage for the SG in renal allograft survival (HR=0.67, 95% CI 0.48-0.94, p=0.02). This gene association study identified a beneficial effect of FOXP3 genetic variants on graft survival in kidney transplant patients., (Copyright © 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Background: Innate immunity plays a role in controlling adaptive immune responses., Methods: We investigated the clinical relevance of single nucleotide polymorphisms in 22 genes encoding innate, secreted, and signaling pattern recognition receptors in a total of 520 donor-recipient pairs of postmortem, human leukocyte antigen-DR-compatible kidney transplantations. Associations with rejection incidence were tested in an a priori randomized training set and validation set., Results: Polymorphisms in TLR-3 (rs3775296) in the recipients and in ficolin-2 (rs7851696; Ala258Ser) and C1qR1 (rs7492) in the donors showed the strongest association with severe rejection. In multivariate analysis, presence of the ficolin-2 Ala258Ser variant in the donor predicted lower incidence of severe rejection (odds ratio=0.3; 95% confidence interval, 0.1-0.9; P=0.024) and of graft loss (hazard ratio=0.5; 95% confidence interval, 0.2-1.0; P=0.046) independently of clinical risk factors. Ficolin-2 messenger RNA expression was detected in pretransplantation biopsies from 69 donor grafts. Serum and tissue ficolin-2 levels were unaffected by genotype. Ficolin-2 protein, which bound to dying cells, was detected in donor kidneys in a passenger leukocyte-like pattern. Indeed, monocytes, monocyte-derived macrophages, and peripheral blood mononuclear cells expressed ficolin-2. Donor grafts with the ficolin-2 Ala258Ser variant contained significantly elevated expression of interleukin 6, having ascribed cytoprotective effects. It has been described that Ala258Ser leads to increased binding capacity of ficolin-2 to N-acetylglucosamine., Conclusions: Presence of the ficolin-2 Ala258Ser polymorphism in the donor independently predicts improved graft outcome. Based on mechanistic data, we propose that this functional polymorphism leads to more efficient handling of injured cells by phagocytozing cells, resulting in decreased intragraft exposure to danger signals and dampened alloimmune responses.

Background and Objectives: Forkhead box P3 regulatory T cells control inflammatory responses, but it remains unclear whether they inhibit brain death-initiated inflammation and tissue injury in deceased kidney donors. DESIGN, SETTING, PARTICIPANTS, MEASUREMENT: To study the actions of regulatory T cells at various stages of the donation and transplantation procedure, forkhead box P3, regulatory and inflammatory cytokine expression, and tissue injury markers were determined in time 0 kidney biopsies from deceased and living donors. Additionally, the interaction between forkhead box P3+ T cells and kidney injury molecule-1 by activated primary tubular epithelial cells was studied., Results: After cold storage, the deceased donor kidneys expressed the higher mRNA levels of kidney injury molecule-1 and CD3ε. In these samples, the inflammatory cytokines IL-8 and IFN-γ and markers associated with regulation (forkhead box P3, TGF-β, and IL-10) were highly expressed compared with living donor kidneys. Correlations were found between mRNA expression levels of forkhead box P3 and kidney injury molecule-1 and forkhead box P3 and IFN-γ. Immunohistochemical analysis confirmed the presence of forkhead box P3+ T cells in donor kidneys. Renal function (analyzed by serum creatinine levels) at the first week posttransplantation correlated with kidney injury molecule-1 and forkhead box P3 mRNA levels. In vitro studies showed that kidney injury molecule-1 expression by primary tubular epithelial cells was 63% (mean) lower when cocultured with regulatory T cells compared with control T cells., Conclusions: These results show that donor forkhead box P3+ T cells infiltrate the deceased donor kidney, where they may control inflammatory and injury responses.

Background: The transcription factor FOXP3 has been identified as the molecule associated with the regulatory function of CD25+ T cells., Methods: To understand the biology of FOXP3+ T cells in allogeneic reactions, we measured FOXP3 mRNA expression levels in allostimulated CD25 cells and CD25 cells and in peripheral blood mononuclear cells (PBMC). The effect of immunosuppressive drugs on FOXP3 expression was studied in mixed lymphocyte reactions (MLR) in the presence and absence of calcineurin inhibitors (CNI), alphaCD25 mAb, and Rapamycin (Rapa), and analyzed in biopsies from cardiac allograft recipients during acute rejection by quantitative (Q)-PCR., Results: FOXP3 mRNA expression was restricted to the CD25 population that inhibited the proliferation of allostimulated CD25 cells. In the MLR FOXP3 was readily induced after allostimulation. Kinetic examination of the MLR showed a 10-20-fold higher FOXP3 mRNA expression level after 5 days of culture. The CNI Cyclosporin and Tacrolimus, and alphaCD25 mAb inhibited in vitro induced FOXP3 gene transcription (range 70%-90%), whereas Rapa did not inhibit the induction. After clinical heart transplantation the highest FOXP3 mRNA expression levels were measured in biopsies during acute rejection (P=0.03)., Conclusions: The high FOXP3 mRNA levels during allogeneic responses in vivo and in vitro suggests that regulatory activities of CD25 T cells or the generation of these cells is an intrinsic part of activation. CNI and alphaCD25 mAb in contrast to Rapa, did interfere with this immunosuppressive counter-mechanism and as a result might have an inhibitory effect to tolerance induction after transplantation.

The extent of graft damage after ischemia-reperfusion reflects the balance between deleterious events and protective factors. Heme oxygenase-1 (HO-1) and vascular endothelial growth factor (VEGF) may contribute to cytoprotection by their anti-inflammatory and antiapoptotic properties. For investigating whether HO-1 and VEGF play a role in the adaptive response to ischemia-reperfusion injury after renal transplantation, kidney biopsies were analyzed from living (n = 45) and cadaveric (n = 16) donors, obtained at three time points: at the end of cold storage T(-1), after warm ischemia T(0), and after reperfusion T(+1). The mRNA expression levels of HO-1, VEGF(165), Bcl-2, Bax, and hypoxia inducible factor-1alpha were quantified by real-time reverse transcriptase-PCR, and the HO-1 and VEGF proteins were analyzed by immunohistochemistry. Cadaveric donor kidneys presented higher mRNA expression levels of hypoxia inducible factor-1alpha. In contrast, mRNA expression levels of HO-1, VEGF(165), and Bcl-2 were significantly lower in kidneys from cadaveric donors. Overall, a significant correlation was observed between mRNA expression of Bcl-2 and VEGF(165), between Bcl-2 and HO-1, and between HO-1 and VEGF(165). Moreover, protein expression of HO-1 and VEGF was detected in the same anatomical kidney compartments (glomerulus, arteries, and distal tubules). Renal function at the first week posttransplantation (analyzed by serum creatinine levels) showed a significant correlation with both HO-1 and VEGF mRNA expression, reinforcing the protective role of both genes in the early events of transplantation. It is concluded that the lower expression of HO-1, VEGF(165), and Bcl-2 in cadaveric donor kidneys can reflect a defective adaptation against ischemia-reperfusion injury that may affect their function in the short term.

Background: To examine whether genetic factors are involved in the development of acute rejection (AR), we investigated a (CA)m(CT)n repeat in the 3'-flanking region of the interleukin (IL)-2 gene., Method: We genotyped 290 heart transplant recipients with and without AR (International Society for Heart and Lung Transplantation criteria > or =3A) and 101 controls., Results: The frequency of allele 135 of the repeat and its genotype distribution (carriers/noncarriers) were significantly associated with freedom from AR (P=0.03 and P=0.02, respectively). We also found interaction between allele 135 and HLA-DR matching. More carriers of allele 135 with no or one mismatch remained free from AR compared to patients without the allele (P=0.01). This was not found in the HLA-DR group with two mismatches., Conclusion: HLA-DR matching might only be effective in reducing AR after heart transplantation in recipients who carry allele 135 of the (CA)m(CT)n repeat in the 3'-flanking region of the IL-2 gene.