Tissue-resident Lymphocytes Are Released During Hypothermic and Normothermic Machine Perfusion of Human Donor Kidneys.

BACKGROUND: Machine perfusion is the preferred preservation method for deceased donor kidneys. Perfusate fluid, which contains a complex mixture of components, offers potential insight into the organ's viability and function. This study explored immune cell release, particularly tissue-resident lymphocytes (TRLs), during donor kidney machine perfusion and its correlation with injury markers. METHODS: Perfusate samples from hypothermic machine perfusion (HMP; n = 26) and normothermic machine perfusion (NMP; n = 16) of human donor kidneys were analyzed for TRLs using flow cytometry. Residency was defined by expressions of CD69, CD103, and CD49as. TRL release was quantified exclusively in NMP. Additionally, levels of cell-free DNA, neutrophil gelatinase-associated lipocalin, and soluble E-cadherin (sE-cadherin) were measured in NMP supernatants with quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: Both HMP and NMP samples contained a heterogeneous population of TRLs, including CD4 + tissue-resident memory T cells, CD8 + tissue-resident memory T cells, tissue-resident natural killer cells, tissue-resident natural killer T cells, and helper-like innate lymphoid cells. Median TRL proportions among total CD45 + lymphocytes were 0.89% (NMP) and 0.84% (HMP). TRL quantities in NMP did not correlate with donor characteristics, perfusion parameters, posttransplant outcomes, or cell-free DNA and neutrophil gelatinase-associated lipocalin concentrations. However, CD103 + TRL release positively correlated with the release of sE-cadherin, the ligand for the CD103 integrin. CONCLUSIONS: Human donor kidneys release TRLs during both HMP and NMP. The release of CD103 + TRLs was associated with the loss of their ligand sE-cadherin but not with general transplant injury biomarkers.

Donor-Derived Cell-Free DNA for the Detection of Heart Allograft Injury: The Impact of the Timing of the Liquid Biopsy.

Background: In heart transplant recipients, donor-derived cell-free DNA (ddcfDNA) is a potential biomarker for acute rejection (AR), in that increased values may indicate rejection. For the assessment of ddcfDNA as new biomarker for rejection, blood plasma sampling around the endomyocardial biopsy (EMB) seems a practical approach. To evaluate the effect of the EMB procedure on ddcfDNA values, ddcfDNA values before the EMB were pairwise compared to ddcfDNA values after the EMB. We aimed at evaluating whether it matters whether the ddcfDNA sampling is done before or after the EMB-procedure. Methods: Plasma samples from heart transplant recipients were obtained pre-EMB and post-EMB. A droplet digital PCR method was used for measuring ddcfDNA, making use of single-nucleotide polymorphisms that allowed both relative quantification, as well as absolute quantification of ddcfDNA. Results: Pairwise comparison of ddcfDNA values pre-EMB with post-EMB samples (n = 113) showed significantly increased ddcfDNA concentrations and ddcfDNA% in post-EMB samples: an average 1.28-fold increase in ddcfDNA concentrations and a 1.31-fold increase in ddcfDNA% was observed (p = 0.007 and p = 0.03, respectively). Conclusion: The EMB procedure causes iatrogenic injury to the allograft that results in an increase in ddcfDNA% and ddcfDNA concentrations. For the assessment of ddcfDNA as marker for AR, collection of plasma samples before the EMB procedure is therefore essential.

A Novel High-throughput Droplet Digital PCR-based Indel Quantification Method for the Detection of Circulating Donor-derived Cell-free DNA After Kidney Transplantation.

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.

The FCGR3A 158 V/V-genotype is associated with decreased survival of renal allografts with chronic active antibody-mediated rejection.

Natural killer (NK) cells express the Fc-gamma receptor CD16 (FCGR3A) and could therefore mediate renal endothelial cell damage in cases of chronic-active antibody mediated rejection (c-aABMR). The V/V-genotype of the FCGR3A 158 F/V polymorphism is associated with increased CD16 expression and cytotoxicity by NK cells. This study evaluated whether this genotype is associated with the diagnosis of c-aABMR and renal allograft loss. The distribution of the FGCR3A 158 F/V-genotypes was not different for c-aABMR cases (N = 133) compared to control kidney transplant recipients (N = 116, P = 0.65). The V-allele was associated with increased median fluorescence intensity (MFI) of CD16 by NK cells (MFI 3.5 × 104 versus 1.3 × 104 for V/V and F/F-genotype, P < 0.001). Increased expression of CD16 correlated with CD16-dependent degranulation of NK cells (R = 0.4; P = 0.02). Moreover, the V/V-genotype was significantly associated with a higher glomerulitis score and an independent risk factor (HR 1.98; P = 0.04) for decreased allograft survival. Death-censored graft survival in c-aABMR cases at 3 years follow-up was 33% for the FCGR3A 158 V/V-genotype versus 62% for the F/F-genotype. In conclusion, the FCGR3A V/V-genotype increases CD16-mediated NK cell cytotoxicity and is associated with a higher glomerulitis score and decreased graft survival in cases with c-aABMR.

Circulating cell-free nucleosomes as biomarker for kidney transplant rejection: a pilot study.

BACKGROUND: There is an unmet need for noninvasive markers specific for kidney transplant rejection. Such a marker may eventually overcome the need for a transplant biopsy. In this pilot study, the potential of circulating cell-free nucleosomes (CCFN) to serve as a biomarker for kidney transplant rejection was evaluated. METHODS: Forty de novo kidney transplant recipients were prospectively followed as part of a randomized, controlled clinical trial. Total CCFN (H3) and CCFN with the histone modifications H3K36me3 and H3 citrulline were measured in patients at four fixed time points: before transplantation and on days 3-6, 30 and 180 after kidney transplantation. In addition, serum collected at times of transplant rejection (n = 14) was analyzed. CCFN were measured with a Nu.Q™ Assay kit (VolitionRx), an ELISA-based assay using antibodies directed against nucleosomes. RESULTS: For total CCFN (H3), H3K36me3, and H3 citrulline, the same pattern was seen over time: Concentrations were elevated shortly after transplantation (day 3-6) followed by a decline reaching baseline (pre-transplantation) values at days 30 and 180. At times of acute rejection, the median concentration of total CCFN (H3) was significantly higher compared to the stable situation (day 30): 4309 (3435-5285) versus 2885 (1668-3923) ng/mL, p < 0.05, respectively. Total CCFN (H3) had an acceptable ability to discriminate rejection from no rejection (AUC-ROC = 0.73) with a negative predictive value of 92.9%. For both histone modifications (H3K36me3 and H3 citrulline), there was no significant difference between episodes of acute rejection and the stable situation (day 30). CONCLUSION: In this pilot study, total CCFN (H3) concentrations are increased at times of acute kidney transplant rejection. The high negative predictive value implies that whenever a patient experiences loss of renal transplant function and the total CCFN (H3) is not increased, causes other than acute rejection should be considered. Clinical implementation of total CCFN (H3) measurement may avoid unnecessary and potentially harmful kidney transplant biopsies.

Inhibition of T Helper Cell Differentiation by Tacrolimus or Sirolimus Results in Reduced B-Cell Activation: Effects on T Follicular Helper Cells.

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.

Characterization of donor and recipient CD8+ tissue-resident memory T cells in transplant nephrectomies.

Tissue-resident memory T (TRM) cells are characterized by their surface expression of CD69 and can be subdivided in CD103+ and CD103- TRM cells. The origin and functional characteristics of TRM cells in the renal allograft are largely unknown. To determine these features we studied TRM cells in transplant nephrectomies. TRM cells with a CD103+ and CD103- phenotype were present in all samples (n = 13) and were mainly CD8+ T cells. Of note, donor-derived TRM cells were only detectable in renal allografts that failed in the first month after transplantation. Grafts, which failed later, mainly contained recipient derived TRM cells. The gene expression profiles of the recipient derived CD8+ TRM cells were studied in more detail and showed a previously described signature of tissue residence within both CD103+ and CD103- TRM cells. All CD8+ TRM cells had strong effector abilities through the production of IFNγ and TNFα, and harboured high levels of intracellular granzyme B and low levels of perforin. In conclusion, our results demonstrate that donor and recipient TRM cells reside in the rejected renal allograft. Over time, the donor-derived TRM cells are replaced by recipient TRM cells which have features that enables these cells to aggressively respond to the allograft.

Differentially methylated regions in T cells identify kidney transplant patients at risk for de novo skin cancer.

Background: Cutaneous squamous cell carcinoma (cSCC) occurs 65-200 times more in immunosuppressed organ transplant patients than in the general population. T cells, which are targeted by the given immunosuppressive drugs, are involved in anti-tumor immune surveillance and are functionally regulated by DNA methylation. Prior to kidney transplantation, we aim to discover differentially methylated regions (DMRs) in T cells involved in de novo post-transplant cSCC development. Methods: We matched 27 kidney transplant patients with a future de novo cSCC after transplantation to 27 kidney transplant patients without cSCC and studied genome-wide DNA methylation of T cells prior to transplantation. From 11 out of the 27 cSCC patients, the DNA methylation of T cells after transplantation was also examined to assess stability of the observed differences in DNA methylation. Raw methylation values obtained with the 450k array were confirmed with pyrosequencing. Results: We found 16 DMRs between patients with a future cSCC and those who do not develop this complication after transplantation. The majority of the DMRs were located in regulatory genomic regions such as flanking bivalent transcription start sites and bivalent enhancer regions, and most of the DMRs contained CpG islands. Examples of genes annotated to the DMRs are ZNF577, coding for a zinc-finger protein, and FLOT1, coding for a protein involved in T cell migration. The longitudinal analysis revealed that DNA methylation of 9 DMRs changed significantly after transplantation. DNA methylation of 5 out of 16 DMRs was relatively stable, with a variation in beta-value lower than 0.05 for at least 50% of the CpG sites within that region. Conclusions: This is the first study demonstrating that DNA methylation of T cells from patients with a future de novo post-transplant cSCC is different from patients without cSCC. These results were obtained before transplantation, a clinically relevant time point for cSCC risk assessment. Several DNA methylation profiles remained relatively stable after transplantation, concluding that these are minimally affected by the transplantation and possibly have a lasting effect on post-transplant cSCC development.

Interferon-Gamma DNA Methylation Is Affected by Mycophenolic Acid but Not by Tacrolimus after T-Cell Activation.

Immunosuppressive drug therapy is required to treat patients with autoimmune disease and patients who have undergone organ transplantation. The main targets of the immunosuppressive drugs tacrolimus and mycophenolic acid (MPA; the active metabolite of mycophenolate mofetil) are T cells. It is currently unknown whether these immunosuppressive drugs have an effect on DNA methylation-an epigenetic regulator of cellular function. Here, we determined the effect of tacrolimus and MPA on DNA methylation of the gene promoter region of interferon gamma (IFNγ), a pro-inflammatory cytokine. Total T cells, naive T cells (CCR7+CD45RO-), and memory T cells (CD45RO+ and CCR7-CD45RO-) were isolated from CMV seropositive healthy controls and stimulated with α-CD3/CD28 in the presence or absence of tacrolimus or MPA. DNA methylation of the IFNγ promoter region was quantified by pyrosequencing at 4 h, days 1, 3, and 4 after stimulation. In parallel, T-cell differentiation, and IFNγ protein production were analyzed by flow cytometry at days 1 and 3 after stimulation. Our results show that MPA induced changes in IFNγ DNA methylation of naive T cells; MPA counteracted the decrease in methylation after stimulation. Tacrolimus did not affect IFNγ DNA methylation of naive T cells. In the memory T cells, both immunosuppressive drugs did not affect IFNγ DNA methylation. Differentiation of naive T cells into a central-memory-like phenotype (CD45RO+) was inhibited by both immunosuppressive drugs, while differentiation of memory T cells remained unaffected by both MPA and tacrolimus. IFNγ protein production was suppressed by tacrolimus. Our results demonstrate that MPA influenced IFNγ DNA methylation of naive T cells after stimulation of T cells, while tacrolimus had no effect. Both tacrolimus and MPA did not affect IFNγ DNA methylation of memory T cells.

Natural regulatory T cells from patients with end-stage renal disease can be used for large-scale generation of highly suppressive alloantigen-specific Tregs.

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.

Thymus-Derived Regulatory T Cells Infiltrate the Cardiac Allograft Before Rejection.

BACKGROUND: FOXP3+ regulatory T cells (Treg) either originate in the thymus (natural [n]Treg) or are induced in the periphery by antigen exposure and cytokines (induced [i]Treg). It is currently not elucidated which and to what extent these Treg subsets regulate intracardiac allogeneic responses in transplant patients. METHODS: By using demethylation of the Treg-specific demethylated region in the FOXP3 gene as a marker for nTreg and FOXP3 messenger RNA expression as a marker for the total Treg population, we examined Treg in endomyocardial biopsies (EMBs) of both patients who developed an acute rejection necessitating therapy (rejectors; International Society for Heart and Lung Transplantation rejection grade ≥ 2R) and patients who remained free from rejection (nonrejectors). RESULTS: In the presence of comparable messenger RNA levels of CD3, IL-10, TGFß, IL2, IFNγ, and IL17A, the percentage of nTreg was significantly higher in EMB with histological signs of mild rejection (rejection grade 1R) collected before rejection than in 1R EMB of nonrejectors. The total Treg population was comparable in 1R EMB of nonrejectors and 1R EMB collected before rejection, which suggests the presence of iTreg in the EMB of nonrejectors. The relative high percentage of nTreg after rejection was not related to the number of rejections, whereas the total Treg population was inversely related to the number of rejections the first year after transplantation. CONCLUSIONS: Our data indicate that intragraft nTreg are unable to restrain alloreactivity leading to rejection. Moreover, the indirect evidence of the presence of intragraft iTreg suggests a possible role of iTreg in the regulation of alloreactivity.

Allogeneic Mature Human Dendritic Cells Generate Superior Alloreactive Regulatory T Cells in the Presence of IL-15.

Expansion of Ag-specific naturally occurring regulatory T cells (nTregs) is required to obtain sufficient numbers of cells for cellular immunotherapy. In this study, different allogeneic stimuli were studied for their capacity to generate functional alloantigen-specific nTregs. A highly enriched nTreg fraction (CD4(+)CD25(bright)CD127(-) T cells) was alloantigen-specific expanded using HLA-mismatched immature, mature monocyte-derived dendritic cells (moDCs), or PBMCs. The allogeneic mature moDC-expanded nTregs were fully characterized by analysis of the demethylation status within the Treg-specific demethylation region of the FOXP3 gene and the expression of both protein and mRNA of FOXP3, HELIOS, CTLA4, and cytokines. In addition, the Ag-specific suppressive capacity of these expanded nTregs was tested. Allogeneic mature moDCs and skin-derived DCs were superior in inducing nTreg expansion compared with immature moDCs or PBMCs in an HLA-DR- and CD80/CD86-dependent way. Remarkably, the presence of exogenous IL-15 without IL-2 could facilitate optimal mature moDC-induced nTreg expansion. Allogeneic mature moDC-expanded nTregs were at low ratios (<1:320), potent suppressors of alloantigen-induced proliferation without significant suppression of completely HLA-mismatched, Ag-induced proliferation. Mature moDC-expanded nTregs were highly demethylated at the Treg-specific demethylation region within the FOXP3 gene and highly expressed of FOXP3, HELIOS, and CTLA4. A minority of the expanded nTregs produced IL-10, IL-2, IFN-γ, and TNF-α, but few IL-17-producing nTregs were found. Next-generation sequencing of mRNA of moDC-expanded nTregs revealed a strong induction of Treg-associated mRNAs. Human allogeneic mature moDCs are highly efficient stimulator cells, in the presence of exogenous IL-15, for expansion of stable alloantigen-specific nTregs with superior suppressive function.

Interleukin-17-producing CD4(+) cells home to the graft early after human heart transplantation.

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.

Genetic variants of FOXP3 influence graft survival in kidney transplant patients.

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.

Interaction between adipose tissue-derived mesenchymal stem cells and regulatory T-cells.

Mesenchymal stem cells (MSCs) exhibit immunosuppressive capabilities, which have evoked interest in their application as cell therapy in transplant patients. So far it has been unclear whether allogeneic MSCs and host regulatory T-cells (Tregs) functionally influence each other. We investigated the interaction between both cell types using perirenal adipose tissue-derived MSCs (ASCs) from kidney donors and Tregs from blood bank donors or kidney recipients 6 months after transplantation. The immunomodulatory capacity of ASCs was not prejudiced by both Tregs from healthy donors and Tregs from graft recipients, indicating that ASCs were not targeted by the inhibitory effects of Tregs and vice versa. In addition, Tregs supported ASC function, as they did not alter the secretion of IFN-γ by immune cells and hence contributed to ASC activation and efficiency. ASCs exerted their suppressive role by expressing IDO, reducing levels of TNF-α, and by inducing the production of IL-10 in effector cells and Tregs. In conclusion, this study presents evidence that donor ASCs and acceptor Tregs do not impair each other's function and therefore encourages the use of MSC therapy for the prevention of graft rejection in solid organ transplantation.

The functional polymorphism Ala258Ser in the innate receptor gene ficolin-2 in the donor predicts improved renal transplant outcome.

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.

FoxP3 T cells and the pathophysiologic effects of brain death and warm ischemia in donor kidneys.

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-ß, 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.

Discontinuation of calcineurin inhibitors treatment allows the development of FOXP3+ regulatory T-cells in patients after kidney transplantation.

This study investigated specific gene expression profiles in patients with donor-specific cytotoxic-hyporesponsiveness, reflected by cytotoxic T-lymphocyte precursor frequency (CTLpf). The effect of calcineurin inhibitor (CNI) withdrawal was studied on markers for cytotoxicity (perforin, granzyme B), apoptosis (Fas,FasL), Th1 and Th2 cytokines (IL-2, IL-10), Th1 and Th2 transcription factors (T-bet, GATA 3), Th17 transcription factor and cytokine (RORγt, IL-17), and for immune regulation/activation (CD25, FOXP3). Peripheral blood samples from renal allograft recipients (n = 18) more than two yr after transplantation with stable renal function were analyzed before and four months after CNI withdrawal. Additionally, systolic and diastolic blood pressure, cholesterol, serum creatinine and proteinuria were evaluated, and no significant differences were measured before and after CNI withdrawal. However, CNIs' discontinuation influenced peripheral gene expression profiles. After CNI withdrawal, the mRNA expression of Granzyme B, Perforin, Fas, FasL, T-bet, GATA3 and CD25 were significantly lower than during CNI treatment. After CNI discontinuation, donor-specific CTLpf decreased, while FOXP3 expression discriminated between detectable and non-detectable donor-specific cytolysis reactivity; FOXP3 transcript values were highest in absence of donor-specific cytotoxicity (p < 0.01). Our study shows CNI withdrawal in stable kidney transplant recipients two yr after transplantation is safe. Moreover, discontinuation of CNIs' treatment allows FOXP3+ regulatory T-cells development, resulting in a significant decrease of anti-donor immune reactivity.