Endothelial precursor cell cross-match using Tie-2-enriched spleen cells.

BACKGROUND: Non-HLA antibodies against human endothelial progenitor cells (EPC) in pre-transplant recipient serum can have a deleterious influence on the graft. EPC enriched from peripheral blood have been commonly used for EPC cross-matching. In the present study, we describe cross-matches using EPC enriched from fresh or frozen-thawed spleen cell preparations, thereby widening the sample source for deceased-donor cross-matching and retrospective studies. METHODS: EPC cross-matches were performed retrospectively using spleen cells and the flow cytometric XM-ONE cross-match test kit. RESULTS: Healthy controls (n = 28) showed no IgG antibodies against EPC. When sera of 11 random dialysis patients were studied, 2 patients (18%) exhibited IgG EPC antibodies. When pre-transplant sera of 20 kidney graft recipients with good long-term graft outcome (serum creatinine 1.0 ± 0.2 mg/dL measured 2463 ± 324 days post-transplant) were investigated using frozen-thawed and then separated Tie-2-enriched spleen cells of the original transplant donor, 3 patients (15%) had pre-transplant IgG EPC antibodies. When pre-transplant sera of 5 patients with intra-operative graft loss were studied employing the original donor spleen cells, 4 (80%) patients showed IgG EPC antibodies. CONCLUSIONS: Cross-matches with spleen cell-derived EPC using the XM-ONE assay are technically possible. Our very preliminary experience suggests clinical relevance.

Correction: Helios expression and Foxp3 TSDR methylation of IFNy+ and IFNy- Treg from kidney transplant recipients with good long-term graft function.

[This corrects the article DOI: 10.1371/journal.pone.0173773.].

Helios expression and Foxp3 TSDR methylation of IFNy+ and IFNy- Treg from kidney transplant recipients with good long-term graft function.

BACKGROUND: There is circumstantial evidence that IFNy+ Treg might have clinical relevance in transplantation. IFNy+ Treg express IFNy receptors and are induced by IFNy. In the present study we investigated in kidney transplant recipients with good long-term stable graft function the absolute cell counts of IFNy+ Treg subsets and whether their expression of Foxp3 is stable or transient. METHOD: Helios expression determined by eight-color-fluorescence flow cytometry and methylation status of the Foxp3 Treg specific demethylation region (TSDR) served as indicators for stability of Foxp3 expression. Methylation status was investigated in enriched IFNy+ and IFNy- Treg preparations originating from peripheral blood using high resolution melt analysis. A total of 136 transplant recipients and 52 healthy controls were studied. RESULTS: Proportions of IFNy+ Treg were similar in patients and healthy controls (0.05% and 0.04% of all CD4+ lymphocytes; p = n.s.). Patients also had similar absolute counts of IFNy producing Helios+ and Helios- Treg (p = n.s.). Most of the IFNy+ and IFNy- Treg in transplant recipients had a methylated Foxp3 TSDR, however, there was a sizeable proportion of IFNy+ and IFNy- Treg with demethylated Foxp3 TSDR. Male and female patients showed more frequently methylated IFNy+ and IFNy- Treg than male and female controls (all p<0.05). CONCLUSIONS: Kidney transplant recipients with good long-term stable graft function have similar levels of IFNy+ Treg as healthy controls. IFNy+ and IFNy- Treg subsets in patients consist of cells with stable and cells with transient Foxp3 expression; however, patients showed more frequently methylated IFNy+ and IFNy- Treg than controls. The data show increased levels of Treg subsets with stable as well as transient Foxp3 expression in patients with stable allograft acceptance compared to healthy controls.

IFNy+ and IFNy- Treg subsets with stable and unstable Foxp3 expression in kidney transplant recipients with good long-term graft function.

BACKGROUND: Treg are a heterogenous cell population. In the present study we attempted to identify Treg subsets that might contribute to stable and good long-term graft function. METHOD: Lymphocyte and Treg subsets were studied in 136 kidney transplant recipients with good long-term graft function and in 52 healthy control individuals using eight-color-fluorescence flow cytometry. Foxp3 TSDR methylation status was investigated in enriched IFNy+ and IFNy- Treg preparations using high resolution melt analysis. RESULTS: Compared with healthy controls, patients showed strong associations of IFNy secreting Helios+ and Helios- Treg with Treg that co-expressed perforin and/or CTLA4 (CD152; p<0.01). Moreover they showed associations of IFNy-Helios+ Treg with Treg that produced TGFß and/or perforin and of IFNy-Helios- Treg with TGFß production (all p<0.01). Only in patients, but not in healthy controls, were IFNy- Helios+ and Helios- Treg associated with higher CD45+, CD3+, (CD4+), CD19+ lymphocyte counts (p<0.001). In addition IFNy-Helios+ Treg were associated with CD16+56+ lymphocytes (p<0.001). Enriched IFNy- Treg from female but not male patients showed an association of Foxp3 methylation with higher total Treg and higher Helios+IFNy-, CXCR3+Lselectin+ (CD183+CD62L+), CXCR3-Lselectin+ and CD28+HLADR+ Treg subsets (p<0.01). Enriched IFNy+ Treg from male patients showed an association of demethylated Foxp3 with total Treg and IL10-TFGß+ Treg counts, and in enriched IFNy- Treg an association of methylated Foxp3 with APO1/FasR+FasL+ (CD95+CD178+) Treg (p<0.01). CONCLUSIONS: Kidney recipients with good long-term graft function possess IFNy+ and IFNy- Treg with stable and unstable Foxp3 expression in the blood. They co-express CD28, HLADR, CTLA4, CXCR3, Lselectin, TGFß, perforin and FasL and might contribute to the establishment and maintenance of good long-term graft function.

Low-dose oral cholecalciferol is associated with higher numbers of Helios(+) and total Tregs than oral calcitriol in renal allograft recipients: an observational study.

BACKGROUND: Regulatory T cells (Tregs) are a cornerstone of graft acceptance. High numbers of Tregs are associated with better long-term graft survival. Recently, Vitamin D was suggested as an immunomodulator, in addition to its classical role in calcium metabolism. Vitamin D modulates Tregs and might, thereby, promote graft acceptance and long-term graft survival. METHODS: One hundred twenty-three renal allograft recipients attending either Heidelberg nephrology or Giessen internal medicine clinic were enrolled in this cross- sectional study. Sixteen healthy controls were studied in addition. Sixty-nine patients were receiving no vitamin D, 38 calcitriol, and 16 cholecalciferol supplementations. We evaluated whether there was a difference in the absolute numbers of Helios(+), Helios(-), CTLA-4(+), IFNg(+), and total Tregs among the patient groups. RESULTS: Cholecalciferol supplementation was associated with higher absolute numbers of Helios(+), CTLA-4(+), and total Tregs than calcitriol (p < 0.001, p = 0.004, p = 0.001 respectively). Helios(+) Tregs were also higher in cholecalciferol than no vitamin D supplementation patients (p = 0.001), whereas CTLA-4(+) and total Tregs were similar in both groups (p = NS). Helios(+), Helios(-), CTLA-4(+), IFNg(+), and total Tregs were similar in the cholecalciferol and healthy control groups (p = NS). CONCLUSION: Our findings indicate that cholecalciferol, even when administered at low dosages, has a stabilizing effect on Tregs (particularly the Helios + subset), in contrast to calcitriol which showed neither a stabilizing nor a proliferation-inducing effect on the same cell population.

Immunosuppressive drugs affect induction of IFNy+ Treg in vitro.

BACKGROUND: We reported previously that patients with poor long-term graft function are able to form IFNy+ Treg in vitro pretransplant, but late posttransplant have more frequently undetectable or lower levels of IFNy+ Treg in the peripheral blood than patients with good long-term graft outcome. In the present study, we investigated the induction of IFNy+ and Tbet+ Treg subsets in the presence of immunosuppressants in vitro. METHODS: PBL of 10 healthy individuals were stimulated with PMA/Ionomycin in the presence of different immunosuppressive drugs at 2 different concentrations that were chosen to approximately mirror the blood levels in renal transplant recipients. IFNy+, Tbet+, CD119+, and Helios+ CD4+CD25+CD127-Foxp3+ Treg subsets were analyzed using 8-color-fluorescence-flow-cytometry. RESULTS: Cyclosporine (p<0.01) and 6α-methylprednisolone (p<0.05) at both concentrations as well as high doses of azathioprine (p<0.05) and mycophenolate mofetil (p<0.05) inhibited the induction of IFNy+ and Tbet+ Treg, whereas lower concentrations of azathioprine and mycophenolate mofetil tended to increase IFNy+, Tbet+ and CD119+ Treg (p⩽0.05). CONCLUSIONS: Drug-induced inhibition of Treg induction might result in low IFNy+ Treg levels with the consequence of T effector activation and impaired graft function. Further studies will show whether monitoring of IFNy+ Treg might help to prevent clinical complications provoked by an inappropriate immunosuppressive protocol.

Clinical relevance of preformed IgG and IgM antibodies against donor endothelial progenitor cells in recipients of living donor kidney grafts.

BACKGROUND: Literature reports suggest that non-HLA-antibodies against human endothelial progenitor cells (EPC) can be detected in pre-transplant recipient serum and that EPC antibodies can have a deleterious influence on the graft. METHODS: We investigated 71 renal transplant recipients from living donors for a possible influence of pre-transplant donor-specific IgG and/or IgM recipient antibodies against EPC of the donor using the flow cytometric XM-ONE cross-match. RESULTS: Eight of the 71 patients developed acute biopsy-proven rejection. Two of these patients showed IgM antibodies against EPC prior to transplantation while the other six patients had neither IgG nor IgM EPC antibodies. Conversely, pre-transplant IgG or IgM antibodies against EPC were detected in 19 patients without acute rejection (3 × both IgG and IgM, 1 × IgG and 15 × IgM). The remaining 44 patients had neither EPC antibodies nor experienced rejection. Comparing serum creatinine levels at one month and one yr post-transplant within and among the three patient groups revealed that serum creatinine levels were similar in patients with or without EPC antibodies (p > 0.05). CONCLUSION: In this series of 71 recipients with living donor kidneys, pre-transplant EPC antibodies detected with the XM-ONE test kit were neither associated with acute rejection nor with graft function at one month or one yr.

IFNγ+ Treg in-vivo and in-vitro represent both activated nTreg and peripherally induced aTreg and remain phenotypically stable in-vitro after removal of the stimulus.

BACKGROUND: IFNγ-producing CD4+CD25+Foxp3+CD127- Treg represent the first line of Treg during an immune response. In the present study we determined whether IFNγ+ Treg in-vivo and in-vitro are Helios-positive representing activated natural (nTreg) or Helios-negative representing adaptive Treg (aTreg) and whether they originate from CD4+CD25+ and/or CD4+CD25- PBL. Furtheron, we investigated whether they are inducible by recombinant IFNγ (rIFNγ) as a single stimulus, decrease in-vitro after elimination of the stimulus, and have a demethylated Foxp3 Treg-specific demethylated region (TSDR) which is associated with stable Foxp3 expression. METHOD: Subsets of IFNγ+ Treg were determined in peripheral blood of healthy controls using eight-color flow cytometry and were further investigated in-vitro. Foxp3 TSDR methylation status was determined using bisulphite polymerase chain reaction (PCR) and high resolution melt (HRM) analysis. RESULTS: Nearly all Treg in the peripheral blood were Helios+IFNγ- (1.9 ± 1.1/µl) and only few were Helios+IFNγ+ or Helios-IFNγ+ Treg (both 0.1 ± 0.1/µl). Enriched IFNγ+ Treg subsets showed in part strong Foxp3 TSDR demethylation. In-vitro, rIFNγ was unable to induce Treg. CD4+CD25+ enriched PBL stimulated with PMA/Ionomycin in the presence of rIFNγ were rather resistant to the effect of rIFNγ, in contrast to CD4+CD25- enriched PBL which showed increasing total Treg with Helios+ Treg switching from IFNγ- to IFNγ+ and increasing Helios-IFNγ+ Treg. The data indicate that rIFNγ, in combination with a polyclonal stimulus, activates nTreg and induces aTreg. When phorbol 12-myristate 13-acetate (PMA)/Ionomycin was washed out from the cell culture after 6 h stimulation, Treg induction continued for at least 96 h of cell culture, contradicting the hypothesis that removal of the stimulus results in significant decrease of IFNγ- and IFNγ+ CD4+CD25+Foxp3+CD127- Treg due to loss of Foxp3 expression. CONCLUSIONS: IFNγ+Helios- aTreg as well as IFNγ+Helios+ nTreg are detectable in the blood of healthy individuals, show in part strong Foxp3 TSDR demethylation and are inducible in-vitro. The present data provide further insight concerning the in-vivo and in-vitro characteristics of IFNγ+ Treg and help to understand their role in immunoregulation. Alloantigen-specific demethylated IFNγ+Helios+ nTreg might represent a suitable marker for monitoring graft-specific immunosuppression in renal transplant recipients.