Analytical evaluation of a real-time PCR-based DNA demethylation assay to assess the frequency of naturally occurring regulatory T cells in peripheral blood.

OBJECTIVES: Regulatory T cells (Tregs) which may indicate operational tolerance provide a promising biomarker for individualization of immunosuppression. Naturally thymus-derived Tregs (nTregs) represent the major suppressive phenotype and can be identified by their demethylation status in the Tregs Specific Demethylated Region (TSDR) of the Forkhead-Box-P3 (FOXP3) gene using quantitative PCR (qPCR). DESIGN AND METHODS: The analytical performance of a TSDR demethylation qPCR assay was assessed in whole blood of healthy individuals (HI) and kidney transplant recipients (KTR). The assay was compared to conventional flow cytometry and the agreement of results between two laboratories using a comparable qPCR protocol was assessed. In addition, the effect of gender, age, and medications was investigated. RESULTS: Within and between series imprecision was <20% (n=6). Whole blood samples are suitable for analysis within 3days when stored at room temperature; both whole blood and DNA samples - within 12months when frozen at -80°C. A significant correlation between the qPCR results and flow cytometry was lacking both with samples from HI and KTR. qPCR results between laboratories showed a bias of 76% but correlated well (r=0.645; p=0.0002, n=29). nTregs determined by qPCR were significantly (p<0.05) higher in HI (0.73%±0.23%, n=60) than in KTR (0.45%±0.21%, n=60) and in female HI (1.0%±0.27%, n=30) than in male HI (0.45%±0.23%, n=27). No effect of drugs or age was observed. CONCLUSIONS: The qPCR assay for nTregs provides reproducible results and is of sufficient quality for working with patient samples although inter-laboratory differences can be encountered due to a lack of method standardization. It was confirmed that gender-specific reference ranges are required.

CD26/dipeptidyl peptidase IV: a comparative study of healthy persons and kidney transplant recipients before and early after transplantation.

OBJECTIVES: Human CD26 is co-stimulatory for lymphocytes, circulates in a soluble form in blood (sCD26), and has intrinsic dipeptidyl peptidase IV (DPPIV) activity. Associations between CD26 expression on the surface of T cells (CD26+/CD3+) and acute rejection and between (CD26+/CD3+)/DPPIV and clinical immunosuppression have been reported. These results encouraged the investigation of CD26 as a potential biomarker to optimize immunosuppressive therapy. To better understand the significance of CD26, a comparative study of CD26 expression on CD3+ cells, sCD26 concentration, and DPPIV activity in healthy persons (HP) and kidney transplant recipients (KTR) was performed. DESIGN AND METHODS: Thirty-one HP and 34 KTR were included in the study. CD26+/CD3+ was determined by FACS, sCD26 concentration was determined by ELISA, and DPP activity was determined by spectrophotometry. For KTR, these parameters were studied on the day before transplantation (preTx) and 7±1days after transplantation (postTx). RESULTS: There was no significant difference in the CD26+/CD3+, sCD26, and DPPIV data regarding gender, donor type (16 living donors), delayed graft function (n=8), or presence of ≥4HLA mismatches (n=16). Compared to the HP data, preTx CD26+/CD3+ was 4.5-fold higher, sCD26 was 1.2-fold higher, and DPPIV showed no significant difference. PostTx, CD26+/CD3+ was 3.8-fold higher, and sCD26 and DPPIV decreased significantly, reaching lower values than that observed in HP. Re-transplanted patients (n=5) showed significantly lower preTx CD26+/CD3+ expression than patients receiving their first transplant. Patients with preemptive transplantation (n=7) showed higher postTx CD26+/CD3+ expression than patients on dialysis. CONCLUSIONS: CD26 expression on CD3+ cells was strongly increased in patients with end stage kidney disease compared to HP and remained high early postTx. The differences in sCD26 and DPPIV behavior compared to that of CD26+/CD3+ postTx may reflect a regulatory response to the new immunological situation and the effects of therapy.