We have previously shown that a high PD-1 expression on CMV-specific CD4 T-cells defines an anergic phenotype that is associated with CMV viremia in patients after renal transplantation. In addition, CD28^–CD27^– CD4 T-cells have been shown to be related to a positive CMV serostatus. This study assessed whether CD28^–CD27^– CD4 T-cells may be used as reliable surrogate for stimulation-induced CMV-specific CD4 T-cells, and whether an increase in the expression of PD-1 on CD28^–CD27^– CD4 T-cells allows for a rapid, stimulation-independent risk-assessment for CMV-complications in transplant recipients.

79 healthy controls, 51 hemodialysis patients, 60 renal transplant recipients with (n=41) and without (n=19) CMV reactivation, and 13 patients with primary infection were recruited in a cross-sectional and longitudinal manner. CMV specific CD4 T-cell levels quantified using intracellular cytokine staining after CMV antigen stimulation were compared to levels of CD28^–CD27^– CD4 T-cells. PD-1 expression levels on CD28^–CD27^– CD4 T-cells were related to viremic episodes.

A ROC-analysis among 79 serologically defined healthy controls identified a percentage ≥0.44% CD28^–CD27^– CD4 T-cells as an accurate indicator of CMV-seropositivity (93.3% sensitivity, 97.1% specificity), and a strong correlation was found between frequencies of CD28^–CD27^– CD4 T-cells and CMV-specific CD4 T-cells after stimulation (R²=0.40; p<0.0001). Co-staining of PD-1 on CD28^–CD27^– CD4 T-cells revealed significantly higher expression levels in patients with both primary CMV-viremia and -reactivation compared to non-viremic patients, controls or hemodialysis patients (p<0.0001). Longitudinal data showed a significant increase in PD-1 expression during the first two months after transplantation, which was most pronounced in patients with high-level viremia. This increase was significant not only during but already prior to viremia (p<0.001).

In conclusion, analysis of CD28^–CD27^– CD4 T-cells from whole blood allows for a rapid stimulation-independent quantitation of CMV-associated cellular immunity. As upregulation of PD-1 on CD28^–CD27^– CD4 T-cells is found both during and prior to viremia, patients at highest risk of CMV-reactivation after transplantation can be identified by simple cell-surface staining from as little as 100¯o;l of blood within a processing time of less than 1 hour.

Sester, U.: Other, Patent Pending, Patent Pending. Sester, M.: Other, Patent Pending, Patent Pending.

« Back to 2013 American Transplant Congress