*Purpose: Cytomegalovirus (CMV)-specific T-cells express IFNγ, measure cell-mediated immunity to CMV and can guide the timing and duration of antiviral therapy. CMV-specific T-cells also co-express CD154, which can substitute for IFNγ and is induced rapidly, but needs clinical validation.

*Methods: CMV-specific CD154+T-cells and their memory (CD45RO+) and naïve (CD45RO-) CD4 and CD8 subsets were measured with flow cytometry after <6-hour stimulation of cross-sectional single peripheral blood leukocyte samples with pp65 CMV antigen. Samples were obtained from a) 28 healthy adults to establish reference range, b) from 142 liver (LT) or intestine transplant (IT) recipients with or without CMV infection. All cross-sectional samples were obtained within the one month period before or the two week period after CMV viral load testing. CMV infection status was based on the presence or absence of CMV viremia measured with polymerase chain reaction. Recipients were not excluded based on CMV seropositive or seronegative status or any type of donor-recipient CMV serologic mismatch. Logistic regression was used to identify threshold frequencies of CMV-specific CD154+T-cells which were associated with absence of CMV viremia.

*Results: In 28 healthy adults, mean (SD) frequency (%) of CMV-specific CD154+T-cells and their memory and naïve subsets was 5.0 (0.3), 5.4 (0.3) and 4.7 (0.3), respectively and showed similar trends for CD4 and CD8 subsets. The 142 recipients included 117 LT and 25 IT recipients. Median (range) age was 10.7 years (0.4-30) and male: female distribution was 65: 77. Samples were obtained at median 1140 (0-7556) days after LT or IT. Twenty five children developed CMV viremia (n=24) or CMV hepatitis (n=1) with median viral load of 2320 copies/ml (range 341-429426) and demonstrated significantly lower (mean+/-SEM) frequencies of CMV-specific CD154+T-cells, compared with 117 children without CMV viremia (1.6+/0.2% vs 4.2+/-0.3%, p=2.4E-11). In 129 of 142 samples, all of which were obtained after transplantation, the threshold frequency of 1.73% CMV-specific T-cells, if exceeded, predicted absence of CMV viremia with negative predictive value or NPV (95% confidence interval) of 92% (85-97%). Corresponding positive predictive value (PPV), specificity and sensitivity were 54% (34-72%), 88% (80%-93%) and 65% (43-84%). In 13 remaining samples, all of which were obtained before transplantation, CMV-specific T-cells of 1.73% or greater were seen in 8 of 11 children who were free of CMV viremia (specificity 73%). Both children with viremia demonstrated frequencies below this threshold (sensitivity 100%). The corresponding NPV was 100% and PPV was 40%.

*Conclusions: Transplant recipients at low risk for CMV viremia can be identified rapidly and with reasonable accuracy using CMV-specific T-cells, which express CD154 .

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