Evaluation of Cytomegalovirus (CMV)-Specific Cytotoxic T Lymphocyte (CTL) Monitoring Procedures with Tetramer and Intracellular IFN-γ Assay after Allogeneic Hematopoietic Stem Cell Transplantation (SCT).
Authors: Morita Yuriko , Hosokawa Mami , Heike Yuji , Sasaki Tohru , Ebisawa Michiko , Mori Shin-ichiro , Tanosaki Ryuji , Kami Masahiro , Ohnishi Mutsuko , Miura Osamu , Tobinai Kensei , Takaue Yoichi 
Published: April 2020

Abstract
Previous studies on the tetramer-based quantification of CMV-specific CTL have shown that the reconstitution of CMV-specific CTL to levels greater than 10/μL may protect against CMV reactivation. To evaluate the usefulness of CMV-specific tetramer for monitoring the number of CMV-specific CTL, assays with CMVpp65 495–503 tetramer were performed in 34 patients with HLA-A02 while CMVpp65 328–336 tetramer was used in 47 HLA-A24 patients who received non-T-cell-depleted SCT from a serologically full-matched donor after a myeloablative or nonmyeloablative regimen without ATG. Patients were assessed after recovery from CMV reactivation. Although the average number of CTL detected in patients with HLA-A02 [23.5/μL (1.33%/lymphocytes)] was significantly higher than that in patients with HLA-A24 [0.48/μL (0.02%)], the CMV reactivation rate was similar (67.6% and 62.5% in HLA-A02 and A24, respectively). This result demonstrates that these assays are of limited value since the number of CTL detected varies among different HLA-restricted epitopes. To further evaluate whether there is any relationship between CMV-specific CTL and CMV reactivation, the number of CTL in HLA-A02 patients was assessed in detail. The average number of CTLs in 11 patients without CMV reactivation, 23 with CMV reactivation, 13 with a peak CMV antigenemia of &gt;10/50000, and 3 who developed CMV disease was 12.3/μL (0.85%), 29.3/μL (1.35%), 13.8/μL (0.8%) and 16.3/μL (1.33%), respectively. No significant correlation was observed between the number of CTL and CMV reactivation after the reconstitution of CMV immunity. Since CMV reactivation usually occurs within 100 days after SCT, tetramer was assessed biweekly until day 100 in 13 HLA-A02 patients. In those who had CMV reactivation, simultaneous intracellular IFN-γ staining was performed with the same peptide used for tetramer. CMV reactivation was observed in 10 patients between day 23 and day 56 (median, day 34); among them, 5 had a peak antigenemia of &gt;10/50000, and required GCV therapy, and 3 developed CMV colitis. The average number of CTL at CMV reactivation was 5.67 (0.08–22.65) /μL in 10 patients who had reactivation, with 3 showing &gt;10/μL, while this was 1.08 (0–1.98) /μL at day 30 in those who did not. Two of the 3 patients who developed CMV colitis had &gt;10/μL CTL at the time of disease onset, while among 8 who did not require GCV therapy, only 1 and 2 patients recovered CTL &gt;10/μL at day 30 and day 60, respectively. The number of intracellular IFN-γ-secreting cells among those with CMV colitis was 18.2/μL (1.8%) at the time of disease onset, and this increased to 47.3/μL (3.5%) after recovery from CMV disease. These results suggest that tetramer-based monitoring of CTL is of limited value in predicting CMV reactivation compared to intracellular IFN-γ assay that assesses the functional properties of CTL.