Detection of Cytomegalovirus Specific Cytotoxic T Lymphocyte in Allogeneic Hematopoietic Stem Cell Transplant Recipients.

T lymphocyte reconstitution of allogeneic hematopoietic stem cell transplantation (alloHSCT) recipients was limited in the counts of T lymphocyte subgroup or T receptor recombination exclusion circles (TRECs). The function of T lymphocyte reconstitution is more important. Cytomegalovirus (CMV)-specific CD8 cytotoxic T lymphocytes (CTL) have the predominant role in protection against CMV. HLA-peptide Pentamer (Pentamer) and enzyme-linked immunospot (ELISPOT) have allowed direct visualization of CMV specific CTL. Both assays are highly specific and sensitive and had been applied to other areas.

29 patients with ANLL (9 cases), ALL (4 cases), CML (14 cases) and NHL (2 cases) were eligible for study. All patients received alloHSCT with 19 sibling-donors and 10 unrelative-donors in follow-up median 25 months, median age is 29 (range 11–52) and male/female is 16/13. CMV-IgG serologic status: 30 pairs were donor negative /recipient negative and 2 pairs were donor positive /recipient negative. The blood samples were collected from alloHSCT recipients with HLA-A2 at pre-HSCT and at 3, 6, 9, 12 month after alloHSCT. Detection of CMV-specific CTL in CD8 using HLA-A2-pp65_495–503 pentamer complexes was performed with the fluorescence activated cell-sorter flow cytometer. IFN-gamma ELISPOT assay was performed in 96 bottomed-well microplates in which added 4×10⁵ MNC /well and incubated with CMV pp65 (10ug/ml). T lymphocyte subgroup reconstitution was detected at same time.

24/29 (82.8%) recipients suffered CMV antigenemia once or more times after alloHSCT. The median time of onset CMV antigenemia is at 78d (range 31–476d) after alloHSCT. The counts of HLA-A2-pp65-specific CTL were continuously increased in 12 month after alloHSCT. The counts of HLA-A2-pp65-specific CTL at 3 month after alloHSCT were lower than at pre-HSCT and 6 month (p=0.000), 9 month (p=0.022), 12 month (p=0.013) post-HSCT obviously. The numbers of spots within 6 month post-HSCT detected for CMV-specific CTL by IFN-gamma ELISPOT was lower than that after 6 month post-HSCT (p=0.004). It mean that CMV-specific CTL grew more quick from 3–6 month than after 6 month. But analysis the reconstitution of CMV-specific CTL in every recipients were showed increasing or decreasing individually, which affected only by aGVHD (r=0.725, p=0.042) and cGVHD (r=0.843, p=0.036).