Analysis of TREC (T Cell Receptor Excision Circles) Levels in CD94 Expressing CD8 T Cells in Chronic GVHD.

CD94 is one of the C-type lectin family members, forms a heterodimer with NKG2 gene family, and CD94 /NKG2A are inhibitory receptors. Not only NK cells but a subset of T cells express CD94/NKG2A, and previously we revealed the proportion of CD94/NKG2A expressing CD8 T cells were higher in patients with chronic graft versus host disease (GVHD), and CD94 expressing T cells have suppressive effects on mixed lymphocyte culture(MLC). We focus on CD94 positive T cell during T cell reconstitution after allogeneic hematopietic stem cell transplantation (allo-HSCT). T cell receptor excision circles (TREC) are suggested to be a useful marker of recent thymic output. In this study, we attempt to study TREC-containing CD8 T cell subset expressing CD94, and to examine the relation of TREC DNA level in CD94 expressing CD8 T cell and GVHD. We analyzed peripheral blood mononuclear cells (PBMCs) isolated from 24 patients (82 samples) undergone allo-HSCT including 15 patients with bone marrow transplantation and 9 patients with non-myeloablative stem cell transplantation. Informed consent was obtained from all patients. CD4 positive T cells were separated from PBMCs by magnetic cell sorting, and CD4 negative cell population was divided into CD94 positive CD8 T cells and CD94 negative CD8 T cells by fluorescence activated cell sorter. Genomic DNA was extracted from these separated T cell subsets. TREC DNA copy numbers per 10⁵ isolated T cells (TREC level) were quantified by real time PCR. We investigated TREC levels in clinical status with pre-allo-HSCT, no episodes of GVHD or before manifestation of GVHD (No GVHD), chronic GVHD on disease (C-GVHD), and no symptoms and remission status of GVHD after immunosuppressive therapy (R-GVHD). Statical analyses were carried out by Mann-Whitney U test. There were no significant differences in TREC level of sorted CD4 positive T cells in C-GVHD compared with No GVHD (p=0.75) and R-GVHD (p=0.61), and also CD94 negative CD8 T cells in C-GVHD compared with No GVHD (p=0.79) and R-GVHD (p=0.20). On the other hand, TREC level of CD94 positive CD8 T cells in C-GVHD decreased in comparison with No GVHD (p=0.015) and R-GVHD (p=0.0019). The reduction of TREC level is thought to be induced not only by low thymic output but also by dilution of TREC concentration due to peripheral T cell expansion without duplications of TREC. These results may suggest that CD94 positive T cells play a role in modulation of GVHD, and proliferate during chronic GVHD with dilution of TREC in CD94 positive CD8 T cells. It is suggested that TREC level of CD94 expressing CD8 T cells may be useful markers of chronic GVHD.