Expansion of Inhibitory NK Receptor (CD94/NKG2A)-Expressing Cytolytic CD8 T Cells and CD4+CD25+ Regulatory T Cells from the Same Cord Blood.
Authors: Tanaka Junji , Sugita Junichi , Kato Naoko , Toubai Tomomi , Ibata Jun , Shono Yusuke , Ota Shuichi , Asaka Masahiro , Imamura Masahiro 
Published: October 2019

Abstract
It has recently been shown that inhibitory natural killer cell receptors (NKRs) on not only NK cells but also on T cells negatively regulate NK cell and T cell functions through their binding to MHC class I molecules. The C-type lectin superfamily inhibitory NKR CD94/NKG2A heterodimer recognizes an HLA-E that preferably bound to a peptide derived from the signal sequences of most HLA class I. Therefore, CD94-expressing cells can monitor the global status of HLA class I on the tumor and leukemic cells and induce cytolytic attack without inhibitory signal against HLA class I decreased target cells resulting induction of graft-versus-leukemia (GVL) effect but does not attack normal cells with HLA class I expression resulting no enhancement of graft-versus-host disease (GVHD). On the other hand, CD4+ CD25+ regulatory T cells (Treg) contribute to suppress allogeneic immune responses and prevent transplant rejection and GVHD. In this study, we tried to expand CD94-expressing T cells and Treg cells from the same cord blood cells and then investigated their cytolytic characteristics and immunoregulatory function in order to develop a potential strategy of cell therapy for hematological malignancy.
After CD4 enrichment by negative selection using magnetic cell sorting (MACS) (Miltenyi Biotec)(CD4-enriched fraction) from cord blood, CD4+ CD25+ cells were isolated by positive selection with anti-CD25 magnetic microbeads. We could get more than 1,000 fold expansion of CD94-expressing CD8 T cells from CD4-depleted fraction after 8 days culture with immobilized anti-CD3 monoclonal antibody (mAb) (1 μg/mL) and IL-15 (5 ng/mL). Isolated CD4+ CD25+ cells were cultured with anti-CD3/CD28 mAb-coated dynabeads and IL-15 (5 ng/mL) and we could get about 50 fold expansion of Treg cells for 8 days. These expanded Treg cells could suppress allogeneic mixed lymphocyte culture more than 80% (effector cells: Treg cells= 2:1) and expressed FoxP3 mRNA about 100 fold compared with isolated CD25-negative cells. Cytolytic activities of purified CD94-expressing cells (CD94 > 90%) detected by 4 hours 51Cr release assay against K562 were 68.8 ± 16.8 % (n=5). Coculture of CD94-expressing cells with expanded Treg cells (CD94-expressing cell: Treg cells= 1:1, preincubation 4 hours) did not have any effect on cytolytic activities of purified CD94-expressing cells against K562 cells (66.1 ± 19.8 %, n=5). CD94-expressing CD8 T cells with cytolytic activity could be expanded from CD4-deplted fractions and Treg cells with immunosuppressive activity and increased expression level of FoxP3 mRNA could be expanded from CD4-enriched fractions of the same cord blood. Expanded these cytolytic CD94-expressing CD8 cells might be able to induce GVL effect without enhancing GVHD and Treg cells might be able to suppress allogeneic response including GVHD and graft rejection. Therefore, this strategy may be useful to differentiate lymphocytes in cord blood to two different kinds of effector cells exhibiting cytolytic or immunoreguratoly characters.