CD4+CD25+ Invariant Natural Killer T Cells Are Enriched in Cord Blood: Implication on Immune-Regulation

CD1d-restricted invariant Natural Killer (iNK) T cells are rare but powerful regulatory T cells that can produce a large amount of both inflammatory (Th-1 type) and regulatory (Th-2 type) cytokines upon activation. Therefore, they can potentially shape subsequent adaptive immune responses towards inflammation or immune tolerance. Unlike the other regulatory immune cells, iNK T cells are heterogeneous in phenotype (CD4+ vs CD4- vs CD8a+), and display a spectrum of function ranging from regulatory to inflammatory responses. Given the potential regulatory function, iNKT cells are thought to play a role in preventing Graft Versus Host Disease (GVHD) in allogeneic stem cell transplant (SCT). Yet, the precise mechanism of immune regulation by iNKT cells in allogeneic SCT setting has not been clearly elucidated. As the cord blood stem cell transplantation is known to have a reduced incidence of GVHD as compared to matched adult donor allogeneic SCT, we hypothesize that iNK T cells in cord blood possess the regulatory phenotype and function as compared to those in healthy adults, thus play a role in preventing GVHD. To test this hypothesis, we first characterized the phenotype of iNK T cells from 25 cord blood and 25 peripheral blood of healthy adults using multi-color Flow Cytometry. We found that iNK T cells were present in variable frequencies ranging from 0.01% to 2% of T cells from healthy adults but rather in similar frequencies around 0.1% of T cells from cord blood. Interestingly, the extremely high percentage of CD4+CD25+CD161- iNK T cells was present in cord blood, while iNK T cells from healthy adults were heterogeneous in phenotype with a trend towards higher percentage of CD4-CD25-CD161+ iNK T cells. Next, we investigated whether CD4+CD25+ iNK T cells from cord blood indeed display regulatory property, and assessed cytokine production profile of ex vivo expanded polyclonal iNK T cells stimulated by autologous or allogeneic dendritic cells in the presence of alpha-GalCer, agonist glycolipid antigen. We found that the iNK T cells from cord blood preferentially produced Th-2 type cytokines (GM-CSF, IL-13, IL-4, IL-5) as compared to iNK T cells from healthy adults. Lastly, polyclonal iNK T cells showed significant cytokine production to autologous or allogeneic dendritic cells in the absence of alpha-GalCer, suggesting that iNK T cells can be activated by endogenous glycolipid antigens in physiologic condition. In summary, we demonstrated that CD4+CD25+CD161-iNK T cells were enriched in cord blood, and indeed displayed regulatory property. Our results sugest that CD4+CD25+ iNK T cells may play role in immunoregulation to prevent GVHD via regulatory function.