The Different Effects Of IL-2 And IL-15 On Cord Blood Innate Immune Cell Differentiation

The innate immune system is our first line of defense against invading organisms. Natural killer(NK)cell is one of the important components of innate immune cells.In contrast to T or B lymphocytes,NK cell does not express clonally distributed receptors for antigen and can cytolysis target cell without preactivation.NK cells are critical for host immunity for their ability of quickly cytotoxic response and to produce a wide variety of cytokines and chemokines to mudulate other cellular components of the immune system.Natural killer T(NKT)cell is another important component of innate immune cells.NKT cell coexpresses the CD56 and CD3-T cell receptor(TCR)complex.They rapidly produce many cytokines after stimulation and thus influence diverse immune responses and pathogenic processes.IL-2 and IL-15 are members ofγc family cytokines.They are important cell growth factors and regulate the proliferation,activation and survival of lymphocytes. IL-2/15R use the sameβandγchains for signal transduction,thus the biological activities of these two cytokines,at least partly,overlap.However,in many immune responses,IL-2 and IL-15 have different,even contrasting roles.The immature and hematopoietic precursors in cord blood are abundant.The study of the differentiation of these potential progenitors under the culture with IL-2 or IL-15 is beneficial to understand the development of innate immune cells and different effects of IL-2 and IL-15.NK92 cell is an IL-2-dependent human NK cell line derived from the peripheral blood lymphocytes of a non-Hodgkin's lymphoma patient.They have the functional and phenotypic characteristics of activated NK cells.NK92 cells were shown to express a relatively large number of activating receptors,while lack most inhibitory receptors,and display high cytolytic activity against a wide range of tumor cells.The ease of NK92 maintenance and expansion in culture,high cytolytic activity and wide target range make it a promising candidate for use as an immune effector cell.The mechanism of NK92 cytolysis tumor cells was investigated in expectation of offering some clues in the development of hopeful NK cell therapies.Cord blood mononuclear cells(CBMCs)were isolated and cultured with IL-2 or IL-15.The cell phenotype and percentage were analyzed by flow cytometry during the following culture process.ELISA and intracellular cytokine staining were used to examine the secretion of IFN-γand IL-4.Annexin V staining and intracellular Bcl-2 and Bcl-xL staining were used to detect the apoptosis of cell subtypes.MACS and FACS sorting methods were used to analyze the change of purified cells under different stimuli.Cytotoxic activity of cultured cells was determined in a standard 4-hour ⁵¹Cr release assay against the NK-sensitive cell line K562.The cell conjugate formation and signal phosphorylation were detected by flow cytometry.Confocal microscopy was used to observe the immunological synapse,the distribution of related molecules and signal molecule activation.In the following,we summarize the major results of our study and the conclusion.1.High expression of NKG2A and low expression of granzyme B are associated with reduced cord blood NK cell activityThe detection of NK cell percentage and cytotoxicity showed that although the Percentages of cord blood NK cells and CD8⁺ NK cells were higher than peripheral blood NK cell,the cytotoxicity of cord blood NK cells was lower.Then we analyzed the expression of some NK cell receptors and cytotoxicity-related molecules in cord blood and peripheral blood NK cells.The expressions of activating NK receptors, CD16,NKG2D and NKp46,did not show significant difference between cord blood and peripheral blood NK cells.But the expression of inhibitory receptor NKG2A was significantly higher on cord blood NK cells.As to the effector function molecules, granzyme B was expressed significantly lower in cord blood NK cells,but the expressions of intracellular perforin,IFN-γ,TNF-αand cell surface FasL and TRAIL did not show difference between cord blood and peripheral blood NK cells.The results indicated that the high expression of NKG2A and low expression of granzyme B may be related with the reduced activity of cord blood NK cells.2.Bcl-xL is associated with the anti-apoptotic effect of IL-15 on the survival of CD56^dimnatural killer cellsWe comparatively analyzed the effects of IL-2 and IL-15 on two NK cell subsets. IL-15 improved the proliferation and activation of CD56^brightNK and CD56^dimNK cells in long term cord blood mononuclear cell culture,but IL-2 only maintained the survival of CD56^brightNK cells.The percentage of CD56⁺Annexin V⁺ NK cells cultured with IL-15 was lower than that with IL-2;moreover,most of Annexin V⁺ NK cells were primarily in the CD56^dimNK cells.And the results from purified CD56^dim NK cells were similar.IL-2 and IL-15 cultured CD56^brightNK and CD56^dimNK cells expressed similar levels of Bcl-2.But IL-15 cultured NK cells expressed higher level of Bcl-xL than IL-2 cultured cells.Furthermore,IL-15 more strongly upregulated CD25 expression and better maintained the expression of IL-15Rαthan IL-2.These results suggest that CD56^dimNK cells undergo apoptosis when cultured with IL-2,but IL-15 inhibits their apoptosis and Bcl-xL is associated with the anti-apoptotic effect of IL-15.So IL-15 played a crucial role in sustaining long-lasting functions of CD56^dimNK cells.3.Different roles of IL-15 from IL-2 in differentiation and activation of human CD³⁺CD56⁺ NKT cells from cord blood in long term cultureIn this study,we explored the different effects of exogenous IL-2 and IL-15 on the expansion of CD3⁺CD56⁺ NKT cells by in vitro long term culture of CBMCs.The results showed that CD3⁺CD56⁺ NKT cells were derived from CD34^-CD56^- CBMCs and IL-2 improved CD3⁺CD56⁺ NKT cell expansion more strongly than IL-15. Interestingly,CD3⁺CD56⁺ NKT cells from IL-15-cocultured CBMCs had significantly lower apoptotic frequency and higher levels of activation markers(CD94, CD161,CD25,and IFN-γ)than those from IL-2-cocultured CBMCs.The anti-apoptotic and activating effects of IL-15 on CD3⁺CD56⁺ NKT cells from CBMCs might possibly explain the pathogenic role of IL-15 in GVHD during allogeneic transplantation.4.ERK1/2 was involved in NK92 cytolysis target cells based on the adhesion of CD11a and CD2In this study,NK92 cell was used to investigate the possible mechanism of NK cytolysis target cell.The binding rate of NK92-target cell was associated with NK92 cytotoxicity.And lost of cell membrane flexibility decreased the stable binding of NK92-target cell.The immunostaining results showed that adhesion molecules, LFA-1 and CD2 were accumulated at the interface of NK92-K562 contact.K562 ligation activated ERK1/2 signal pathway of NK92 cells and the inhibition of the phosphorylation of ERK1/2 decreased the cytolysis activity of NK92 cells.Thus,conjugate formation of NK92-target cell was prerequisite to NK cell activation and the following signal transduction was also required for NK cell cytotoxicity.