The Effects Of γc Cytokine-Family Members On The Development And Regulatory Function Of NK Cells

Natural killer (NK) cells were originally identified on a functional basis because of their ability to lyse certain tumors in the absence of previous stimulation. They represent a lymphoid population that, in contrast to T or B lymphocytes, does not express clonally distributed receptors for antigen, eg, TCR and BCR. NK cells are normally confined to peripheral blood, spleen and bone marrow, but can migrate to inflamed tissues in response to different chemoattractants. They play important roles in the control of tumor growth in vivo and prevent the rapid dissemination of metastatic tumors, at least in a murine model. In addition, NK cells are fundamental in defenses against certain cytopathic viruses, primarily herpesviruses. Upon activation, NK cells release cytokines and chemokines that induce inflammatory responses, modulate hematopoiesis, control monocyte and granulocyte cell growth and function and influence the type of subsequent adaptive responses. Besides cytotoxicity, NK cells play important immunoregulatory roles in innate and adaptive immune responses.ObjectiveNK cells played a critical role in the innate and adaptive immune system. NK cell development and function deficiency resulted in more lethal damage than T and B lymphocytes. The differentiations of myeloid-line and lymphoid-line progenitors were detailedly documented. But the development of NK cells was not well studied yet. It was known that the development stage of NK cells in fetation was earlier than those of T and B lymphocytes. More evidence (relationship between MHCs, surface markers, cytokine secretion, and so on) indicated that the development of NK cells was similar to T lymphocytes. To study the changes in functions, phenotype, proliferation characteristics, and signal transduction pathways when NK cells exposed to different stimuli is beneficial to understand the essence of NK cells and the related diseases.It had been known for decades that NK cells develop within the bone marrow (BM) microenvironment, as its ablation in mice resulted in the loss of lytic NK cells. Multiple cytokines were involved in the maturation of NK cells. And IL-2 and IL-15 played the critical roles in the development of NK cells. IL-2 and IL-15 are produced by different cell types with overlapping functions in immune responses. Both cytokines co-stimulate lymphocyte proliferation, activation, and cytokine release and induce CD34⁺ HSCs (hematopoietic stem cells) to differentiate into CD56⁺ cells. Although IL-2 and IL-15 share IL-2Rβandγchains for signal transduction, each cytokine has a private a receptor. In many adaptive immune responses, IL-2 and IL-15 have contrasting roles. IL-2^-/- and IL-2R^-/-mice displayed normal NK cell development, in contrast, IL-15^-/- and IL-15R^-/- mice were characterized by a reduction in innate immune cell lineages, including NK cells, NKT cells, andγδT cells. We suggested that IL-2 and IL-15 exhibit differential effects regarding the development of NK cells from the cord blood.Based upon the density of surface CD56 molecules, human peripheral blood NK cells can be divided into two subsets, CD56^bright and CD56^dim, and they represent two functionally and phenotypically distinct subsets. CD56^dim NK cells occupy more than 90% of whole NK cells and are more cytotoxic, whereas CD56^bright NK cells typically produce greater amounts of cytokines. The exact differentiation stages of these two subtypes of NK cells are not well known. It was important to note that the NK cells resulting from CD34⁺ HSCs in stroma-free cultures with IL-15 were CD56⁺, lysed tumor target cells, and produced immunoregulatory cytokines and chemokines upon stimulation. However, these NK cells had high-density expression of CD56, closely resembling the minor CD56^bright NK cell population in peripheral blood, as they had little surface expression of CD16, with most cells expressing C-type lectin CD94/NKG2 NK receptors and only small percentage of cells expressing killer-cell immunoglobulin-like receptor (KIR). CD56^dim NK cells, the majority subset in peripheral blood, had never been generated convincingly in vitro, from either HSCs or CD56^bright NK cells. There were several conceivable possibilities concerning the development of CD56^dim NK cells. First, CD56^dim NK cells might have unique precursors not identified yet. Second, other soluble or cell contact signals might be required for the acquisition of expression of KIRs and additional characteristics of CD56^dim NK cells by common CD56^bright or CD56^dim NK-cell progenitors. Finally, CD56^bright NK cells, under the influence of unknown stimuli, might differentiate into CD56^dim NK cells in the periphery or BM.Functionally similar to CD4⁺ T cells, CD56^bright NK cells, were able to proliferate in response to IL-2 and IL-15, possibly played important roles in immunoregulation as Treg cells. On the other hand, functionally similar to CD8⁺ T cells, CD56^dim NK cells were strongly cytotoxic effector cells. Surprisingly, IL-15 protected CD56^dim cells from apoptosis, as did on CD8⁺ T cells.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 NK cell.NK cells are multifunctional cells, which are not only the effectors in the innate immune response to various infections and transformed cells, but also the important regulators in innate and adaptive immune responses. It's commonly accepted that NK cells are involved in the immune regulation by secreting various cytokines, especially IFN-γ. More and more attentions are paid to the immunoregulation function of NK cells.Recently, it became more and more seriously for people suffering the M. tuberculosis infection. There is an ascending incidence of tuberculosis world-widely.γδT cells are the important undertaker of the anti-M. tuberculosis immune response. It's urgent to develop an effective approach to promote the survival and activation ofγδT cells to increase the anti-M. tuberculosis immune response. It is widely accepted that natural killer cells are both the undertaker of innate immune response and the regulator of innate and adaptive immune responses. But there is little direct evidence to identify whether NK cells regulate the response ofγδT cells to M. tuberculosis.Several studies have suggested thatγδT cells play an important role in the immune response to M. tuberculosis, and M. tuberculosis antigens are able to induce the proliferation ofγδT cells. Supernatant from heat-treated H37Ra is a widely accepted stimulant forγδT cells. NK cells were also involved in the anti-M. tuberculosis immune response and the activity of NK cells was reduced, consistent with the loss ofγδT cells in the multidrug-resistant-pulmonary-tuberculosis patients. Based on above, we guess that there may be some interaction between NK cells andγδT cells. So we investigated the regulation effect of NK cells onγδT cells, employing the cellular immune response model ofγδT cell response to M. tuberculosis.Methods1. Cord blood mononuclear cells were isolated and cultured with IL-2 or IL-15. The expressions of some surface molecules, such as CD3, CD16, CD25, CD34, CD56, CD94, CD117, CD122, CD132, CD158a, CD161, IL-15Rαwere analyzed by flow cytometry during the following culture process.2. CFSE staining was used to detect the proliferation of specific cell population.3. Intracellular cytokine staining was used to analyze the secretion of IFN-γin specific cell population.4. MACS and FACS sorting methods were used to analyze the change of purified cells under different stimuli.5. Annexin V staining and intracellular Bcl-2 and Bcl-xL staining were used to detect the apoptosis of cell subtypes.6. Cytotoxic activity of cultured cells was determined in a standard 4-hour ⁵¹Cr release assay against the NK-sensitive cell line K562.7. Peripheral blood mononuclear cells were isolated and cultured with M.tb Ag and/or IL-2. The expressions of some surface molecules were analyzed by FACS.8. ELISA assay was used to detect the cytokine secretion on the culture supematants.9. Confocal microscopy was used to observe the immunological synapse and the distribution of related molecules.Results1. The different effects of IL-2 and IL-15 on cord blood NK cells.In the long term culture with IL-2 or IL-15, the percentages of NK cells in CB were different. IL-15 exerted stronger effect on NK cell expansion in magnitude and survival interval than IL-2. In the presence of IL-15, there was a steady increase both in the percentage and the absolute number of CD56⁺CD3^-NK cells. The doubling time of NK cells in the presence of IL-15 was shorter than that of IL-2. IL-2 prompted the proliferation of CB NK cells in short term and exhibited stronger effect on CD56^bright NK cells than CD56^dim NK cells. In addition, IL-2 could induce CD56^dim NK cell apoptosis. IL-15 upregulated the expression of CD25 on NK cells, especially on CD56^bright NK cells, but IL-2 did not. The cytotoxicity of NK cells in CB could be upregulated by both IL-2 and IL-15. IL-2 acted quickly and shortly, while IL-15 acted reversely. Both IL-2 and IL-15 could prompt CBMC secrete IFN-γ. IL-2 exerted stronger effect on T lymphocytes than on NK cells, but the effect of IL-15 was inversely.2. The different effects on T lymphocytes between IL-2 and IL-15.IL-2 exerted stronger effect on T lymphocyte proliferation than IL-15, and upregulated the expression of CD25 on T lymphocytes quickly. 3. The phenotypic difference of NK cells between CBMC and PBMC.Phenotypic analyses of mononuclear cells in freshly isolated CBMC or PBMC were performed by two- and three-color flow cytometry. In contrast to PBL, NK cells in CBMC expressed high-density of CD16. The higher proportion of NK cells was found in CBMC (25.26%±7.34%, n = 30) than that in PBL (13.27%±3.84%, n = 10). CD94, CD122 (IL-2Rβ), CD132 (IL-2Rγ), CD158a, CD161 and low level of CD25 were expressed on CB NK cells. There were 2-4% CD34⁺ cells in CBMC, but not in PBL.4. The similar effects of IL-2 and IL-15 on adult PBMC.Compared with those in cord blood, the effects of IL-2 and IL-15 on the proliferation of NK and T cells in PBMC were not obvious, and there were no significant difference between IL-2 and IL-15.5. The positive relation between NK cells andγδT cells.The function of NK cells was positively related to the proliferation ofγδT cells. The proliferation ofγδT cells was inhibited when selectively depleting NK cells from PBMC.6. M.tb Ag specifically activated NK cells with the cooperation of low dose of IL-2.NK cells, which were specifically activated by M.tb Ag, promoted the response ofγδT cells to M.tb Ag, and the regulation of NK cells was dependent on IL-2.7. NK cells regulated the function ofγδT cells through secreting cytokines and CD54/LFA-1 interaction.M.tb Ag stimulated NK cells to secrete IL-12, GM-CSF, TNF-α, but not IL-4 or IL-10. IL-12, GM-CSF and TNF-αcould individually or combined prompt the proliferation ofγδT cells, but could not substitute the NK cells to enhance the response ofγδT cells to M.tb Ag. IFN-γwas not involved in the process. The experiment with Transwell showed that besides secreting cytokines, NK cells could regulate the specific response ofγδT cells to M.tb Ag by cell-cell contact. Confocol microscopy and inhibition experiments showed that NK-γδT cell interaction by CD54 molecules might play a critical role in the regulation ofγδT cells by NK cells.Conclusions1. IL-15 prompted the proliferation of cord blood CD56^bright and CD56^dim NK cells, sustained their long-term survival, and enhanced the IFN-γproduction and cytotoxicity of NK cells. IL-2 only stimulated cord blood NK cell expansion at the early stage and induced CD56^dim NK cell apoptosis at the late stage, resulting in the decline of NK functions.2. The effect of IL-2 on cord blood T cells was stronger, for the positive feedback loop between IL-2 and IL-2Ra on T cells.3. The phenotype of NK cells from cord blood was different from that from adult peripheral blood. NK cells from cord blood might be immature NK cells or the precursors for mature NK cells.4. IL-2 and IL-15 showed similar effects on mature NK cells.5. NK cells, which were specifically activated by M.tb Ag, promoted the immune response of y6T cells to M.tb Ag. The regulation of NK cells toγδT cells was dependent on IL-2.6. NK cells regulated the immune response ofγδT cells to M.tb Ag through secreting cytokines (IL-12, GM-CSF, TNF-α) and cell-cell contact (CD54/ LFA-1).