| CD69 gene, used to be named as AIM/Leu23/EA1/MLR3, clustered within the natural killer (NK) gene complex on mouse chromosome 6 and the long branch of human chromosome 12. The genetic and biological characteristics of mouse CD69 have a much higher similarity with its human analogues. This gene codes for a type II C type lectin, which ascribes to the family of NK receptors, and present at the cell surface as a disulfide-linked homodimer. Mature CD69 molecule comprises of 199 amino acids. The cytoplasmic domain constitutes Ser/Thr phosphorylation sites contains 38 acids. And the out-membrane domain of CD69 may be involved in pathogen recognition via its C terminal unit , contributing to induction of tumor necrosis factor-alpha (TNF-α) production and elimination of invading bacteria .CD69 is an early leukocyte activation molecule expressed at sites of chronic inflammation. The rapid and transient induction of CD69 expression can be detected within 1 to 2 hours after lymphocyte activation, and reaches its peak after 18 to 24 hours. CD69 is widely expressed in all myeloid cells including monocytes, eosinophils and mast cells, constitutively expressed by platelets, mature thymocytes, bone marrow lymphoid precursors. CD69 is not detected in peripheral blood lymphocytes or resting lymphocytes, but is expressed by small subsets of T and B cells in peripheral lymphoid tissues. Although previous data suggested CD69 as an activation marker, the reason to be named as activation-induced molecule (AIM), recent studies revealed a novel role for CD69 in downregulation of immune response via the production of transforming growth factor-beta (TGF-β). Though a full pattern of spatial and temporal regulation by CD69 will require the detailed characterization of its ligands, the function of CD69 at cellular and molecular levels in negative immune regulation has been attracted much more attention now. Recent data suggested that the precise role of CD69 in immune response tended to be more as a co-stimulator, rather than a merely activator or inhibitor. On the different cellular context, CD69 maybe has a dual role and mediates the secretion of different cytokines. Mast cells expressed CD69 and secreted TNF-αby FcεRI activation, and a more significant production of TNF-αcould be induced by cross-linking CD69 using the agonistic anti-CD69 mAb, which was additive to FcεRI activation. Phorbol esters (PMA) could induce peripheral T lymphocytes to express CD69. In the presence of PMA, anti-CD69 mAbs could stimulate T cell proliferation, which was not achieved when PMA or anti-CD69 used alone. Cross-linking of CD69 by its mAb maybe enhance the activation of PKC signal pathway by PMA. All these data identified a co-stimulatory function for CD69. Though induced rapidly after lymphocyte activation, CD69 does not appear to be required for the early T cell activation and proliferation, at least does not play the deciding role, and this can be proved in CD69 deficient mice. Nevertheless, CD69 may be non-redundantly involved in the late phase of T cell activation, and in the process of T cell differentiation.Although CD69 seems mostly to co-stimulate production of pro-inflammatory cytokines in most condition, CD69 cross-linking could also induce the production of TGF-βin CD4~+ and CD8~+ T cells as well as in NK cells and macrophages, suggesting that CD69 exerts a more wide immune-regulatory function. In vitro and in vivo studies have proved that anti-CD69 mAb could regulate pro-inflammatory cytokines production and tumor immune escape as well through TGF-βsecretion. It has been shown that CD69 deficiency leads to diminished TGF-βlevels in both native and adoptive immune effector cells with an enhanced immune response, also, CD69 deficiency results in a more efficient depletion of tumors in the NK sensitive tumor model or increased inflammation in the collagen induced arthritis (CIA) model. Thus, on one hand, CD69 could increase the synthesis of pro-inflammatory molecules to play a pro-inflammatory role, alternatively it could also act as a anti-inflammatory molecule through the induction of TGF-β. The possible pro- and anti-inflammatory role of CD69 makes it difficult to predict the final outcome of cross-linking by it ligands. Many scientists speculate that this may mainly concern with the particular microenvironment and the target cells interacted with CD69, which has also been supported by our results in current study.CD69 was commonly regarded as the activation marker of both T and NK cells, recent data indicates this molecule may participate in tumor immune escape. As we know, T cell immune response palys a crucial role in tumor control, and accumulating data supported a positive function of CD4~+ T cells in anti-tumor immune responses. Instead, there also are some T cell subsets such as CD4~+CD25~+ regulatory T cells, which have an impaired capacity to respond to proliferative signals and are able to inhibit other immune cell functions through cell-cell contact or through the production of suppressive cytokines. Accordingly, we want to address the following questions: if the CD4~+CD69~+ T cells exist in tumor-bearing host; and what is the exact function they have during tumor development and progression; and what are the precise mechanisms by which they regulate the immune response?We first concluded that in normal mouse and tumor models, CD4~+CD69~+ T cells do exist, and the percentage of CD4~+CD69~+ T cells has an increasing tendency concomitant with tumor progression. With this phenomenon, we designed our experiments and got the results as below:) We prepared orthotopic Hepa tumour, 3LL tumour or B16 tumour-bearing mouse models. Then we analysed the quantity and phenotype of CD4~+CD69~+ T cells in spleen lymphocytes, and examined their cytokine profile. We found that CD4~+CD69~+ T cells represents about 3-6% in CD4~+NK1.1- spleen lymphocytes in normal mice, and this ratio rises to approximately 12-50% concomitant with tumor progression. The CD4~+CD69~+ T cell population expressed high level of CD122 (the IL-2 receptorβchain), but almost did not express IL-2 receptorαchain CD25--which was regarded as a comparative marker of na?ve CD4~+ regulatory T cells. The CD25-CD122~+ phenotype distingushed CD4~+CD69~+ T cells from CD4~+CD25~+ Treg. By intracellular staining and ELISA, we also identified that this CD4~+CD69~+ T cell subset did not secret IL-4,IL-10,IFN-γ,or TGF-β. We did not observe whether they would secret any cytokines with CD69 cross-linking, after they were sorted.2) To further identify the role of CD4~+CD69~+ T cells in immune response , we used the splenic stormal cell line 107B to partially mimic the immune microenvironment in vivo ( we have previously shown that this stromal microenvironment could induce mature DC to proliferate for an extended period of time and further differentiate into a type of regulatory DC . Zhang M et al: Nat Immunol, 2004). Cocultured with the stromal cells, we could not detect any cytokine production of CD4~+CD69~+ T cells by intracellular staining , and ELISA results further confirmed our observation. As we have mentioned above, CD69 is now considered not a merely stimulatory or inhibitory receptor , but more likely serves as a co-stimulator , its pro- or anti- inflammatory outcome mainly depends on the microenvironment it exists. As the detailed characterization of its ligands has not been unveiled nowadays, anti-CD69 mAb was widely used as an agonist in vitro and in vivo so as to elucidate the function of CD69. Our experiments proved that in such kind of inhibitory stromal surroundings, CD4~+CD69~+ T cells could produce TGF-βafter cross-linking by anti-CD69 mAb, suggesting a possible suppressive role for this T cell subtype in immune response in vivo.3) Recent data from a number of groups have demonstrated an immune suppressive myeloid cells in a wide variety of unrelated pathologic conditions (tumor, infection, immune stress and organ transplantation), can be isolated from mouse spleen , bone marrow and tumor infiltrate . Extensive studies indicated that these cells share the CD11b and Gr-1 markers, represent a heterogeneous population of myeloid cells comprising immature macrophages, granulocytes, dendritic cells and other myeloid cell progenitors. These cells, recently named myeloid derived suppressive cell (MSC), increase in accordance with tumor progression and can contribute greatly to promote tumor evasion from immune attack by inhibiting development of adaptive immune response and other unknown mechanisms. As 107B splenic stromal cell line was established by us through long term in vitro culture, which maybe could not exactly mimic the immune microenvironment in vivo, we replaced the 107B stromal cells with the MSCs abundantly distributed in tumor-bearing mice to repeat the above experiments. Our results showed that under the dual stimulation of anti-CD69 mAb and unknown signals delivered by MSCs, CD4~+CD69~+ T cells could produce TGF-βmore significantly.Our experiments showed that under suppressive immune microenvironment, CD69 cross-linking of CD4~+CD69~+ T cells could induce the production of suppressive molecule TGF-β. CD69 serves just as a co-stimulator to amplify the inhibitory signals, and CD69 alone could not drive this process. In tumor-beating mice, this type of T cells accumulated as the tumor progression and started to produce TGF-β. All these data supported the possibility of CD4~+CD69~+ T cells function as a novel mechanism for tumor evasion. Additionally, the precise and high efficient interaction between immune suppressive cells such as MSCs, CD4~+CD69~+ T cells and Treg induced in tumor-bearing hosts should be further studied. Better understanding of the precise mechanisms, by which tumors escape from immune attack, could help to develop new approaches for breaking tumor immune tolerance and substantially improve the cancer therapy strategies.
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