| BackgroundAny biological system involves negative feedback, and it is now recognized that the regulatory T cells (Treg cells) play key roles in the maintenance of lymphoid homeostasis in a number of immune circumstance. These cells maintain tolerance to self and control autoimmune deviation, prevent runaway response to pathogens or allergens, help maintain a balance with obligate microbial flora,and facilitate tumor’s escape from immune monitoring, promoting the growth and metastasis of tumor.An important population was initially identified in the mouse as CD4+CD25+. Naturally arising CD4+CD25+regulatory T cells are produced by the thymus as a functionally mature and distinct T cell population and express the forehead-winged helix transcription factor Foxp3, which was reported to be critical to the development and function of Treg cells. Foxp3+Treg cells use the aβ T cell antigen receptor (TCR) for antigen recognition and have a broad TCR repertoire similar in size but largely distinct in composition relative to that of CD4+conventional T cells.The mechanism of actions of Treg cells are clearly pleomorphic, and several modes and mediators of their activity have been described:suppression by inhibitory cytokines, suppression by cytolysis, and suppression by metabolic disruption and suppression by modulation of dendritic-cell maturation or function.Defining the mechanisms of Treg-cell function is clearly of crucial importance. Recent studies about regulatory T cell were mainly built on inflammation or tumor environment, as inflammatory bowel disease and inflammation-associated cancer in GI tract. Building on hygiene hypothesis model in which GI infections lead to change in regulatory T cell that reduce immune-mediated disease. Other study also pointed out that depending on IL-10bacteria-stimulated regulatory T cells were able to downregulate inflammation, restored immune homeostasis and reduced the risk of cancer.CD24is cell surface glycosylphosphatidylinositol-anchored protein with highly variable glycosylation.Due to its heat resistance; mouse CD24has been called the heat stable antigen (HSA). CD24is expressed on a variety of cell types, including hematopoietic cells as developing T and most B lymphocytes and a variety of APCs, and non-hematopoietic cells as neural cells, epithelia cells and many types of cancer cells. As a rule CD24tends to be expressed at higher levels in progenitor cells and metabolically active cells and to a lesser extent in terminally differentiated cells.The function of CD24is unclear for most cell types; however, diverse immunological functions for CD24have been reported. Although CD24is down-regulated when T cells reach maturity, it is rapidly induced after the engagement of the TCR-CD3complex [13]. It has been demonstrated that CD24on APC mediated costimulatory pathway for both CD4and CD8T cell response, and CD24expression on T cells was found to be essential for T cell homeostatic proliferation.The first evidence linking CD24to autoimmune disease was reported when CD24-deficient mice were found to be highly resistant to experimental autoimmune encephalomyelitis. Further studies found that polymorphisms of CD24are associated with the risk and the progression of autoimmune disease, including multiple sclerosis, rheumatoid arthritis and systemic lupus erythematous[21]. The mechanism by which CD24regulates autoimmune disease remains to be fully elucidated.CD24is extensively studied on cancer as it is broadly overexpressed on many types of tumor tissues, including small-cell lung cancer,hepatocellular carcinoma and breast cancer and so on.CD24is an important marker for cancer diagnosis and prognosis. In breast cancer, cell-surface and cytoplasmic expression of CD24correlates to poor prognosis, histology grades, tumor sizes and lymph node positivity.Additional studies are needed to demonstrate the function of CD24in cancer pathogenesis ant the underlying mechanisms involved.There are still some controversies in the mechanism of CD24in immune system; the role of CD24on T cells is unclear. Few researches refer to the effect of CD24on regulatory T cells. Here, based on the correlation between the expression of CD24and the function of T cells, we try to investigate the role of CD24on the suppressive function of CD4+CD25+Treg cells. Taking advantage of CD24deficient transgenic mice, CD4+CD25+T cells were purified as target cells which we tried to study in this project; an autoimmune disease mice model, experimental autoimmune encephalomyelitis (EAE), were used to test the function of CD24deficient regulatory T cells in vivo. Aiming at investigating the molecule mechanism of regulatory T cell mediated suppression; we hope to discover a novel therapeutic target for autoimmune disease.Materials and Methods1Characterization of the expression of Foxp3and TCR repertoire in regulatory T cells between wild type (WT) and CD24deficient (CD24-/-) mice by FlowCytometry.1.1Sample grouping:samples were grouped on the basis of mouse strain (BALB/c and C57BL6), genotype (WT and CD24-/-) and organ (spleen and thymus).1.2Count the total cell number of spleen and thymus by hematocytometry.1.3Single cells were obtained and treated by fluorescence staining (anti-CD4and anti-Foxp3Abs) to compare the expression of Foxp3in different groups, based on the percentage and total number of CD4+Foxp3+cells.1.4Samples were stained and compared the difference in the TCR repertoire (anti-TCR vβ2,3,4,5,6,7,8,9,10,11,12,13,14Abs) of CD4+and CD8+T cells.1.5Single cells were obtained and treated by fluorescence staining (anti-CD4, anti-CD24and anti-Foxp3Abs), to test the expression of CD24in CD4+Foxp3+cells.1.6Samples were stained and compared the difference in the TCR repertoire of CD4+Fopx3+regulatory T cells.2Identify the in vitro suppressive activity of CD24-/-Treg.2.1Sample grouping:WT Treg, CD24-/-Treg from BALB/c及C57BL6mice respectively.2.2Isolation of CD4+CD25+Treg by FACS or MACS methods.Single cells were obtained from the spleen and draining lymph nodes of different groups, and purified CD4+CD25+cells by FACS sorting (Fluorescence-activated cell sorting, FACS)or by magnetic method (MACS microbeads), according the manufacturer’s instructions. Purified CD4+CD25+cells were taken as regulatory T cells (Treg cells, target cells), and CD4+CD25-cells as conventional T cells (Tconv cells, effector cells) in these experiments.2.3Comparison of the different suppressive activity of two kinds of Treg cells by [3H] thymidine incorporated suppression assay.Using2000rad X-ray irradiated Rag-1(C57BL6) or Rag-2(BALB/c) splenocytes as antigen presenting cells, around50000of Tconv cells were seeded and cultured with titrated Treg cells in96-well-plate for around60h.[3H]-TdR was incorporated at0.1μ Ci/well and incubated for another8-16h; and then the CPM value was tested and analyzed.2.4Identification of IL-2, IL-10, TGF-β, P35and EBI-3by quantitative RT-PCR within cell subtypes.Using the QIAGEN RNeasy Mini Kit, total RNA were purified from Tconv and Treg cell; and then reverse-transcribed to cDNA by SuperScript(?)Ⅲ First-Strand Synthesis System. With QuantiTect SYBR Green PCR kit, the real time PCR were set up in triplicate (95℃15s,56℃30s,72℃30s,40cycles).2.5Identification of IL-2, IFN-γ and IL-10in cell culture by ELISA assay.3Comparison of the different suppressive function of two kinds of Treg cells by MOG-induced EAE mouse model.3.1Mice grouping:WT Treg cell transferred group, CD24-/-Treg cell transferred group and blank control/positive control group (CD4+CD25-T transferred only).3.2Isolation of CD4+CD25+Treg cells by MACS method mentioned above.3.3Induction of EAE mouse model and T cell adoptive transfer.On day0, mice of8-12weeks of age were immunized subcutaneously with200μg MOG peptide (Myelin Oligodendrocyte Glycoprotein) in CFA (400μg of Mycobacterium tuberculosis per milliliter) in a total volume of100μL. They received150ng of pertussis toxin in200μL PBS in the tail vein immediately after the immunization. Spontaneously, they were intravenously injected1×106CD24-/-Tconv cells with or without0.5×106WT or CD24-/-Treg cells.48hours later, they received another injection of150ng of pertussis toxin.The EAE mice were monitored every day and scored on a scale of0-5:0, no clinical signs;1, loss of tail tone or weakness of hind limb;2, totally loss of tail tone and wobbly gait;3, hind limb paralysis;4, hind and fore limb paralysis; and5, death.4StatisticsThe results of EAE model were only described medically without statistical analysis due to the small sample size. All other quantitative results were presented as mean±SD (x±s). Mean values were tested by2-tailed Student’s t test, including paired sample t test, two independent sample t test and one sample t test. In all cases, the a level was set at P<0.05.Results1The role of CD24in the development of lymphoid organs of mice.1.1The effect of CD24on the development of mouse thymus.Balb/c.CD24-/-mice had physically smaller size of thymus than the WT mice; by hematocytometry, the total cell numbers of thymus in Balb/c.CD24-/-mice were reduced compared with thymus in WT mice, t=2.529、P=0.045and t=4.642、P=0.002respectively for<10week group and>10week group.Howerver, such reduction was not observed in mice of C57BL6background, t=0.061、P=0.954and t=1.606、P=0.169respectively for<10week group and>10week group.1.2The role of CD24in the clone selection in the thymus.Globally, CD24-/-mice had similar TCR repertoire in thymus and spleen cells, just had scatted quantitative difference in percentage. For example, the proportion of TCR vβ11、v(312T cell at CD4+cells from thymus decreased slightly in C57BL6.CD24-/-mice, t=3.183、P=0.010and t=4.5178、P=0.002respectively; which indicated that the CD24in WT mice may have an effect on the escape of thymic clonal depletion.2Characterization of the expression of Foxp3and TCR repertoire in regularoty T cell.2.1Foxp3intracellular staining. Based on percentage in CD4+cells, Balb/c.CD24-/-mice had lower expression of Foxp3in the spleen than WT control group, t=4.932and P<0.001; accordingly, they had less Foxp3+cells in the spleen, t=5.144and P<0.001. At the same time, Balb/c.CD24-/-thymus had less Foxp3+cells in number (t=2.574, P=0.022) while similar in percentage (t=0.140, P=0.890). Of C57BL6mouse, both percentage and absolute number of Foxp3+cells were observed higher in the CD24-/-mice, but only the difference in total number was statistically significant (t=3.394, P=0.009).2.2The TCR repertoire in Foxp3+regularoty T cell.Similar with the global TCR repertoire result above, Foxp+cells from CD24-/-mice had similar repertoire in thymus and spleen as those from WT mice, except for some scatted quantitative difference, as vβ4、vβ7TCR in Balb/c mice and vβ3、 vβ12in C57BL6mice.3The expresssion of surface CD24in Treg cells.Setting the staining of CD24-/-cells and IgG2b isotype staining in WT cells as controls, Treg cells from thymus, spleen and lymph nodes all had certain level of CD24expression in the cell surface.4The role of CD24in the in vitro suppressive activity of Treg cells.4.1Regulatory T cell suppressive assay.In Balb/c mice, CD24-/-Treg cells had more powerful suppression activity than WT Treg, and this enhancement occurred mayjorly when in contact with CD24-/-. Tconv cells at1:1ratio, the suppression rate of WT.Treg and CD24-/-.Treg cells for WT.Tconv cells were31.134±5.67%and45.479±3.955%respectively, t=3.594, P=0.023; and33.122±4.931%'57.345±2.595%respectively for CD24-/-.Tconv cells, t=4.931, P=0.002. Meanwhile, CD24-/-and WT Treg cells from C57BL6mice displayed identical inhibitory function in the proliferation of WT.Tconv cells (P>0.05). But increased suppressive rate was found in the CD24-/-Treg when co-cultured with CD24-/-.Tconv cells at higher Treg concentration (6.873±5.439%and43.156±6.629%for WT.Treg and CD24-/-.Treg cells respectively, t=4.981, P=0.016), which was consistant with the result in Balb/c mice. Further more, using CD24-/-.Tconv cells and CD24-/-APC, anti CD24blocking antibody (a-CD24) was introduced to the co-culture system at the right beginning at two different concentrations (10μg/ml、5μg/ml), which increased the suppsssion rate of WT.Treg cell, and the increase seemed to be antibody concentration-dependent. However, such increase was not significant in statistics (P>0.05)4.2The effect of CD24on the production of cytokines.In quantitative real time PCR, setting the expression level of mRNA in WT.Tconv cells as control, CD24-/-.Treg subset expressed more IL-10(t=10.457, P<0.001), TGF-P (t=16.332, P<0.001) and EBI-3(t=5.846, P=0.004), which might be one of the mechanisms of the enhanced suppressive activity observed in CD24-/-Treg cells.Within the culture supernatant of each population, identical amount of IL-2and IFN-y were detected in Tconv and Treg between both mice (P>0.05). But higher concentration of IL-10were found in the supernatant of CD24-/-Treg cells, t=5.416, P=0.006, which suggested that CD24-/-Treg cells could produce more IL-10than WT Treg.In the Tconv and Treg co-culture system, less IL-2and IFN-y were detected in CD24-/-.Treg co-culture supernatant:t=101.717,P<0.001and t=5.800, P=0.004respectively in the CD24-/-.Tconv co-culture system. However, no difference was found in the production of IL-10, P>0.05.5The role of CD24in the suppressive activity of Treg cells in EAE model.Upon adoptive transfer of5×106CD24-/-.Tconv and0.37x106Treg cells to C57BL6.Rag-1-/-recipients, mice which received WT.Treg cells developed more early and severe EAE than those which got CD24-/-.Treg injection. And when monoclonal2D2.CD24-/-.Tconv cells were injected at1x106/mouse (Treg0.50x106/mouse), similar observation was found. Taken together, the in vivo data indicated that CD24-/-.Treg cells could display higher suppressor activity to partially pretect mouse from EAE。DiscussionCD24play a role in the development of thymocytes; globally, CD24-/-T cells have similar TCR repertoire with WT T cells, only scatted quantitative changes of some subtypes can be found.Upon the quantitative analysis of Foxp3+cells, CD24has different effect on Balb/c and C57BL6mice. In vitro suppressor function, CD24-/-Treg cells display increased inhibitory function to the proliferation of Tconv cells, but this increase seems to be concentration dependent and could be compensated by the CD24expression on the Tconv cell surface. CD24-/-.Treg might produce more IL-10, TGF-β or IL-35to enhance the suppression activity; meanwhile CD24-/-.Treg could influence the amount of IL-2or IFN-y in contact with Tconv, then affect the proliferation of Tconv. Taking advantage of EAE model of MS, no matter upon polyclonal Tconv or monoclonal Tconv injection, CD24-/-.Treg cells show stronger ability to protect recipient from EAE, by slowering the disease breakout and relieving the clinical symptoms, but still not enough to prevent the development or change the prognosis of EAE. This research on CD24might provide a novel clue for the therapy of autoimmune disease on the basis of regulatory T cells. |
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