Research On Role Of CD4～+CD25～+ Regulatory T Cells In Malaria

ObjectiveMalaria is a kind of infectious diseases with high concern all over the world. The prevention and therapy of such infectious diseases as Malaria, AIDS and tuberculosis have been taken as the focus of researches by WHO. However, up till now, the mechanisms of immune response to malaria parasite infection are not fully understood yet. The development of effective vaccine and anti - malaria drug was seriously impeded.Models of murine malaria have proved that preventive immunity during intra - erythrocyte stage is set up with the involvement of CD4~+ T cells and some antibodies. Successive activation of Th1 - Th2 is the key to preventive immunity, and IFN - γ plays an important role in the production and maintenance of Th1 in early response. Previous studies have shown that Thl response is essential for resistant mice to clear parasites during intra - erythrocyte stage. IFN - γ, as an important regulatory Thl cytokine, can activate such immunocytes as macropha-ges which can secrete non - specific effector molecules such as NO. Since effective Thl response can not be set up in susceptible mice, different outcomes -death or spontaneous cure occur between mice from different heritable backgrounds. What is involved in early immune response remains unclear. Recently, the mechanisms of immunoregulation in the early stage of malaria have been one of the foci of researches.Regulatory T cells (Treg cells) were firstly reported by Sakaguchi in 1995.CD25 is the a subunit of membrane interleukin - 2 receptor ( mIL - 2R) , which is currently the best marker for CD4* regulatory T cells. Some studies have shown that CD4 * CD25 * regulatory T cells could suppress proliferation of many types of immunocytes including CD4 * T cells and CD8 + T cells and inhibit secretion of cytokines. Recently, it is reported that depletion of CD4*CD25* T cells could protect mice from death when they were infected with a lethal strain of Plasmodium yoelii , and this protection was associated with specific T cell responsiveness , suggesting that CD4 * CD25 + regulatory T cells might contribute to immune suppression during malaria infection.In this study, we investigated the different Thl immune responses between resistant (DBA/2) and susceptible ( BALB/c) mouse strains during the early stage of P. yoelii 17XL, and then determined the numbers of CD4 * CD25 + regulatory T cells and levels of cytokines as IL - 10, IFN - -y and TGF - |3l and percentage of apoptotic cells, to investigate the role and the possible immunosup-pressive mechanisms of CD4 + CD25 + regulatory T cells. On the basis of it, in vitro blockades of CD25 and (TLA - 4 were performed to further determine the role and mechanisms of CD4 + CD25 * regulatory T cells in early stage of malaria infection.Method1. Determination of contents of IFN - 7 and NO2" in DBA/2 and BALB/c mice by double antibody sandwich ELISA and Griess reactionSpleen cells were harvested from mice on day 0, 3, 4, 5, 6 post - infection. Concentration of cells were adjusted to 1 x 10 /ml, and plated 500uJ /per well in a 24 - well plastic flat - bottomed tissue culture plates. Plates were incubated 48 hours at 37 *￡ in 95% air - 5% C02. Part of cells from day 3 post - infection were incubated with PRBC, LPS and RPMI 1640 medium supplemented with 10% heat - inactivated FCS in 5% C02 at 31°C for 48 hours.Supernatants were collected after centrifugation at room temperature, 350g. Supernatants above were detected in duplicate using ELISA kits for IFN - -y and Griess reaction for NO.2. Determination by flow cytometry of the percentage of the CD4 * CD25 *T cells and apoptotic cells in DBA/2 and BALB/c mice.Spleen ceUs were aliquoted into staining tubes at approximately 1 x 106 cells per tube and incubated with Mouse - block to block non - specific binding of flu-orochrome - labeled antibodies. Two - color staining was then performed using FITC - labeled anti - mouse CD4 mAbs and PE - labeled anti - mouse CD25 mAbs simultaneously. The antibodies were diluted in FASC buffer and incubated with the cells for 30 min on ice without light. FACS analyses were performed on a FACSCalibur instrument run with the CELLQUEST programme.Spleen cells diluted with binding buffer were aliquoted into staining tubes at approximately 1x10 cells per tube. Two - color staining was then performed u-sing FITC - labeled Annexin V and PI simultaneously, and incubated with the cells for 15 min on ice without light. FACS analyses were performed on a FACSCalibur instrument run with the CELLQUEST programme.Flow cytometric analysis utilized linear forward light scatter ( FS) , linear side light scatter (SS) and log fluorescence parameters.3. Determination of the contents of TGF - pi and IL - 10 in DBA/2 and BALB/c mice by double antibody sandwich ELJSAAll cells used were spleen cells harvested from mice on day 0, 3 and 6 post - infection. Concentration of cells were adjusted to 1 x lOVml and 500ui cells per well were plated on the plastic 24 - well plates with RPMI 1640 medium supplemented with 10% heat - inactivated FCS in 95% air - 5% CO2 at 371 for 48 hours. Supernatants were collected after centrifiigation at room temperature, 350g. Supematants above were tested in duplicate using ELISA kits for TGF-plandIL-10.4. In vitro study of change of Thl response after CD25 and CTLA - 4 blockadelOOfxl of 1 x lOVml spleen cells per well on the plastic 96 - well plates were incubated with RPMI 1640 medium supplemented with 10% heat - inactivated FCS in 95% air - 5% CO2 at 37^ for 44 hours. lOjxl MTT per well at 37^ for 4 hours. Aspirated and dimethyl sulphoxide were added lOOjxl per well after centrifugation at room temperature, 350g.500 ui of 1 x lOVml cells per well on the plastic 24 - well plates with RPMI 1640 medium supplemented with 10% heat - inactivated FCS in 95% air -5% CO2 at 37 Xi for 48 hours. Supernatants were collected after centrifugation at room temperature, 35Og. Supernatants above were tested in duplicate using ELISA kits for IFN - 7, TGF - pi and IL - 10.Results1. Comparison of the concentration of IFN - 7 and NO from spleen supernatants in DBA/2 and BALB/c mice.The level of IFN - -y increased rapidly at day 3 post — infection and then decreased slowly in DBA/2 mice, while in BALB/c mice, the level of IFN - -y increased significantly and peaked at day 3, which was followed by a sharp reduction. The levels of IFN - y and NO increased markedly after the stimulation of two non - specific stimulators in DBA/2 mice, and no change was detected in BALB/c mice.2. Comparison of differences in percentages of CD4 + CD25 +T cells and ap-optotic cells between DBA/2 and BALB/c mice.The proportion of CD4 + CD25 + T lymphocytes increased markedly at day 3 -4 and then decreased sharply in BALB/c mice, but for DBA/2 mice, significant increase was seen only at day 5 - 6. The percentage of apoptotic cells increased obviously at day 3 and further increased till day 6 in BALB/c mice, while there is no change of it in DBA/2 mice.3. Comparison of the contents of TGF - pi and IL - 10 in DBA/2 and BALB/c mice.The concentration of IL - 10 increased rapidly and kept an elevated level from day 3 to day 6 in BALB/c mice, whereas there was no change of IL - 10 in DBA/2 mice. There was no notable change of TGF - pi by day 6 post - infection in both strains of mice.4. Comparison of change of immune response after in vitro blockade of CD25 andCTLA-4.There was a decrease in the level of IL - 10 after in vitro blockade of CD25in both kinds of mice. After in vitro blockade of CTLA -4, proliferation of lymphocytes was promoted and level of IL - 10 decreased.Conclusions1. During early infection of lethal strain of Plasmodium yoelii , immune responses in Thl cells from BALB/c mice and DBA/2 mice are different.2. During early infection of lethal strain of Plasmodium yoelii , Thl cell immunity is crucial for control of parasitemia.3. CD4 + CD25+T cells might be one of the mechanisms that Thl cell immunity fail to be set up in BALB/c mice during early infection of lethal strain of Plasmodium yoelii.4. Apoptosis might be a kind of pattern for immunosuppressive function of CD4+ CD25+ regulatory T cells during early infection of lethal strain of Plasmodium yoelii.5. During early infection of lethal strain of Plasmodium yoelii , CD4+ CD25 + regulatory T cells can exert an immunosuppressive function by means of IL-10.6. During early infection of lethal strain of Plasmodium yoelii , CD4 + CD25 + regulatory T cells can exert an immunosuppressive function by means of CTLA-4.