Low Dose Of IL-2 Through Upregulation Of SOCS-3 In Na(?)ve CD4+T Cell Inhibits The Polarization To Th1 Cell And Prevents Mice Acute GVHD

Objectives: To explore the method of the suppression of acute GVHD by using low dose of IL-2 pretreat allogeneic lymphocytes, and elucidate the potential role and the underlying mechanisms of the change of SOCS-3 after IL-2 pretreatment in the above responses.Methods:1. Na(?)ve T lymphocytes purified from DO11.10 TCR transgenic mice and splenic cells from C57BL/6N mice, as well as Na(?)ve CD4+T lymphocytes from C57BL/6N mice were pretreated with 50U/mL of IL-2, and coclutured with the OVA323-329 peptide or the allogeneic antigens from BALB/C mice. The proliferation responses of the T lymphocytes were determined.2. Na(?)ve T lymphocytes purified from DO11.10 TCR transgenic mice and splenic cells from C57BL/6N mice, as well as Na(?)ve CD4+T lymphocytes from C57BL/6N mice were pretreated with IL-2 and the expression of SOCS-3 was detected. The expression changes of SOCS-3 in the OVA323-329 peptide treated and untreated T lymphocytes purified from DO11.10 mice were determined.3. The DS cells, which were generated by transfecting SOCS-3 into DO11.10 cells, were stimulated with the OVA323-329 peptide and the proliferation response was determined.4. We got T lymphocytes from C57BL/6N mouse and used inactivated splenic cells from BABL/C mouse as stimulating cells. T lymphocytes from C57BL/6N mouse were incubated in IL-2 for 4 hours before being stimulating by inactivated splenic cells from BABL/C mouse. They were incubated with either IL-12 or IL-4.We detected the expression of IL-12R_β1 andβ2 on the cell surface and intracellular IFN-γand IL-4 on day 14 and compared with the controlled group (T lymphocytes from C57BL/6N mouse were not incubated in IL-2 for 4 hours.)5. Female BALB/C mice given 5GY TBI were divided into five groups: A, B, C, D, E(n=12), which received male C57BL/6N spleen lymphocytes 5×10⁷, 1×10⁷ and male BALB/C×C57BL/6→F1 spleen lymphocytes 0.5×10⁷, 1×10⁷, 5×10⁷ (cells/mouse), respectively. The chimerism of doner lymphocytes was detected by PCR after recipient mice survived over 21 days. The survival time and the manifestations of acute GVHD were observed.6. The host is female BALB/C. The donor is male C57BL/6N. The conditioning regimen is 5GY of ¹³⁷Se TBI. 3×10⁷ splenic cells/mouse was injected intraperitoneally. All of mice were divided into four groups——Group A (the splenic cells were transferred directly), Group B(the transferred splenic cells were pretreated 4 hours by IL-2(50U/ml), Group C(the transferred splenic cells were cocultured 72hours with inactived BALB/C splenic cells), Group D(the transferred splenic cells were pretreated 4 hours by IL-2(50U/ml), and then were cocultured 72hours with inactived BALB/C splenic cells). We observed the survival time and the symptoms of acute GVHD during 60 days of the observation period. The liver and small bowel of the dead hosts were taken for the acute GVHD histologic examination. After the observation period, the splenic cells of the survival mice were used to detect the chimerism and the liver and small bowel were taken for the GVHD histologic examination.Results:1. The proliferation responses of Na(?)ve T lymphocytes purified from DO11.10 TCR transgenic mice and splenic cells from C57BL/6N mice, as well as Na(?)ve CD4+T lymphocytes from C57BL/6N mice to the specific and allogeneic antigens were significantly abrogated by the IL-2 pretreatment.2. The expression of SOCS-3 in Na(?)ve T lymphocytes and splenic cells purified from DO11.10 TCR transgenic mice and C57BL/6N mice were dramatically elevated by the pretreatment with IL-2, which reached the peak after 4-6h treatment. However, the SOCS-3 expression was significantly decreased by the stimulation with the OVA323-329 specific antigen and the expression reached the lowest level following 2-3d stimulation.3. The proliferation response of DS cells, which express high levels of SOCS-3, to OVA323-329 peptide was robustly abrogated.4. CD4+ T lymphocytes incubated in IL-2 before being stimulated by allogeneic antigen expressed less IL-12R_β1 and IL-12R_β2 with either IL-12 or IL-4.Under IL-12 condition, the ratio of intracellular IFN-γand IL-4 was lower than control group. And under IL-4 condition, T lymphocytes produced more IL-4 than control group.5. With 5GY TBI, the allogeneic lymphocytes could be engraftted successfully. The time of engraftment in MHC all-mismatached group (group B) was shorter than halpoidentical group(group D)(P<0.01). The time of engraftment prolonged with the increase of the cell dose infused in C, D, E group, gradually(P<0.05). After the engraftment of allogeneic lymphocytes, acute GVHD symptoms were observed. Acute GVHD in MHC all-mismatached group (group A) was more serious than halpoidentical group (group E) (P<0.01).6. The intension of acute GVHD in Group D in which the transferred splenic cells were pretreated 4 hours by IL-2(50U/ml), and then were cocultured 72 hours with inactived BALB/C splenic cells was suppressed obviously.Conclusions:Using the low dose of IL-2 to pretreat the allogeneic lymphocytes can suppress obviously the intension of acute GVHD, the mechanism of which is that IL-2 can suppress the proliferative response and blockage Th0 differentiate to Th1 through up-regulating the express of SOCS-3.