Study On Follicular Helper T Cells In The Pathogenesis Of Myelodysplastic Syndromes

ObjectiveThis study was to investigate the changes of peripheral blood and bone marrow, the amount of follicular helper T(Tfh) cells from spleen and bone marrow, the level of inducible costimular(ICOS), programmed death-1(PD-1), CD40 L and OX40 on surface of spleen and bone marrow Tfh cells from NUP98-HOXD13 transgenic mice(NHD13 mice). To explore the role of Tfh cells in the pathogenesis of the myelodysplastic syndrome.To prove the relationship between immune tolerance, immune surveillance and the disease of myelodysplastic syndrome, and to provide the academic basis for targeted immune therapy of myelodysplastic syndrome.MethodNUP98-HOXD13 transgenic mice were obtained from Jackson Laboratory,(Strain Name:C57BL/6-Tg(Vav-NUP98/HOXD13);Stock Number:010505) same aged non-transgenic wild type(WT) C57BL/6J mice were used as controls. NUP98-HOXD13 transgenic mice and WT controls’ amounts were 6-13 respectively.All mice were male and 6-8 weeks old. Mice were housed in the Animal Center of Institute of Radiation Medicine Chinese Academy of Medical Sciences under SPF conditions.First section Complete blood count was performed using a automatical blood cell analyzer from 13 male NHD13 mice and 8 male WT controls, cell smears of peripheral blood and bone marrow were observed using a microscope after Wright’ stain.Second section To observe the structure of lymphoid follicles of spleen and the pathological feature of bone marrow from NHD13 mice, to explore the lever of CXCR5 and PD-1 on surface of spleen cells from 5 NHD13 mice and 5 WT controls using the Immunohistochemistry method.Third section The amount of Tfh cells and the expression of related signaling molecules(ICOS, PD-1, CD40 L and OX40) of spleen and bone marrow from 10 NUP98-HOXD13 transgenic mice and WT controls were detected by flow cytometry.Fourth section The m RNA expression of Tfh cells’ signaling molecules(ICOS, PD-1, CD40 L and OX40) from spleen and bone marrow mononuclear cells of 8 NUP98-HOXD13 transgenic mice and WT controls were analyzed by real-time PCR. ResultsFirst section The count of red blood cell, white blood cell, platelet, neutrophil and lymphocyte from NHD13 mice was significantly lower than those of wild mice(both P<0.05). The mean corpuscular volume(MCV) and mean platelet volume(MPV) of NHD13 mice were both significantly higher than those of WT mice,(both P<0.05). The red blood width(RDW) and the platelet distribution width(PDW)of NHD13 mice were significantly higher than those of wild mice,( both P<0.05). The NHD13 mice peripheral blood smear shows that the counts of red blood cell, white blood cell and platelet were significantly lower than those of wild mice. The peripheral blood smear shows lager volume red blood cell in NHD13 mice. The NHD13 mice marrow smear characterized of the binucleated erythrocytes,ring neutrophils and erythroblastic island.Second section Compared to wild type mice, lymphoid follicles and germinal centers in the spleen of NHD13 mice are both less. The red pulp in the spleen of NHD13 mice is more widened than that of wild type mice. Result of Immunohistochemistry showed that CXCR5 on cell from spleen of NHD13 mice was decreased significantly than that of wild type mice,and PD-1 on spleen cells of NHD13 mice was increased significantly than that of wild type mice.Third section The cell from spleen of NHD13 mice had significantly lower proportion of Tfh/CD4+cell than that of wild type mice( P<0.05). ICOS,CD40 L and OX40 on Tfh cell from spleen of NHD13 mice were decreased significantly than those of wild type mice( All P<0.05). PD-1 on Tfh cell from spleen of NHD13 mice was increased significantly than that of wild type mice( P<0.05). The bone marrow mononuclear cells(BMMNC) from NHD13 mice had significantly lower proportion of Tfh/CD4+T cell than that of wild type mice( P<0.05). OX40 on Tfh of BMMNC from NHD13 mice was decreased significently than that of wild type mice( P<0.05). PD-1 on Tfh of BMMNC from NHD13 mice was increased significently than that of wild type mice( P<0.05). There were no significantdifference between of ICOS and CD40 L on Tfh of BMMNC from NHD13 mice and on Tfh of BMMNC from wild type mice( both P﹥0.05).Fourth section The expression of ICOS and CD40 L mRNA in spleen cells from NHD13 mice were lower compared with wild type mice( both P<0.05). The expression of PD-1 m RNA was up-regulated in spleen cells from NHD13 mice compared with wild type mice( P<0.05). There were no significant difference in expression of OX40 m RNA in spleen cells from NHD13 mice and wild type mice. The expression of ICOS,CD40 L,OX40 m RNA in BMMNC from NHD13 mice were lower compared with wild type mice( All P<0.05). The expression of PD-1 m RNA was up-regulated in BMMNC from NUP98-HOXD13 transgenic mice compared with wild type mice( P<0.05).Conclusion1. Peripheral blood cells including WBC, RBC, PLT were lower in the NHD13 mice compared to wild type controls, with larger volume and morphologic abnormalities. The bone marrow cells of NHD13 mice have morphological abnormalities, bi-nucleated erythrocytes, ringed nucleated neutrophil and erythroblastic island and so on.2. The amounts of lymphoid follicles and germinal centers in the spleen of NHD13 mice are reduced significantly than those of wide type mice, the red pulp in the spleen of NHD13 mice is more widened than that of wild type mice, and result of Immunohistochemistry showed that CXCR5 on spleen cells of NHD13 mice was decreased significantly than that of wild type mice, and PD-1 on spleen cells of NHD13 mice was increased significantly than that of wide type mice. All of these results show that functional structures of spleen and the amount of CXCR5+T cell from NUP98-HOXD13 transgenic mice are reduced, which can infer the dysfunction of the NUP98-HOXD13 transgenic mice’ spleen.3. The amount of Tfh cell and moleculars including ICOS,CD40 L and OX40 on Tfh cell from spleen of NUP98-HOXD13 transgenic mice are decreased significantly than that of wide type mice, In contrast, the expression of PD-1 is higher than that of wide type mice. The amount of Tfh cell and the OX40 on Tfh cell from bone marrow of NUP98-HOXD13 transgenic mice are decreased significantly than that of wide typemice, and PD-1 on Tfh cell is increased significantly. Compared to wide type mice, NUP98-HOXD13 transgentic mice has worse immune function, its dysfunction of Immune surveillance and immune tolerance promotes the proliferation of the malignant clone.