骨髓增生异常综合征小鼠滤泡辅助T细胞及表面分子PD-1表达的研究
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摘要
目的:观察骨髓增生异常综合征(MDS)小鼠血象和骨髓象变化、骨髓和脾脏滤泡辅助T细胞(Tfh)数量及表面分子程序化死亡分子-1(PD-1)的表达情况,探讨其在MDS发病机制中的作用。方法:分别选取10只雄性NUP98-HOXD13转基因小鼠及其同源的野生型C57BL/6J小鼠作为实验对象,行外周血细胞计数,瑞氏染色观察外周血细胞变化、骨髓细胞涂片检查细胞形态;流式细胞术检测骨髓单个核细胞(BMMNC)及脾脏来源的Tfh细胞数量及其表面分子PD-1表达;实时定量PCR法分析BMMNC及脾脏来源细胞PD-1 mRNA的表达。结果:上述转基因小鼠的红细胞、中性粒细胞和血小板数均低于野生型C57BL/6J小鼠;与野生型C57BL/6J小鼠比较,转基因小鼠外周血红细胞及血小板形态相对大小不均,骨髓中可见双核红细胞、环状核粒细胞和红系造血岛等;转基因小鼠骨髓及脾脏Tfh数量较野生型小鼠少,且表面分子PD-1表达率增高;还发现该小鼠BMMNC PD-1 mRNA表达量明显高于野生型小鼠。结论:NUP98-HOXD13转基因小鼠出现全血细胞减少和病态造血,Tfh数量减少,PD-1表达量升高,可能是导致机体免疫功能抑制,促进恶性克隆逃逸免疫监视的重要原因之一。
Objective:To investigate the complete blood count,morphological changes,follicular T helper(Tfh)cells and expression of PD-1 in bone marrow and spleen of mice with myelodysplastic syndrome(MDS) and to explore their significance in pathogenesis of MDS.Methods:The 10 male NUP98-HOXD13 transgenic mice and 10 male homologous wild-type C57BL/6J mice were used for experments.The complete blood count,morphological change of NUP98-HOXD13 transgenic mice and wild-type C57BL/6J were detected by routine methods.The level of Tfh cells and expression of PD-1 in bone marrow and spleen were measured by flow cytometry.The PD-1 mRNA of bone marrow mononuclear cells and spleen cells were analyzed by real-time PCR method.Results:The counts of RBC,neutrophile and platelet in above-mentioned transgenic mice were less than that in wild type C57BL/6J mice.As compared with wild type C57BL/6J mice,the morphology of RBC and platelet in transgenic mice was some abnormal,including binucleated erythrocytes,ringed mucleated neutrophil and erythroblastic islands.The count of Tfh cells in transgenic mice was less than that in wild type mice,but the expression of PD-1 was higher.The expression of BMMNC PD-1 mRNA was obviously higher than that in wild type mice.Conclusion:The pancytopenia and dysplasia,decrease of Tfh cells and increase of PD-1 expression have been observed in NUP98-HOXD13 transgenic mice,which may be one of important reasons for promoting malignant clone and leading to impair anti immune respones.
Objective:To investigate the complete blood count,morphological changes,follicular T helper(Tfh)cells and expression of PD-1 in bone marrow and spleen of mice with myelodysplastic syndrome(MDS) and to explore their significance in pathogenesis of MDS.Methods:The 10 male NUP98-HOXD13 transgenic mice and 10 male homologous wild-type C57BL/6J mice were used for experments.The complete blood count,morphological change of NUP98-HOXD13 transgenic mice and wild-type C57BL/6J were detected by routine methods.The level of Tfh cells and expression of PD-1 in bone marrow and spleen were measured by flow cytometry.The PD-1 mRNA of bone marrow mononuclear cells and spleen cells were analyzed by real-time PCR method.Results:The counts of RBC,neutrophile and platelet in above-mentioned transgenic mice were less than that in wild type C57BL/6J mice.As compared with wild type C57BL/6J mice,the morphology of RBC and platelet in transgenic mice was some abnormal,including binucleated erythrocytes,ringed mucleated neutrophil and erythroblastic islands.The count of Tfh cells in transgenic mice was less than that in wild type mice,but the expression of PD-1 was higher.The expression of BMMNC PD-1 mRNA was obviously higher than that in wild type mice.Conclusion:The pancytopenia and dysplasia,decrease of Tfh cells and increase of PD-1 expression have been observed in NUP98-HOXD13 transgenic mice,which may be one of important reasons for promoting malignant clone and leading to impair anti immune respones.
引文
1 Tefferi A,Vardiman JW.Myelodysplastic syndromes.N Engl J Med,2009;361(19):1872-1885.
2 Braun T,Fenaux P.Myelodysplastic Syndromes(MDS)and autoimmune disorders(AD):cause or consequence?Best Pract Res Clin Haematol,2013;26(4):327-336.
3 王化泉,邵宗鸿.滤泡辅助T细胞的研究进展.中国实验血液学杂志,2009;17(5):1384-1389.
4 Hutchinson L.Haematological cancer:PD-1 blockade:opening the door to attack.Nat Rev Clin Oncol,2015;12(2):65.
5 Barber DL,Wherry EJ,Masopust D,et al.Restoring function in exhausted CD8 T cells during chronic viral infection.Nature,2006;439(7077):682-687.
6 Iwai Y,Ishida M,Tanaka Y,et al.Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade.Proc Natl Acad Sci USA,2002;99(19):12293-12297.
7 Dong H,Strome SE,Salomao DR,et al.Tumor-associated B7-H1promotes T-cell apoptosis:a potential mechanism of immune evasion.Nat Med,2002;8(8):793-800.
8 王化泉,邵宗鸿,邢莉民,等.骨髓增生异常综合征患者外周血CD4+CD25+调节性T细胞和Th3细胞变化.中华血液学杂志,2008;29(07):482-484.
9 Lin YW,Slape C,Zhang Z,et al.NUP98-HOXD13 transgenic mice develop a highly penetrant,severe myelodysplastic syndrome that progresses to acute leukemia.Blood,2005;106(1):287-295.
10 Dulos J,Carven GJ,van Boxtel SJ,et al.PD-1 blockade augments Th1and Th17 and suppresses Th2 responses in peripheral blood from patients with prostate and advanced melanoma cancer.J Immunother.2012;35(2):169-178.
11 Ansell SM,Lesokhin AM,Borrello I,et al.PD-1 blockade with nivolumab in relapsed or refractory Hodgkin's lymphoma.N Engl J Med,2015;372(4):311-319.
12 Robert C,Long GV,Brady B,et al.Nivolumab in previously untreated melanoma without BRAF mutation.N Engl J Med,2015;372(4):320-330.
13 Brahmer JR,Tykodi SS,Chow LQ,et al.Safety and activity of antiPD-L1 antibody in patients with advanced cancer.N Engl J Med,2012;366(26):2455-2465.
14 Topalian SL,Hodi FS,Brahmer JR,et al.Safety,activity,and immune correlates of anti-PD-1 antibody in cancer.N Engl J Med,2012;366(26):2443-2454.
15 Robert C,Ribas A,Wolchok JD,et al.Anti-programmed-deathreceptor-1 treatment with pembrolizumab in ipilimumab-refractory advanced melanoma:a randomised dose-comparison cohort of a phase 1trial.Lancet,2014;384(9948):1109-1117.
2 Braun T,Fenaux P.Myelodysplastic Syndromes(MDS)and autoimmune disorders(AD):cause or consequence?Best Pract Res Clin Haematol,2013;26(4):327-336.
3 王化泉,邵宗鸿.滤泡辅助T细胞的研究进展.中国实验血液学杂志,2009;17(5):1384-1389.
4 Hutchinson L.Haematological cancer:PD-1 blockade:opening the door to attack.Nat Rev Clin Oncol,2015;12(2):65.
5 Barber DL,Wherry EJ,Masopust D,et al.Restoring function in exhausted CD8 T cells during chronic viral infection.Nature,2006;439(7077):682-687.
6 Iwai Y,Ishida M,Tanaka Y,et al.Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade.Proc Natl Acad Sci USA,2002;99(19):12293-12297.
7 Dong H,Strome SE,Salomao DR,et al.Tumor-associated B7-H1promotes T-cell apoptosis:a potential mechanism of immune evasion.Nat Med,2002;8(8):793-800.
8 王化泉,邵宗鸿,邢莉民,等.骨髓增生异常综合征患者外周血CD4+CD25+调节性T细胞和Th3细胞变化.中华血液学杂志,2008;29(07):482-484.
9 Lin YW,Slape C,Zhang Z,et al.NUP98-HOXD13 transgenic mice develop a highly penetrant,severe myelodysplastic syndrome that progresses to acute leukemia.Blood,2005;106(1):287-295.
10 Dulos J,Carven GJ,van Boxtel SJ,et al.PD-1 blockade augments Th1and Th17 and suppresses Th2 responses in peripheral blood from patients with prostate and advanced melanoma cancer.J Immunother.2012;35(2):169-178.
11 Ansell SM,Lesokhin AM,Borrello I,et al.PD-1 blockade with nivolumab in relapsed or refractory Hodgkin's lymphoma.N Engl J Med,2015;372(4):311-319.
12 Robert C,Long GV,Brady B,et al.Nivolumab in previously untreated melanoma without BRAF mutation.N Engl J Med,2015;372(4):320-330.
13 Brahmer JR,Tykodi SS,Chow LQ,et al.Safety and activity of antiPD-L1 antibody in patients with advanced cancer.N Engl J Med,2012;366(26):2455-2465.
14 Topalian SL,Hodi FS,Brahmer JR,et al.Safety,activity,and immune correlates of anti-PD-1 antibody in cancer.N Engl J Med,2012;366(26):2443-2454.
15 Robert C,Ribas A,Wolchok JD,et al.Anti-programmed-deathreceptor-1 treatment with pembrolizumab in ipilimumab-refractory advanced melanoma:a randomised dose-comparison cohort of a phase 1trial.Lancet,2014;384(9948):1109-1117.