小鼠配对免疫球蛋白样受体及其介导的T细胞阶联与树突状细胞免疫耐受作用关系的研究
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摘要
移植物抗宿主病(GVHD)仍然是临床骨髓移植治愈血液系统恶性疾病的主要障碍之一。供者异基因淋巴细胞的异常活化是GVHD发生、发展的重要因素,诱导受者产生针对供者异基因主要组织相容性抗原(MHC)特异性耐受是预防GVHD、保存GVL的最佳途径。配对免疫球蛋白样受体(Paired immunoglobin-like receptor,PIR)是近年发现的主要表达在小鼠树突状细胞(DCs)上的免疫抑制性调节受体,包括免疫抑制性受体(PIR-B)及免疫活化性受体(PIR-A),其配体均是MHC-Ⅰ,二者与其配体的结合的水平是决定DCs免疫活化程度的重要分子标志之一。由于DC不仅是调控体内T细胞活化的关键,也是诱导免疫耐受的理想的靶细胞,可以直接或间接通过诱导供者抗原特异性调节性T细胞的生成、在体内形成T细胞阶联效应而抑制异基因T淋巴细胞的活化,因此研究PIR在树突状细胞的表达、T细胞阶联的关系及与免疫耐受的关系,可能为诱导供者MHC特异性耐受,实现GVHD及GVL的分离提供新的途径。
第一部分免疫球蛋白样受体在小鼠树突状细胞上的表达及与免疫耐受关系的研究
目的配对的免疫球蛋白样受体A、B(paired immunoglobulin-like receptor A、B, PIR-A、PIR-B)属于小鼠免疫球蛋白超家族成员。研究配对免疫球蛋白样受体PIR-A、B在小鼠DCs上的表达及与表面共刺激分子变化的关系,探讨PIR与免疫耐受的关系,探索诱导耐受性DCs的有效途径。
方法以C57BL/6小鼠来源DC系DC2.4细胞为研究对象,分别以重组小鼠白介素-10(recombinant mouse interleukin-10,rmIL-10)、重组人转化生长因子β1 (recombinant human transforming growth factorβ1,rhTGF -β1)诱导DC2.4细胞为耐受性DC(tolerogenic DC,T-DC),脂多糖(LPS)刺激48h为成熟DC2.4细胞(LPS-DC),体外化学合成特异PIR-B小RNA干扰片段(small interfering RNA,siRNA),以Lip2000转染DC2.4细胞(Si-DC)。分别应用半定量RT-PCR、流式细胞仪(Flow cytometry, FCM)及Western blot检测IL-10、TGF-β1、LPS及小干扰RNA对DC2.4细胞上PIR-A/B表达的影响;检测LPS刺激Si-DCs后表面共刺激分子CD80、CD86、MHC-Ⅱ及PIR-A的变化;以2-△△Ct表示各目的基因cDNA转录的相对表达量。分别以上述各组DCs细胞为刺激细胞,以异基因BALB/c小鼠脾淋巴细胞为反应细胞,应用3H-TdR标记法检测同种异体淋巴细胞的增殖能力(MLR),ELISA方法测MLR上清中IFN-γ的分泌水平变化。
结果FCM检测DC2.4细胞上PIR-A、PIR-B的共同的胞外区PIR表达的阳性率为(28.65±8.12)%,IL-10、TGF-β1及LPS诱导后PIR表达均上调(P<0.05),分别为(54.21±6.34)%,(58.78±4.70)%,(48.24±6.75)%,但IL-10、TGF-β1及LPS各组间无显著性差别(P>0.05)。半定量RT-PCR及Western blot显示,IL-10、TGF-β1诱导DC2.4细胞后PIR-B的mRNA及蛋白表达升高,而PIR-A表达则降低,而LPS刺激时则相反,PIR-A的mRNA及蛋白表达升高、PIR-B的表达则降低。流式细胞仪检测SiRNA阳性对照组的转染率为93.12%,SYBR greenⅠRealtime-PCR检测,LPS刺激后Si-DCs CD80、CD86、MHC-Ⅱ及PIR-A的表达高于正常DCs组。LPS-DCs组CD80、CD86、MHC-Ⅱ及PIR-A的2-△△Ct分别为5.02±1.09、4.69±1.75、5.46±1.79、6.02±2.13;LPS刺激后Si-DC组TAI分别为8.79±2.2、11.03±1.96、10.26±2.55、12.10±2.83,同LPS刺激的正常组相比,Si-DC组分别增加了3.72、6.34、4.8、6.08倍(P<0.05)。混合淋巴细胞反应显示:正常DC2.4细胞可刺激异基因淋巴细胞反应,IL-10、TGFβ1诱导的T-DC组MLR明显受抑(P<0.05),MLR上清中IFN-γ水平也相应降低(P<0.05)。LPS-DC及Si-DCs组MLR明显增强(P<0.05),MLR上清中IFN-γ水平明显增高(P<0.05);
结论上调免疫抑制性受体PIR-B、下调活化性受体PIR-A是小鼠DCs获得耐受的普遍表型特征及分子生物学机制,沉默PIR-B的表达可使PIR-A及CD80、CD86、MHC--Ⅱ及PIR-A过表达,使DCs活化的机制,PIR-A和PIR-B构成了小鼠树突状细胞耐受的新靶点。
第二部分高度表达免疫球蛋白样受体B耐受性树突状细胞可诱导CD4+CD25+调节性T细胞生成
目的研究配对免疫球蛋白样受体B在树突状细胞上表达与调节性T细胞的生成的关系,探讨耐受性DCs诱导耐受的详细机制,为体内诱导耐受性DCs及调节性T细胞(Treg)生成提供实验依据。
方法免疫磁珠分选BALB/c小鼠CD4+T脾淋巴细胞。以rmIL-10(50ng/ml)、rhTGF-β1(50ng/ml)联合诱导C57BL/6小鼠来源的DC2.4细胞3天生成耐受性DCs(T-DC),同时设正常DC2.4细胞(DC)及LPS刺激48h后成熟的DC2.4细胞(mDC)干扰PIR-B组(Si-DCs)为对照组,各组DCs分别与BALB/c小鼠CD4+T脾淋巴细胞混合培养48h,检测Treg生成。RT-PCR检测转录因子Foxp3mRNA的表达变化,流式细胞仪检测CD4+CD25+Treg细胞的比例,PI检测CD4+T细胞的凋亡。磁珠分选的CD4+CD25+Treg与CD4+T细胞按照不同的比例加入MLR体系中,3H检测Treg对异基因DCs刺激的同基因淋巴细胞的增殖能力影响。
结果磁珠分选BALB/c小鼠脾CD4+T及CD4+CD25+T淋巴细胞纯度>95%,正常DCs、T-DCs、Si-DC及mDCs各组细胞同BALB/c小鼠脾细胞CD4+T细胞混合培养3天,RT-PCR检测表明,T-DCs组诱导后CD4+T细胞Foxp3mRNA表达明显高于正常DCs、LPS-DC及Si-DC组。而Si-DC及LPS-DC刺激的CD4+T细胞Foxp3mRNA的表达明显降低(P>0.05)。流式细胞仪检测表明,正常DCs、T-DCs、Si-DC及mDCs诱导的CD4+CD25+Treg细胞比例分别为(5.19±1.2)%、(28.29±2.36)%、(1.06±0.55) %,(2.01±0.66) %,以T-DC组诱导的Treg细胞比例明显增高(P>0.01)。PI检测正常DCs、T-DCs、mDCs及Si-DC组诱导48h后CD4+T细胞的的凋亡率分别为(8.3±0.7)%、(21.56±2.32)%、(2.5±0.8)%、(1.9±0.7)%。3H检测异基因混合淋巴细胞增殖反应细胞刺激的增殖效应,且呈剂量依赖性。
结论诱导Treg细胞生成、促进异基因淋巴细胞凋亡是PIR-B介导性DCs耐受的分子机制,为临床应用耐受性DCs诱导免疫耐受提供理论依据,也为Treg的诱导提供新的途径。
第三部分CD8+CD28-T细胞对小鼠树突状细胞上配对免疫球蛋白样受体A和B表达的影响及与耐受的关系
目的诱导宿主产生供者主要组织相容性抗原的特异性耐受是临床骨髓移植的最终目标,CD8 + CD28-T(Ts)细胞是具有免疫抑制作用的调节性T细胞亚群之一,体外诱导异基因抗原特异性Ts生成,以研究Ts细胞与小鼠树突状细胞(DCs)上配对免疫球蛋白样受体A和B表达的关系,探讨其诱导免疫耐受的分子机制及特点,为临床抗原特异性免疫治疗的诱导提供理论基础。
方法体外诱导Ⅰ类主要组织相容性抗原(H-2b)抗原特异性Ts细胞群,以C57BL/6小鼠(H-2b)骨髓来源的树突状细胞系DC2.4细胞为刺激细胞,同BALB/c小鼠(H-2d)脾淋巴细胞混合培养,连续两次,每次培养7天,第10天于培养体系中加入IL-2(10u/ml),第14天结束培养。以生物素标记的CD28、CD8标记上述细胞群,以链亲和素标记的免疫磁珠分两步分选Ts细胞,首先负选CD8+T细胞,再正选CD28+T细胞,阴选细胞悬液为Ts细胞群。Ts同C57BL/6小鼠DC2.4(H-2b)细胞混合培养48h,RT-PCR检测DCs细胞PIR-A、PIR-BmRNA的表达,Westernblot检测DCs细胞PIR-A、B的表达。3H标记检测混合淋巴细胞增殖反应(MLR),体外诱导培养KM鼠骨髓来源的树突状细胞,分别以DC2.4(H-2b)及第三者主要组织相容性抗原无关的KM供鼠DCs细胞为刺激细胞,以BALB/c小鼠来源的脾CD4+T淋巴细胞为反应细胞,加入Ts细胞,以CPM检测异基因淋巴细胞增殖反应能力。
结果体外以C57BL/6小鼠DCs诱导并在体外应用免疫磁珠分选的CD8+、CD8+CD28-Ts >90%,以BALB/c小鼠Ts细胞(H-2d)与异基因C57BL/6小鼠DCs细胞(H-2b)混合培养48h后,RT-PCR及Western blot检测PIR-BmRNA及蛋白表达上调、PIR-A的mRNA及蛋白表达则下调。3H掺入标记检测显示,DC2.4细胞及KM鼠DCs细胞均可刺激BALB/c小鼠(H-2d)脾CD4+T淋巴细胞增殖,加入Ts细胞后,可以明显抑制DC2.4细胞刺激的BALB/c小鼠脾CD4+T淋巴细胞(H-2d)的增殖,而并不抑制KM鼠DCs细胞刺激的CD4+T脾淋巴细胞(H-2k)的增殖反应。
结论体外诱导的Ts呈现Ⅰ类主要组织相容性抗原特异性抑制异基因反应性淋巴细胞的增殖。其机制与Ts细胞上调PIR-B mRNA、下调PIR-A mRNA的表达有关。升高供体移植物中抗原特异性Ts细胞比例或受体树突状细胞表面免疫抑制性受体的表达可能成为诱导受者产生针对供体抗原特异性免疫耐受的有效途径。
Graft-versus-host diseases(GVHD) still are one of the obstacles for clinical bone marrow transplantation in treating malignant hematologic diseases. The activation of allogenetic lymphocytes promote the occurrence and development of GVHD. So induction immune tolerance specific for host major histocompatibility antigens is the optimization for prevention GVHD and preservation the graft-versus-leukemia(GVL). Recently, Paired immunoglobin-like receptor (PIR) , including PIR-A and PIR-B, are found to be inhibiting receptor expressed on dendritic cells with typeⅠmajor histocompatibility antigens as its ligand. PIR-A and B and its ligand constituted the system for deciding the dendritic cells active. For the dendritic cells can not only induct the immunue cells active but also induct the immune tolerant by inducing the regulatory T cells by T cells link, the study of PIR-B on dendritic cells may provide new way for inducing specific immune tolence for host antigen with the molecular PIR-B.
PartⅠStudy of the relationship between paired immunoglobin-like receptors expression and immune tolerant on dendritic cells in mouse
Objective Paired immunoglobulin-like receptor A,B are numbers of immunoglobin superfamily. The study of the relationship of paired immunoglobin-like receptor expression and co-stimlation molecules on tolerant dendritic cells may provide utility way for inducting immune tolerant.
Methods The DC2.4 cells derived from the C57BL/6 mouse were inducted by re recombinant mouse interleukin-10(rmIL-10) and recombinant human transforming growth factorβ1(rhTGF -β1) and serve as tolerant DC(T-DC). Lipopolysaccharide(LPS) stimulated the dendritic cells mature for 48 hours as mature DC(LPS-DC). The small interfering RNA (siRNA) was chemical synthesis in vitro and transduced into the DC2.4 cells by Lip 2000(Si-DC). RT-PCR, Flow cytometry and Western blot were used to examine the PIR-A/B expression on T-DCs, LPS-DCs and Si-DCs. Syb green 1 realtime-PCR were used to examined transcription expression of co-stimulation molecule such as CD80,CD86, MHC-Ⅱand PIR-A. 2-△△Ct was used for evaluating the relative quantity of gene cDNA transcrition. Mixing lymphocytes reaction(MLR) was used to examined the allogenetic reactive lymphocytes proliferation for the DC2.4 cells as stimulating cells and the BALB/c spleen lymphocytes as the reactive cells. Enzyme linked immunosorbent assay(ELISA) was used detect the interference-γlevels in the supernatant in the MLR.
Results FCM examination demonstrated that positive reate of PIR, which is the common extramembrane district of PIR-A and B, were (28.65±8.12)% on DC2.4 cells, and the rate added up to (54.21±6.34)%,(58.78±4.70)%,(48.24±6.75)% respectively for IL-10, TGF-β1 and LPS. But there was no significant between the IL-10, TGF-β1 or LPS-DC(P>0.05). RT-PCR and Western blot demonstrated that the mRNA and protein level of PIR-B increased and the level of PIR-A were decreased by IL-10 and TGF-β1 inducton. On the contrary, the mRNA and its protein level for PIR- A mRNA and pritein expression increased and that for PIR- B were decreased by the LPS stimulation. The positive rate of RNAi transfection was 93.12% by FCM detection. SYBR greenⅠRealtime-PCR results showed that the CD80, CD86, MHC-Ⅱa nd PIR-A were over-expressed on Si-DCs other than the mormal DCs by the same stimulation of LPS. The 2-△△Ct CD80, CD86, MHC-Ⅱand PIR-A in LPS-DCs were respectively 5.02±1.09, 4.69±1.75, 5.46±1.79, 6.02±2.13 and the 2-△△Ct were 8.79±2.2, 11.03±1.96, 10.26±2.55, 12.10±2.83 in Si-DC with the same LPS stimulation. CD80, CD86, MHC-Ⅱa nd PIR-A transcription were increased by 3.72、6.34、4.8、6.08 times in Si-DCs than the normal DCs(P<0.05). MLR were suppressed in T-DCs for IL-10 and TGF-β1 and IFN-γlevel were decreased in MLR supernatant(P<0.05). MLR were intensive in LPS-DCs and Si-DCs groups and the IFN-γlevel were increased(P<0.05).
Conclusions Up-regulating PIR-B or down-regualuting PIR-A expression are the common characteristics and molecules mechanism. Silencing the PIR-B expression promote the PIR-A, CD80, CD86 and MHC-Ⅱoverexpression and that makes the DCs immune active. PIR-A and B constitute molecule targets for inducting dendritic cells immune tolerance in mouse.
PartⅡDendritic cells with higher expressed paied immunoglobin- like receptors induce the CD4+CD25+T cells development
Objective To investigate the relationship between the paired immunoglobin receptor B(PIR-B) and the CD4+CD25+ regulatory T cells(Treg) development, suggest the tolerant dendritic cells detail mechanisms and provide the base for induction Treg development by the tolerant DCs.
Methods CD4+T and CD4+CD25+T cells were sorted by immune magcellect from the BALB/c spllen cells. rmIL-10(50ng/ml) and rhTGF-β1(50ng/ml) were combinated to induced the DC2.4 cells tolerant(T-DCs) for three days. The small interfering RNA (siRNA) transfected into the DC2.4 cells by Lip2000 to made the silencing PIR-B DCs(Si-DC). Mature DCs( mDC) were stimulated by the LPS for 48h. The nomal DC2.4 cells(DCs), T-DCs, siRNA and mDC were mixed and culcured with the CD4+T cells for five days for inducing the Treg cells. RT-PCR were used for detecting the the transcript factor Foxp3 mRNA expression. FCM examined the CD4+CD25+T cells percentage in mixed culture system. PI were detect the apoptosis of CD4+T cells after cultured with the different group DCs. Different percentage of CD4+CD25+T cells were co-cultured with the syngennetic cells proliferation with the allogenetic dendritic cells as stimulating cells. 3H test were used to detect the proliferation ability in MLR.
Results CD4+T and CD4+CD25+T cells of BALB/c mouse were sorted by immune magnetic beads and the purity of CD4+T and CD4+CD25+T cells were aboved 95%. Different groups of DCs such as normal DCs, T-DCs, Si-DCs and mDCs derived from C57BL/6(H-2b) were co-cultured with the CD4+T BALB/c cells for three days to developing the Treg cells. RT-PCR detection showed that the the Foxp3 mRNA expression were higher in T-DCs than the othe groups(P>0.01). Si-DCs and the LPS-DC with lower PIR-B expression induced the lower Foxp3 mRNA level(P>0.01). FCM demonstrated that CD4+CD25+T cells percentage were (5.19±1.2)%, (28.29±2.36)%, (1.06±0.55)%, (2.01±0.66) % respectively for DCs, T-DCs, Si-DC and mDCs groups. It was demonstrated that T-DC also induced a higher rate CD4+CD25+T cells from the CD4+T cells than the other groups(P<0.05). PI detection demonostrated that apoptosis rate of CD4+T cells were (8.3±0.7)%, (21.56±2.32)%, (2.5±0.8)% and (1.9±0.7)%, respectively for DCs, T-DCs, mDCs and Si-DC. 3H incoupration test showed that CD4+CD25+T cells mixed with CD4+T cells at different percentage and inhibited CD4+T cells proliferation after stimulated by the allogenetic cells.the inhibition was paralleveling to the quantity of CD4+CD25+T cells.
Conclusion Induction CD4+CD25+T cells development and apoptosis of allogenegtic lymphocyte cells may be associated with the tolerant DCs with higher expression inhibited receptor PIR-B. This may provide base for transplamtation immune tolerenat and new pathway for Treg induction.
PartⅢCD8+CD28-T cells changes PIR-A/B expression and promotes the allogentic immune tolerance
Objective Specific suppression of the host’s immune response to donor human leucocyte antigen(HLA) antigens remains the ultimate goal for clinical transplantation. CD8 + CD28-T(T suppression cell, Ts)is a subset of regulatory T cells which play regulatory role in immune reaction. Allogenetic antigen specific Ts were induced and to study the influnce for the expression of paired immunoglubin-like receptor A and B on the dendritic cells in mouse. The study aim to explore the molecular mechanism, immune tolerance characteristic of Ts and to provide a basis for the development of specific immunosuppressive therapy.
Methods Ts was induced in vitro for specific MHC-Ⅰ(H-2b) antigen in vitro. DC2.4 cells derived from the C57BL/6 mouse(H-2b) was used to prime allogenetic lymphocyte spleen cells of the BALB/c mouse in mixed lymphocyte cultures repetedly for twice. Every culture prolonged for seven days. IL-2(10u/ml) was added to the culture at day 10 and finished the culture at day 14. Biotin-CD28 and Biotin-CD8 antigen and streptavidin magnetic bead were used to sorting the CD8+CD28-T cells. CD8+T cells were positive sorted firstly and then CD28-T cells were negatively sorting. CD8+CD28-T cells co-cultured wth the DC2.4 cells for 48h. RT-PCR were used to determin the PIR-A/B mRNA level and Westernblot was use to detect the PIR-A/B protein leve. 3H-incorporation test was used to detect the allogenetic cells proliferation. The myeloid DCs from KM mouse were cultured in vitro for a MHC-Ⅰnon-asscociated antigen doner. Both DC2.4 cells and the DCs of KM mouse served as stimulating cells. The CD4+T cells of BALB/c spleen cells served as reacting cells. Ts cells were added into the mixing lymphocyte reaction. CPM was used for determing the proliferation.
Results CD8+CD28-Ts were inducted with the allogenetic DC2.4 cells derived from C57BL/6 mouse and sorted by magnetic bead in vitro. Purity of CD8+ and CD8+CD28-Ts cells was above >90%. After cultured mixingly Ts cells and DC2.4 cells for 48h, PIR-B mRNA and protein level increased and the PIR-A mRNA and protein level decreased examined by RT-PCR and Westernblot. 3H-incorporation test demonstrated that the DC2.4 cells and DCs from KM mouse stimulated the allogenetic CD4+T cells of BALB/c proliferation. Ts inhibited CD4+T cells(H-2d) proliferation stimulated by the DC2.4 cells but did not inhibited CD4+T cells(H-2d) proliferation stimulated by DCs from KM mouse.
Conclusion Ts induced in vitro specifically inhibited the allogenetic areactive cells proliferation with MHC-Ⅰconstraction. This may be associated with up-regulation of the PIR-B expression and down-regulating of PIR-A expression. Increasing the Ts cells ratio in donator's grafts or the immune inhibiting receptor on receptor’s DCs may provide utility pathway for a specific antigen tolerance for donator in transplantation.
第一部分免疫球蛋白样受体在小鼠树突状细胞上的表达及与免疫耐受关系的研究
目的配对的免疫球蛋白样受体A、B(paired immunoglobulin-like receptor A、B, PIR-A、PIR-B)属于小鼠免疫球蛋白超家族成员。研究配对免疫球蛋白样受体PIR-A、B在小鼠DCs上的表达及与表面共刺激分子变化的关系,探讨PIR与免疫耐受的关系,探索诱导耐受性DCs的有效途径。
方法以C57BL/6小鼠来源DC系DC2.4细胞为研究对象,分别以重组小鼠白介素-10(recombinant mouse interleukin-10,rmIL-10)、重组人转化生长因子β1 (recombinant human transforming growth factorβ1,rhTGF -β1)诱导DC2.4细胞为耐受性DC(tolerogenic DC,T-DC),脂多糖(LPS)刺激48h为成熟DC2.4细胞(LPS-DC),体外化学合成特异PIR-B小RNA干扰片段(small interfering RNA,siRNA),以Lip2000转染DC2.4细胞(Si-DC)。分别应用半定量RT-PCR、流式细胞仪(Flow cytometry, FCM)及Western blot检测IL-10、TGF-β1、LPS及小干扰RNA对DC2.4细胞上PIR-A/B表达的影响;检测LPS刺激Si-DCs后表面共刺激分子CD80、CD86、MHC-Ⅱ及PIR-A的变化;以2-△△Ct表示各目的基因cDNA转录的相对表达量。分别以上述各组DCs细胞为刺激细胞,以异基因BALB/c小鼠脾淋巴细胞为反应细胞,应用3H-TdR标记法检测同种异体淋巴细胞的增殖能力(MLR),ELISA方法测MLR上清中IFN-γ的分泌水平变化。
结果FCM检测DC2.4细胞上PIR-A、PIR-B的共同的胞外区PIR表达的阳性率为(28.65±8.12)%,IL-10、TGF-β1及LPS诱导后PIR表达均上调(P<0.05),分别为(54.21±6.34)%,(58.78±4.70)%,(48.24±6.75)%,但IL-10、TGF-β1及LPS各组间无显著性差别(P>0.05)。半定量RT-PCR及Western blot显示,IL-10、TGF-β1诱导DC2.4细胞后PIR-B的mRNA及蛋白表达升高,而PIR-A表达则降低,而LPS刺激时则相反,PIR-A的mRNA及蛋白表达升高、PIR-B的表达则降低。流式细胞仪检测SiRNA阳性对照组的转染率为93.12%,SYBR greenⅠRealtime-PCR检测,LPS刺激后Si-DCs CD80、CD86、MHC-Ⅱ及PIR-A的表达高于正常DCs组。LPS-DCs组CD80、CD86、MHC-Ⅱ及PIR-A的2-△△Ct分别为5.02±1.09、4.69±1.75、5.46±1.79、6.02±2.13;LPS刺激后Si-DC组TAI分别为8.79±2.2、11.03±1.96、10.26±2.55、12.10±2.83,同LPS刺激的正常组相比,Si-DC组分别增加了3.72、6.34、4.8、6.08倍(P<0.05)。混合淋巴细胞反应显示:正常DC2.4细胞可刺激异基因淋巴细胞反应,IL-10、TGFβ1诱导的T-DC组MLR明显受抑(P<0.05),MLR上清中IFN-γ水平也相应降低(P<0.05)。LPS-DC及Si-DCs组MLR明显增强(P<0.05),MLR上清中IFN-γ水平明显增高(P<0.05);
结论上调免疫抑制性受体PIR-B、下调活化性受体PIR-A是小鼠DCs获得耐受的普遍表型特征及分子生物学机制,沉默PIR-B的表达可使PIR-A及CD80、CD86、MHC--Ⅱ及PIR-A过表达,使DCs活化的机制,PIR-A和PIR-B构成了小鼠树突状细胞耐受的新靶点。
第二部分高度表达免疫球蛋白样受体B耐受性树突状细胞可诱导CD4+CD25+调节性T细胞生成
目的研究配对免疫球蛋白样受体B在树突状细胞上表达与调节性T细胞的生成的关系,探讨耐受性DCs诱导耐受的详细机制,为体内诱导耐受性DCs及调节性T细胞(Treg)生成提供实验依据。
方法免疫磁珠分选BALB/c小鼠CD4+T脾淋巴细胞。以rmIL-10(50ng/ml)、rhTGF-β1(50ng/ml)联合诱导C57BL/6小鼠来源的DC2.4细胞3天生成耐受性DCs(T-DC),同时设正常DC2.4细胞(DC)及LPS刺激48h后成熟的DC2.4细胞(mDC)干扰PIR-B组(Si-DCs)为对照组,各组DCs分别与BALB/c小鼠CD4+T脾淋巴细胞混合培养48h,检测Treg生成。RT-PCR检测转录因子Foxp3mRNA的表达变化,流式细胞仪检测CD4+CD25+Treg细胞的比例,PI检测CD4+T细胞的凋亡。磁珠分选的CD4+CD25+Treg与CD4+T细胞按照不同的比例加入MLR体系中,3H检测Treg对异基因DCs刺激的同基因淋巴细胞的增殖能力影响。
结果磁珠分选BALB/c小鼠脾CD4+T及CD4+CD25+T淋巴细胞纯度>95%,正常DCs、T-DCs、Si-DC及mDCs各组细胞同BALB/c小鼠脾细胞CD4+T细胞混合培养3天,RT-PCR检测表明,T-DCs组诱导后CD4+T细胞Foxp3mRNA表达明显高于正常DCs、LPS-DC及Si-DC组。而Si-DC及LPS-DC刺激的CD4+T细胞Foxp3mRNA的表达明显降低(P>0.05)。流式细胞仪检测表明,正常DCs、T-DCs、Si-DC及mDCs诱导的CD4+CD25+Treg细胞比例分别为(5.19±1.2)%、(28.29±2.36)%、(1.06±0.55) %,(2.01±0.66) %,以T-DC组诱导的Treg细胞比例明显增高(P>0.01)。PI检测正常DCs、T-DCs、mDCs及Si-DC组诱导48h后CD4+T细胞的的凋亡率分别为(8.3±0.7)%、(21.56±2.32)%、(2.5±0.8)%、(1.9±0.7)%。3H检测异基因混合淋巴细胞增殖反应细胞刺激的增殖效应,且呈剂量依赖性。
结论诱导Treg细胞生成、促进异基因淋巴细胞凋亡是PIR-B介导性DCs耐受的分子机制,为临床应用耐受性DCs诱导免疫耐受提供理论依据,也为Treg的诱导提供新的途径。
第三部分CD8+CD28-T细胞对小鼠树突状细胞上配对免疫球蛋白样受体A和B表达的影响及与耐受的关系
目的诱导宿主产生供者主要组织相容性抗原的特异性耐受是临床骨髓移植的最终目标,CD8 + CD28-T(Ts)细胞是具有免疫抑制作用的调节性T细胞亚群之一,体外诱导异基因抗原特异性Ts生成,以研究Ts细胞与小鼠树突状细胞(DCs)上配对免疫球蛋白样受体A和B表达的关系,探讨其诱导免疫耐受的分子机制及特点,为临床抗原特异性免疫治疗的诱导提供理论基础。
方法体外诱导Ⅰ类主要组织相容性抗原(H-2b)抗原特异性Ts细胞群,以C57BL/6小鼠(H-2b)骨髓来源的树突状细胞系DC2.4细胞为刺激细胞,同BALB/c小鼠(H-2d)脾淋巴细胞混合培养,连续两次,每次培养7天,第10天于培养体系中加入IL-2(10u/ml),第14天结束培养。以生物素标记的CD28、CD8标记上述细胞群,以链亲和素标记的免疫磁珠分两步分选Ts细胞,首先负选CD8+T细胞,再正选CD28+T细胞,阴选细胞悬液为Ts细胞群。Ts同C57BL/6小鼠DC2.4(H-2b)细胞混合培养48h,RT-PCR检测DCs细胞PIR-A、PIR-BmRNA的表达,Westernblot检测DCs细胞PIR-A、B的表达。3H标记检测混合淋巴细胞增殖反应(MLR),体外诱导培养KM鼠骨髓来源的树突状细胞,分别以DC2.4(H-2b)及第三者主要组织相容性抗原无关的KM供鼠DCs细胞为刺激细胞,以BALB/c小鼠来源的脾CD4+T淋巴细胞为反应细胞,加入Ts细胞,以CPM检测异基因淋巴细胞增殖反应能力。
结果体外以C57BL/6小鼠DCs诱导并在体外应用免疫磁珠分选的CD8+、CD8+CD28-Ts >90%,以BALB/c小鼠Ts细胞(H-2d)与异基因C57BL/6小鼠DCs细胞(H-2b)混合培养48h后,RT-PCR及Western blot检测PIR-BmRNA及蛋白表达上调、PIR-A的mRNA及蛋白表达则下调。3H掺入标记检测显示,DC2.4细胞及KM鼠DCs细胞均可刺激BALB/c小鼠(H-2d)脾CD4+T淋巴细胞增殖,加入Ts细胞后,可以明显抑制DC2.4细胞刺激的BALB/c小鼠脾CD4+T淋巴细胞(H-2d)的增殖,而并不抑制KM鼠DCs细胞刺激的CD4+T脾淋巴细胞(H-2k)的增殖反应。
结论体外诱导的Ts呈现Ⅰ类主要组织相容性抗原特异性抑制异基因反应性淋巴细胞的增殖。其机制与Ts细胞上调PIR-B mRNA、下调PIR-A mRNA的表达有关。升高供体移植物中抗原特异性Ts细胞比例或受体树突状细胞表面免疫抑制性受体的表达可能成为诱导受者产生针对供体抗原特异性免疫耐受的有效途径。
Graft-versus-host diseases(GVHD) still are one of the obstacles for clinical bone marrow transplantation in treating malignant hematologic diseases. The activation of allogenetic lymphocytes promote the occurrence and development of GVHD. So induction immune tolerance specific for host major histocompatibility antigens is the optimization for prevention GVHD and preservation the graft-versus-leukemia(GVL). Recently, Paired immunoglobin-like receptor (PIR) , including PIR-A and PIR-B, are found to be inhibiting receptor expressed on dendritic cells with typeⅠmajor histocompatibility antigens as its ligand. PIR-A and B and its ligand constituted the system for deciding the dendritic cells active. For the dendritic cells can not only induct the immunue cells active but also induct the immune tolerant by inducing the regulatory T cells by T cells link, the study of PIR-B on dendritic cells may provide new way for inducing specific immune tolence for host antigen with the molecular PIR-B.
PartⅠStudy of the relationship between paired immunoglobin-like receptors expression and immune tolerant on dendritic cells in mouse
Objective Paired immunoglobulin-like receptor A,B are numbers of immunoglobin superfamily. The study of the relationship of paired immunoglobin-like receptor expression and co-stimlation molecules on tolerant dendritic cells may provide utility way for inducting immune tolerant.
Methods The DC2.4 cells derived from the C57BL/6 mouse were inducted by re recombinant mouse interleukin-10(rmIL-10) and recombinant human transforming growth factorβ1(rhTGF -β1) and serve as tolerant DC(T-DC). Lipopolysaccharide(LPS) stimulated the dendritic cells mature for 48 hours as mature DC(LPS-DC). The small interfering RNA (siRNA) was chemical synthesis in vitro and transduced into the DC2.4 cells by Lip 2000(Si-DC). RT-PCR, Flow cytometry and Western blot were used to examine the PIR-A/B expression on T-DCs, LPS-DCs and Si-DCs. Syb green 1 realtime-PCR were used to examined transcription expression of co-stimulation molecule such as CD80,CD86, MHC-Ⅱand PIR-A. 2-△△Ct was used for evaluating the relative quantity of gene cDNA transcrition. Mixing lymphocytes reaction(MLR) was used to examined the allogenetic reactive lymphocytes proliferation for the DC2.4 cells as stimulating cells and the BALB/c spleen lymphocytes as the reactive cells. Enzyme linked immunosorbent assay(ELISA) was used detect the interference-γlevels in the supernatant in the MLR.
Results FCM examination demonstrated that positive reate of PIR, which is the common extramembrane district of PIR-A and B, were (28.65±8.12)% on DC2.4 cells, and the rate added up to (54.21±6.34)%,(58.78±4.70)%,(48.24±6.75)% respectively for IL-10, TGF-β1 and LPS. But there was no significant between the IL-10, TGF-β1 or LPS-DC(P>0.05). RT-PCR and Western blot demonstrated that the mRNA and protein level of PIR-B increased and the level of PIR-A were decreased by IL-10 and TGF-β1 inducton. On the contrary, the mRNA and its protein level for PIR- A mRNA and pritein expression increased and that for PIR- B were decreased by the LPS stimulation. The positive rate of RNAi transfection was 93.12% by FCM detection. SYBR greenⅠRealtime-PCR results showed that the CD80, CD86, MHC-Ⅱa nd PIR-A were over-expressed on Si-DCs other than the mormal DCs by the same stimulation of LPS. The 2-△△Ct CD80, CD86, MHC-Ⅱand PIR-A in LPS-DCs were respectively 5.02±1.09, 4.69±1.75, 5.46±1.79, 6.02±2.13 and the 2-△△Ct were 8.79±2.2, 11.03±1.96, 10.26±2.55, 12.10±2.83 in Si-DC with the same LPS stimulation. CD80, CD86, MHC-Ⅱa nd PIR-A transcription were increased by 3.72、6.34、4.8、6.08 times in Si-DCs than the normal DCs(P<0.05). MLR were suppressed in T-DCs for IL-10 and TGF-β1 and IFN-γlevel were decreased in MLR supernatant(P<0.05). MLR were intensive in LPS-DCs and Si-DCs groups and the IFN-γlevel were increased(P<0.05).
Conclusions Up-regulating PIR-B or down-regualuting PIR-A expression are the common characteristics and molecules mechanism. Silencing the PIR-B expression promote the PIR-A, CD80, CD86 and MHC-Ⅱoverexpression and that makes the DCs immune active. PIR-A and B constitute molecule targets for inducting dendritic cells immune tolerance in mouse.
PartⅡDendritic cells with higher expressed paied immunoglobin- like receptors induce the CD4+CD25+T cells development
Objective To investigate the relationship between the paired immunoglobin receptor B(PIR-B) and the CD4+CD25+ regulatory T cells(Treg) development, suggest the tolerant dendritic cells detail mechanisms and provide the base for induction Treg development by the tolerant DCs.
Methods CD4+T and CD4+CD25+T cells were sorted by immune magcellect from the BALB/c spllen cells. rmIL-10(50ng/ml) and rhTGF-β1(50ng/ml) were combinated to induced the DC2.4 cells tolerant(T-DCs) for three days. The small interfering RNA (siRNA) transfected into the DC2.4 cells by Lip2000 to made the silencing PIR-B DCs(Si-DC). Mature DCs( mDC) were stimulated by the LPS for 48h. The nomal DC2.4 cells(DCs), T-DCs, siRNA and mDC were mixed and culcured with the CD4+T cells for five days for inducing the Treg cells. RT-PCR were used for detecting the the transcript factor Foxp3 mRNA expression. FCM examined the CD4+CD25+T cells percentage in mixed culture system. PI were detect the apoptosis of CD4+T cells after cultured with the different group DCs. Different percentage of CD4+CD25+T cells were co-cultured with the syngennetic cells proliferation with the allogenetic dendritic cells as stimulating cells. 3H test were used to detect the proliferation ability in MLR.
Results CD4+T and CD4+CD25+T cells of BALB/c mouse were sorted by immune magnetic beads and the purity of CD4+T and CD4+CD25+T cells were aboved 95%. Different groups of DCs such as normal DCs, T-DCs, Si-DCs and mDCs derived from C57BL/6(H-2b) were co-cultured with the CD4+T BALB/c cells for three days to developing the Treg cells. RT-PCR detection showed that the the Foxp3 mRNA expression were higher in T-DCs than the othe groups(P>0.01). Si-DCs and the LPS-DC with lower PIR-B expression induced the lower Foxp3 mRNA level(P>0.01). FCM demonstrated that CD4+CD25+T cells percentage were (5.19±1.2)%, (28.29±2.36)%, (1.06±0.55)%, (2.01±0.66) % respectively for DCs, T-DCs, Si-DC and mDCs groups. It was demonstrated that T-DC also induced a higher rate CD4+CD25+T cells from the CD4+T cells than the other groups(P<0.05). PI detection demonostrated that apoptosis rate of CD4+T cells were (8.3±0.7)%, (21.56±2.32)%, (2.5±0.8)% and (1.9±0.7)%, respectively for DCs, T-DCs, mDCs and Si-DC. 3H incoupration test showed that CD4+CD25+T cells mixed with CD4+T cells at different percentage and inhibited CD4+T cells proliferation after stimulated by the allogenetic cells.the inhibition was paralleveling to the quantity of CD4+CD25+T cells.
Conclusion Induction CD4+CD25+T cells development and apoptosis of allogenegtic lymphocyte cells may be associated with the tolerant DCs with higher expression inhibited receptor PIR-B. This may provide base for transplamtation immune tolerenat and new pathway for Treg induction.
PartⅢCD8+CD28-T cells changes PIR-A/B expression and promotes the allogentic immune tolerance
Objective Specific suppression of the host’s immune response to donor human leucocyte antigen(HLA) antigens remains the ultimate goal for clinical transplantation. CD8 + CD28-T(T suppression cell, Ts)is a subset of regulatory T cells which play regulatory role in immune reaction. Allogenetic antigen specific Ts were induced and to study the influnce for the expression of paired immunoglubin-like receptor A and B on the dendritic cells in mouse. The study aim to explore the molecular mechanism, immune tolerance characteristic of Ts and to provide a basis for the development of specific immunosuppressive therapy.
Methods Ts was induced in vitro for specific MHC-Ⅰ(H-2b) antigen in vitro. DC2.4 cells derived from the C57BL/6 mouse(H-2b) was used to prime allogenetic lymphocyte spleen cells of the BALB/c mouse in mixed lymphocyte cultures repetedly for twice. Every culture prolonged for seven days. IL-2(10u/ml) was added to the culture at day 10 and finished the culture at day 14. Biotin-CD28 and Biotin-CD8 antigen and streptavidin magnetic bead were used to sorting the CD8+CD28-T cells. CD8+T cells were positive sorted firstly and then CD28-T cells were negatively sorting. CD8+CD28-T cells co-cultured wth the DC2.4 cells for 48h. RT-PCR were used to determin the PIR-A/B mRNA level and Westernblot was use to detect the PIR-A/B protein leve. 3H-incorporation test was used to detect the allogenetic cells proliferation. The myeloid DCs from KM mouse were cultured in vitro for a MHC-Ⅰnon-asscociated antigen doner. Both DC2.4 cells and the DCs of KM mouse served as stimulating cells. The CD4+T cells of BALB/c spleen cells served as reacting cells. Ts cells were added into the mixing lymphocyte reaction. CPM was used for determing the proliferation.
Results CD8+CD28-Ts were inducted with the allogenetic DC2.4 cells derived from C57BL/6 mouse and sorted by magnetic bead in vitro. Purity of CD8+ and CD8+CD28-Ts cells was above >90%. After cultured mixingly Ts cells and DC2.4 cells for 48h, PIR-B mRNA and protein level increased and the PIR-A mRNA and protein level decreased examined by RT-PCR and Westernblot. 3H-incorporation test demonstrated that the DC2.4 cells and DCs from KM mouse stimulated the allogenetic CD4+T cells of BALB/c proliferation. Ts inhibited CD4+T cells(H-2d) proliferation stimulated by the DC2.4 cells but did not inhibited CD4+T cells(H-2d) proliferation stimulated by DCs from KM mouse.
Conclusion Ts induced in vitro specifically inhibited the allogenetic areactive cells proliferation with MHC-Ⅰconstraction. This may be associated with up-regulation of the PIR-B expression and down-regulating of PIR-A expression. Increasing the Ts cells ratio in donator's grafts or the immune inhibiting receptor on receptor’s DCs may provide utility pathway for a specific antigen tolerance for donator in transplantation.
引文
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