Study Of The Relationship Of Paired Immunoglubin-like Receptors And Its T Cells Link In Induction Dendritic Cells Tolerant In Mouse

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 mouseObjective 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 developmentObjective 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 toleranceObjective 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.