The Research Of The Immune Modulation Effect And Mechanism Of Bone Marrow Mesenchymal Stem Cells On CD8+T Lymphocytes

Stem cells (SC) have unlimited proliferation and multi-directionaldifferentiation potential. They can be divided into embryonic stem cells (ESC),induced pluripotent stem cells (IPSC）and adult stem cells (ASC) according tothe differentiation ability. ASC include hematopoietic stem cells（HSCs）andmesenchymal stem cells (MSCs). MSCs can be derived from a variety of tissuesor organs include bone marrow, fat, placenta, umbilical cord and dental pulp.Bone marrow mesenchymal stem cells (BMSCs) have been studied deeply andapplied widly due to its relatively abundant sources and matured isolation andculture methods.MSCs have four major functions: supporting the hematopoieticreconstruction of HSCs, promoting the tissue repair and regeneration, targetingtherapy of tumor and immune modulation. Immune modulation is a hotspot ofMSCs research. MSCs cannot activate the reaction of allogeneic lymphocytesbecause of their low immunogenicity. In addition, MSCs can inhibit theproliferation, activation and effect of a variety of immune cells include T cells,natural killer cells (NK), dendritic cells (DC) and B lymphocytes. Therefore,MSCs have important application value in the prevention and treatment ofimmune disease. BMSCs are widely used in the prevention or treatment of graftversus host disease (GVHD). However, the basic research and clinical trialsabout MSCs are far from enough and many problems have not been answered.The problems existed are: The mechanism of the treatment of immune diseaseby MSCs, especially the effect and mechanism of MSCs on all kinds of effector cells involved in the immune disease; How to ensure the effect of the clinicalapplication of MSCs; The safety of the clinical application of MSCs.CD8+T lymphocytes and activated cytotoxic T lymphocytes (CTL) are themain effector lymphocytes in GVHD. The mechanism of the treatment ofGVHD by MSCs is particularly important because MSCs were mostly used inthe prevention and treatment of GVHD. The immune modulation andmechanism of MSCs on CD8+T lymphocytes which is the main effector cells inGVHD is important. Nowadays, the reports about MSCs-CD8+T lymphocytesinteraction is less, and the mechanism research about MSCs-mediatedmodulation on CD8+T lymphocytes is lacking. In order to further clarify theimmune rmodulation and mechanism of MSCs on CD8+T lymphocytes, wefinished this study with three parts as follows.Part1: Isolation, culture and identification of human bone marrowmesenchymal stem cellsObject: To isolate, culture, amplify and identify BMSCs in vitro, andprovide sufficient cell source for subsequent experiments.Method: First, we separate the bone marrow mononuclear cells throughficoll density gradient centrifugation, cultured and amplified the cells useing thebone marrow mesenchymal stem cells cultured medium (BMSC-GM). Then, weget the cell growth curve through quantitative inoculation and counting. Inaddition, we detect the BMSCs surface molecular markers through flowcytometry detection. Moreover, we investigate the multi-directionaldifferentiation of BMSCs by cultured BMSCs with osteoblast and adipocyteinduced medium. Finally, we identify the cells by the combining use of cellmorphology, growth characteristics, surface molecular markers and multi- directional differentiation ability.Result: The BMSCs cultured in vitro were fibrous cells and have spiralgrowth performance. The growth curve of BMSCs was "S" type which hastypical incubation period, growth period and platform period. The surfacemolecular markers of BMSCs were positive with CD29, CD105and CD166,negative with CD34and CD45. BMSCs can be differentiated into osteoblast andadipocyte by inducing cultured in vitro. The combining use of cell morphology,growth characteristics, surface molecular markers and multi-directionaldifferentiation ability can determine the cells as MSCs. The cells of P3-P6generation were suitable for subsequent experiments because of their relativelystable characteristics.Conclution: We can obtain plenty of BMSCs through isolation, culture,amplification and identification in vitro. The cells of P3-P6generation weresuitable for subsequent experimentsPart2: Research of the immune modulation of BMSCs on CD8+TlymphocytesObject: To study the immune modulation of BMSCs on CD8+Tlymphocytes and preliminary discuss the effective concentration range ofBMSCs in modulating CD8+T cells.Method: First, we purified the peripheral blood CD8+T lymphocytes byimmune magnetic beads technology and negative selection method, and detectthe proportion of CD3+CD8+T lymphocytes by flow cytometry detection. Then,we observe the ability of BMSCs to activated CD8+T lymphocytes byco-cultured BMSCs with CD8+T lymphocytes and then detected the CD8+Tlymphocytes proliferation by carboxyfluorescein diacetate succinimidyl ester (CFSE) method. In addition, we established the co-cultred system of BMSCsand CD8+T lymphocytes, use the phytohaemagglutinin A (PHA) as polyclonalT lymphocytes stimulator, and detected the proliferation of CD8+Tlymphocytes. Finally, we detected the mRNA expression of interleukin2(IL-2),interferon-γ (IFN-γ) and particle enzyme B (GZMB) of CD8+T lymphocytes inthe co-cultured system by real time-polymerase chain reaction(real time-PCR)method.Result: The propotion of CD3+CD8+T lymphocytes in the cells obtainedfrom magnetic bead separation was88.1%. When CD8/BMSCs ratio is1:1-100:1, the CD8+T lymphocytes proliferation rate has no obvious rise afterco-cultured with BMSCs. BMSCs could inhibit the CD8+T lymphocytesproliferation in a dose-dependent form when the CD8/BMSCs ratio is1:1-20:1and the effect is more obvious when the concentration of BMSCs is higher.When CD8/BMSCs ratio is50:1-100:1, the CD8+T lymphocytes proliferationwas not inhibited by BMSCs. BMSCs can inhibit the IL-2, IFN-γ and GZMBexpression in CD8+T lymphocytes.Conclution: BMSCs lack the stimulatory capacity toward CD8+Tlymphocytes because of their low immunogenicity. BMSCs can suppress theCD8+T lymphocty activation in a certain concentration range.Part3: Research of the mechanism of BMSCs-mediated immunemodulation on CD8+T lymphocytesObject: To clarify the BMSCs-mediated immune modulation mechanismand provide a massy foundation for clinical application.Method: First, we detect the CD8+T lymphocytes proliferation in atranswell system which can avoid the direct contact between BMSCs and CD8+ T lymphocytes. Then we detect the NKG2D expression on CD8+T lymphocytesand the MIC A/B expression on BMSCs by flow cytometry detection. Inadditiom, we detect the NKG2D expression on CD8+T lymphocytes in theco-cultured system seted up by CD8+T lymphocytes and BMSCs with differentCD8/BMSCs ratios. Moreover, we blocked the MIC A/B molecule on BMSCswith MIC A/B monoclonal antibody before co-cultured the BMSCs with CD8+T lymphocytes and then detect the proliferation and the IL-2, IFN-γ and GZMBexpression of CD8+T lymphocytes. We also detected the mRNA expression ofIDO and HGF in BMSCs which was co-cultured with CD8+T lymphocytes byreal time-PCR method and detected the PGE2and TGF-β1content in thesupernatant of co-cultured system. Finally, we detect the inhibition of BMSCson CD8+T lymphocytes proliferation after blocked the soluble factors PGE2,IDO, TGF-β and HGF respectively and simultaneously.Result: The CD8+T lymphocytes proliferation was higher but did notcompletely recovered in the transwell system when compared with the directcontact system. CD8+T lymphocytes express NKG2D receptor and BMSCsexpress MIC A/B ligand. BMSCs can suppress the NKG2D expression on CD8+T lymphocytes in a dose-dependent from when the CD8/BMSCs ratio is1:1-20:1and the effect is more obvious when the concentration of BMSCs ishigher. When the CD8/BMSCs ratio is50:1-100:1, BMSCs can not suppress theNKG2D expression. The proliferation and cytokine expression of CD8+Tlymphocytes were increased when co-cultured CD8+T lymphocytes withBMSCs which was blocked the MIC A/B molecule. The IDO expression wasobviously increased and the HGF expression was not changed in BMSCs whenco-cultured with CD8+T lymphocytes. The content of PGE2and TGF-β1wasincreased in the supernatant of co-cultured system. The proliferation of CD8+T lymphocytes was increased when blocked the PGE2, IDO and TGF-βrespectively, while was not changed when blocked the HGF alone. Whenblocked the PGE2, IDO and TGF-β simultaneously, the proliferation wasobviously increased but significantly lower then the mean expected valuecalculated for an additive model.Conclution: The immune inhibition of BMSCs on CD8+T lymphocyteswas mediated by cell-cell contact. Soluble factors were involved in a certaindegree. CD8+T lymphocytes expree NKG2D receptor and BMSCs expressedMIC A/B ligand. BMSCs can down-modulate the NKG2D expression on CD8+T lymphocytes in a dose-dependent from. The MIC A/B of BMSCs wasinvolved in the immune modulation of BMSCs on CD8+T lymphocytes. Theinteraction of NKG2D and MIC A/B was one of the mechanisms ofBMSCs-mediated immune modulation on CD8+T lymphocytes. The solublefactors PGE2, IDO and TGF-β were involved in the immune modulation ofBMSCs on CD8+T lymphocytes, but they did not have synergistic effect.In conclution, our study reveals the immune modulation and mechanism ofBMSCs on CD8+T lymphocytes. First, we proved that the NKG2D receptor onCD8+T lymphocytes and the MIC A/B ligand on BMSCs were involved in theimmune modulation of BMSCs on CD8+T lymphocytes. We indicate that theBMSCs suppress the NKG2D expression on CD8+T lymphocytes through theMIC A/B and so they can inhibit the proliferation and founction of CD8+Tlymphocytes. The interaction of NKG2D and MIC A/B which mediated thecell-cell contacte of BMSCs and CD8+T lymphocytes was one of the importantmechanisms of BMSCs-mediate immune modulation on CD8+T lymphocytes.Second, we proved that the PGE2, IDO and TGF-β were involved in the immune modulation of BMSCs on CD8+T lymphocytes. We indicate that thePGE2, IDO and TGF-β were one of the mechanisms of BMSCs-mediateimmune modulation on CD8+T lymphocytes, but they did not have synergisticeffect. These results further enriched the molecular mechanism research of theclinical application with BMSCs in immune disease. In addition, these resultsprovide a massy foundation for the clinical application of BMSCs. Furthermore,these results provide a new thinking and enlightenment to improve the clinicalapplication effect of BMSCs in autoimmune disease.