High Expression Of PD-L1on B Cells In NOD Mice And The Research Of It’s Inhibition Effect On Autoreactive T Cells

BackgroundType1diabetes（T1D）is one of the most important autoimmune diseases whichthreatens human’s health. As the mainly onset is among young people, the influence ofT1D is very serious. The disease occurs as a resμlt of the organ-specific immunedestruction of the insμlin-producing beta cells in the islets causing diabetes. So in theory,if we can suppress the activation of antigen-specific autoreactive T cells, sustain immunetolerance to islet beta cell antigens, which maybe an ideal therapy to cure the disease.The NOD (non-obese diabetic) mouse is an excellent animal model of autoimmunedisease and an important tool for dissecting tolerance mechanisms. And the biggestadvantage is that it develops spontaneous autoimmune diabetes, which shares manysimilarities to T1D in human. So far, there are many resμlts in the understanding type Idiabetes based on research in NOD mice. Recent studies have shown that PD-1(programmed death-1) molecμle is an important receptor to maintain immune tolerance, lossing function of PD-1molecμles will directly break the immune tolerance to selfantigens, leading to the occurrence of autoimmune diseases. Research from Ansari showthat PD-1/PD-L1(programmed death ligand1) signal pathway plays an important roleduring the pathogenesis of type I diabetes. The PD-1/PD-L1pathway is not only involvedin the onset of immune tolerance but also in the maintenance of immune tolerance. It hasbeen reported that B cell serves as the mainly APC (antigen presenting cell) when theNOD mouse having autoimmune diabetes, activating auto-reactive CD4+T cells. But thereis a fact that there is no expression of PD-L1on the surface of B lymphocytes infiltratedinto the NOD mouse pancreatic island. Thus in this project we plan to establish a PD-L1transgenic NOD mouse model to provide a stable PD-L1/PD-1inhibitory pathway,expecting the delaying or reduction of the diabetes incidence, providing new strategies fortreating human type1diabetes.Objective1. By establishing a PD-L1transgenic NOD mouse model and then promoting theexpression of PD-L1on the surface of B cells to establish a stable PD-1/PD-L1pathway;2. Inhibiting autoreactive T cells especially CD4+T cells in NOD mice through theinteraction between B cells and T cells, in order to prevent or delay diabetes in NODmice.Methods1. Cloning PDL1cDNA by using genetic methods, and then the PDL1fragment wasinserted into the eukaryotic expression vector pCAGGS to construct a transgenicvector pCAG-PDL1, after that, the vector was linearized and microinjected intozygotes to get a transgenic mouse TgPDL1-B6; and then we establish aTgPDL1-NOD (PDL1transgenic mice in the NOD genetic background) mouse modelby mating strategy and detect the expression of PD-L1. 2. The biological characteristics of B cells in transgenic mice were compared withwild-type mice by flow cytometry: the expression of some surface molecμles of Bcells such as IgM、MHCⅡ、CD80and CD86were identified. Then we compared theratio of CD4+T cells and CD8+T cells in spleen，thymus and pancreatic lymph nodebetween transgenic mice and wild-type mice. And insμlitis of transgenic mice andwild-type mice were analyzed and compared through histological observation.3. We observed the effect of of BPDL1cells and wild-type B cells on CD4+T cells in NODmice in vitro; then an adoptive transfer experiment is ongoing：first，weseparately isolated BPDL1from TgPDL1-NOD mice and B cells from wild-type NODmice mixed with T cells from diabetic NOD Mice and then ip injection to NOD-scidmice (severe combined immunodeficiency animal model called NOD/LtSzPrkdcscid/Jwith T, B, NK cell defection).Next,we observe the incidence of diabetic NOD.SCIDmice, in order to clear the role of B cells.Results1. Through molecμlar cloning techniques, we successfμlly cloned the PDL1cDNA andconstructed a pCAG-PDL1transgenic vector; and we got3TgPDL1-B6foundertransgenic mice by pronuclear microinjection, the founder mice were mated withwild-type mice to maintain the transgenic lines; finally we successfμlly establishedthe NOD genetic background of PDL1transgenic mice—TgPDL1-NOD mice bymating strategy.2. Compared with the wild-type NOD mice, the surface of TgPDL1-NOD mouse B cellscan highly express PD-L1, this high expression a didn’t affect the expression ofsurface molecμles on B cells, the biological characteristics of B cells from transgenicmice were not affected; in addition, flow cytometry resμlts showed that the highexpression of PD-L1didn’t affect the ratio of CD4+T cells, CD8+T cells in spleen,thymus, pancreatic lymph node lymphocytes.3. We analysed the Insμlitis among8weeks,12weeks,16weeks,20weeks,28weeks inTgPDL1-NOD mice and NOD mice by histologically staining, we found that as compared to wild type mice, the islet damage degree and the severity of insμlitissignificantly weakened in transgenic mice at different ages and different sex.4. In mixed lymphocyte cμlture，we detect and compare the level of IL-2, IFN-γ in thesupernatant secreted by CD4+T cells，the resμlt revealed that BPDL1cells can inhibitthe proliferation of CD4+T cells, and the CFSE labeled T lymphocytes proliferationassay also showed the inhibition effect.Conclusions1. Through molecμlar cloning techniques, we successfμlly establish TgPDL1-B6transgenic mice by pronuclear microinjection. PD-L1is highly expressed in manytissues, including T、B lymphocytes. PD-L1gene is inherited through hemizygosity.2. Then we successfμlly established the NOD genetic background of PDL1transgenicmice—TgPDL1-NOD mice by mating strategy. And the high expression of PD-L1didn’t affect the expression of surface molecμles on B cells and the ability to secretantibody. Besides, the islet damage degree and the severity of insμlitis significantlyweakened in transgenic mice.3. Further research showed that the resμlt revealed that BPDL1cells can inhibit theproliferation of CD4+T cells and the secretion of cytokines.4. NOD mice are also prone to developing other autoimmune syndromes. PD-L1/PD-1isan important inhibitory pathway to sustain peripheral immune tolerance, regμlates theoccurrence and development of mμltiple autoimmune diseases in mammalian animals.Thus, the transgenic mouse model we founder can also be used in other autoimmunediseases.