Regulation of B cells by E2/MSC and IL-12immunothempy of SCC
|Keywords||B-cells estrogen TLR9 MSC IL-12|
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies against nucleic acid-associated antigens especially anti-double strand DNA (dsDNA). B cells play a cardinal role in SLE as they can produce antibodies, inflammatory cytokines and present antigens. In SLE, women are more likely to be affected than men. The disease status worsens during pregnancy and menstrual cycle, when circulating level of estrogen changed. It is reported that oral contraceptives may increase the rick of flare. Many evidences have proved that estrogen contribute to the gender bias in SLE.17β-estradiol (E2) is the most common and important form of estrogen. B cells have estrogen receptors. It is showed that E2can alter the apoptosis and activation of B cells, and suggested E2accelerate the disease by regulating molecular signaling in B cells. However, the mechanism is still not clear. B cells can express Toll like receptor9(TLR9), which can recognize dsDNA and mediate innate and adaptive immunity. It is demonstrated the generation of anti-dsDNA is specifically inhibited in the TLR9deficient mice. Therefore, it played a critical role in the pathogenesis of SLE. However, the crosstalk between estrogen and TLR9in B cells remains unknown and need to be understood.So we investigated E2’s effects in the presence of the TLR9ligand CpG-ODN on spleen B cells. In vitro studies, we isolated and purified the mice naive B cells from mice spleen, using a negative selection immuno-magnetic beads. The we investigated the E2and CpG stimulations on naive B cells. We first found the morphology of B cells changed obviously after24h of stimulations. Then we use the CCK-8kit to test the cell viability, trypan blue staining for cell counting, CFSE labeled the proliferation, Annexin-PI double staining to determine the apoptosis, flow cytometry to analyze expression of co-stimulation molecules and cytokines, ELISA to detect the levels of IgM. And we found that the up-regulation of cell growth (viability, survival), expression of co-stimulation molecules (CD40, CD86) and IgM secretion were more significant in the group stimulated with both E2and CpG compared to CpG alone, while E2alone could not arise the changes.Furthermore, in order to investigate E2effects on spleen B cells in vivo, we established the mouse model:ovariectomized mice or made the sham operation two weeks before a two-week continuous s.c. injection of E2/CpG to female Balb/c mice. Then we determined the serums and lymphocytes from mice spleen. We found the proportion of B cells in spleen lymphocytes were not changed among each group, thus analyzed the B cells by gating CD19+/B220+cells. Consistent with in vitro studies, we proved the synergies of E2and CpG on the promotions of CD40and IgM secreting in the in vivo studies. In addition, E2was showed to play a more important role on these activation of B cells, which indicated that E2’s regulation to B cells was more complicated in vivo. The expressions of cytokines IL-6, IL-10, IL-12on spleen B cells were also enhanced by E2.In order to understand the mechanism of how TLR9signal modulated by E2, we also determined whether E2could effect the expression of TLR9directly. The results showed that E2up-regulated the expression of TLR9on B cells both in vivo and in vitro. For the purpose of founding new modulated molecules, we did a gene microarray assay. And we found a molecular MCM6regulated by E2. Then we verified the involvement of MCM6in the modulation of E2on the TLR9signal in in vivo experiment, as the expressions of MCM6were up-regulated by both E2and CpG stimulations. In summary, this study suggested that E2could enhance the activation of B cells though TLR9. And this study might provide a new way to investigate the mechanism of how E2modulate the B cells functions in SLE.In addition to E2, Mesenchymal stem cells (MSCs) were considered to regulate the activation of B cells in SLE. MSCs are arising as a potent therapeutic tool in autoimmune disease and cancer due to their regenerative, low immunogenicity and immunomodulatory properties. And MSCs derived from Umbilical cord (UC-MSCs) has been proved to be effective in the treatment of SLE both on mice disease model and clinical trial. Although many studies have proved the immusuppression role of MSC on T cells and other immune cells, including DC and NK cells, the studies on the effects of MSC on B cells were scarce and contradictory.We used three human non-Hodgkin’s lymphoma B cell line Daudi, Namalwa and Raji as the research models. All these cell lines express TLR9and IgM. We first investigate UC-MSCs modulations on the cell cycle, apoptosis, proliferation, co-stimulation molecules and surface IgM (slgM) expression, signal pathways of B cells in the presence or absence of CpG. We found MSC arrested cell cycles of Namalwa and Raji cells, but no influence of apoptosis was observed. The results showed that MSCs inhibited the expression of CD86(all three cell lines), CD40(Raji) and sIgM (Daudi and Namalwa) on B cell lines.Next, we determined whether the suppression of CD86and slgM on Daudi was cell-contact dependent. According to the transwell experiments, we found MSCs modulated B cells via soluble factors. Many soluble factors have been suggested secreted by MSCs, Cox2is one of the most important. We examined the involvement of Cox2by adding a COx inhibitor Indomethacin into the medium of co-culture system, and the results the suppression effect of CD86and IgM was Cox2-independent. As it is suggested MSC:lymphocyte ratio may be an important point to have different results, we used two different ratio1:1and1:10to confirm the above effect of MSCs to B cells. To further understand how MSCs modulate the B cell lines though soluble factors, we tested some immunomodulators might produced by B cells by real-time PCR. The results showed that MSC up-regulated the expression of COX2, CCL2, IL-6, IL-8, CXCL1sustained but not proinflammatory TNF-a in Daudi. It is thought MSCs need to be activated to exert their immunomodulation abilities by factors like IFN-γ、IL-1β, and IL-1β is also involved in the pathogenesis of SLE, we used IL-1β to pretreated MSCs for24h, and found the mRNA levels of CCL2, CCL5, CXCL1, Cox2, IL-6and IL-8, but not TGF-β、SDF-1、IL-10were increased by the treatment. The further results showed the up-regulations of immunomodulators were improved by the treatment of IL-1β to MSCs, in addition to inducing anti-inflammatory IL-10by IL-1β treated MSC.Above results suggested MSCs impacted B cell lines though soluble factor, but how did this work? What cell signals in B cells involved in the modulation? Therefore, we investigated the PI3K, STAT, MAPK pathways which may involved in the regulation of excellular soluble factors by western blots. The cell signal results showed a sustained phosphorylation of ERK in all three cell lines by MSCs with or without the stimulation of CpG. Using the ERK inhibitor U0126, we verified the activation of ERK was involved the suppression of sIgM and CD86, and the up-regulation of CCL2, as the effects by MSCs were attenuate when treated Daudi with U0126.Combined all the results, UC-MSCs played a suppression role on human B cell lines, including inhibition of the cell cycle, expression of co-stimulation molecules and immunoglobulin, whereas promoting the immunomodulatory factors. This study provided a model for further investigation on the mechanism of MSCs to primary B cells.Interleukin-12is the key cytokine in the initiation of a Thl response and has shown promise as an anti-cancer agent. However, clinical trials involving IL-12have been unsuccessful due to toxic side-effects. To address this issue, lentiviral vectors were used to transduce tumor cell lines that were injected as an autologous tumor cell vaccine. The focus of the current study was to test the efficacy of this approach in a solid tumor model.A murine squamous cell carcinoma cell line SCCVII cells were transduced to produce IL-12, and clones that secreting IL-12at different concentrations were then isolated. Subcutaneous (s.c.) injection of parental SCCVII cells resulted in tumor development, while the IL-12producing cells were not. It suggested IL-12secreting SCC could be recognized and removed by immune system.Then we considered if T cells participated the recognition and remove of tumor cells. And what types of T cells involved in the immune responses to SCC? Thus, we injected depletion antibodies of CD4and CD8respectively or together at day-14,-1,3,7,14,21to deplete the T cells from mice. The results showed tumor grown out in the mice depleted T cells, which indicated T cells were crucial for the therapy, both CD4+and CD8+T cells were necessary for the remove of SCC.Furthermore, we investigated whether immunizing mice with IL-12producing cells could lead to a long term immunity. Therefore, we immuned mice with IL-12SCC via both s.c. and i.p. injections, then s.c. injected SCCVII on the same or opposite side after fifty days and seventy days respectively. The result showed a100%depletion of tumor cells in IL-12SCC injected mice, which meant the establishment of both local and systemic immunity against challenges with SCCVII.Our previously works suggested that the a high local concentration of IL-12but not high amount of entire IL-12delivered is more important for the elimination of tumors, and caused less toxic side-effects. In this study, we found mixture of IL-12producing (IL-12SCC) and non-producing cells (SCCVII) results in tumor clearance. Interestingly, when comparing mice injected with a mixture of SCCVII and either high IL-12producing tumor cells or low IL-12producing tumor cells, we observed that mixtures containing small amounts of high producing cells leads to tumor clearance whereas mixtures containing large amounts of low producing cells fails to elicit protection; despite the production of equal amounts of total IL-12in both mixtures. Above all, this study results showed that the delivery of IL-12by cancer cells can be an effective route for immune activation.