Enhanced Anti-tumor Immune Effect And Mechanisms Of Tlr2 Agonist Blp

TLR is a member of pattern recognition receptor family of innate immunity molecules. Recent studies have indicated that TLRs not only expressed on the innate immune cell surface, but also on the adaptive immune cells, such as T-cell subsets. The surface TLRs on T cells can regulate the function of T cells by binding their ligands. Just like in Leishmania major infection induced autoimmune colitis model, TLR2 agonist BLP enhanced CD4+CD25-Teff cell proliferation and cytokine secretion, down regulated CD4+CD25+Treg cell proliferation suppression function, thereby increased the auto-immune disease condition. TLR2 agonists on autoimmune disease model inspired us that the regulating role of TLR2 agonist can regulate tumor model immune status.Based on the above hypothesis, we first detected the expression of TLR2 on T lymphocyte surface in 3LL tumor-bearing mice. In the premise of TLR2 expression on T cells, we treated tumor bearing mice with BLP. BLP stimulate immune cells, specialy the T cells, through its ligand-receptor binding and its antitumor effect were observed. Further, through the analysis of T cell subsets in immune pattern and its immune function, we explore the immune mechanism of BLP enhanced anti-tumor immunol response and provided theoretical and experimental basis for BLP as an immunoenhancer used in tumor biological therapy.Part I The anti-tumor effect of TLR2 agonist BLP3LL tumor cell line is a murine non-small cell lung cancer tumor cell line. In order to verify whether the TLR2 agonist BLP has the characteristic of tumor growth inhibition, we subcutaneously inoculated 3LL tumor cells in right groin of C57BL/6 mice. After tumor inoculation, the experimental group intraperitoneally injected 50μg BLP every five days, injected a total of 5 times. The control group was injected PBS. The size of tumor growth and mice survival rate was measured every 2-3 days during the period. Mice were sacrificed, when the tumor area extended more than 144mm2. The results showed that BLP treated mice could be completely inhibited tumor growth in vivo, and mice survival rate was 100%. In the control group, tumor kept on growing until mice were put to death. Tumor growth curves and survival rate are significantly different.In order to understand the anti-tumor effect of BLP by acting on tumor cells directly, or through the host's immune system play a role, firstly, we detected whether TLR2 expressed on 3LL cell surface by FACS. FACS result confirmed the expression of TLR2 on 3LL tumor cells. Then, we detected whether BLP could directly inhibit tumor cell growth in vitro. Various concentrations of BLP (0,0.5,1,2,4μg/ml) were incubated with 3LL cells for 48 hours. After stimulation, tumor cell proliferation and apoptosis did not change significantly. BLP treatment either did not affect the ability of 3LL tumor cell secreting TNF-a and IL-6.Further, we subcutaneously inoculated 3LL tumor cells in right groin of T cell immune deficiency nude mice and intratumorally injected BLP every 5 days. Tumor growth was observed each 2-3 days in order to discover weather BLP could influence 3LL tumor in vivo growth. The results showed that BLP had no direct effect on 3LL tumor growth in nude mice. These results indicate that BLP induced anti-tumor effect did not generated by directly affecting tumor cells biological characters, but may be act on the host's immune system.PartⅡTLR2 agonist BLP induced enhancing specific anti-tumor immune responseIn vitro and in vivo experiments of the first part confirmed that the TLR2 agonist BLP does not change 3LL tumor cell biology characters. TLR2 agonist BLP may be ruled out a direct role in tumor cells. In this section, we focused on BLP impaction on the immune system. Firstly, we dealt spleen lymphocytes with BLP in vitro to observe the influence of BLP on lymphocyte subsets proportion. These dates showed that BLP major changed the T cell subsets ratio, specially the CD8+T cells. Then we used the method of FACS to detect TLR2 expression on various subgroups of T cells. The results suggest that, after activated by CD3/CD28 monoclonal antibodies, TLR2 expression were detected on CD4+CD25-Teff, CD4+CD25+Treg and CD8+CTL cell surface, in which TLR2 expression on CD4+CD25-Teff is higher than Treg and CTL Next, we directly incubated TLR2 agonists with three T cell subsets in vitro to observed impact of BLP on T cells function. The results showed that the in vitro proliferation capacity of CD4+CD25-Teff, treated by the BLP, improved much more than the control group. Its IFN-y secretion level was markedly improved (3.215±0.063μg/ml vs 0.6538±0.005μg/ml), while, the concentration of IL-4 was decreased significantly (19.24±1.552pg/ml vs 40.08±2.439pg/ml). These results indicate that BLP stimulated Teff in vitro increased its proliferation capacity and induced Thl-type immune response. Similarly, BLP stimulated CTL also upregulated its proliferation ability and promoted its effect molecular secretion, like Greanzyme B and IFN-y. The above results show that the BLP can directly combined with T cell surface TLR2 and changed T cells function.Further, we studied the impact of BLP on the T lymphocytes of tumor-bearing mice. The results showed that Teff source from tumor-bearing mice obtained same effect with normal mice cells treated by the BLP. Reflected as follows:Cell proliferation ability was markedly improved. Cytokine IFN-y level significantly increased, while IL-4 levels decreased significantly, showing a Thl-type pattern of cell-mediated immune response. Proliferation capacity and effect molecules expression of CTLs were also significantly upregulated by BLP treated in vitro, which suggested the enhanced CTL function.Based on the above In vitro studies, we deeply studied the immune function of BLP treated tumor-bearing mice. Splenocytes from BLP treated mice were marked by CFSE and cultured with inactivated 3LL tumor cell for 7 days. Cell proliferation were detected by FACS. Results showed that after BLP in vivo treatment, lymphocytes against tumor antigen-specific proliferation significantly regulated. We further tested CTL in vitro specific cytolysis after BLP treatment. Tumor-specific CTL cells (effector cells) were applied as the method above. CFSE marked 3LL tumor cells were target cells. effector cells and target cells were incubated 4 hours in 96-well plates at different ratio (100:1,50:1,25:1,5:1). After the response, added 1ml PI in each system, CFSE positive target cell death rate was detected by FACS rapidly, so as to reflect the CTL cytolysis ability. The results showed that BLP treatment significantly upregulated CTL's cytolysis function. When E:T ratio at 100:1, Special cytolysis ratio of CTLs from BLP treated mice was 41.90±0.905%. The control group was 15.38±0.62%. The results indicate that after BLP treatment the cytolysis function of CTL markedly increased.To sum up, regardless of normal mice or tumor-bearing mice T cells, BLP treatment could enhance CD4+CD25-Teff cells proliferation and regulated cytokine secretion type tended to Thl-type bias. At the same time, BLP treatment also enhanced the ability of CTL proliferation and its effector molecule expression. Both tumor-specific CTL proliferation and cytolysis ability was enhanced by BLP treatment. Thus make the body produced enhanced anti-tumor-specific immune response.PartⅢPossible mechanisms of TLR2 agonist BLP enhanced anti-tumor immune responseThe above experimental results confirmed that TLR2 agonist BLP induced enhanced anti-tumor immune response. we discussed relative mechanisms in this part through the detection of CD4+CD25+ Treg cell function, the ability of anti-specific CTL in vivo and the generation of anti-tumor immune memory.CD4+ CD25+Foxp3+Treg cells played a key role in tumor immune tolerance. Did Treg function change after BLP treatment? We sorted CD4+CD25+Treg cells from normal or tumor-bearing mice splenocyte by MACS. Then, Treg cells were activation by CD3/CD28 and join the final concentration of 5μg/ml of BLP in vitro for 72 hours. After treatment, Treg were incubated with CFSE labeled Teff cells and activated by CD3/CD28. Proliferations of Teff cells were detected by FACS after incubation. Fresh sorted Treg were used as control. The results showed that proliferation inhibit function of normal or tumor-bearing mice Treg cells were significantly reduced after BLP treatment.To verify the in vivo CTL function, high and low concentration of CFSE fluorescent respectively marked 3LL target antigen and control cells. Two groups of cells were inoculated via the tail vein to tumor-bearing mice or BLP treated tumor-bearing mice. The Changes of proportion of two CFSE positive cells groups reflected tumor-specific CTL cytolysis ability in vivo. The results show that BLP treatment induced enhanced CTL function in vivo.Finally, we choose mice that had BLP treated and survived over 100 days.3LL tumor cells were inoculated into the left groin of mice again, while 1×106 unrelated tumor cells FBL3 were inoculated in the right inguinal. Normal mice were chose as control. The results showed that BLP treated of mice could completely reject the second attack of 3LL tumor cells,3LL tumor persistently grown in normal mice. However, FBL3 tumor continued growth in both groups of mice. There were not significant differences in two groups. BLP treated mice received special immune memory for 3LL tumors. In summary, TLR2 agonist BLP induced enhanced anti-tumor immune response may be related to the mechanism of reduced Treg proliferation suppressed function, special CTL cytolysis capacity and tumor associated immune memory.