TLR2 Agonist BLP Improves Adoptive T Cell Immunotherapy In Glioma-bearing Mice And The Underlying Mechanisms

Gliomas are malignant tumors that derived from glial cells. They are divided into astrocytomas, oligodendrogliomas and ependymomas according to the specific cell type of glial cells. And astrocytomas are the most aggressive with 80% of all incidence of gliomas. Clinical treatment consists of maximal surgical resection, radiotherapy and chemotherapy However, the prognosis of patients with malignant gliomas remains dismal with modest improvement in median survival, and five-year survival is less than 10%. One important reason is the infiltrative growth of gliomas so that it is difficult to remove the tumor completely. Moreover, glioma is insensitive to both radiotherapy and chemotherapy. In addition, the blood-brain barrier in the brain also limits the treatment. Thus, there is an urgent need for novel potent strategies for the gliomas treatment.Adoptive immunotherapy is a promising strategy for cancer therapy. Lymphocytes derived from patients are activated, expanded and antigen-specific induced in vitro. Then, these cells are adoptively transferred back into patients. Clinical trials that using autologous tumor-infiltrating lymphocytes have been shown amazing therapeutic effect for patients with acute leukemia, chronic leukemia and metastatic melanoma. However, there is no breakthrough in gliomas treatment, which is probably due to the unique structure and function of central nervous system. First of all, central nervous system is considered as an immunologically quiescent site. Unique brain features such as the lack of conventional lymphatics and professional antigen presenting cells profoundly influence immune cells trafficking and function in the central nervous system. Secondly, gliomas micro-environment participates in immunosuppression by secreting TGF-β2 and inducing immunosuppressive cells, such as myeloid-derived suppressor cells, and regulatory T cells, which potentially impair anti-tumor immunity. Thus, therapeutic strategy to break gliomas-associated immune tolerance would account for gliomas immunotherapy.Toll-like receptors (TLRs), a type of pattern recognition receptor, recognize pathogen-associated molecular patterns to play a key role in anti-infection, anti-virus and anti-tumor immune responses. Recent studies found that TLRs not only take part in innate immune responses, but also mediate adaptive immune responses directly in some pathological states, such as malignant tumor. Bacterial lipoprotein (BLP) is a kind of cell wall components, which is TLR1/2 agonist. Our previous study have uncovered that BLP could enhance T cell-mediated anti-tumor immunity as well as decrease tumor-associated immunosuppression. So we wonder whether BLP would improve adoptive T cell immunotherapy, and if so, what are the mechanisms? Based on the above results, the aim of our project is to figure out whether and how BLP could facilitate immunotherapy in orthotopic gliomas mouse model.Part Ⅰ TLR2 agonist BLP improves adoptive T cell therapyFirst of all, GL261 tumor cells were implanted into the caudate nucleus of mice by using a stereotactic head frame to establish murine GL261 malignant glioma model. In order to validate whether BLP exhibits anti-tumor efficacy inthe GL261-bearing model, mice bearing five-day old intracranial tumors were treated with or without adoptive T cell transfer, combined with PBS or BLP i.p. every other day for total four times. The adoptive T cell transfer plus four systemic injections of BLP eradicated intracranial gliomas in 33% of mice. MRI imaging of the GL261 tumor further confirmed the tumor eradication in tumor-bearing mice treated with adoptive T cell transfer plus BLP, whereas no survival benefit was observed in other groups. After that, the treated mice that had survived for more than 90 days were re-challenged by GL261. These mice successfully resisted glioma re-challenge, suggesting that the combination therapy induced long-term immune protection to prevent gliomas relapse. Finally, to assess the function of TLR2 signal in combination therapy, TLR2 deficiency mice were treated with adoptive T cells and BLP. Results showed no survival benefit could be observed TLR2-/-tumor-bearing host. Therefore, BLP improve adoptive T cell therapy in TLR2-dependent manner.Then, we tried to determine whether the anti-tumor activity is directly towards tumor itself, since GL261 cells have TLR1 and TLR2 expression in both mRNA and protein levels. GL261 tumor cells were stimulated with different doses of BLP for 48 hours in vitro. However, no matter how much BLP was used, there was no detectable change in the viability and apoptosis of GL261 tumor cells. Furthermore, TNF-α and IL-6 production by GL261 tumor cells were also not altered after BLP stimulation. So the improvement of adoptive T cell therapy by BLP is not attributed to the direct tumoricidal activity of BLP, which has been proved by the above data.Part Ⅱ TLR2 agonist BLP enhances anti-glioma immunity of adoptive T cellsSince adoptive T cell transfer combined BLP therapy exhibited effective anti-glioma efficacy, and we also excluded the possibility of the direct tumoricidal activity of BLP. Next, we wonder if BLP could maintan and enhance anti-tumor immunity of adoptive T cells in vivo, so this part will pay attention to the effect of BLP on adoptive T cells.Firstly, tumor-infiltrating leukocytes (TILs) were isolated from GL261-bearing mice treated with adoptive T cells and BLP. The phenotype of TILs were analyzed by FACS. Expectedly, TILs, specifically T cells, were much more increased after combined therapy. Then, tumor-specific T cells from GFP transgenic mice were used for BLP combined therapy. More tumor-infiltrating GFP+T cells were detected after combined therapy, among which 92.8% was CD8+T cells. Also, brain slices confirm the result. Next, the capacity of IFN-γ-secreting activity by GFP+CD8+T cells was significantly enhanced. And higher transcript and.secretion level of CXCL10 were detected by real-time PCR and ELISA.Moreover, when CFSE-labeled T cells were used for adoptive transfer, a more obvious cell division in the brains could be observed after adoptive therapy combined with BLP. Finally, tumor-infiltrating GFP+T cells were shown an increased anti-tumor activity in terms of enhanced specific cytotoxicity. Taken together, these observations suggested that BLP could facilitate T cells migration across the blood-brain barrier, but also promote T cell proliferation, as well as maintain T cell survival and cytotoxicity in tumor-bearing host to improve adoptive T cell therapy.Part III TLR2 agonist BLP overcomes MDSC-mediated T cell immunosuppressionGliomas induce immunosuppressive tumor micro-environment (TME) by multiple mechanisms. MDSCs and Treg are the key cells in maintaining immunosuppression in TME. In order to assess whether anti-glioma effect by BLP combined therapy was due to the overcome of TME, MDSCs and Treg infiltration in spleen, blood, cervical lymph nodes, inguinal lymph nodes and brain were detected by FACS. Results showed remarkably decreased infiltration of MDSCs in brain and other lymph tissues after combined treatment with BLP and T cells. While changes of Treg were undetectable. Since CCL2 plays a role in MDSCs acccumlation, CCL2 was analyzed by real-time PCR and ELISA, which showed notably decreased expression in brain after combined treatment. What’s more, TLR2 deficiency significantly increased the expression of CCL2 and the infiltration of MDSCs, while attenuated the infiltration of IFN-γ-secreting CD8+ T cells. Taken together, these results showed that BLP overcomes tumor microenvironment by decreasing CCL2 level to reduce MDSCs accumulation, as a result, adoptive T cell therapy is remarkably improved.Concludsions:1. TLR2 agonist BLP improves adoptive T cell therapy for murine gliomas, which is not attributed to the direct tumoricidal activity of BLP.2. TLR2 agonist BLP enhances T cells infiltration, proliferation and cytotoxicity in tumor-bearing host to improve adoptive T cell therapy.3. TLR2 agonist BLP targets tumor microenvironment by decreasing CCL2 level to reduce MDSCs accumulation, as a result, adoptive T cell therapy is remarkably improved.