Experimental Research Of Tim-3, STim-3 On Antitumor Immunity And Other Antitumor Gene Therapy

The search for cell-surface markers that can distinguish T_H1 from T_H2 cells has lead to the identification of a new molecular---Tim-3. It has been reported that administration of anti-Tim-3 antibody and Tim-3 Ig fusion protein can abrogate the induction of peripheral tolerance and promote T_H1-mediated autoimmune and alloimmune disease, which indicated that Tim-3 has important function in the regulation of immune-mediated disease. It was found that Tim-3 mRNA could be alternatively spliced to produce two mRNA molecules. The longer one directs the synthesis of full-length Tim-3 (i.e., Tim-3), the shorter one, without the region encoding the mucin domain and transmembrane domain, was supposed to direct the synthesis of a splice variant of sTim-3.Many co-stimulatory molecules and immunoregulatory receptors, such as CD86, Fas and CTLA-4, have native soluble molecules, and these soluble variants were important in controlling immune response as well as in susceptibility and resistance to autoimmune disease. The discovery of sTim-3 raised several question to be answer. The first one was the expression pattern of Tim-3 and sTim-3 mRNA in immune response. The second one was the existence of the translated product of sTim-3 and whether sTim-3 could bind to Tim-3 ligands. The third one was the regulatory role of Tim-3 and sTim-3 in antitumor immune response.At present, tumor immunotherapy efforts are focused on the generation of strong T cell response against tumor antigens. However, strong T cell response does not always coincide with tumor rejection, for which upregulated expression of irnmunoinhibitory molecules--- may be responsible. Emerging evidences suggest that B7 homolog 1 (B7-H1) is important in the mechanisms of resistance against tumor associated antigen-specific immunity. Blockade of B7-H1 by sPD-1 can enhance tumor specific CTL response and cause tumor rejection.Part IEctopic expression of Tim-3 induces tumor-specific antitumor immunityObjective: To express Tim-3 in tumor microenvironment and investigate the role of Tim-3 in regulation of lymphocyte activity and antitumor effect. Methods: Total RNA of concanavalin A stimulated splenocytes was extracted and Tim-3 cDNA was amplified by RT-PCR. The amplified fragment was digested by restriction enzymes and inserted into compatible enzyme restriction sites of pcDNA3.1 to generate murine Tim-3 eukaryotic expression plasmid (pTim-3). CHO cells were transfected with pTim-3 with Dosper liposomal transfection reagent, the transfected cells were screened by G418. And the expression of Tim-3 in the transfected cells were detected by FACS. Tumors were established by injection of H22 into right hind thigh muscle and pTim-3 plasmid was injected intratumorally. The expression of Tim-3 on H22 cells was examined by immunohistochemistry, and tumor volume and the survive was recorded. Hematoxylin and eosin (H&E) staining was performed to detect tumor-infilating T lymphocytes. Cytolytic activity against H22 was determined in 4h - ⁵¹Cr release assay. Tim-3 expression on freshly isolated CD3⁺ T and CD8⁺ T cells and HSP70-tumor peptide complex stimulated CD3⁺ T and CD8⁺ T cells were examined by FACS analysis. Results: The constructed plasmid, designated as pTim-3, was confirmed by restriction enzymes digestion and DNA sequencing. Flow cytometric analysis revealed that Tim-3 antibody stained the surface of cells transfected with pTim-3 but not the surface of cells transfected with mock plasmid (pcDNA3.1). Intratumor injection of pTim-3 plasmid potently inhibited tumor growth and results in prolonged survival. There was microscopic evidence of dense lymphocyte infiltration in pTim-3 treated mice. In addition, Splenocytes from the pTim-3 treated group showed a strong CTL response against H22 tumor cells, which suggested that ectopic Tim-3-mediated antitumor responses were dependent on a functional adaptive lymphocyte response. We also demonstrate endogenous Tim-3 expression on lymphocyte is relevant in antitumor immune response. Since considerable number of CD3⁺ T cell including CD8⁺ T cell can express Tim-3 after stimulate with tumor-peptide complex. Conclusion: Expression of Tim-3 on H22 tumor cells can enhance the proliferation and other functions of T lymphocytes. Tim-3 expression is not limit on CD4⁺ T cell but also on CD8⁺ T cells after stimulation with tumor- peptides complex. Our results thus gain further insight into the function of Tim-3 expressed on nonlymphoid tissues in regulating antitumor immune response. Part IISoluble Form of Tim-3 Is An Inhibitory Molecule in T Cell-Mediated Immune ResponseIn initial experiments, we demonstrated that Tim-3 mRNA was existent in the cells from thymus, bone marrow and spleen, whereas sTim-3 mRNA was expressed only in spleen cells but not in bone marrow, thymus and non-lymphoid organs. To investigate the expression pattern of sTim-3 and Tim-3 mRNA in immune response, we examined the expression of these mRNAs in freshly isolated and activated splenocytes. Real-Time PCR revealed that both Tim-3 mRNA and sTim-3 mRNA were significantly increased after each rounds of stimulation, whereas there appeared to be a higher increase of sTim-3 product than that of Tim-3 product, suggesting that the expression pattern of Tim-3 mRNA and sTim-3 relied on the activation of splenic lymphocyte.We next proceed to examine the existence of sTim-3's protein product by Western blot. The results showed that sTim-3's protein was detected in the cell-free supernatants of the stimulated splenocyts and CHO cells transfected with sTim-3 eukaryotic expression plasmid. Fluorescence microscope analysis revealed that Green fluorescence was found in CHO cells transfected with pTim-3-GFP but scarcely in CHO cells transfected with psTim-3-GFP, indicating that sTim-3 is mostly secreted out, which further conformed that sTim-3 is a soluble molecule. The binding of sTim-3-GFP to its putative ligand(s) on splenic T cells was determined by FACS analysis. The fluorescence intensity on T cells incubated with sTim-3-GFP was significantly increased as compared with control. These results clearly indicated that sTim-3 could bind to putative Tim-3 ligand(s).In vitro, the response of T cells to antigen-specific stimulation or anti-CD3 mAb plus anti-CD28 mAb costimulation was significantly suppressed in the presence of sTim-3, and the blockade of sTim-3 with anti-Tim-3 antibody recovered the response of T cells to these stimuli, supporting the notion that sTim-3 mediates the inhibitory effect in immune response. We then attempt to further understand the function of sTim-3 in vivo, in tumor rejection model, sTim-3 facilitated the growth of tumor in vivo and shorten survival of tumor-bearing mice. The proliferation of T cells from sTim-3 treated mice was significantly reduced when they were re-stimulated by antigen-loaded DC in vitro, Moreover, the antitumor CTL activity was significantly reduced by sTim-3 and the amount of T cells in tumor tissue was also significantly decreased. Pathologic and immunohistological findings revealed the reduced tumor-infilating T lymphocytes. To sum up, these results confirmed the inhibitory effect of sTim-3 on the generation of antigen-specific T cell immune response.Since both effector CD4⁺CD25^- T cells and regulatory CD4⁺CD25⁺ T cells express a putative Tim-3 ligand, the impairment of antitumor immunity by sTim-3 might be the result of either impairing the function of effector CD4⁺ T cells or activating the function of CD4⁺ regulatory T cells. To understand the underlying cellular mechanism contributing to the impaired antitumor immunity by sTim-3, we analyzed the expression of genes related to the function of tumor-infiltrating lymphocytes by real-time quantitative PCR. The results indicated that, in sTim-3 treated mice, the expression of IL-2, IFN-γ and TNF-β mRNA was down-regulated, whereas, the expression of forkhead transcription factor 3 (Foxp3), IL-10 and TGF-p mRNA was not influenced as compared with control, theses results suggested that sTim-3 didn't influence Treg cells but otherwise resulted in the profound immune unresponsiveness of CD4⁺ T effector cells. Part IIIHSP70 Vaccine in Combination with Gene Therapy with PlasmidDNA Encoding sPD-1 Overcomes Immune Resistance and Suppressesthe Progression of Pulmonary Metastatic MelanomaB7-H1 (also known as PD-L1) is a cell surface glycoprotein belonging to the B7 family of costimulatory molecules. It was founded that PD-1 (programmed death-1), which is expressed on the activated lymphocytes, is the receptor of B7-H1. B7-H1 is thought to contribute to immune escape of cancer by interacting with PD-1 receptor. In this study, the treatment with HSP70 vaccine induced an infiltration of T cells into the tumor site as well as the expression of IFN-γ and IL-2, and delayed lung metastases of tumor, but the tumor progression nonetheless occur finally. We incubated B16F1 cells with different doses of IFN-γ in vitro. FACS analysis revealed that the percentage of B7-H1-positive B16F1 cells was significantly increased by IFN-γ stimulation. By contrast, B7-H1 was hardly detectable on the untreated B16F1 cells. Consistent with this result, the upregulatd expression of B7-H1 in vivo in tumor microenvironment was also confirmed by Real-time PCR. We demonstrated that B7-H1 expressed by residual tumor cells was responsible for the resistance of tumor to the therapy with HSP70 vaccine. Blockade of B7-H1 by i.v. injection pPD-1A, a plasmid encoding the extracellular domain of PD-1 (sPD-1) could reverse this resistance and enhance the therapeutic efficacy. To complement these findings, we investigated the gene expression of TILs by Real-time PCR analysis, which revealed that the expression of TH1 cytokines IFN-γ and IL-2 by TIL in the mice treated with HSP70 vaccine in combination with sPD-1 was increased and the expression of negative regulatory molecules IL-10, TGF-β and foxp3 was decreased, demonstrating that multifunctional properties afforded by the combination therapy can effectively overcome tumor resistance and promote effective antitumor immunity. In vivo transfection with pPD-1A could be performed as infrequently as once a week and still produce a significant antitumor effect. These findings suggest that the treatment with HSP70 vaccine followed by blockade of tumor-B7-H1 with sPD-1 may provide a promising approach for tumor immunotherapy.