The Functions And Molecular Mechanisms Of SLC- CCR7 Axis In The Immune Microenvironment Of Hepatocellular Carcinoma

Primary liver cancer is a malignant tumor with severely poor prognosis, among which about 90% is hepatocellular carcinoma (HCC). HCC is the third leading cause of cancer-related death worldwide and the second leading cause in China. Most patients, about 60-80%, are diagnosed at intermediate or advanced stage of HCC, who can only receive palliative treatment with a very poor prognosis related with tumor recurrence. Therefore, it is emergent for developing a new treatment for these HCC patients.Immunotherapy is considered to be a promising therapy in treating cancer, especially for HCC. Immunotherapy mainly modulates the immune system of patients either by active intervention or passive intervention, resulting in re-activation of the immune system and eradication of tumor cells. Among the available immunotherapies, treatments based on SLC (secondary lymphoid-tissue chemokine, SLC/CCL21) have demonstrated effective anti-tumor effects. SLC is mainly secreted by endothelial cells, fibroblast cells and activated T cells. SLC can bind to its specific receptor CCR7, which is expressed on various immune cells including dendritic cells (DCs) and T cells, and the activation of SLC-CCR7 axis causes immune cells chemotactic migration and maintains the normal migration and homing of immune cells. In murine HCC model, local expression of SLC significantly increases the intratumoral CD4+, CD8+T cells and DCs, and the growth of tumor is effectively inhibited.Among the various immune cells, regulatory T cells (Tregs) and DCs are the core components of the tumor immune microenvironment. Tregs are the major immune cells that suppress the immune response in the microenvironment. While they are heterogeneous cells consisting of many sub-populations, the CD4+CD25+Foxp3+T cells are the predominant population. Once activated, Tregs can efficiently suppress other immune cells either through cell-cell contact or through secretion of inhibitory cytokines. Various studies have confirmed that the frequency of Tregs is significantly increased in the peripheral blood of HCC patients, tumor tissue and peri-tumor tissue, and Tregs derived from HCC patients exhibit enhanced suppressive functions. These alterations are closely correlated with patients’ prognosis. In contrast, depletion of Tregs by anti-CD25 monoclonal antibodies (mAbs) leads to a significant increase of tumor specific CD4+ and CD8+ T cells and finally manifests good anti-tumor effects, demonstrating that depletion of Tregs in the HCC is crucial to improve the immune microenvironment.SLC-CCR7 axis is indispensable for Tregs in vivo; besides, SLC-CCR7 signaling has additional functions in T cells. Activation of SLC-CCR7 axis can prevent apoptosis of T cells and function as co-stimulation molecules in CD4+ T cells after activation, affecting the secretion of IL-2 and IL-4. These data imply that SLC-CCR7 axis might serve as key co-stimulation molecules in Tregs, provide necessary co-stimulation signals and finally affect the functions of Tregs. As Tregs have been demonstrated to be widely modulated by Foxp3-miRNAs network, it is highly possible that the SLC-CCR7 axis provides co-stimulation signals for the Foxp3-miRNAs network, and they finally synergistically modulate the functions of Tregs.DCs are differentiated from progenitors derived from the bone marrow, including myeloid DCs and plasmacytoid DCs, and they are the most important antigen presenting cells (APCs) in vivo, which play an indispensable role in initiating immune responses. DCs in the immune microenvironment of HCC show reduced number and impaired functions, representing an immature phenotype. These DCs induce and maintain tumor tolerance instead of anti-tumor immune responses. In addition to chemotaxis, SLC-CCR7 signaling has additional functions on DCs by influencing the maturation and proliferation both in vitro and in vivo, resulting in enhanced functions of antigen presenting. Therefore the maturation and proliferation of DCs induced by SLC critically contributes to SLC mediated anti-tumor effects. However, the molecular mechanisms underlying the SLC-induced maturation and proliferation are still unclear.In order to improve the anti-tumor effects of SLC-based immunotherapies in HCC, and to clarify the molecular mechanisms how SLC-CCR7 axis affects Tregs and DCs, we performed experiments to explore:1) whether intratumoral injection of SLC and anti-CD25 mAbs would elicit better anti-tumor effects in murine HCC model; 2) how SLC-CCR7 axis and Foxp3-miRNAs network affected Tregs in the context of HCC; 3) the molecular mechanisms underlay SLC-induced maturation and proliferation of DCs.Methods1. SLC and anti-CD25 mAbs were intratumorally injected in the Hepa 1-6 murine model. Tumors were isolated and prepared for 1HC and FACs analysis. The number of Tregs, CD4+ and GD8+ T cells and CD11c+ DCs was quantified. Intratumoral levels of IL-10, TGF-1β, IFN-y and IL-12 were determined by ELISA on day 5 post-treatment.2. Frequency of Tregs, CD4+ and CD8+ T cells in the lymph nodes, spleen and liver was quantified by IHC and FACs in different groups. Tumor volume and weight was monitored in all groups after the treatments.3. Tregs were isolated from the spleens of Hepa 1-6 murine models. The purity of Tregs was then checked by FACs. miRNAs were prepared from Tregs and used for miRNAs microarray by mouse miRCURYTM LNA Array (v.16.0). Array data were normalized and filtered through volcano plots, and differentially expressed miRNAs were validated by real-time RT-PCR. Tregs isolated from peripheral blood of HCC patients were used for further validation of differentially expressed miRNAs.4. Tregs isolated from HCC-bearing mice and control mice were cultured in vitro and transfected with siRNAs against Foxp3. Real-time RT-PCR and FACs were performed to examine the efficiency of Foxp3 silencing. miRNAs from Tregs after Foxp3 silencing were used for miRNAs microarray.5. Target genes of miRNAs were predicted by TargetScan Mouse and target genes list were then imported into the software EGAN to construct Foxp3-miRNAs network; genes in SLC-CCR7 axis were retrieved from MeSH database in EGAN. Array data of tumor-activated Tregs were retrieved from GEO database and analyzed by GSEA, with gene sets consisting of genes from literature and SABiosciences database.6. BMDCs were cultured and stimulated in the presence of SLC/ELC for 12 or 24 hours. Cells were then harvested and mRNAs were prepared and used for microarray by mouse G Protein-coupled Receptors Signaling PathwayFinder Gene Array. Western blot was performed to validate differentially expressed genes in response to SLC/ELC.Results1. The number of intratumoral Tregs in SLC groups was much lower than that in control groups, and anti-CD25 mAbs further synergistically reduced Tregs, resulting in the lowest number of Tregs. The two treated groups showed increased CD4+, CD8+T cells and CD11c+ DCs on day 5 post-treatment, but anti-CD25 mAbs only manifested an effective synergy in increasing the CD8+ T cells; increased intratumoral IL-12 and decreased IL-10 and TGF-β1 was found in SLC groups, and anti-CD25 mAbs synergistically increased the level of IFN-γ.2. In lymph nodes, the frequency of Tregs in the two treated groups was lower than control groups and combination therapy groups had much lower frequency of Tregs (day 7 and 9). The two treated groups had higher levels of CD8+and CD4+T cells (day 3 to 9); combination therapy groups had a significantly higher level of CD8+T cells (day 3 and 5), and a significantly higher level of CD4+T cells on day 5.3. In the spleen the two treated groups had a lower percentage of Tregs, and anti-CD25 mAbs showed significant synergies (day 3 to 9) and maintained the lowest level of Tregs. SLC groups showed increased levels of CD4+and CD8+T cells (5,7 and 9), and anti-CD25 mAbs showed potent synergies as demonstrated by increased CD4+ and CD8+T cells at all time points.4. The two treated groups showed decreased frequency of Tregs and increased CD8+ and CD4+T cells in the liver; anti-CD25 mAbs only enhanced this effect of SLC on day 9. SLC increased CD8+T cells and anti-CD25 mAbs significantly synergized with SLC (day 1,3 and 9). Control groups showed the lowest level of CD4+T cells; SLC group had the highest level of CD4+T cells.5. SLC effectively inhibited the increase of tumor volume and this effect was enhanced by anti-CD25 mAbs from day 5 post-treatment. SLC significantly inhibited the increase of tumor weight on day 7 and 9; anti-CD25 mAbs efficiently synergized to inhibit the increase of tumor weight (day 3 to 9).6. HCC-primed Tregs had significantly up-regulated mmu-miR-487b-5p, mmu-miR-709, mmu-miR-182-5p, mmu-miR-214-3p and mmu-miR-467a-3p and down-regulated mmu-miR-142-5p, mmu-miR-30b-5p, mmu-miR-409-3p and mmu-miR-129-5p, and among them, mmu-miR-214-3p and mmu-miR-30b-5p were significantly down- regulated and mmu-miR-409-3p were up-regulated after Foxp3 silencing; Tregs from peripheral of HCC patients manifested significant up-regulation of hsa-miR-182-5p, hsa-miR-214-3p, hsa-miR-129-5p and hsa-miR-30b-5p.7. There existed obvious intersection between genes down-stream of SLC-CCR7 axis and genes modulating IL-10, TGF-β1, IFN-y and IL-12. GSEA showed genes modulating IL-10 and TGF-β1 significantly enriched in tumor-primed Tregs. Foxp3-miRNAs network had crosstalk with SLC-CCR7 axis in modulating IL-10, TGF-β1, IFN-y and IL-12.8. SLC/ELC induced significant phosphorylation of NF-kB p65 subunit after stimulation by SLC for 12 and 24 hours, along with a significant down-regulation of total p65 in the cytoplasma, but ELC induced phosphorylation of p65 in a far less potent way than SLC. PDTC almost completely blocked SLC/ELC-induced phosphorylation of p65; Cyclin D1, E1, E2 and p21 was significantly up-regulated after stimulating by SLC for 12 hours and maintained at this high level when stimulated for 24 hours, whereas the expression levels of E2F-1 and E2F-2 were not significantly changed.Conclusions1. Intratumoral injections of SLC and anti-CD25 mAbs efficiently improved the tumor immune microenvironment, with decreased Tregs, IL-10 and TGF-β1, and with increased CD8+T cells, CD4+T cells, CD11c+ DCs, IFN-γ and IL-12 levels; in the mean time it systemically optimized composition of the immune cells in peripheral lymph organs including the lymph nodes, spleen, and liver, with decreased Tregs and increased CD8+T cells and CD4+T cells. This optimization contributed to the enhanced anti-tumor effects.2. HCC-primed Tregs manifested specific signatures of miRNAs with 9 miRNAs differentially expressed, among which four (hsa-miR-182-5p, hsa-miR-214-3p, hsa-miR-129-5p and hsa-miR-30b-5p) were found to be up-regulated in Tregs from peripheral blood of HCC patients; in mice Foxp3 specifically modulated mmu-miR-214-3p, mmu-miR-30b-5p and mmu-miR-409-3p in Tregs and this Foxp3-miRNAs network had crosstalk with SLC-CCR7 axis in modulating IL-10, TGF-β1, IFN-γ and IL-12.3. Stimulation of BMDCs by SLC resulted in activation of the NF-kB signaling pathway and subsequent phosphorylation of NF-κB p65 subunit, which mediated the maturation of BMDCs; in the mean time, up-regulated Cyclin D1, Cyclin E1, Cyclin E2 and p21 participated in the proliferation process of BMDCs.