The Mohawk (mkx) gene functions as a transcriptional repressor for tendon morphogenesis during embryonic development. Previous research showed that mkx KO mice overexpressed the osteogenic gene Runx2. Runx2 plays a role in recognition and long-term immune memory. A study showed Runx2 KO mice had a significantly lower number of CD8 T cells specific to lymphocytic choriomeningitis virus (LCMV) and CD8 memory precursor T cells. To determine the direct effects of Mohawk expression on the immune system, development, acute response, and immune memory of innate, B and T cells were compared between WT and mkx KO mice after LCMV infection. Paired t-test analyses were performed between KO and WT data. We first found significantly higher numbers of granulocytes and dendritic cells in the periphery but lower numbers of B cells in the bone marrow and T cells in the thymus of KO mice. When analyzing immune response, we observed a significantly high number of activated CD8 T cells that proliferated in the KO mice in response to the infection. Next, we found no difference in cytokine production for TNF and IFNγ which shows Mohawk does not impair acute immune response. Finally, we found no significant difference between WT and KO mice in the CD8 T cells' ability to make an immune memory. In the present study, we found that, fewer immune cells continued their maturation. However, Mohawk expression did not impact their acute response or ability to become memory cells once the T cells matured and became activated. Rather, T cells specific for LCMV were present in higher numbers in mkx KO mice. Further research will study the impact Mohawk has on both B and T cell memory.

Osteosarcoma is the most common bone cancer in children and adolescents. Although 70% of patients with localized disease are cured with chemotherapy and surgical resection, patients with metastatic osteosarcoma are typically refractory to treatment. Numerous lines of evidence suggest that cytotoxic T lymphocytes (CTLs) limit the development of metastatic osteosarcoma. We have investigated the role of PD-1, an inhibitory TNFR family protein expressed on CTLs, in limiting the efficacy of immune-mediated control of metastatic osteosarcoma. We show that human metastatic, but not primary, osteosarcoma tumors express a ligand for PD-1 (PD-L1) and that tumor-infiltrating CTLs express PD-1, suggesting this pathway may limit CTLs control of metastatic osteosarcoma in patients. PD-L1 is also expressed on the K7M2 osteosarcoma tumor cell line that establishes metastases in mice, and PD-1 is expressed on tumor-infiltrating CTLs during disease progression. Blockade of PD-1/PD-L1 interactions dramatically improves the function of osteosarcoma-reactive CTLs in vitro and in vivo, and results in decreased tumor burden and increased survival in the K7M2 mouse model of metastatic osteosarcoma. Our results suggest that blockade of PD-1/PD-L1 interactions in patients with metastatic osteosarcoma should be pursued as a therapeutic strategy.

Background: Osteosarcoma is one of the most common bone cancers in children. Most patients with metastatic osteosarcoma die of pulmonary disease and limited curative therapeutic options exist for such patients. We have previously shown that PD-1 limits the efficacy of CTL to mediate immune control of metastatic osteosarcoma in the K7M2 mouse model of pulmonary metastatic disease and that blockade of PD-1/PD-L1 interactions can partially improve survival outcomes by enhancing the function of osteosarcoma-specific CTL. However, PD-1/PD-L1 blockade-treated mice eventually succumb to disease due to selection of PD-L1 mAb-resistant tumor cells. We investigated the mechanism of tumor cell resistance after blockade, and additional combinational therapies to combat resistance.

Methods: We used an implantable model of metastatic osteosarcoma, and evaluated survival using a Log-rank test. Cellular analysis of the tumor was done post-mortem with flow cytometry staining, and evaluated using a T-test to compare treatment groups.

Results: We show here that T cells infiltrating PD-L1 antibody-resistant tumors upregulate additional inhibitory receptors, notably CTLA-4, which impair their ability to mediate tumor rejection. Based on these results we have tested combination immunotherapy with α-CTLA-4 and α-PD-L1 antibody blockade in the K7M2 mouse model of metastatic osteosarcoma and show that this results in complete control of tumors in a majority of mice as well as immunity to further tumor inoculation.

Conclusions: Thus, combinational immunotherapy approaches to block additional inhibitory pathways in patients with metastatic osteosarcoma may provide new strategies to enhance tumor clearance and resistance to disease.