Ewing's sarcoma (EwS) is a pediatric solid tumor entity with low somatic mutational burden and a low rate of tumor-infiltrating T cells, indicating a low extent of immunogenicity. In EwS, immunogenicity may furthermore be significantly diminished by a predominantly M2 macrophage driven pro-tumorigenic tumor microenvironment. In the past, we demonstrated that CHM1319-specific TCR-transgenic T cells are able to control EwS growth in a preclinical mouse model as well as in a patient with metastatic disease. However, new adjuvant techniques to induce long lasting and curative CHM1319-specific TCR-transgenic T cell-mediated anti-tumor responses are needed. In this work, we sought to identify a technique to improve the cytotoxic effect of CHM1319-specific TCR-transgenic T cell by altering the immunogenic cell surface marker expression on EwS cell lines using different cytokines. We demonstrate that TNF, IL-6, IL-1β and PGE2 cause pro-immunogenic CD83, MHC class I and II as well as ICAM-1 upregulation in EwS cell lines. This observation was associated with significantly improved recognition and killing of the tumor cells by EwS-specific CHM1319/HLA-A*02:01-restricted TCR-transgenic T cells. Conclusively, we demonstrate that the induction of an inflammatory signature renders EwS more susceptible to adoptive T cell therapy. TNF, which is upregulated during inflammatory processes, is of particular translational interest as its secretion may be induced in the patients e.g., by irradiation and hyperthermia in the clinical setting. In future clinical protocols, this finding may be important to identify appropriate conditioning regimens as well as point of time for adoptive T cell-based immunotherapy in EwS patients.
Ewing sarcoma (EwS) is an aggressive pediatric cancer of bone and soft tissues characterized by scant T cell infiltration and predominance of immunosuppressive myeloid cells. Given the important roles of extracellular vesicles (EVs) in cancer-host crosstalk, we hypothesized that EVs secreted by EwS tumors target myeloid cells and promote immunosuppressive phenotypes. Here, EVs were purified from EwS and fibroblast cell lines and exhibited characteristics of small EVs, including size (100-170 nm) and exosome markers CD63, CD81, and TSG101. Treatment of healthy donor-derived CD33+ and CD14+ myeloid cells with EwS EVs but not with fibroblast EVs induced pro-inflammatory cytokine release, including IL-6, IL-8, and TNF. Furthermore, EwS EVs impaired differentiation of these cells towards monocytic-derived dendritic cells (moDCs), as evidenced by reduced expression of co-stimulatory molecules CD80, CD86 and HLA-DR. Whole transcriptome analysis revealed activation of gene expression programs associated with immunosuppressive phenotypes and pro-inflammatory responses. Functionally, moDCs differentiated in the presence of EwS EVs inhibited CD4+ and CD8+ T cell proliferation as well as IFNγ release, while inducing secretion of IL-10 and IL-6. Therefore, EwS EVs may promote a local and systemic pro-inflammatory environment and weaken adaptive immunity by impairing the differentiation and function of antigen-presenting cells.
<p>Supplemental Figure 2: Combination of XVir-N-31 (XVir) and CDK4/6 inhibitor LEE011 (LEE) increases viral replication and immunogenicity of xenografted EwS tumors in vivo.</p>
Introduction Pediatric sarcomas, including osteosarcoma (OS), Ewing sarcoma (EwS) and rhabdomyosarcoma (RMS) carry low somatic mutational burden and low MHC-I expression, posing a challenge for T cell therapies. Our previous study showed that mediators of monocyte maturation sensitized the EwS cell line A673 to lysis by HLA-A*02:01/CHM1 319 -specific allorestricted T cell receptor (TCR) transgenic CD8 + T cells (CHM1 319 CD8 + T cells). Methods In this study, we tested a panel of monocyte maturation cytokines for their ability to upregulate immunogenic cell surface markers on OS, EwS and RMS cell lines, using flow cytometry. xCELLigence, SRB and ELISpot assays were used to assess whether TNF pretreatment increases CD8 + T cell cytotoxicity. Results We observed that TNF and IL-1β upregulated MHC class I, ICAM-1 as well as CD83 and PD-L1 on the surface of pediatric sarcoma cell lines, while IL-4, GM-CSF, IL-6 and PGE 2 failed to induce respective effects. Although pretreatment of pediatric sarcoma cell lines with TNF did not improve unspecific peripheral blood mononuclear cells (PBMCs) cytotoxicity, TNF enhanced specific lysis of 1/3 HLA-A2 + EwS cell lines by CHM1 319 CD8 + T cells depending on MHC-I expression and ICAM-1 upregulation. Discussion Our study supports utilization of TNF or TNF-inducing regimens for upregulation of MHC-I and costimulatory surface molecules on pediatric sarcoma cells and for enhancing recognition of responsive HLA-A2 + EwS tumor cells by antigen-specific CD8 + T cells.
<div>AbstractPurpose:<p>Ewing sarcoma (EwS) is a highly malignant pediatric tumor characterized by a non-T-cell-inflamed immune-evasive phenotype. When relapsed or metastasized, survival is poor, emphasizing the need for novel treatment strategies. Here, we analyze the novel combination approach using the YB-1-driven oncolytic adenovirus XVir-N-31 and CDK4/6 inhibition to augment EwS immunogenicity.</p>Experimental Design:<p><i>In vitro</i>, viral toxicity, replication, and immunogenicity were studied in several EwS cell lines. <i>In vivo</i> tumor xenograft models with transient humanization were applied to evaluate tumor control, viral replication, immunogenicity, and dynamics of innate as well as human T cells after treatment with XVir-N-31 combined with CDK4/6 inhibition. Furthermore, immunologic features of dendritic cell maturation and T-cell-stimulating capacities were assessed.</p>Results:<p>The combination approach significantly increased viral replication and oncolysis <i>in vitro</i>, induced HLA-I upregulation, and IFNγ-induced protein 10 expression and enhanced maturation of monocytic dendritic cells with superior capacities to stimulate tumor antigen-specific T cells. These findings were confirmed <i>in vivo</i> showing tumor infiltration by (i) monocytes with antigen-presenting capacities and M1 macrophage marker genes, (ii) T<sub>Reg</sub> suppression in spite of adenovirus infection, (iii) superior engraftment, and (iv) tumor infiltration by human T cells. Consequently, survival was improved over controls with signs of an abscopal effect after combination treatment.</p>Conclusions:<p>The joint forces of the YB-1-driven oncolytic adenovirus XVir-N-31 and CDK4/6 inhibition induce therapeutically relevant local and systemic antitumor effects. Innate as well as adaptive immunity against EwS is boosted in this preclinical setting, pointing toward high therapeutic potential in the clinic.</p></div>
