HLA-G and HLA-E Immune Checkpoints Are Widely Expressed in Ewing Sarcoma but Have Limited Functional Impact on the Effector Functions of Antigen-Specific CAR T Cells

SIMPLE SUMMARY: Solid cancers can effectively counteract immune attack by inhibitory checkpoints in the tumor microenvironment. Blockade of relevant immune checkpoints could be a useful tool for enhancing the efficacy of antitumor T cell therapies. Here, we studied the capacity of two nonclassical HLA molecules with known immunosuppressive function, HLA-G and HLA-E, to prevent antigen-specific immune effector functions of gene-engineered T cells against Ewing sarcoma. Inflammatory conditions and interactions of Ewing sarcoma cells with antitumor T cells reliably induced upregulation of the two molecules on the tumor cells. Moreover, as previously shown for HLA-G, HLA-E was detected in a high proportion of human Ewing sarcoma biopsies. However, artificial expression of either of the two molecules on Ewing sarcoma cells failed to reduce cytolytic and activation responses of antigen-specific T cells. We conclude that blockade of HLA-G and HLA-E immune checkpoints is not a promising strategy for enhancing T cell therapies in Ewing sarcoma. ABSTRACT: Immune-inhibitory barriers in the tumor microenvironment of solid cancers counteract effective T cell therapies. Based on our finding that Ewing sarcomas (EwS) respond to chimeric antigen receptor (CAR) gene-modified effector cells through upregulation of human leukocyte antigen G (HLA-G), we hypothesized that nonclassical HLA molecules, HLA-G and HLA-E, contribute to immune escape of EwS. Here, we demonstrate that HLA-G isotype G1 expression on EwS cells does not directly impair cytolysis by G(D2)-specific CAR T cells (CART), whereas HLA-G1 on myeloid bystander cells reduces CART degranulation responses against EwS cells. HLA-E was induced in EwS cells by IFN-γ stimulation in vitro and by G(D2)-specific CART treatment in vivo and was detected on tumor cells or infiltrating myeloid cells in a majority of human EwS biopsies. Interaction of HLA-E-positive EwS cells with G(D2)-specific CART induced upregulation of HLA-E receptor NKG2A. However, HLA-E expressed by EwS tumor cells or by myeloid bystander cells both failed to reduce antitumor effector functions of CART. We conclude that non-classical HLA molecules are expressed in EwS under inflammatory conditions, but have limited functional impact on antigen-specific T cells, arguing against a relevant therapeutic benefit from combining CART therapy with HLA-G or HLA-E checkpoint blockade in this cancer.