ABSTRACT Mutations of the isocitrate dehydrogenase (IDH) enzymes IDH1 and IDH2 are early and frequent genetic alterations in WHO grade II or III lower grade diffuse gliomas (LGG), as well as in secondary glioblastomas, which progress from LGG. Although IDH mutant (IDH-MUT) LGGs are associated with longer survival compared to IDH wild type (IDH-WT) LGGs, most IDH-MUT LGG patients eventually succumb to the disease. The median age at diagnosis is younger in IDH-MUT patients than IDH-WT patients, suggesting more therapeutic opportunities for IDH-MUT patients, such as immunotherapy. All IDH1 and IDH2 mutations confer a novel gain-of-function activity by converting α-ketoglutarate (αKG) to (R)-enantiomer of 2-hydroxyglutarate (R- 2HG), and coordinate genome-wide epigenetic changes. Approximately 90% of all IDH mutations are a single base mutation at position 132 of IDH1 replacing arginine (R) with histidine (H; R132H). As a way to target the IDH1 (R132H)-derived epitope by immunotherapy, we recently cloned cDNAs for T-cell receptors (TCR) that are specifically reactive to the IDH1(R132H)-derived peptide epitope (IDH1-TCR). Without immunotherapy, LGGs are known to be infiltrated by relatively few T-cells, implying that LGG may exhibit a unique immunosuppression mechanism. To support this, we have recently demonstrated that IDH mutations and R- 2HG lead to decreased effector T-cell-attracting chemokines, such as CXCL10, thereby suppressing infiltration of effector T-cells in gliomas. Our data also show that a mutant IDH1-specific inhibitor recovered CXCL10 and enhanced the efficacy of T-cell-based immunotherapy in preclinical mouse models of IDH-MUT gliomas. Finally, our data indicate that R-2HG also suppresses CXCL10 production by myeloid cells. In the current proposal, we will build on these data to evaluate our central hypothesis that IDH-MUT gliomas could be susceptible to immunotherapy by proper modulation of the LGG microenvironment and targeting of the IDH1(R132H)-derived neoantigen epitope. We will test the following Specific Aims: Aim 1. Determine the effects of IDH mutations on glioma-infiltrating myeloid cells. As myeloid cells could function as antigen-presenting cells (APCs), we will delineate the effects of IDH mutations on glioma-infiltrating myeloid cells and determine whether inhibition of mutant IDH can promote their APC functions. Aim 2. Determine how the IDH(1R132H) epitope is presented and recognized by the IDH1-TCRs. We will determine antigen-specificity and avidity of TCRs reactive to the IDH1(R132H) epitope. We will also evaluate how IDH1-TCRs recognize the epitope presented by a variety of HLA-class II molecules. Aim 3. Determine the efficacy and mechanisms of ACT with IDH1-TCR-Th1 cells in preclinical models. Using HLA-A2.DR1 transgenic mice, we will evaluate two mutually non-exclusive mechanistic hypotheses: 1) Th1-cells function as cytotoxic T lymphocytes (CTLs) and directly kill HLA-class II+ IDH1(R132H)+ glioma cells, and 2)...