Glioblastoma Multiforme (GBM) tumors are the most aggressive malignant glioma and are highly heterogeneous and refractory to current modes of treatment. The immune system in cancer patients has been shown to be in a highly suppressed state, making it difficult to elicit anti-tumor responses. In addition to tumor production of anti-inflammatory TGF, PGE2 and IL-10 molecules, numerous reports have documented that the circulation and tumors in cancer patients are highly enriched for the immunosuppressive population of T cells referred to as natural regulatory T cells or nTregs. These intra-tumor nTregs can inhibit the induction of anti-tumor responsive cells as well as the activity of matured anti-tumor effector cells, thereby decreasing the efficacy of both arms of immune-targeted therapies.
We have recently demonstrated that there are three functionally distinct subsets of CD4+CD25highFoxP3high human nTregs that differ in their ex vivo expression of HLA Class II and CD127 (the IL-7 receptor, IL-7R). These subsets exhibit marked differences in their ability to suppress, proliferate, produce IL-10 and IL-17, and express granzyme B (GzmB), a protease typically found in the cytotoxic granules of NK and CD8 T cells and implicated in the killing of tumor cells. Our initial studies indicate that GBM tumors are significantly enriched for the presence of CD25high nTregs that express HLA Class II proteins (“DR+nTregs”) as compared to meningiomas, or healthy donor and patient peripheral blood. Our hypothesis is that the preferential enrichment of DR+nTregs in GBMs may contribute to their extremely poor prognosis and resistance to therapy, as peripheral blood derived DR+nTregs are the most potent nTreg subset. However, the identity of the tumor-enriched nTregs needs to be supported by functional criteria as strong activation can also induce the expression of HLA Class II by “IL-7R+” or “IL-7Rneg” nTregs, which differ in their capacity to secrete the inhibitory cytokine IL-10, and to inhibit the expression of GzmB in effector T cells. Additional preliminary data indicate that IL-10 enhances the proliferation of all three types of nTregs. Thus, as IL-10 is strongly produced in GBM tumors, these data may suggest a mechanism for the observed tumor-associated increase in nTregs.
The studies outlined in this proposal will determine both the ex vivo frequency and gene expression signatures of the functionally distinct types of nTregs that are FACS-sorted from different grade or types of CNS tumors that include GBM, meningiomas, pilocytic astrocytomas, and CNS metastases, and from PBLs from these same patients as well as healthy donors. Subsequently, we will examine the functional capacity of the distinct nTreg subsets isolated from these ex vivo tumor samples to determine how they differ in frequency or activity from those isolated from healthy donor or from patient peripheral blood. Due to the importance of Granzyme B in the effector arm of anti-tumor immunity, we will identify molecules and pathways that are utilized by nTregs to inhibit its expression in effector T cells. Lastly, using in vitro multi-component co-culture models, we will examine how communication between the nTreg subsets, and effector T cells is modulated by the presence of GBM tumor cells. The ultimate goal of these studies is to identify therapeutic targets to selectively inactivate the specific subsets of nTregs that may be differentially associated with particular types of tumors, and provide an alternative to the current nTreg-targeted treatments that kill all IL-2R expressing cells which can also deplete beneficial anti-tumor effector T cells.