IMMU-32. NON-METABOLIC IDO ACTIVITY INCREASES COMPLEMENT FACTOR H LEVELS WHICH ENHANCES IMMUNOSUPPRESSION IN HUMAN GLIOBLASTOMA

Indoleamine 2,3 dioxygenase 1 (IDO) is an immunosuppressive factor expressed in ≥90% of patient resected glioblastoma (GBM). Canonically, IDO suppresses the immune response by metabolizing the essential amino acid, tryptophan, into the downstream metabolite, kynurenine. Based on the in vivo finding that the genetic knockout of tumor cell IDO does not change brain tumor tryptophan and kynurenine levels in syngeneic mice, we recently questioned the mechanistic role of IDO in GBM. To determine whether tumor cell tryptophan metabolism is responsible for the pathogenic effects of IDO, we created IDO-deficient murine GBM cells that were reconstituted with either (i) empty expression vector, (ii) a vector expressing wild-type IDO, (iii) or a vector expressing enzyme-inactive IDO. GBM cell IDO expression increased Tregs and decreased survival, but did so independent of tryptophan metabolism. To understand how IDO causes these effects, we performed Clariom D microarray analysis of human GBM cells and discovered that complement factor H (CFH) is significantly increased in GBM cells expressing IDO but decreased by treatment with IDO siRNA. Notably, CFH siRNA decreased CFH- but not IDO-expression in GBM cells. We next found that GBM cell IDO regulates CFH levels independent of tryptophan metabolism. To determine the effects of CFH on tumor outgrowth, we intracranially-engrafted syngeneic mice with IDO-deficient murine GBM cells reconstituted with either empty expression vector or a vector expressing CFH splice variant 2. Tumor CFH significantly decreased survival from GBM bearing mice similar to that of patient-resected GBM with high CFH levels whereby patients present with a faster rate to tumor recurrence. In summary, our study is the first to report an IDO-dependent non-metabolic regulation of CFH in tumor cells and highlights a new immunotherapeutic target in patients with incurable GBM.

Stable inhibition of interleukin 1 receptor type II in Ishikawa cells augments secretion of matrix metalloproteinases: possible role in endometriosis pathophysiology

Our previous studies showed a marked deficiency in interleukin 1 receptor type II (IL1R2) in the endometrial tissue of women with endometriosis, particularly in epithelial cells. We believe that such a deficiency in IL1R2, a potent and specific IL1 inhibitor, makes endometrial cells more sensitive to IL1 and less capable of buffering the cytokine’s effects, which may lead to functional changes that favor endometriosis development. The main objective of our study was to stably inhibit IL1R2 expression in endometrial cells in order to evaluate the role of IL1R2 deficiency in endometriosis pathophysiology. Stable clones of Ishikawa adenocarcinoma endometrial cells transfected with IL1R2 antisense and showing downregulation of IL1R2 protein expression, or with the empty expression vector alone and showing no noticeable difference in IL1R2 expression, were selected. The downregulation of IL1R2 expression in IL1R2 antisense transfectants when compared with control cells was confirmed by ELISA, Western blot and immunofluorescence. In these cells, IL1R2 expression was markedly reduced, compared with non-transfected cells or cells transfected with the empty vector, and there was a significant increase in the basal and the IL1-β (IL1B)-induced levels of matrix metalloproteinase (MMP)-2 and MMP-9 secretion. Furthermore, a significant decrease in IL1B-induced secretion of tissue inhibitor of MMPs-1, a known MMP-9 inhibitor, was observed. These in vitro data make plausible a role for IL1R2 deficiency in the capability of endometrial cells to invade the host tissue and develop in ectopic locations.