GITR Ligation with GITR Ligand On Leukemic Cells Suppresses Induction of Leukemia-Associated Antigen Specific T Cells by Increasing Indoleamine 2, 3-Dioxygenase (IDO) Activity Leading to Kynurenine Secretion.
Authors: Kondo Yukio , Espinoza Luis , Katagiri Takamasa , Qi Zhirong , Nakao Shinji 
Published: October 2019

Abstract
Abstract 3557
Poster Board III-494
Immunization of allogeneic stem cell transplant (SCT) recipients with leukemia-associated antigens (LAAs) is an attractive approach to the augmentation of graft-versus-leukemia (GVL) effect. However, the induction of CTLs specific to LAAs is hampered by various inhibitory molecules expressed on leukemic cells that restrain the T cell function in connection with their receptors on T cells. Even if the cellular immunity is rebuilt after SCT by T cells of the donor origin, overcoming such an escape mechanism is required to effectively induce the CTLs specific to TAAs by vaccination after allogeneic SCT. Glucocorticoid-induced TNFR-related protein (GITR) belongs to the TNF receptor superfamily and is expressed on NK cells, CD25+ regulatory T cells and activated T cells. The binding of GITR ligand (GITRL) on leukemic cells to GITR on NK cells restrains NK cell activity but the influence on T cells of the GITR/GITRL binding has not been clarified. Myeloid dendritic cells derived from myeloid leukemic cells express GITRL which inhibits induction of LAA-specific CTLs (Blood 2008; 112:817a). The mechanisms of the negative effect on the induction of LAA-specific T cells through the GITR/GITRL interaction was investigated to improve the efficiency of the CTL induction. The expression of GITRL was observed on leukemic cells from 9 of 16 patients with myeloid leukemia and a monocytic leukemia cell line THP-1, and soluble GITRL (sGITRL) was detectable in the serum from 3 of 5 patients as well as in the culture supernatant of THP-1 cells. CFSE-labeled pan T cell, CD4+ T cell and CD8+ T cell proliferation in response to microbeads coated with anti-CD3 and anti-CD28 monoclonal antibodies (CD3/CD28 microbeads) was suppressed to 55.0%, 63.6%, 65.8% of the controls in a culture supernatant of THP-1 cells, and was restored to 86.9%, 65.1% and 76.8% respectively by addition of sGITR to block the binding of sGITRL in the supernatant and GITR on T cells. Flow cytometry detected GITRL in exosomes, which express HLA class II, purified from the culture supernatant of THP-1 with anti-HLA class II antibody-coated microbeads, and CFSE-labeled pan T cell, CD4+ T cell and CD8+ T cell proliferation was restrained as well by the addition of GITRL+ exosomes in a dose dependent manner (27.6%, 54.1%, 27.9% reduction of proliferation with 10 μl exosome, respectively). Indoleamine 2, 3-dioxygenase (IDO) activity in plasmacytoid DC (pDC) is negatively correlated with the activity of CD4+ T cells induced by their interaction with the pDC through the GITR/GITRL interaction in a mouse model. Kynurenine (Kyn), a metabolite of tryptophan in leukemic cells that is broken down by IDO, suppressed CFSE-labeled pan T cell, CD4+ T cell and CD8+ T cell proliferation in response to CD3/CD28 microbeads in a dose dependent manner (24.5%, 12.3%, 18.3% reduction in the proliferation at 100 μM, respectively). Significantly higher concentrations of Kyn were detected in the supernatant of THP-1 cells after incubation in the presence of sGITR than a control, and the production of Kyn was suppressed by the addition of an IDO inhibitor, 1-Methyl Tryptophan (1MT) (Fig). Moreover, the addition of sGITR to leukemic cells from five patients with AML induced Kyn (Fig). These findings indicate that GITRL on leukemic cells and sGITRL secreted by leukemic cells as an exosome protein suppress the induction of LAA-specific CTLs by directly binding GITR on LAA-specific CTLs, increasing the IDO activity in leukemic cells and inducing Kyn secretion from leukemic cells. The administration of anti-IDO agents or anti-GITRL blocking Abs combined with LAA vaccination may therefore effectively induce LAA-specific T cells in SCT recipients.
Fig GITR/GITRL binding induces kyn secretion from THP-1 cell and primary AML cells. Fig. GITR/GITRL binding induces kyn secretion from THP-1 cell and primary AML cells.

Disclosures:
No relevant conflicts of interest to declare.