The Roles of Aryl Hydrocarbon Receptor in T Cells at IDO-Positive Tumor Microenvironment

Introduction:Aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor well known to generate biological responses to dioxins. A recent study indicated that kynurenine was an endogenous ligand of AhR and was associated with suppression of the antitumor response. The essential amino acid tryptophan metabolism proceeds via the enzymes IDO, which generates the immunosuppressive metabolites, such as kynurenine. Accumulation of these tryptophan derivatives block antigen-specific T-cell proliferation and induces T-cell death through the AhR. IDO is known to be produced by tumor cells and by some immune cells, such as dendritic cells and macrophages, which reside in tumor-draining lymph nodes or are recruited to tumors. IDO is overexpressed in several human cancers, including prostate, breast, brain, and hematologic malignancies.We previously showed that both IDO expression by tumor cells and high serum kynurenine levels correlate with poor prognosis in non-Hodgkin lymphoma patients. We also revealed that tumor IDO activity can inhibit CD19-specific chimeric antigen-receptor (CAR) T-cells (CARTs) therapy through the action of tryptophan metabolites (Blood. 2015; 125(25): 3905-3916). Therefore, we focused on the AhR in T-cells at IDO-positive tumor microenvironment.

Methods and Results:We collected PBMCs from healthy blood donors, and activated the cells with anti-CD3 and anti-CD28 Abs, which mimic physiological T-cell activation. We measured the expression of AhR in activated T-cells using by flow cytometry, and found that the range of expression were from 4.5% to 38%. We then studied the effect of kynurenine on the proliferation of T-cells. The proliferation was assessed by MTS assay. We found that kynurenine significantly inhibited the proliferation of T-cells with high expression of AhR. The percentage inhibition with kynurenine 25µM was 33%. While T-cells with low expression of AhR were not inhibited with even 50µM of kynurenine. To investigate potential mechanisms of T-cell inhibition by kynurenine, we assessed the effect of kynurenine on T-cell apoptosis. We found that kynurenine was also associated with increased the apoptosis of T-cells with high AhR, as assessed by Annexin-V staining (percent Annexin-V positive and 7-AAD negative cells in 0 and 25 µM kynurenine was 11.8% and 36%, respectively). To evaluate the effect of inhibiting AhR in T-cells, we used AhR inhibitor (CH-223191). We found that CH-223191 could blocked the inhibitory effects of kynurenine on T-cells. The percent Annexin-V positive and 7-AAD negative cells in 25 µM kynurenine plus CH-223191 was 22.5%. Next, to assess the functional effects of tryptophan metabolites on the T-cells, we cocultured T-cells (1x10⁶) with GFP-transduced wild-type Raji cells or IDO-transduced Raji cells (1x10⁶). After 72 hours, cells were stained with CD8 antibody to distinguish tumor (GFP+ CD8-) and T-cells (GFP- CD8+). We found that the percentage of T-cells with high AhR in culture with IDO negative and positive Raji cells was 46.7% and 61.4%, respectively, while the percentage of T-cells with low AhR was 41.6% and 39.4%. These data showed that only T-cells with high AhR were inhibited at IDO-positive tumor microenvironment. Next, bone marrow (BM) samples were obtained from multiple myeloma (MM) patients at diagnosis. We could generated the activated T-cells from 10 of 12 BM samples. The expression of AhR in T-cells was ranged from 3.3% to 26.6%. We found that there was a positive correlation between the expression of AhR and the proportion of plasma cells in BM (r=0.76, P=0.04).

Discussion:The expression of AhR in activated T cells play an important role in the effect of kynurenine, which is a metabolite produced by IDO. A few clinical trials are evaluating the effects of IDO inhibitors that competitively block the degradation of tryptophan to kynurenine by the enzyme. Because anti-PD-1 and anti-CTLA-4 therapies, which block directly the inhibitory signal in T-cells, have been getting some clinical benefits against such as melanoma and Hodgkin lymphoma. Therefore, AhR in T cells might be a target for IDO-positive hematological malignancies.