T Cells Expressing CD19-Specific Chimeric Antigen Receptors Are Inhibited By Indoleamine 2,3-Dioxygenase in Tumors
Authors: Ninomiya Soranobu , Huye Leslie E , Savoldo Barbara , Dotti Gianpietro , Heslop Helen E. , Brenner Malcolm K. , Rooney Cliona M , Ramos Carlos A. 
Published: November 2019

Abstract
Indoleamine 2,3-dioxygenase (IDO) is an intracellular enzyme that mediates the metabolism of tryptophan to kynurenines, which have an inhibitory activity on immune cells. IDO-positive tumors thus establish a microenvironment in which NK and T cells are inactivated, and high IDO expression by tumor cells and high serum kynurenine levels correlate with poor prognosis in diffuse large B-cell lymphoma patients.
CD19-specific chimeric antigen receptor T cell (CART) therapy is a promising new approach against B-cell malignancies but not every tumor responds. To determine whether the presence of IDO in tumors affects CART activity, we first investigated the expression of IDO by the human B-cell lymphoma cell lines Raji, Daudi, BJAB and Jeko-1. We found that only Jeko-1 expresses IDO and produces kynurenine natively. IDO was not expressed by the other cell lines, even after exposure to IFNγ, a known IDO inducer. Based on these results, we chose Raji as a baseline IDO-negative cell line and made an IDO-positive Raji clone by retroviral transduction with human IDO cDNA (Raji-IDO); a clone transduced with an empty vector served as control (Raji-control). We injected SCID mice subcutaneously in opposite flanks with luciferase-transduced Raji-control and Raji-IDO cells. Seven days later, we injected human non-transduced T cells (NTs) or CARTs intravenously. In the NT group, tumors had similar growth on both sides. In contrast, in the CART group, Raji-control tumors had significantly slower growth than Raji-IDO tumors (3.1 ± 1.1×108 and 20 ± 7.3×108 bioluminescence units [BU] at day 7, respectively, P = 0.03). We also found that CARTs significantly inhibited Raji-control tumor growth (NT: 27 ± 6.8×108 vs CART: 3.1 ± 1.1×108 BU at day 7, P = 0.02), but did not affect Raji-IDO tumors (NT: 24 ± 5.4×108 vs CART: 20 ± 7.3×108 BU at day 7, P = 0.35). In another experiment, Raji-IDO cells were injected subcutaneously, and mice were treated with an IDO inhibitor (1-methyl-tryptophan, 1-MT), CARTs, or both. The combination treatment produced significantly better tumor control than either single therapy (1-MT: 45 ± 6.8×108 and CART: 22 ± 4.6×108 vs both: 8.2 ± 3×108 BU at day 7, P = 0.001 and 0.04, respectively). Thus, the IDO inhibitor protects CARTs against the deleterious effects of IDO-positive tumors.
To investigate potential mechanisms for CART inhibition by IDO, we assessed the effect of kynurenine and found that even low concentrations (12.5 µM) inhibited CART proliferation in response to IL-2, IL-7, IL-15 or CD19-positive targets, although there was no effect on proliferation of B-cell lymphoma cell lines at this kynurenine concentration. CART apoptosis was also increased by kynurenine (8.6 ± 0.6%, 13.9 ± 2.2%, and 33.5 ± 10.6% annexin V-positive cells with 0, 12.5, or 25 µM kynurenine). In coculture of CARTs with wild-type Raji cells, the latter were eliminated by day 6 in the absence of kynurenine, but increased in numbers (in parallel with a decrease in CARTs) in its presence. Kynurenine also inhibited the release of IFNγ (13,143 ± 848 pg/mL vs 2,663 ± 1,873 pg/mL, P = 0.02) and IL-2 (718 ± 355 pg/mL and 199 ± 165 pg/mL, P = 0.03) by CARTs. Expression of granzyme B, PD-1 and CTLA-4 on CARTs was not significantly affected.
Fludarabine has been reported to downregulate IDO expression in tumors and this drug is used in many lymphodepleting regimens that are administered before CART infusion in an effort to augment the efficacy of these therapies. However, the beneficial mechanism of lymphodepleting chemotherapy drugs is not fully understood. Therefore, we also measured the effect of fludarabine and mafosfamide (a cyclophosphamide analog) on IDO expression by Jeko-1 cells. We found that both drugs downregulate IDO expression by Jeko-1, even in the presence of IFNγ.
In summary, expression of IDO by tumor cells inhibits CART activity, likely because kynurenine is produced and has negative effects on proliferation and cytokine secretion by CARTs. Fludarabine and cyclophosphamide downregulate IDO expression in tumors and this effect may contribute to the benefits of lymphodepletion before CART therapy. Direct IDO inhibitors may further improve clinical CART activity against IDO-positive tumors.

Disclosures
No relevant conflicts of interest to declare.