IDO downregulation sensitized A549 xenografts to pemetrexed treatment.

<p><b>A</b>: Ten million A549 cells transfected with control shRNA (clone NC-3) or anti-IDO shRNA (clone 2–18) were injected into flanks of SCID mice. Once tumors reached ~300 mm³ animals were injected with 100,000 IU IFNγ i.p. (twice a week for four weeks) and 50 mg/kg pemetrexed i.p. (once per week for four weeks). Animals transplanted with A549 clonal cells transfected with control shRNA or anti-IDO shRNA received four weeks of IFNγ treatment alone <b>(B)</b> or IFNγ and pemetrexed <b>(C)</b>. Tumor growth was monitored with caliper measurement to estimate tumor volume. *Significant difference, Student's <i>t</i>-test, <i>p</i><0.05.</p

Concurrent IDO and TS downregulation sensitizes A549 cells to 5FUdR more than TS knockdown alone.

<p>A549 cells were transfected with control or anti-thymidylate synthase (anti-TS) siRNA, treated with IFNγ (25 ng/ml) for 48 h, with 5FUdR (40 nM) for 72 h, and then enumerated. Bars indicate mean proliferation relative to appropriate controls ± SD (n = 3). <b>A</b>: Proliferation of clonal A549 cell populations induced with IFNγ and then treated with 5FUdR, but untransfected with siRNA of any kind. <b>Gray bars</b>: clones containing anti-IDO shRNA. <b>White bars</b>: clones containing non-targeting control shRNA. <b>B</b>: Proliferation of the same clonal A549 cell populations transfected with control non-targeting siRNA, TS siRNA #3, or TS siRNA #4, induced with IFNγ, and then treated with 5FUdR. Bars represent values normalized to values obtained from clones treated with IFNγ but untreated with pemetrexed or siRNA; those cells were considered to have a proliferation value of 100% after IFNγ treatment. <b>Gray bars</b>: clones containing anti-IDO shRNA. <b>White bars</b>: clones containing non-targeting control shRNA. *Significant difference, Student's <i>t</i>-test, <i>p</i><0.05. Data presented a representative experiment from two independent experiments. Results are normalized to control cells not treated with 5FUdR, but with treated with IFNγ. <b>Panel C</b>: shows the pooled results from panel A and B.</p

1. ON-Target Plus^® TS and BRCA2 siRNA target mRNA sequences.

<p>1. ON-Target Plus^® TS and BRCA2 siRNA target mRNA sequences.</p

A549 clone sensitivity to methoxyamine (3 mM) before and after IDO induction.

<p>Proliferation of each of 5 individual A549 cell clonal populations before (<b>Panel A</b>) and after (<b>Panel B</b>) IDO induction with IFNγ. A549 clonal populations were cultured with or without IFNγ (25 ng/ml) for 48 h. Cultured medium was then replaced with fresh growth medium containing Methoxyamine (MX) (3 mM) and cells were allowed to proliferate for 72 h. Cells were then trypsinized and live cells were enumerated. <b>White bars</b>: A549 clones transfected with scrambled, non-targeting control shRNA. <b>Gray bars</b>: A549 cells transfected with anti-IDO shRNA. Each bar represents the mean of 3 values (<i>n</i> = 3 for determination of each value) ± SD. Results are normalized to control cells not treated with methoxyamine, without (panel A) or with (panel B) IFNγ treatment. <b>Panel C</b>: Induction of IDO in A549 clonal cell populations induces resistance to MX (3 mM). Results were obtained from 3 or 2 independent clonal cell populations with scrambled, non-targeting control shRNA or anti-IDO shRNA, respectively. Each bar represents a mean of 9 (white bars) or 6 (black bars) values ± SEM, *Significant difference, Student's <i>t</i>-test, <i>p</i><0.05. <b>Panel D</b>: Relationship between IDO protein level (relative to actin) and resistance to methoxyamine (MX)(proliferation relative to untreated control cells). The R² value of 0.83 represents a moderate positive relationship.</p

Suppression of Immunodominant Antitumor and Antiviral CD8⁺ T Cell Responses by Indoleamine 2,3-Dioxygenase

<div><p>Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-degrading enzyme known to suppress antitumor CD8⁺ T cells (T_CD8). The role of IDO in regulation of antiviral T_CD8 responses is far less clear. In addition, whether IDO controls both immunodominant and subdominant T_CD8 is not fully understood. This is an important question because the dominance status of tumor- and virus-specific T_CD8 may determine their significance in protective immunity and in vaccine design. We evaluated the magnitude and breadth of cross-primed T_CD8 responses to simian virus 40 (SV40) large T antigen as well as primary and recall T_CD8 responses to influenza A virus (IAV) in the absence or presence of IDO. IDO^−/− mice and wild-type mice treated with 1-methyl-D-tryptophan, a pharmacological inhibitor of IDO, exhibited augmented responses to immunodominant epitopes encoded by T antigen and IAV. IDO-mediated suppression of these responses was independent of CD4⁺CD25⁺FoxP3⁺ regulatory T cells, which remained numerically and functionally intact in IDO^−/− mice. Treatment with L-kynurenine failed to inhibit T_CD8 responses, indicating that tryptophan metabolites are not responsible for the suppressive effect of IDO in our models. Immunodominant T antigen-specific T_CD8 from IDO^−/− mice showed increased Ki-67 expression, suggesting that they may have acquired a more vigorous proliferative capacity <i>in vivo</i>. In conclusion, IDO suppresses immunodominant T_CD8 responses to tumor and viral antigens. Our work also demonstrates that systemic primary and recall T_CD8 responses to IAV are controlled by IDO. Inhibition of IDO thus represents an attractive adjuvant strategy in boosting anticancer and antiviral T_CD8 targeting highly immunogenic antigens.</p></div

Concurrent downregulation of IDO and BRCA2 did not sensitize A549 to 5FUdR.

<p>A549 clonal cells transfected with either scrambled shRNA (NC-3) or anti-IDO shRNA (2–18) were transiently transfected with breast cancer type-2 susceptibility protein (BRCA2) siRNA, induced with IFNγ (25 ng/ml) for 24 h, and then treated with 5FUdR (40 nM) for 72 h, at which time live cells were enumerated. Simultaneous downregulation of IDO and BRCA2 did not sensitize A549 to the TS-targeting drug 5FUdR to a greater degree than the knockdown of either gene alone. Bars represent the means of 3 independent measurements of cells (with or without downregulation of IDO) after BRCA2 siRNA transfection + 5FUdR treatment (<i>n</i> = 3 for each measurement) ± SD. Bars were normalized to values obtained from clones treated with IFNγ but untreated with 5FUdR or siRNA; those cells were considered to proliferate at a 100% level after IFNγ treatment. *Significant difference, Student's <i>t</i>-test, <i>p</i><0.05.</p

The cross-primed T_CD8 response to T Ag is augmented in the absence of IDO.

<p>WT and IDO^−/− mice were injected <i>i.p.</i> with allogeneic T Ag⁺ KD2SV cells. Nine days later, the frequencies (A) and absolute numbers (B) of T Ag-specific T_CD8 recognizing site IV, total T Ag-specific T_CD8 that synthesize IFN-γ after incubation with C57SV cells, and total alloreactive T_CD8 that produce IFN-γ after incubation with KD2SV cells were determined by ICS as described in Materials and Methods. Background obtained from wells receiving no peptide was subtracted and values are presented as mean ± SEM of 8 mice/group pooled from independent experiments.</p

A549 clone sensitivity to Gemcitabine and 5FUdR before and after IDO induction.

<p>Proliferation of each of 5 individual A549 cell clonal populations before <b>(Panel A and C)</b> and after <b>(Panel B and C)</b> IDO induction with IFNγ. A549 clonal populations were cultured with or without IFNγ (25 ng/ml) for 48 h, treated with gemcitabine (10 nM) for 72 h, and then enumerated. <b>White bars</b>: A549 clones transfected with scrambled, non-targeting control shRNA. <b>Gray bars</b>: A549 cells transfected with anti-IDO shRNA. Each bar represents a mean of 9 (white bars) or 6 (black bars) values ± SD for Panels A and B and SEM for panel C, (*<i>p</i><0.05). Results are normalized to control cells not treated with Gemcitabine, without (panel A) or with (panel B) IFNγ treatment. <b>Panels D-F</b>: Proliferation of each of 5 individual A549 cell clonal populations before and after IDO induction with IFNγ. A549 clonal populations were cultured with or without IFNγ (25 ng/ml) for 48 h and, 5FUdR (200 nM) for 72 h, and then enumerated. <b>White bars</b>: A549 clones transfected with scrambled, non-targeting control shRNA. <b>Gray bars</b>: A549 cells transfected with anti-IDO shRNA. Each bar represents a mean of 9 (white bars) or 6 (black bars) values ± SD for Panels D and E and SEM for panel F ± SD. *Significant difference, Student's <i>t</i>-test, <i>p</i><0.05. Results are normalized to control cells not treated with 5FUdR, without (panel A) or with (panel B) IFNγ treatment.</p

Concurrent IDO and TS downregulation sensitizes A549 cells to pemetrexed more than IDO knockdown alone.

<p>A549 cells were transfected with scrambled, non-targeting control siRNA or anti-thymidylate synthase (anti-TS) siRNA, treated with IFNγ (25 ng/ml) for 48 h, pemetrexed (30 nM) for 72, and then enumerated. Bars indicate mean relative cell numbers (n = 3 ± SD). <b>A</b>: Proliferation of clonal A549 cell populations induced with IFNγ and then treated with pemetrexed, but untransfected with siRNA of any kind. <b>Gray bars</b>: clones containing anti-IDO shRNA. <b>White bars</b>: clones containing scrambled, non-targeting control shRNA. <b>B</b>: Proliferation of the same clonal A549 cell populations transfected with scrambled, non-targeting control siRNA, TS siRNA #3, or TS siRNA #4, induced with IFNγ, and then treated with pemetrexed. Bars represent values normalized to values obtained from clones treated with IFNγ but untreated with pemetrexed or siRNA; those cells were each considered to have a proliferation value of 100% after IFNγ treatment. <b>Gray bars</b>: clones containing anti-IDO shRNA. <b>White bars</b>: clones containing non-targeting control shRNA. *Significant difference, Student's <i>t</i>-test, <i>p</i><0.05. Data presented a representative experiment from two independent experiments. Results are normalized to control cells not treated with pemetrexed, but with treated with IFNγ. <b>Panel C</b>: shows the pooled results from panel A and B.</p

nTreg cells do not mediate the suppressive effect of IDO on the site IV-specific response.

<p>(A) Splenocytes from indicated numbers of naïve WT and IDO^−/− mice were stained for surface CD4 and intracellular FoxP3. In separate experiments, WT and IDO^−/− mice were inoculated with C57SV cells followed, 9 days later, by cytofluorimetric determination of their splenic nTreg cell frequencies, which were used to calculate the absolute number of nTreg cells within each spleen. Representative FACS plots are shown in addition to mean nTreg cell frequencies and absolute numbers ± SEM for each group. (B) WT CD4⁺CD25⁻ conventional T cells were co-cultured with γ-irradiated bone marrow-derived DCs and stimulated with an anti-CD3 mAb in the presence of varying numbers of CD4⁺CD25⁺ nTreg cells magnetically purified from WT and IDO^−/− mice. T cell proliferation was measured by tritiated thymidine incorporation after 72 hours. (C) WT and IDO^−/− mice were injected with an anti-CD25 mAb (clone PC61) or PBS 3 days before they were inoculated with C57SV cells. Nine days later, site IV-specific T_CD8 were enumerated by ICS for IFN-γ. Values are presented as mean ± SEM for indicated numbers of mice per group pooled from 3 independent experiments.</p