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

Pharmacological inhibition of IDO amplifies the T_CD8 response to T Ag’s most immunodominant epitope.

<p>WT mice were treated with 1-D-MT (10 mg/mouse total) or vehicle and injected <i>i.p.</i> with C57SV cells. Nine days later, splenic T_CD8 were examined <i>ex vivo</i> for IFN-γ accumulation following restimulation with C57SV cells (A and C) or synthetic peptides corresponding to T Ag epitopes (B and D). T Ag-specific T_CD8 frequencies were determined after subtracting background and expressed as mean ± SEM of 7 mice per group (A and B). These values were used to calculate the absolute number of T Ag-specific T_CD8 present within each spleen (C and D).</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

Genetic deficiency of IDO enhances the T_CD8 response to T Ag’s most immunodominant epitope.

<p>WT and IDO^−/− mice were injected <i>i.p.</i> with syngeneic, SV40-transformed C57SV cells. Nine days later, splenic T_CD8 were examined <i>ex vivo</i> for IFN-γ accumulation following brief restimulation (of 5 hours in duration) with C57SV cells used at 2×10⁵ cells/well (A) or synthetic peptides corresponding to T Ag-derived epitopes (B). Background obtained from wells receiving no peptide was subtracted and values are expressed as mean ± standard error of the mean (SEM) of multiple mice per group (n = 12 and n = 10 for WT and IDO^−/− mice, respectively) pooled from independent experiments yielding similar results.</p

Ki-67 expression by T Ag-specific T_CD8 and non-specific proliferative responses of IDO^−/− T cells.

<p>(A) Splenocytes from WT and IDO^−/− mice inoculated with C57SV cells were restimulated for 5 hours with synthetic peptides corresponding to site I or site IV. Cells were then stained for surface CD8, intracellular IFN-γ and intracellular Ki-67 as described in Materials and Methods. Representative FACS plots after live gating on CD8⁺ events are shown. Quadrants’ positions were set based on staining with an isotype control. (B) WT and IDO^−/− splenocytes were left untreated or stimulated with PHA, ConA or a mitogenic anti-CD3 mAb. T cell proliferation was measured by tritiated thymidine incorporation after 72 hours.</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

Primary and recall T_CD8 responses of WT and IDO^−/− mice to influenza A virus.

<p>Mice were injected with the PR8 strain of IAV. Seven days later, splenocytes were prepared and restimulated <i>ex vivo</i> with IAV-infected DC2.4 cells (A) or with synthetic peptides corresponding to IAV epitopes (B). T_CD8 responses were then quantified by ICS for IFN-γ. To assess recall responses, WT and IDO^−/− mice were primed with PR8 (H1N1) and boosted, one month later, with the X31 reassortant virus (H3N2). Seven days after the boost, the frequencies of total T_CD8 synthesizing IFN-γ in response to IAV-infected DC2.4 cells (C) and those recognizing individual IAV-derived epitopes (D) were determined by ICS. Data are shown as mean IFN-γ⁺ cells as a percentage of CD8⁺events (± SEM) obtained from indicated numbers of mice per group, which were pooled from independent experiments.</p

Peptides used in this study.

a<p>SV40: simian virus 40;</p>b<p>IAV: influenza A virus (Puerto Rico/8/34 strain);</p>c<p>HSV-1: herpes simplex virus-1.</p