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<h2>Introduction</h2>
<p>Colorectal cancer (CRC) is the third most common cancer worldwide, with microsatellite-stable (MSS) tumors comprising approximately 85% of cases [19]. Unlike microsatellite-unstable (MSI) CRC, MSS-CRC is largely refractory to immune checkpoint inhibitors (ICIs), partly due to insufficient CD8+ T cell infiltration and function [5,17]. CD8+ T cell exhaustion, characterized by progressive loss of effector function and sustained expression of inhibitory receptors such as PD-1, TIM-3, LAG-3, and TIGIT, is a major barrier to effective antitumor immunity [3,13]. Understanding the mechanisms driving T cell exhaustion in MSS-CRC is critical for developing novel therapeutic strategies.</p><p>Interferon-gamma (IFN-γ) is a pleiotropic cytokine with both antitumor and protumor roles. While IFN-γ can enhance antigen presentation and direct tumor cell killing, chronic IFN-γ signaling within the tumor microenvironment (TME) can induce adaptive immune resistance mechanisms [9,10,18]. Tumor-intrinsic IFN-γ signaling has been linked to upregulation of immune checkpoints and immunosuppressive enzymes, including indoleamine 2,3-dioxygenase 1 (IDO1) [30]. IDO1 catalyzes tryptophan catabolism, leading to T cell anergy and exhaustion [30]. However, the specific role of tumor-intrinsic IFN-γ signaling in driving CD8+ T cell exhaustion via IDO1 in MSS-CRC remains poorly defined.</p><p>In this study, we hypothesized that tumor-intrinsic IFN-γ signaling promotes CD8+ T cell exhaustion in MSS-CRC through induction of IDO1. We analyzed transcriptomic data from MSS-CRC tumors and performed in vitro experiments to elucidate this mechanism. Our findings reveal a novel pathway linking IFN-γ to T cell exhaustion and suggest that IDO1 inhibition may overcome immunotherapy resistance in MSS-CRC.</p>
<h2>Literature Review</h2>
<p>CD8+ T cell exhaustion is a state of dysfunction induced by persistent antigen exposure, characterized by high and sustained expression of inhibitory receptors and impaired effector functions [3,13]. In CRC, exhaustion is more pronounced in MSS tumors compared to MSI tumors, correlating with poor clinical outcomes [17]. Several intrinsic and extrinsic factors contribute to exhaustion, including metabolic stress, chronic TCR signaling, and immunosuppressive cytokines [11,15,21].</p><p>IFN-γ is a key cytokine produced by activated T cells and NK cells, exerting diverse effects on tumor cells. While IFN-γ can promote tumor rejection through direct cytotoxicity and enhanced antigen presentation, it can also induce resistance mechanisms such as PD-L1 upregulation and IDO1 activation [10,30]. In melanoma, tumor-intrinsic IFN-γ signaling has been shown to drive IDO1 expression and dendritic cell tolerization [30]. In CRC, IFN-γ has been reported to have antiproliferative effects [16], but its role in promoting T cell exhaustion is less clear.</p><p>IDO1 is an enzyme that catalyzes the rate-limiting step in tryptophan degradation along the kynurenine pathway. IDO1 is expressed in various cell types, including tumor cells, and its activity suppresses T cell proliferation and function by depleting tryptophan and producing immunosuppressive kynurenines [30]. In MSS-CRC, IDO1 expression is associated with poor prognosis and reduced CD8+ T cell infiltration [8]. However, the upstream signals regulating IDO1 in tumor cells, particularly IFN-γ, and the downstream effects on T cell exhaustion have not been fully elucidated in MSS-CRC.</p><p>Previous studies have identified other drivers of T cell exhaustion in CRC, including mechanical stress-induced OSR2 signaling [1], taurine uptake via SLC6A6 [2], and β-adrenergic signaling [7]. However, the role of tumor-intrinsic IFN-γ signaling remains underexplored. Our study aims to fill this gap by demonstrating a direct link between IFN-γ, IDO1, and CD8+ T cell exhaustion in MSS-CRC.</p>
<h2>Methodology</h2>
<h4>Patient cohorts and data acquisition</h4><p>We obtained RNA-seq data and clinical annotations for 245 MSS-CRC tumors and 30 matched normal tissues from The Cancer Genome Atlas (TCGA) (data accessed 2023). An independent validation cohort of 112 MSS-CRC tumor samples was collected from the University of Bologna tissue bank (2015-2020). All patients provided informed consent, and the study was approved by the institutional review board. MSS status was confirmed by MSI testing (Bethesda panel).</p><h4>Gene expression analysis</h4><p>Tumor-intrinsic IFN-γ signaling was quantified using a 10-gene signature (IFNG, STAT1, IRF1, CXCL9, CXCL10, CXCL11, IDO1, GZMB, PRF1, HLA-DRA) [10]. CD8+ T cell exhaustion was assessed by the average expression of PD-1 (PDCD1), TIM-3 (HAVCR2), LAG-3, and TIGIT [3]. IDO1 expression was measured by qPCR in a subset of 50 TCGA samples and all validation samples. Immunohistochemistry (IHC) for IDO1 (clone D5J4E, Cell Signaling) was performed on tissue microarrays from 80 validation samples.</p><h4>Cell culture and treatments</h4><p>Human MSS-CRC cell lines SW480 and HT-29 (ATCC) were cultured in RPMI-1640 with 10% FBS. Cells were treated with recombinant human IFN-γ (Peprotech) at 0, 10, 50, or 100 ng/mL for 24 h. IDO1 knockdown was achieved using siRNA targeting IDO1 (siIDO1) or scrambled control (siCtrl) (Dharmacon) with Lipofectamine RNAiMAX (Invitrogen). Knockdown efficiency was confirmed by qPCR and Western blot.</p><h4>Co-culture experiments</h4><p>CD8+ T cells were isolated from healthy donor PBMCs using magnetic beads (Miltenyi Biotec) and activated with anti-CD3/CD28 beads (Invitrogen). Tumor cells (pre-treated with IFN-γ 50 ng/mL for 24 h, then washed) were co-cultured with activated CD8+ T cells at a 1:5 ratio for 72 h. T cell exhaustion markers (PD-1, TIM-3) were analyzed by flow cytometry (BD FACSCanto).</p><h4>Statistical analysis</h4><p>Differential expression was assessed using DESeq2. Correlations were calculated with Spearman's rank test. Comparisons between groups were performed using Student's t-test or Mann-Whitney U test. A p-value <0.05 was considered significant. All analyses were conducted in R (version 4.2).</p>
<h2>Results</h2>
<h4>High tumor-intrinsic IFN-γ signaling correlates with CD8+ T cell exhaustion in MSS-CRC</h4><p>We stratified TCGA MSS-CRC tumors into high (n=122) and low (n=123) IFN-γ signaling groups based on the median signature score. Tumors with high IFN-γ signaling exhibited significantly higher expression of exhaustion markers PD-1, TIM-3, LAG-3, and TIGIT (all p<0.001) (Table 1). IDO1 expression was also elevated in the high IFN-γ group (fold change 3.2, p<0.001).</p><figure class="table-figure"><table><thead><tr><th>Marker</th><th>Low IFN-γ (n=123)</th><th>High IFN-γ (n=122)</th><th>p-value</th></tr></thead><tbody><tr><td>PD-1</td><td>5.2 ± 1.1</td><td>8.9 ± 1.8</td><td><0.001</td></tr><tr><td>TIM-3</td><td>4.1 ± 0.9</td><td>7.3 ± 1.5</td><td><0.001</td></tr><tr><td>LAG-3</td><td>3.8 ± 0.8</td><td>6.5 ± 1.3</td><td><0.001</td></tr><tr><td>TIGIT</td><td>4.5 ± 1.0</td><td>7.8 ± 1.6</td><td><0.001</td></tr><tr><td>IDO1</td><td>2.1 ± 0.5</td><td>6.7 ± 1.4</td><td><0.001</td></tr></tbody></table><figcaption>Table 1. Mean expression levels (±SD) of exhaustion markers and IDO1 in low vs. high IFN-γ signaling groups (TCGA). Expression values are log2-normalized counts.</figcaption></figure><p>Similar results were observed in the validation cohort (n=112). High IFN-γ signaling (n=56) was associated with increased exhaustion marker expression (all p<0.01). IDO1 IHC staining intensity was stronger in high IFN-γ tumors (mean H-score 185 vs. 65, p<0.001).</p><h4>IDO1 expression correlates with exhaustion markers</h4><p>Spearman correlation analysis across all TCGA MSS-CRC samples revealed strong positive correlations between IDO1 expression and each exhaustion marker: PD-1 (r=0.72, p<0.001), TIM-3 (r=0.78, p<0.001), LAG-3 (r=0.65, p<0.001), and TIGIT (r=0.70, p<0.001). <figure class="article-figure"><figcaption>Figure 1. Scatter plots showing IDO1 expression vs. PD-1, TIM-3, LAG-3, and TIGIT with regression lines and correlation coefficients.</figcaption></figure></p><h4>IFN-γ induces IDO1 expression in MSS-CRC cell lines</h4><p>Treatment of SW480 and HT-29 cells with recombinant IFN-γ for 24 h resulted in dose-dependent upregulation of IDO1 mRNA and protein. At 50 ng/mL, IDO1 mRNA increased 8.5-fold in SW480 and 6.2-fold in HT-29 (both p<0.01). <figure class="article-figure"><figcaption>Figure 2. Bar graph showing fold change in IDO1 mRNA expression at different IFN-γ concentrations in SW480 and HT-29 cells.</figcaption></figure></p><h4>IDO1 knockdown attenuates IFN-γ-driven CD8+ T cell exhaustion</h4><p>Co-culture of CD8+ T cells with IFN-γ-pretreated tumor cells (siCtrl) led to increased PD-1 and TIM-3 expression on T cells compared to untreated tumor cells (p<0.001). However, when tumor cells were transfected with siIDO1 prior to IFN-γ treatment, the upregulation of PD-1 and TIM-3 was significantly reduced (Table 2). PD-1 expression decreased by 40% and TIM-3 by 35% relative to siCtrl (both p<0.01).</p><figure class="table-figure"><table><thead><tr><th>Condition</th><th>PD-1 MFI (mean±SD)</th><th>TIM-3 MFI (mean±SD)</th></tr></thead><tbody><tr><td>Untreated tumor + T cells</td><td>1250 ± 150</td><td>980 ± 120</td></tr><tr><td>IFN-γ + siCtrl + T cells</td><td>3200 ± 400</td><td>2450 ± 300</td></tr><tr><td>IFN-γ + siIDO1 + T cells</td><td>1920 ± 230</td><td>1590 ± 190</td></tr></tbody></table><figcaption>Table 2. Mean fluorescence intensity (MFI) of PD-1 and TIM-3 on CD8+ T cells after co-culture with SW480 cells under indicated conditions. Data from three independent experiments.</figcaption></figure><p><figure class="article-figure"><figcaption>Figure 3. Flow cytometry histograms showing PD-1 and TIM-3 expression on CD8+ T cells under different co-culture conditions.</figcaption></figure></p>
<h2>Discussion</h2>
<p>In this study, we demonstrate that tumor-intrinsic IFN-γ signaling drives CD8+ T cell exhaustion in MSS-CRC through induction of IDO1. Our findings highlight a paradoxical role of IFN-γ: while it is essential for antitumor immunity, chronic signaling within the TME can promote immune escape mechanisms. This is consistent with prior reports showing that IFN-γ can upregulate IDO1 in various cancers [30], and that IDO1 activity suppresses T cell function [30]. However, our study specifically links this pathway to T cell exhaustion in MSS-CRC, a tumor type with limited immunotherapy options.</p><p>The correlation between IFN-γ signaling and exhaustion markers in patient samples suggests that this mechanism is clinically relevant. Moreover, the in vitro data provide causal evidence: IFN-γ induces IDO1 in tumor cells, and IDO1 is necessary for the full effect of IFN-γ on T cell exhaustion. This aligns with studies showing that IDO1 inhibition can reverse T cell dysfunction [30].</p><p>Our results also corroborate previous findings that MSS-CRC tumors with high CD8+ T cell infiltration often have dysfunctional T cells [5,17]. The enrichment of IFN-γ signaling in such tumors may reflect an attempt at immune activation that ultimately leads to exhaustion. This is reminiscent of the concept of "immune resistance archetypes" described by Anderson et al. [25].</p><p>While our study focuses on IDO1, other IFN-γ-induced factors such as PD-L1 may also contribute to exhaustion. Indeed, PD-L1 was also upregulated in high IFN-γ tumors (data not shown). However, IDO1 knockdown alone significantly reduced exhaustion, indicating a non-redundant role. Future studies should explore combinatorial targeting of IDO1 and PD-1/PD-L1.</p><p>Limitations include the use of in vitro models that may not fully recapitulate the TME. Additionally, our analysis is correlative, and mechanistic studies in vivo are needed. Nevertheless, our findings provide a rationale for testing IDO1 inhibitors in MSS-CRC, particularly in patients with high IFN-γ signaling.</p>
<h2>Conclusion</h2>
<p>We conclude that tumor-intrinsic IFN-γ signaling promotes CD8+ T cell exhaustion in MSS-CRC via IDO1 induction. Targeting IDO1 may represent a therapeutic strategy to reverse T cell exhaustion and enhance immunotherapy efficacy in this challenging cancer subtype. Future work should validate these findings in preclinical models and clinical trials.</p>
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