AhR blockade rewires myeloid–T cell crosstalk and boosts anti-PD-1 response in preclinical tumor models

Blocking the aryl hydrocarbon receptor (AhR), a transcription factor downstream of tryptophan metabolism, can reprogram the tumor immune microenvironment and strengthen responses to anti-PD-1 immunotherapy, according to a new preclinical study published in Experimental Hematology & Oncology. The research, led by Dong Kwon Kim, Youngtaek Kim and colleagues with senior author Byoung Chul Cho, combined genetic knockout mice, a novel pharmacological inhibitor called DA-4505, and single-cell RNA sequencing across lung, colorectal, and breast cancer models. The work addresses a long-standing problem in cancer immunotherapy: many patients fail to achieve durable benefit, particularly those with immunologically cold tumors dominated by suppressive myeloid cells and regulatory T cells.

The study builds on the failure of IDO1 inhibitors in the clinic. IDO1 catalyzes the degradation of tryptophan into kynurenine, an endogenous AhR ligand, and the phase III ECHO-301 trial of epacadostat combined with immunotherapy failed to demonstrate significant clinical benefit, leading to the suspension or discontinuation of most IDO1 inhibitor trials. Because kynurenine production and downstream immunosuppressive signaling can persist through alternative metabolic pathways, the authors reasoned that direct AhR inhibition might suppress immunoregulatory signaling more completely than blocking IDO1 alone. Upon ligand binding, AhR translocates to the nucleus and regulates genes involved in immune modulation, metabolism, and cell differentiation, including programs that promote regulatory T-cell differentiation and suppressive myeloid populations.

To dissect the role of myeloid-cell AhR signaling, the team generated two conditional knockout models: CD11cCre-AhRLoxP mice targeting the dendritic cell lineage and LysMCre-AhRLoxP mice targeting macrophage-lineage cells. Under steady-state conditions, AhR deletion in either compartment significantly enhanced interferon-γ expression in T cells and increased dendritic cell abundance compared with wild-type mice. The frequency of regulatory T cells and polymorphonuclear myeloid-derived suppressor cells fell significantly in both knockout groups, while M1 macrophage abundance and PD-1-expressing T cells rose in the dendritic cell knockout group. These results established a baseline of systemic immune activation that the authors then traced into tumor-bearing settings.

In tumor models, conditional AhR ablation delayed progression of LLC1 and TC-1 lung cancers. Tumors from knockout mice showed increased effector-memory T-cell infiltration, enhanced PD-1 and interferon-γ expression in CD4-positive T-cell subsets, and reduced monocytic myeloid-derived suppressor cells. When the researchers treated these mice with anti-PD-1 antibody, tumor growth suppression was further enhanced in both macrophage-lineage and dendritic cell knockouts compared with wild-type controls, indicating synergy between AhR loss and PD-1 blockade. Notably, the macrophage-lineage knockout was most effective in the LLC1 model, whereas the dendritic cell knockout showed stronger suppression in the TC-1 model, an early hint that the dominant immune mechanism varies by tumor context.

For pharmacological validation, the team used DA-4505, a novel AhR inhibitor provided by Dong-A ST and selected for in vivo assessment based on the potency, selectivity, and minimal direct cytotoxicity of its active form, SB3181. In an immune checkpoint inhibitor–refractory LLC1 model, the combination of DA-4505 with anti-PD-1 produced significant improvements in survival and tumor control that were exclusive to the combination group. Immune profiling revealed that macrophages accounted for 45 percent of tumor-infiltrating immune cells while CD8 T cells comprised only 2 percent. Depletion experiments confirmed this hierarchy: macrophage depletion produced the most pronounced attenuation of tumor control, with smaller effects from NK-cell and CD4 T-cell depletion and comparatively little from CD8 T-cell depletion.

The picture reversed in the ICI-sensitive TC-1 model, where the tumor microenvironment contained five times the proportion of CD8 T cells seen in LLC1. Here, CD8 T-cell depletion most strongly reversed tumor control, while macrophage depletion had a smaller effect. Although no complete responses occurred with any single or dual immunotherapy group, adding DA-4505 to a standard-of-care regimen of cisplatin, pemetrexed, and anti-PD-1 produced complete responses in both the DA-4505-alone maintenance group (1 of 9 mice) and the DA-4505 add-on group (2 of 10), and these responses persisted after treatment cessation. During maintenance therapy, DA-4505-containing regimens elevated CD8 T-cell and effector CD8 populations between the interim and end-of-treatment time points, and interferon-γ expression in CD4 T cells was significantly higher in the add-on group.

Therapeutic activity extended to other tumor settings. In the CT26 colorectal cancer model, combination treatment showed the strongest tumor growth inhibition, durable complete responses after treatment withdrawal, and a survival benefit, with depletion experiments supporting a major contribution from CD8 T cells. In the aggressive 4T1 orthotopic triple-negative breast cancer model, which spontaneously metastasizes to the lung, combination treatment enhanced primary tumor inhibition, improved survival, and reduced spontaneous lung metastatic burden. In a perioperative setting, combination treatment reduced tumor relapse and improved overall survival after surgical tumor resection, accompanied by increased infiltration of CD4 T cells and macrophages in tissues collected at surgery.

Single-cell RNA sequencing of a humanized patient-derived xenograft model provided translational evidence. The researchers used YHIM-2004, a lung squamous cell carcinoma PDX that did not respond to immune checkpoint inhibitor treatment, engrafted in humanized mice. Both DA-4505 monotherapy and the combination with pembrolizumab significantly reduced tumor growth compared with control and pembrolizumab groups. Sub-clustering 3,767 intratumoral T cells identified 11 phenotypes, and pathway analysis showed that AhR inhibition was linked to T-cell activation, tumor-cell killing, and memory-like differentiation, including antigen processing and interferon-γ production. Flow cytometry confirmed increased CD8 TEMRA cell abundance, proliferation, and activation, while multiplex immunohistochemistry showed higher intratumoral CD8 T-cell density, more 4-1BB-positive T cells, a lower regulatory T-cell to CD4 T-cell ratio, and deeper CD8 infiltration into tumor nests. Among 8,446 intratumoral myeloid cells, AhR inhibition downregulated inflammatory pathways, upregulated chemotaxis-related genes, reduced the frequency of IL6-positive macrophages, and diminished an inferred CCL-mediated interaction between IL6-positive macrophages and CD4-positive regulatory T cells. Plasma levels of IL-6, CCL20, interferon-γ, TNF-α, and granzymes shifted accordingly.

Translational analyses in human tissue supported these observations. In a commercial non-small cell lung cancer tissue microarray analyzed by multiplex immunohistochemistry, the proportion of AhR-positive macrophages correlated positively with the density of FOXP3-positive regulatory T cells, while AhR-negative macrophages showed a negative association, and AhR-positive macrophages in the tumor nest correlated strongly with PD-L1-positive tumor cell density. Survival analyses using TCGA cohorts found that higher expression of an AhR inhibition–associated T-cell signature was significantly associated with improved overall and disease-free survival in colorectal and breast cancers, with a similar trend in lung squamous cell carcinoma, and among patients treated with anti-PD-1 therapy, high signature expression was associated with significantly prolonged overall survival (hazard ratio 0.65, p = 0.024). The authors emphasize that these are correlative analyses that do not establish causality.

Important limitations temper the findings. The causal link between AhR inhibition, IL6-positive macrophage remodeling, and regulatory T-cell recruitment was not directly tested through cytokine blockade or macrophage-specific perturbation, and several in vivo experiments had unequal sample sizes due to feasibility constraints and experimental attrition. In particular, the humanized PDX combination group included only three evaluable animals, so the authors describe those results as preliminary and supportive rather than definitive evidence of therapeutic synergy. NK cells and other suppressive populations may also contribute to the observed antitumor effects, and the study focused primarily on myeloid–T-cell crosstalk. Nonetheless, the authors conclude that AhR inhibition reprograms systemic and local antitumor immunity by enhancing myeloid antigen presentation, strengthening myeloid–T-cell crosstalk, and promoting CD8 T-cell activation, providing a mechanistic rationale for developing AhR inhibitors as combination partners for immune checkpoint blockade, chemotherapy, and perioperative strategies.

Subject of Research: Preclinical evaluation of aryl hydrocarbon receptor inhibition as a strategy to remodel the tumor immune microenvironment and enhance anti-PD-1 immunotherapy

Article Title: Blocking aryl hydrocarbon receptor remodels myeloid–T cell crosstalk and enhances anti-PD-1 response in the tumor microenvironment

Article References: Kim, D. K., Baek, S., Kim, Y., Yang, S. M., Park, S. Y., Choi, S. J., Lee, J. H., Lee, S. H., Cho, J., Cha, D., Kim, D., Kim, S., Kim, J. H., & Cho, B. C. (2026). Blocking aryl hydrocarbon receptor remodels myeloid–T cell crosstalk and enhances anti-PD-1 response in the tumor microenvironment. Experimental Hematology & Oncology, 15(1), Article 112. https://doi.org/10.1186/s40164-026-00843-8

Image Credits: AI Generated

DOI: 10.1186/s40164-026-00843-8

Keywords: aryl hydrocarbon receptor, AhR inhibitor, DA-4505, anti-PD-1 immunotherapy, tumor microenvironment, macrophages, dendritic cells, CD8 T cells, regulatory T cells, IDO1-kynurenine pathway, single-cell RNA sequencing, patient-derived xenograft