Indole-related metabolites suppress the anti-tumor function of CD8+ T cells in the bone marrow microenvironment of multiple myeloma

Abstract Multiple myeloma (MM), the second most common hematologic malignancy, presents significant clinical challenges due to its relapsing/refractory nature and incurability, primarily attributed to the high complexity of the bone marrow (BM) microenvironment. Emerging evidence suggests that metabolic reprogramming within the tumor microenvironment (TME) plays a pivotal role in tumor progression and the anti-tumor function of CD8+ T cells. However, the limited understanding of the metabolic landscape of the MM BM microenvironment has hindered the identification of potential therapeutic targets. In this study, we demonstrate that although CD8+ T cells from MM patients exhibit elevated expression of immune checkpoint molecules, they do not enter a canonical exhausted state. Instead, their dysfunction is reversibly suppressed by microenvironmental alterations. Metabolomic profiling revealed a significant remodeling of the tryptophan metabolic pathway in the MM BM microenvironment, characterized by the accumulation of nitrogen-containing metabolites and indole derivatives, which directly impair CD8+ T cell-mediated anti-tumor activity. Further investigation identified indole-related metabolites as key metabolic regulators in the MM BM niche, promoting immune evasion by functionally suppressing CD8+ T cells. These findings not only uncover a novel mechanism of immune suppression in MM but also provide potential therapeutic targets for developing metabolism-focused immunomodulatory strategies. In multiple myeloma (MM) patients, bone marrow CD8+ T cells exhibit elevated PD-1 expression but comparable TIM-3 levels to non-myeloma controls. Notably, no significant differences were observed in cytokine secretion capacity or exhaustion-related transcription factor expression between PD-1+/TIM-3+ and PD-1-/TIM-3- T cell subsets. Thus, while a substantial population of CD8+ T cells persists in the MM microenvironment, they do not exhibit the canonical exhausted phenotype traditionally defined in chronic viral infections or other malignancies. Unexpectedly, fresh myeloma BM CD8+ T cells show blunted cytokine responses, reversible by overnight culture but inhibited by autologous BM supernatant. This suppression is BM-specific, as peripheral T cells remain unaffected by plasma. The <3 kDa hydrophilic fraction of bone marrow (BM) supernatant significantly impaired the cytotoxic capacity of BCMA CAR T cells. Untargeted metabolomic profiling revealed that the inhibitory BM supernatant exhibited distinct metabolic alterations, primarily enriched in the following pathways：Amino acid metabolism; Energy metabolism ; Nucleotide metabolism. Notably, multiple indole derivatives were upregulated in the tryptophan metabolic pathway. Metabolomic analysis revealed significant upregulation of indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), and kynurenine in the inhibitory bone marrow supernatant. Functional validation demonstrated that indole, indoleacrylic acid, IPA, and IAA all exhibited dose-dependent suppression of CAR T cell-mediated tumor cytotoxicity. Notably, IAA displayed potent immunosuppressive activity even at low concentrations, suggesting enhanced immunomodulatory potential. Mechanistically, both IAA and IPA significantly upregulated aryl hydrocarbon receptor (AHR) expression， indicating that indole-related metabolites may impair antitumor T cell function through AHR-mediated signaling pathways. Overall, we have come to the following conclusions：flow cytometric and transcriptional analyses revealed that CD8+ T cells in the multiple myeloma (MM) bone marrow microenvironment exhibit an activated phenotype rather than classical exhaustion markers, despite their functional impairment. Ultrafiltration and functional assays demonstrated that the <3 kDa hydrophilic fraction of MM bone marrow supernatant significantly inhibits CD8+ T cell effector functions. Indole-3-acetic acid (IAA) and indole-3-propionic acid (IPA) are significantly elevated in the bone marrow of a subset of multiple myeloma (MM) patients, where they potently inhibit the anti-tumor function of CD8+ T cells, likely through activation of the aryl hydrocarbon receptor (AhR) signaling pathway on T cell surfaces.