Driver and mechanism of t-cell dysfunction and immune cold microenvironment in deadly developmental tumor in infants

Neuroblastoma (NB), a prevalent extracranial pediatric malignancy, is characterized by an immunologically “cold” tumor microenvironment (TME), yet the mechanisms underlying its immune evasion remain poorly understood. Here, we identify a novel immunoregulatory role for Retinal Degeneration 3 (RD3) in shaping host immunity and T-cell fate during NB progression. Using immune-competent mouse models, neural crest cell (NCC)-specific RD3 knockout (RD3 ⁻/⁻ ), ALK phosphor-mimetic mutated ( ALKF1174L knock-in), or RD3 ⁻/⁻ ALKF1174L (aggressive NB phenotype), we demonstrate that RD3-loss does not impair thymic T-cell development, but profoundly alters peripheral T-cell maturation and activation. RD3-deficient mice exhibit enhanced thymic commitment of CD3⁺ T-cells, increased TCR rearrangement, and elevated CD8⁺ cytotoxic T lymphocyte (CTL) maturation, while CD4⁺ helper T-cell development is markedly suppressed. In NB-bearing RD3 ⁻/⁻ mice, we observed tumor-driven reprogramming of effector T-cell populations, with increased CD8⁺ CTLs and diminished CD4⁺ activation, indicating a skewed immune response. Splenic microenvironment revealed robust homing of TCR-programmed cells but a blockade in CD4⁺ T-cell activation. Wild-type controls maintain balanced T-cell activation and effector composition, underscoring the immune detour induced by RD3 deficiency. Multiplex immunofluorescence highlights a depletion of CD4⁺ and CD8⁺ effector T-cells, reduced activation markers (CD44, GITR), and elevated exhaustion marker CD244.2. Innate immune compartments are similarly compromised, with diminished CD206⁺, CD86⁺, and STING⁺ populations, reflecting impaired antigen presentation. Notably, RD3 ⁻/⁻ tumors exhibit heightened adenosinergic suppression via the CD73/CD39/A2AR axis, establishing a metabolic barrier to immune activation. New to science, these findings reveal RD3-loss as a central orchestrator of immune collapse in NB, promoting T-cell exhaustion, suppressing antigen presentation, and fostering a metabolically suppressed TME. This study provides mechanistic insight into tumor-intrinsic immune evasion and lays the groundwork for therapeutic strategies to restore immune competence in NB. Funding: Department of Defense CA-210339; OCAST-HR19-04; NIH-P20GM103639; and supported by P30CA225520 and P30GM154635.