Background Immunotherapy with CAR-T cells, as well as immune checkpoint blockade, show limited clinical efficacy in the pediatric solid cancer neuroblastoma, despite the success in various adult cancers. The lacking efficacy may be due to various immune evasion strategies employed by neuroblastoma tumors, leading to altered functionality of tumor-infiltrating immune cells. We aimed to provide a comprehensive overview of the composition and function of the neuroblastoma immune environment, as well as relevant immunoregulatory interactions (=), to identify novel targets for immunotherapy.

Materials and Methods 25 tumor samples from 20 patients (17 with high-risk disease, 6 with MYCN amplification), were collected during diagnostic biopsy pre-treatment (n=10) or during resection surgery after induction chemotherapy (n=15). Samples were enzymatically digested, single-cell FACS sorted and sequenced by Cel-Seq2 protocol.

Results Lymphoid cells in the TME consisted of αβ-, γδ-T cells, NK cells and B cells. Among αβ-T cells we identified CD8⁺ T cells, two functionally distinct clusters of CD4⁺ T cells, naive-like T cells and FOXP3⁺ regulatory T cells (Tregs). CD8⁺ T cells had reduced cytotoxic capacity compared to blood-derived T cells from a reference group. Tregs expressed high levels of PRDM1, LAYN and ICOS, suggesting an effector Treg profile, which is associated with increased inhibitory capacity. Although NK cells expressed the cytotoxic genes NKG7, KLRF1, GNLY, GZMB and PRF1, their expression was significantly lower than in blood-derived reference NK cells. Gene set enrichment analysis (GSEA) confirmed a reduced cytotoxic capacity of tumoral NK cells, which correlated with a decreased expression of activating receptors (r=0.41, p<0.001) and increased TGFβ signaling (r=-0.45, p<0.001). In addition, NK cells highly expressed the heterodimeric receptor KLRC1:KLRD1, which can inhibit NK cell function through HLA-E binding. High HLA-E expression by endothelial, immune and mesenchymal cells confirmed its inhibitory activity in the TME. Within the myeloid compartment we identified various immunosuppressive populations, comprising a cluster of IL10 and VEGFA expressing macrophages, three clusters of M2 differentiated macrophages expressing MMP9 and LGALS3, and dendritic cells with intact antigen presenting capacity, but high expression of numerous genes encoding immunosuppressive molecules such as IDO1, LGALS1, LGALS2, CCL22 and NECTIN2. In MYCN amplified tumors, specifically, we observed even lower cytotoxic capacity of CD8⁺ T and NK cells. We identified increased TGFB1 expression and defective antigen presentation by myeloid and tumor cells as potential causes for reduced cytotoxicity in MYCN amplified tumors. To identify relevant targets for immunotherapy we constructed an unbiased interaction network, which revealed NECTIN1=CD96 and MIF=CD74 as active immunoregulatory interactions between tumor and T/NK cells, and CD80/CD86=CTLA4, CLEC2D=KLRB1, HLA-E=KLRC1/KLRC2, CD99=PILRA, LGALS9=HAVCR2, and NECTIN2=TIGIT between myeloid and T/NK cells.

Conclusions Cytotoxic lymphocytes in the neuroblastoma TME show reduced cytotoxic capacity, likely due to highly immunosuppressive myeloid cells, Tregs and numerous immunoregulatory interactions, which may serve as novel targets for immunotherapy in neuroblastoma.

Disclosure Information J. Wienke: None. W.M. Kholosy: None. L.L. Visser: None. K.M. Keller: None. P. Lijnzaad: None. T. Margaritis: None. K.P.S. Langenberg: None. R.R. De Krijger: None. F.C.P. Holstege: None. J.J. Molenaar: None.