Background Immunotherapy holds great promise for neuroblastoma. Implementation of anti-GD2 therapy improved patient survival rates substantially. However, many patients with neuroblastoma relapse and the 5-year survival rate for high-risk patients is below 50%. Here, we aimed to unravel the immune environment of neuroblastoma using spectral flow cytometry to identify novel targets for immunotherapy.

Materials and Methods Tumor resection material from 18 patients taken after induction chemotherapy was enzymatically digested. Single cells were stained with a lymphocyte antibody panel, containing 35 markers, or a myeloid antibody panel, containing 36 markers, and measured on a Cytek Aurora spectral flow cytometer. Peripheral blood mononuclear cells (PBMCs) from 13 healthy donors were measured as controls. Manual gating of the main immune cell populations was done using FlowJo software. Unsupervised clustering was performed in R.

Results We identified detailed lymphocyte clusters including B cells, 5 populations of NK cells, NKT cells, three populations of γδT cells, CD8+ T cells, two populations of CD4+ Non-Treg cells, and Tregs. Immune cell populations in the tumor expressed Ki-67 at a higher level than cells in blood, indicating a higher proliferation rate. Cytotoxic tumor infiltrating lymphocytes (TILs) had decreased expression levels of Perforin and GZMB compared to blood, indicating a dysfunctional phenotype with reduced cytotoxic potential. In addition, these cells expressed low levels of CD137, a marker for activation c.q. tumor recognition. To explore immunosuppressive mechanisms which could contribute to this effector cell dysfunctionality, we analyzed 1) the functional profile of Tregs, 2) expression of co-inhibitory receptors and 3) suppressive myeloid populations in the tumor. Tumor-infiltrating Tregs had a higher expression of CD137, CD25 and ICOS than blood-derived Tregs, indicating that they were highly activated, with a high suppressive capacity to inhibit effector functions of cytotoxic TILs. Each TIL population had a unique co-inhibitory receptor expression pattern. NK cells had high expression of NKG2A, KLRD1 and KLRB1. Remarkably, a distinct population of CCR7+ γδT cells expressed high levels of exhaustion-related immune checkpoint receptors PD1, CTLA4, TIGIT, LAG3 and TIM3 compared to CD8+ and CD4+ αβ T cells. Also, they expressed high levels of activation marker. This activation/exhaustion profile potentially identifies these cells as the primary tumor reactive cells. Among the myeloid populations we identified dendritic cells and 4 populations of monocytes/macrophages. The latter expressed high levels of CD163, indicating a M2-like, pro-tumorigenic and immunosuppressive phenotype, high levels of checkpoint molecules, including PD-L1 and Nectin-2, and immunoregulatory cytokine IL-10.

Conclusions Effector TILs in neuroblastoma have a dysfunctional phenotype with decreased cytotoxicity, which may explained by inhibition by highly suppressive tumor infiltrating Tregs, co-inhibitory receptor expression on potentially tumor-reactive (CCR7+ γδT cells) T cells and immunosuppressive myeloid cells. These findings may aid the educated development of novel immunotherapies for neuroblastoma.

A.L. Borst: None. M.M. van Noesel: None. J.J. Molenaar: None. J. Wienke: None.