Neuroblastoma grows within an intricate network of different cell types including epithelial, stromal and immune cells. The presence of tumor-infiltrating T cells is considered an important prognostic indicator in many cancers, but the role of these cells in neuroblastoma remains to be elucidated. Herein, we examined the relationship between the type, density and organization of infiltrating T cells and clinical outcome within a large collection of neuroblastoma samples by quantitative analysis of immunohistochemical staining. We found that infiltrating T cells have a prognostic value greater than, and independent of, the criteria currently used to stage neuroblastoma. A variable structural organization and different concurrent infiltration of T-cell subsets were detected in tumors with various outcomes. Low-risk neuroblastomas were characterized by a higher number of proliferating T cells and a more structured T-cell organization, which was gradually lost in tumors with poor prognosis. We defined an immunoscore based on the presence of CD3, CD4 and CD8 infiltrating T cells that associates with favorable clinical outcome in MYCN-amplified tumors, improving patient survival when combined with the v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN) status. These findings support the hypothesis that infiltrating T cells influence the behavior of neuroblastoma and might be of clinical importance for the treatment of patients.

In this study, we show that neuroblastoma (NB) cell conditioning affects the chemokine receptor repertoire of human resting NK cells. In particular, NB cells upregulated the expression of CXCR4 and CXCR3 in all NK cells and downregulated CX3CR1 in the CD56dim subset. On the contrary, the expression of CXCR1 and CCR7 remained unaltered. The phenomenon was dependent on the release by NB cells of TGF-β1, and rTGF-β1 induced a chemokine receptor repertoire identical to that of NB-conditioned NK cells. The immune modulatory role of TGF-β1 appears to be dose dependent because low amounts of the cytokine were sufficient to modulate CXCR4 and CX3CR1 expression, intermediate amounts modified that of CXCR3, and high amounts were necessary to downregulate the expression of the NKp30 activating receptor. Notably, a similar receptor modulation was observed in rTGF-β2–conditioned NK cells. Finally, the analysis of NK cells from patients with stage 4 NB suggests that NB conditioning could exert in vivo an immune modulatory effect resembling that emerged from in vitro experiments. Altogether our data propose a novel tumor escape-mechanism based on the modulation of chemokine receptors that play pivotal roles in NK cells bone marrow homing, egress, or recruitment into peripheral tissues.

The prognosis of high-risk neuroblastoma (NB) remains poor, although immunotherapies with anti-GD2 antibodies have been reported to provide some benefit. Immunotherapies can be associated with an IFNγ storm that induces in tumor cells the "adaptive immune resistance" characterized by the expression of Programmed Death Ligands (PD-Ls). Tumor cells can also constitutively express PD-Ls in response to oncogenic signaling. Here, we analyze the constitutive and the inducible surface expression of PD-Ls in NB cells. We show that virtually all HLA class I NB cell lines constitutively express PD-L1, whereas PD-L2 is rarely detected. IFNγ upregulates or induces PD-L1 both in NB cell lines and in NB engrafted nude/nude mice. Importantly, after IFNγ stimulation PD-L1 can be acquired by NB cell lines, as well as by metastatic neuroblasts isolated from bone marrow aspirates of high-risk NB patients, characterized by different amplification status. Interestingly, in one patient NB cells were poorly responsive to IFNγ stimulation, pointing out that responsiveness to IFNγ might represent a further element of heterogeneity in metastatic neuroblasts. Finally, we document the presence of lymphocytes expressing the PD-1 receptor in NB-infiltrated bone marrow of patients. PD-1 cells are mainly represented by αβ T cells, but also include small populations of γδ T cells and NK cells. Moreover, PD-1 T cells have a higher expression of activation markers. Overall, our data show that a PD-L1-mediated immune resistance mechanism occurs in metastatic neuroblasts and provide a biological rationale for blocking the PD-1/PD-Ls axis in future combined immunotherapeutic approaches.