We investigated the status of the adaptive and innate immune responses in patients with HR or LR neuroblastoma. Patients with HR showed increases in the number of cells involved in adaptive immune responses as well as lower levels of IL-10 and MDSC, compared with LR patients. In addition, cytokines involved in the innate immune responses including IL-1β and MCP-1 were increased in LR patients. Treatment of a HR tumor cell line with IL-1β induced the expression of pro-inflammatory cytokines involved in the innate immune responses, including MCP-1, GM-CSF and TNF-α. Our data suggest a favorable prognostic value of the signatures of immune function genes associated with the innate immune response, such as increased IL-1β and MCP-1 as well as a diminished adaptive immune response, evidenced by increased levels of MDSC, IL-10 and decreased expression of NKG2D and CXCR4.
IL-1β and MCP-1 are involved in pro-inflammatory responses, and are components of non specific innate immune responses. Increased expression of these molecules in LR patients underscores their importance in a favorable prognosis. Since IL-1β has been shown to induce the expression of MCP-1 [21
] leading to chronic inflammation, we hypothesized that IL-1β-may induce the expression of MCP-1 and IL-10 in HR tumor cells. We showed an increased expression of MCP-1, GM-CSF and TNF-α in the HR tumor cell line pulsed with IL-1β, both by flow cytometry and cytokine array analyses. GM-CSF has been shown to promote the development of MDSC and in turn suppress adaptive immune responses [22
]. A higher frequency of CD8+CXCR4+ T cells in the peripheral blood of HR patients may facilitate infiltration of T cells into the tumor site [23
]. However, such an increased infiltration of T cells failed to protect HR patients. This may be because of a dual function of CD8+ T cells, i.e. inducing epigenetic changes in the tumors leading to tumor escape and a worse prognosis [24
] as well as a direct cytotoxic effect on tumor cells. For instance, we have shown that CD8+ T cells can induce epithelial to mesenchymal transition (EMT) as well as HER-2/neu antigen loss, leading to tumor escape in breast cancer model [25
]. Others also reported that T cells can induce tumor escape in a variety of tumor models including CT-26 colon carcinoma [27
], renal cell carcinoma [28
], Uveal melanoma [29
] and breast cancer patients [30
NKG2D is an activating receptor expressed on activated CD8+ T cells and NK cells. Signalling by NKG2D has been shown to be involved in the activation of T cells against the tumors [31
]. Of note was the relative absence of activated NK cells (CD65+NKG2D+) in the circulation of LR patients. NK cells play a key role in innate immunity, and it was surprising to note higher levels of CD56+NKG2D+ cells in HR patients. Higher expression of MCP-1 in LR tumors could induce infiltration of NK cells to the tumor site and as a result reduce circulating NK cells [32
Regrouping the microarray data based on the percentage of infiltrating immune cells would further refine the differential expression of the immune function genes in HR vs. LR patients. However a limitation in our study was that because of the rare nature of the disease and limited access to sufficient number of patients, many of our samples were received from outside the institution. These samples contained RNA so that the percent of infiltrating cells could not be determined and regrouping for microarray analysis was not possible. However our data still show very clear segregation between the two groups.
It has been reported that pro-inflammatory products can cause impairment of DNA synthesis in neuroblastoma cell lines [33
] leading to cell death.
While a single gene or cytokine may not be able to independently be a prognosticator; the combination of differentially expressed immune function genes and the pattern of cellular and cytokine responses can be used to generate a 'signature' pattern for HR vs. LR neuroblastoma. A signature of the innate immune responses is important in lieu of reports showing that pro-inflammatory products can cause impairment of DNA synthesis in neuroblastoma cell lines [33
] leading to cell death. MDSC stand out as a distinct difference between the two groups and it can be added to the current prognostication parameters, though it needs to be further validated by using a large number of samples because we were not able to include more than one LR patient for the detection of MDSC in the circulation due to the rate nature of LR disease. One of our limitations was a small sample size due to the nature of the diseases and the relatively higher frequency of HR disease.