Until recently, no information was available about HLA-G expression in pediatric tumors and their role in the course of the disease. In the frame of a program aimed at identifying the immune escape mechanisms that contribute to NB growth and spreading [62
] we have recently investigated whether membrane-bound and soluble HLA-G play any role in the biology of NB cells and in the clinical course of this disease [17
NB, that represents the most frequent extra-cranial solid tumor and the first cause of lethality in pre-school age children, originates from the sympathetic nervous system and is characterized by heterogeneous pathological and clinical presentation [66
]. A short description of these latter features is provided to facilitate the interpretation of our findings. NB presenting as disseminated disease after the first year of life is one of the most aggressive solid tumors of childhood [66
]. This tumor, classified as stage 4 according to the International Neuroblastoma Staging System (INSS) classification, occurs predominantly in children aged 4 to 6 years and accounts for about 50% of all NB cases. Stage 4 NB usually originates from the adrenal gland and spreads virtually to every organ; the most common metastatic sites are cortex of long bones, skull, lymph nodes and bone marrow. The majority of patients with stage 4 NB have grim prognosis, with 70-75% of them dying in 5 years from diagnosis. Malignant neuroblasts have a special propensity to localize to the bone marrow [66
Localized NB at diagnosis includes stages 1, 2A-B, and 3. Patients with stage 1 disease have a completely resectable tumor, whereas stage 2A-B and 3 patients have a localized tumor that can be completely or partially excised, and may or may not show ipsilateral lymph node involvement. Stage 1 and 2 patients have an overall survival (OS) of approximately 95% at 5 years from diagnosis, while OS of stage 3 patients approximates 75% [66
A third type of NB, the so-called stage 4s, includes patients with less than one year of age who present with metastatic lesions at onset, localized in skin, liver and/or bone marrow. In spite of the disseminated disease, these patients have an OS that approximates 75% at five years. In most of these patients, the tumor regresses spontaneously without any treatment or following supportive treatment only. Regression is probably related to delayed neuroblast cell differentiation and/or late activation of programmed cell death [66
We investigated serum levels of sHLA-G in fifty three untreated NB patients, 25 of whom had localized disease, 20 metastatic disease at diagnosis (stage 4) and 8 stage 4s disease. Altogether, sHLA-G serum levels were significantly higher in NB patients than in age-matched healthy controls. However no difference was detected among patient subgroups. The pattern of expression of serum HLA-G is similar to that of the intercellular adhesion molecule sICAM-1. As shown in , sICAM-1 was significantly increased in serum from the latter patients, and there was a clear trend, that however did not reach statistical significance, towards a parallel increase in serum sHLA-G. Lack of statistical correlation between sICAM-1 and sHLA-G in these patients is likely due to the limited number of patients tested for both parameters.
Figure 2 Serum levels of sHLA-G, sHLA-class I, sHLA-class II and sICAM-1 in NB patients. Black and broken lines indicate the mean of serum levels obtained for each molecule in healthy subjects and in NB patients, respectively. Results are expressed as ng/ml for (more ...)
Whether the increased serum levels of sHLA-G and sICAM-1 play a role in tumor growth and/or in the interactions of NB cells with the host immune system, as well as whether they represent useful tumor markers, warrants further investigation. To the best of our knowledge, NB represents the first example of malignant disease with a combined increase of serum levels of sHLA-G and sICAM-1. Such increase appears to be selective, since it was detected in NB patients with normal serum levels of classical sHLA-I and soluble HLA class II (sHLA-II) antigens (). The latter results also suggest that sHLA-G and classical sHLA-I and sHLA-II are controlled by different regulatory mechanisms, at least in this malignancy. The available experimental evidence argues against the association between presence of sHLA-G in serum and cell surface expression of HLA-G by NB cells is conflicting [17
]. First, the level of sHLA-G was low in the supernatants of five NB cell lines without detectable cell surface expression of HLA-G as well as in those of six NB cell lines with low to moderate cell surface expression of HLA-G. Second, HLA-G was present in sera from NB patients, although its expression could not be detected by immunohistochemical analyses of primary tumors. However the latter results have to be testes to be interpreted with caution, since, in recent studies, flow cytometric analysis of metastatic NB cells isolated from the bone marrow of two stage 4 patients and stained with HLA-G-specific mAb has detected HLA-G expression on neuroblasts gated on the ground of GD2 expression. The results are shown in , panel A. Several mechanisms may account for the different results derived from the analysis of neuroblasts present in primary and metastatic NB lesions. First, the metastatic NB cells and the primary NB lesions we analyzed were derived from different patients. Therefore we cannot exclude that the different results we obtained reflect individual variability in the regulation of HLA-G expression by NB cells. Second, different assay systems with different sensitivity, i.e. flow cytometry and immunohistochemistry, were used to analyze neuroblasts isolated from metastatic lesions and those present in primary NB lesions, respectively. Immunohistochemistry is less sensitive than flow cytometry, raising the possibility that the different sensitivity of the assays contributed to the different results obtained. Lastly, the mechanism we favour is represented by the different epigenetic control of HLA-G gene expression in primary and metastatic lesions because of the diverse environmental conditions. In this regard, previous studies carried out with various human tumor cell lines have shown that both CpG methylation and histone deacetylation play a role in transcriptional silencing of the HLA-G gene, although to a different extent in the individual cell lines analyzed [67
]. Experiments performed with histone deacetylase (HDAC) and DNA methyl transferase (DNMT) inhibitors have demonstrated that demethylation with 5-aza-2′-deoxycytidine (5-AC) is more effective at activating HLA-G mRNA and protein expression than incubation of cell lines with HDAC inhibitors, such as trychostatin A (TSA) [67
]. Along this line our own studies still in progress with five NB cell lines displaying low to absent surface HLA-G expression have shown that their incubation with 5-AC or with TSA upregulated HLA-G expression, as assessed by flow cytometric analysis of cells stained with HLA-G-specific mAb. These experiments also showed that the five NB cell lines analyzed have a differential sensitivity to 5-AC and HDAC inhibitors. The effects of 5-AC and TSA on the representative GI-LI-N NB cell line are shown in , panels B and C, respectively. If epigenetic mechanisms do indeed contribute to the control of HLA-G expression by NB cells, one can envision differential HLA-G expression in tumor lesions located in different anatomic sites, changes in HLA-G expression by NB cells during the course of the disease because of changes in the microenvironment, and differences among patients. If this scenario is correct, such variables should be taken into account when analyzing the influence of HLA-G cell surface expression on the serum level of sHLA-G.
Figure 3 Potential role of epigenetic mechanisms in the differential HLA-G expression by freshly isolated NB cells and by NB cells in long term culture. Panel A. GD2+ cells were purified from BM of two NB patients (right and left panel,), stained with HLA-G1-specific (more ...)
The available evidence strongly suggests that sHLA-G present in NB patients' sera is derived from monocytes, that represent together with mature myeloid and plasmacytoid dendritic cells the major source of sHLA-G in physiological conditions. Indeed, monocytes from NB patients spontaneously release significantly higher levels of sHLA-G than control monocytes, although they express surface HLA-G at the same levels as monocytes from age- and gender-matched healthy donors. It is likely that the synthesis and release of HLA-G by monocytes from NB patients are maximal under basal conditions, since the number of sHLA-G secreting monocytes was not increased following incubation with IFN-γ. In contrast an increase was observed when monocytes from healthy donors were incubated with IFN-γ [17
]. These data altogether suggest that NB patients' monocytes are in an activated state. This possibility is supported by the results of flow cytometry analysis of freshly isolated monocytes from NB patients showing that these cells express de novo
CD69, a marker of recently activated cells [17
The above set of experiments points to monocytes as a major source of sHLA-G in NB patients, but the trigger responsible for their activation is not known. It is our working hypothesis that tumor cells “instruct” monocytes to produce increased levels of sHLA-G that, in turn, protects malignant cells from the attack of the host immune system. Two lines of evidence suggest that this interaction between tumor cells and monocytes takes place through soluble factors released by the former cells rather than through direct cell-to-cell contact. First, monocytes are found in the circulation and their migration to tissues is associated with irreversible differentiation into resident macrophages or dendritic cells. Thus, they have virtually no chance to encounter NB cells. Second, the results of transwell experiments in which monocytes from normal subjects are separated from NB cell lines by a permeable filter demonstrate that sHLA-G production by monocytes takes place in the absence of physical interactions with tumor cells. Accordingly, NB cell line supernatants are able to instruct normal monocytes to produce increased amounts of sHLA-G [17
In our own experiments, following incubation with pooled supernatants from four NB cell lines, the proportions of sHLA-G secreting monocytes was doubled and a number of immunophenotypic changes took place. They include induction of the CD69 and CD71 activation markers, and up-regulation of HLA class II molecules, of the macrophage marker CD68, and of the costimulatory molecule CD86 [17
]. Taken together, these findings indicate that normal monocytes incubated with NB cell supernatants undergo activation and increase their sHLA-G producing capacity. Futhermore, the latter cells are larger in size than control monocytes, undergo spreading on plastic surfaces and display pseudopodia-like structures projecting from the cell surface. All of these changes are consistent with a macrophage-like differentiation of monocytes.
Since tumor cells produce the immunosuppressive cytokines IL-10 and TGF-β1, that are among the best inducers of sHLA-G production [68
], one might postulate that these molecules released by NB cells could mediate the effects of NB cells on normal monocytes. However, the results of experiments in which NB cell line supernatants were incubated with neutralizing anti-IL10 or anti-TGFβ1 antibodies before being tested on monocytes did not support this working hypothesis. Likewise, neutralization of the ganglioside GD2, that is released constitutively by NB cells and possesses immunosuppressive activity, was unsuccessful [17
]. Thus, for the time being, the identity of the soluble factor(s) produced by NB cells and endowed with “arming” activity on monocytes remains unknown.
IL-12 is a pleiotropic cytokine produced predominantly by macrophages and mature myeloid dendritic cells that mediates potent anti-tumor activity by promoting Th1 cell differentiation, and activating cytotoxic effector functions of CTL and NK cells [69
]. IL-10 is the antagonistic cytokine to IL-12; it is produced mainly by macrophages, plasmacytoid dendritic cells and Th2 cells, and, as mentioned above, anergizes the host immune system thus facilitating tumor growth and spreading [70
A series of studies has demonstrated the existence of two distinct lineages of macrophages, named M1 and M2, that exhibit discrete transcriptional profiles. M1 macrophages are potent producers of IL-12 and low producers of IL-10, whereas M2 macrophages display a specular behaviour, i.e. high IL-10 and low IL-12 production. These latter macrophages, that are frequently detected within tumor infiltratates, promote tumor progression, tissue repair and remodeling [71
NB tumors usually contain scarce lymphoid infiltrates, that are detected occasionally in stroma poor tumors [62
] and consistently in stroma rich tumors [63
]. Tumor infiltrating cells are predominantly T and B lymphocytes admixed with macrophages whose M1/M2 profile has not yet been investigated.
In view of the ability of NB cells to activate normal monocytes, it is noteworthy that when normal peripheral blood monocytes were incubated with NB cell line supernatants, IL-10 was not detected under any of the experimental conditions tested, whereas IL-12 was down-regulated in cells exposed to tumor cell supernatants as compared to cells cultured with medium alone [17
]. Although this cytokine profile is unrelated to that of M1/M2 macrophages, NB cell induced downregulation of IL-12 production by monocytes may represent an additional immnosuppressive mechanism utilized by tumor cells to avoid immune recognition and destruction by the host's immune system. Thus, it is our working hypothesis that tumor cell mediated monocyte activation is a sort of “frustrated activation”, characterized simultaneously by activated immunophenotype and diminished IL-12 production. A cartoon depicting this novel mechanism is shown in .
Figure 4 Schematic representation of a novel mechanism utilized by human neuroblastoma cells to elude the control of the host's immune system. Neuroblastoma cells release soluble factors that activate monocytes to upregulate synthesis and release of soluble HLA-G. (more ...)