Fabio Morandi

IMMUNOGENICITY OF HUMAN MESENCHYMAL STEM CELLS IN HLA-CLASS I RESTRICTED T CELL RESPONSES AGAINST VIRAL OR TUMOR-ASSOCIATED ANTIGENS

Human mesenchymal stem cells (MSC) are immunosuppressive and poorly immunogenic but may act as antigen‐presenting cells (APC) for CD4+ T‐cell responses; here we have investigated their ability to serve as APC for in vitro CD8+ T‐cell responses. MSC pulsed with peptides from viral antigens evoked interferon (IFN)‐γ and Granzyme B secretion in specific cytotoxic T lymphocytes (CTL) and were lysed, although with low efficiency. MSC transfected with tumor mRNA or infected with a viral vector carrying the Hepatitis C virus NS3Ag gene induced cytokine release but were not killed by specific CTL, even following pretreatment with IFN‐γ. To investigate the mechanisms involved in MSC resistance to CTL‐mediated lysis, we analyzed expression of human leukocyte antigen (HLA) class I‐related antigen‐processing machinery (APM) components and of immunosuppressive HLA‐G molecules in MSC. The LMP7, LMP10, and ERp57 components were not expressed and the MB‐1 and zeta molecules were downregulated in MSC either unmanipulated or pretreated with IFN‐γ. Surface HLA‐G was constitutively expressed on MSC but was not involved in their protection from CTL‐mediated lysis. MSC supernatants containing soluble HLA‐G (sHLA‐G) inhibited CTL‐mediated lysis, whereas those lacking sHLA‐G did not. The role of sHLA‐G in such inhibition was unambiguously demonstrated by partial restoration of lysis following sHLA‐G depletion from MSC supernatants. In conclusion, human MSC can process and present HLA class I‐restricted viral or tumor antigens to specific CTL with a limited efficiency, likely because of some defects in APM components. However, they are protected from CTL‐mediated lysis through a mechanism that is partly sHLA‐G‐dependent.

Human Neuroblastoma Cells Trigger an Immunosuppressive Program in Monocytes by Stimulating Soluble HLA-G Release

HLA-G is overexpressed in different tumors and plays a role in immune escape. Because no information is available on HLA-G in relation to human neuroblastoma, we have investigated the expression of membrane-bound and secretion of soluble isoforms of HLA-G in neuroblastoma and functionally characterized their immunosuppressive activities. At diagnosis, serum soluble HLA-G (sHLA-G) levels were significantly higher in patients than in age-matched healthy subjects. In addition, patients who subsequently relapsed exhibited higher sHLA-G levels than those who remained in remission. Neuroblastoma patient sera selected according to high sHLA-G concentrations inhibited natural killer (NK) cell and CTL-mediated neuroblastoma cell lysis. Such lysis was partially restored by serum depletion of sHLA-G. In 6 of 12 human neuroblastoma cell lines, low HLA-G surface expression was not up-regulated by IFN-gamma. Only the ACN cell line secreted constitutively sHLA-G. IFN-gamma induced de novo sHLA-G secretion by LAN-5 and SHSY5Y cells and enhanced that by ACN cells. Primary tumor lesions from neuroblastoma patients tested negative for HLA-G. Neuroblastoma patients displayed a higher number of sHLA-G-secreting monocytes than healthy controls. Incubation of monocytes from normal donors with IFN-gamma or pooled neuroblastoma cell line supernatants significantly increased the proportion of sHLA-G-secreting cells. In addition, tumor cell supernatants up-regulated monocyte expression of CD68, HLA-DR, CD69, and CD71 and down-regulated IL-12 production. Our conclusions are the following: (a) sHLA-G serum levels are increased in neuroblastoma patients and correlate with relapse, (b) sHLA-G is secreted by monocytes activated by tumor cells rather than by tumor cells themselves, and (c) sHLA-G dampens anti-neuroblastoma immune responses.

Multiple defects of the antigen-processing machinery components in human neuroblastoma: Immunotherapeutic implications

Low expression of human leukocyte antigen (HLA) class I in human tumors may be related to defects of the antigen-processing machinery (APM) components. Neuroblastoma cells are virtually HLA class I negative, but (i) the underlying mechanisms are unknown, and (ii) expression of the APM components has never been investigated. Here we have used a panel of novel monoclonal antibodies to proteasomal and immunoproteasomal components, chaperons and transporter associated with antigen processing (TAP) to characterize 24 stroma-poor neuroblastoma tumors and six neuroblastoma cell lines. Primary tumors showed defects in the expression of zeta, tapasin, TAP1 or TAP2, HLA class I heavy chain and β2 microglobulin, LMP2 and LMP7, as compared to normal adrenal medulla. Neuroblastoma cell lines displayed roughly similar patterns of APM expression in comparison to primary tumors. Incubation of neuroblastoma cell lines with interferon-γ caused upregulation of HLA class I molecules and reduced lysis by killer inhibitory receptor HLA ligand-matched NK cells. Defects in APM components explain reduced peptide loading on HLA class I molecules, their instability and failure to be expressed on the cell surface. HLA class I upregulation by interferon-γ, although enhancing neuroblastoma cell recognition by cytotoxic T cells, dampens their susceptibility to NK cells.

Emerging topics and new perspectives on HLA-G

Following the Fifth International Conference on non-classical HLA-G antigens (HLA-G), held in Paris in July 2009, we selected some topics which focus on emerging aspects in the setting of HLA-G functions. In particular, HLA-G molecules could play a role in: (1) various inflammatory disorders, such as multiple sclerosis, intracerebral hemorrhage, gastrointestinal, skin and rheumatic diseases, and asthma, where they may act as immunoregulatory factors; (2) the mechanisms to escape immune surveillance utilized by several viruses, such as human cytomegalovirus, herpes simplex virus type 1, rabies virus, hepatitis C virus, influenza virus type A and human immunodeficiency virus 1 (HIV-1); and (3) cytokine/chemokine network and stem cell transplantation, since they seem to modulate cell migration by the downregulation of chemokine receptor expression and mesenchymal stem cell activity blocking of effector cell functions and the generation of regulatory T cells. However, the immunomodulatory circuits mediated by HLA-G proteins still remain to be clarified.

Phenotypic and functional characterisation of CCR7+ and CCR7- CD4+ memory T cells homing to the joints in juvenile idiopathic arthritis

The aim of the study was to characterise CCR7+ and CCR7- memory T cells infiltrating the inflamed joints of patients with juvenile idiopathic arthritis (JIA) and to investigate the functional and anatomical heterogeneity of these cell subsets in relation to the expression of the inflammatory chemokine receptors CXCR3 and CCR5. Memory T cells freshly isolated from the peripheral blood and synovial fluid (SF) of 25 patients with JIA were tested for the expression of CCR7, CCR5, CXCR3 and interferon-γ by flow cytometry. The chemotactic activity of CD4 SF memory T cells from eight patients with JIA to inflammatory (CXCL11 and CCL3) and homeostatic (CCL19, CCL21) chemokines was also evaluated. Paired serum and SF samples from 28 patients with JIA were tested for CCL21 concentrations. CCR7, CXCR3, CCR5 and CCL21 expression in synovial tissue from six patients with JIA was investigated by immunohistochemistry. Enrichment of CD4+, CCR7- memory T cells was demonstrated in SF in comparison with paired blood from patients with JIA. SF CD4+CCR7- memory T cells were enriched for CCR5+ and interferon-γ+ cells, whereas CD4+CCR7+ memory T cells showed higher coexpression of CXCR3. Expression of CCL21 was detected in both SF and synovial membranes. SF CD4+ memory T cells displayed significant migration to both inflammatory and homeostatic chemokines. CCR7+ T cells were detected in the synovial tissue in either diffuse perivascular lymphocytic infiltrates or organised lymphoid aggregates. In synovial tissue, a large fraction of CCR7+ cells co-localised with CXCR3, especially inside lymphoid aggregates, whereas CCR5+ cells were enriched in the sublining of the superficial subintima. In conclusion, CCR7 may have a role in the synovial recruitment of memory T cells in JIA, irrespective of the pattern of lymphoid organisation. Moreover, discrete patterns of chemokine receptor expression are detected in the synovial tissue.

Binding of HLA-G to ITIM-Bearing Ig-like Transcript 2 Receptor Suppresses B Cell Responses

Inhibition of B cells constitutes a rational approach for treating B cell–mediated disorders. We demonstrate in this article that the engagement of the surface Ig-like transcript 2 (ILT2) inhibitory receptor with its preferential ligand HLA-G is critical to inhibit B cell functions. Indeed, ILT2–HLA-G interaction impedes both naive and memory B cell functions in vitro and in vivo. Particularly, HLA-G inhibits B cell proliferation, differentiation, and Ig secretion in both T cell–dependent and –independent models of B cell activation. HLA-G mediates phenotypic and functional downregulation of CXCR4 and CXCR5 chemokine receptors on germinal center B cells. In-depth analysis of the molecular mechanisms mediated by ILT2–HLA-G interaction showed a G0/G1 cell cycle arrest through dephosphorylation of AKT, GSK-3β, c-Raf, and Foxo proteins. Crucially, we provide in vivo evidence that HLA-G acts as a negative B cell regulator in modulating B cell Ab secretion in a xenograft mouse model. This B cell regulatory mechanism involving ILT2–HLA-G interaction brings important insight to design future B cell–targeted therapies aimed at reducing inappropriate immune reaction in allotransplantation and autoimmune diseases.

Therapeutic Targeting of TLR9 Inhibits Cell Growth and Induces Apoptosis in Neuroblastoma

The Toll-like receptor 9 (TLR9) evolved to cope with pathogens, but it is expressed in a variety of tumors for reasons that are unclear. In this study, we report that neuroblastoma (NB) cells express functional TLR9. Liposome-complexed CpG oligonucleotides inhibited the proliferation of TLR9-expressing NB cells and induced caspase-dependent apoptotic cell death. Inhibitory oligonucleotides (iODNs) abrogated these effects. RNA interference reduced TLR9 expression but not to the level where functional responses to CpG were abolished. Compared with free CpG, liposomal formulations of NB-targeted CpG (TL-CpG) significantly prolonged the survival of mice bearing NB tumor xenografts. While CpG alone lacked antitumor efficacy in NOD/SCID/IL2rg(-/-) mice, TL-CpG retained significant efficacy related to direct effects on tumor cells. TLR9 expression in primary human NB specimens was found to correlate inversely with disease stage. Our findings establish functional expression of TLR9 in NB and suggest that TLR9 may represent a novel theranostic target in this disease.

CXCR5 may be involved in the attraction of human metastatic neuroblastoma cells to the bone marrow

Introduction Up-regulation of some chemokine receptors on tumor cells is associated with increased metastatic potential. In this respect, limited information is available on chemokine receptor in human neuroblastoma (NB).Objects Purpose of the study was to identify chemokines/chemokine receptors involved in bone marrow (BM) localization of metastatic NB cells in view of the development of targeted therapeutic strategies. CD45− metastatic NB cells were isolated from the BM of six patients by immunomagnetic bead manipulation. Some experiments were carried out using a panel of human neuroblastoma cell lines (GI-ME-N, GI-LI-N, LAN-5, HTLA-230, SH-SY-5Y and IMR-32). Immunophenotypic analyses were performed by flow cytometry. Cell migration assays were carried out using transwell systems. Calcium ion mobilization, chemokine receptor internalization and cell proliferation were investigated by flow cytometry.Results In all BM samples, CXCR5 was expressed by the majority of primary neuroblasts and mediated their chemotaxis in response to CXCL13. Primary metastatic NB cells from all BM samples expressed CXCR6, but were not attracted by soluble CXCL16. Studies performed with two CXCR6+ NB cell lines showed that the mechanism whereby neuroblasts did not migrate to CXCL16 was likely related to defective calcium ion mobilization.Conclusions CXCR5 is the first chemokine receptor so far identified able to attract in vitro primary metastatic NB cells. CXCR6 may be involved in retention of metastatic neuroblasts in the BM through interaction with CXCL16 expressing stromal cells in the absence of signal transduction.

Immunogenicity of human neuroblastoma

Abstract: Neuroblastoma (NB) is a neuroectodermal tumor that affects children in the first years of life. Half of NB cases present with metastatic disease at diagnosis and have a poor prognosis, in spite of the most advanced chemotherapeutic protocols combined with autologous hematopoietic stem cell transplantation. Among the new avenues for NB treatment that are being explored, immunotherapy has attracted much interest. Emphasis has been placed on monoclonal antibodies directed to tumor‐associated antigens—in particular the disialoganglioside GD2—that have been tested in the clinical setting with promising results. In addition, stimulation of cell‐mediated antitumor effector mechanisms have been attempted—for example, by recombinant interleukin (IL)‐2 administration. Nonetheless, the issue of the immunogenicity of human NB cells has never been thoroughly addressed. Here we shall review the work carried out in our lab in recent years and show that NB cells express tumor‐associated antigens, such as MAGE‐3, but lack constitutive expression of costimulatory molecules and surface HLA class I and II molecules. As such, NB cells are likely to be ignored by the host T cell compartment, since expression of HLA and costimulatory molecules on antigen presenting cells are sine qua non conditions for efficient peptide presentation to T cells and for the subsequent activation and clonal expansion of the latter cells. Notably, in vitro experiments with NB cell lines demonstrated that surface HLA class I molecules and the CD40 costimulatory molecule were upregulated following cell incubation with recombinant interferon‐γ. Interaction of CD40 with recombinant CD40 ligand induced apoptosis of NB cells through a caspase 8‐dependent mechanism. Collectively, these results indicate that the immunogenicity of human NB cells is very low but suggest that manipulation by cytokine administration or gene transfer can increase their immunogenic potential. On the other hand, NB cells represent an excellent target for natural killer cells, the potential role of which in immunotherapy of NB is now being investigated.

CD56brightCD16− NK Cells Produce Adenosine through a CD38-Mediated Pathway and Act as Regulatory Cells Inhibiting Autologous CD4+ T Cell Proliferation

Recent studies suggested that human CD56brightCD16− NK cells may play a role in the regulation of the immune response. Since the mechanism(s) involved have not yet been elucidated, in the present study we have investigated the role of nucleotide-metabolizing enzymes that regulate the extracellular balance of nucleotides/nucleosides and produce the immunosuppressive molecule adenosine (ADO). Peripheral blood CD56dimCD16+ and CD56brightCD16− NK cells expressed similar levels of CD38. CD39, CD73, and CD157 expression was higher in CD56brightCD16− than in CD56dimCD16+ NK cells. CD57 was mostly expressed by CD56dimCD16+ NK cells. CD203a/PC-1 expression was restricted to CD56brightCD16− NK cells. CD56brightCD16− NK cells produce ADO and inhibit autologous CD4+ T cell proliferation. Such inhibition was 1) reverted pretreating CD56brightCD16− NK cells with a CD38 inhibitor and 2) increased pretreating CD56brightCD16− NK cells with a nucleoside transporter inhibitor, which increase extracellular ADO concentration. CD56brightCD16− NK cells isolated from the synovial fluid of juvenile idiopathic arthritis patients failed to inhibit autologous CD4+ T cell proliferation. Such functional impairment could be related to 1) the observed reduced CD38/CD73 expression, 2) a peculiar ADO production kinetics, and 3) a different expression of ADO receptors. In contrast, CD56brightCD16− NK cells isolated from inflammatory pleural effusions display a potent regulatory activity. In conclusion, CD56brightCD16− NK cells act as “regulatory cells” through ADO produced by an ectoenzymes network, with a pivotal role of CD38. This function may be relevant for the modulation of the immune response in physiological and pathological conditions, and it could be impaired during autoimmune/inflammatory diseases.