Doriana Fruci

The putative role of endoplasmic reticulum aminopeptidases in autoimmunity: Insights from genomic-wide association studies

Autoimmune diseases represent a heterogeneous group of conditions whose incidence is increasing worldwide. This has stimulated studies on their etiopathogenesis, derived from a complex interaction between genetic and environmental factors, aimed at finally improving prevention and treatment of these diseases. In the autoimmune process, immune responses are generated against self antigens presented by Major Histocompatibility Complex (MHC) class I on the cell surface. These peptide/MHC class I complexes are generated and assembled through MHC class I antigen processing and presentation machinery. In the endoplasmic reticulum (ER), aminopeptidases ERAP1 and ERAP2 display distinct trimming activity before antigenic peptides are loaded onto MHC class I molecules. The advent of new tools such as genome-wide association studies (GWAS) has provided evidence for new susceptibility loci and candidate genes playing a role in the autoimmune process for the recognized immune function of their transcripts. Genetic linkage has been discovered with MHC antigens and various autoimmune conditions. Recent GWAS showed the importance of ERAP1 and ERAP2 in several autoimmune diseases, including ankylosing spondylitis, insulin-dependent diabetes mellitus, psoriasis, multiple sclerosis, Crohns disease. In this review, we first provide a general overview of ERAP1 and ERAP2 genes, their biological functions and their relevancy in autoimmunity. We then discuss the importance of GWAS and the case-control studies that confirm the relevancy of ERAP single-nucleotide polymorphism associations and their linkage with particular MHC class I haplotypes, supporting a putative functional role in the autoimmune process.

T and NK cells: Two sides of tumor immunoevasion

Natural Killer (NK) cells are known to reject several experimental murine tumors, but their antineoplastic activity in humans is not generally agreed upon, as exemplified by an interesting correspondence recently appeared in Cancer Research. In the present commentary, we join the discussion and bring to the attention of the readers of the Journal of Translational Medicine a set of recent, related reports. These studies demonstrate that effectors of the adaptive and innate immunity need to actively cooperate in order to reject tumors and, conversely, tumors protect themselves by dampening both T and NK cell responses. The recently reported ability of indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2) expressed by melanoma cells to down-regulate activating NK receptors is yet another piece of evidence supporting combined and highly effective T/NK cell disabling. Major Histocompatibility Complex class I (MHC-I) molecules, including Human Leukocyte Antigen E (HLA-E), represent another class of shared activating/inhibitory ligands. Ongoing clinical trials with small molecules interfering with IDO and PGE2 may be exploiting an immune bonus to control cancer. Conversely, failure to simultaneously engage effectors of both the innate and the adaptive immunity may contribute to explain the limited clinical efficacy of T cell-only vaccination trials. Shared (T/NK cells) natural immunosuppressants and activating/inhibitory ligands expressed by tumor cells may provide mechanistic insight into impaired gathering and function of immune effectors at the tumor site.

Major histocompatibility complex class i and tumour immuno-evasion: how to fool T cells and natural killer cells at one time.

Cytotoxic T lymphocytes (ctls) and natural killer (nk) cells lyse tumours expressing and lacking, respectively, properly conformed class i molecules of the major histocompatibility complex [mhc-i (Figure 1)]. In keeping with the “missing self” hypothesis 1, a logical extrapolation would be to postulate that the primary goal of a tumour is to elude both defense lines. FIGURE 1 Activation–inhibition and molecules of major histocompatibility complex, class i (mhc-i). Tumours are killed either when they are able to express mhc-i molecules containing tumour peptide antigens recognized by the rearranging T-cell receptor ... With regard to ctl evasion, tumour losses of mhci have been thoroughly studied (our group has more than 200 papers on file) and have, in most instances (although not invariably), been associated with poor outcome (reviewed in Garrido et al. 2). Interestingly, the principle of mhc-i loss also applies to the members of the so-called antigen-processing machinery, such as the transporter associated with antigen processing (tap), the endoplasmic reticulum aminopeptidase associated with antigen processing (eraap in mice and erap1 and erap2 in humans), and tapasin. These are in charge of, respectively, translocation (into the endoplasmic reticulum), final trimming, and editing of peptide antigens (Figure 1) before loading onto mhc-i. After our initial observation of linked expression patterns between mhc-i and members of the antigen-processing machinery 3, coordinated downregulation of some of these molecules was shown to correlate with poor prognosis 4. Immunotherapeutic approaches, including the massive administration of dominant tumour antigens in peptide-based T-cell therapy (mostly pursued in melanoma and incorrectly called “vaccination”), impose an even greater selective pressure, possibly leading to an increased advantage for tumour cell variants lacking the antigen-presenting mhc-i molecule or the protein antigen that contains the immunogenic peptide epitope (or both) 2,5. Particularly when irreversible, mhc-i loss in cancer patients has been claimed to negatively affect prognosis 2. Assuming that spontaneous and immunotherapy-induced mhc-i losses are drivers and not passengers of tumour progression, it remains to be explained why they do not incite recognition and tumour lysis by nk cells (Figure 1). Porgador et al. 6 described a very high prevalence (5 in 13 cases) of irreversible complete mhc-i losses in patients treated with various immunotherapeutic regimens. Despite the cells being very sensitive targets of autologous nk cells in vitro, clinical outcome was reported to be poor. Likewise, Pende et al. 7 observed that long-term tumour cell lines, even when established from patients not undergoing immunotherapy, do not express enough mhc-i to protect themselves from nk recognition. Why, then, can these tumours evade in the face of a brisk in vitro nk response? A possible interpretation is that simple cytotoxicity readouts do not reflect the lytic behaviour of immune effectors in vivo. After all, if antitumour T-cell counts and activity in vitro are not entirely predictive of clinical responsiveness to vaccination 8, why should nk cell responses in vivo be faithfully recapitulated in an in vitro assay? Alternatively, it might be hypothesized that nk cells have nothing to do with tumour immune surveillance, at least in humans. Indeed, lymphoid cell infiltrates contain many more T cells than nk cells, and only T cells are positively associated with a favorable outcome 9. Whatever the interpretation, a drastic objection is that certain subsets of nk cells may be important at early stages, but may be long gone by the time the tumour becomes clinically evident and hits the pathology slide. If nk cells are indeed important, tumours low in mhc-i may elude them either by exploiting certain “gaps” in the inhibitory nk receptor repertoire 10 or, analogous with viral immuno-evasion strategies 11, by “replacing” mhc-i self-inhibitory signals with other inhibitory ligands such as the non-classical mhc-i human leukocyte antigens G (hla-g) and E (hla-e) 12–14. However, at least hla-e behaves not only as an inhibitory, but also as a triggering ligand 15. In addition, hla-e expression may not be restricted to tumours with mhc-i loss as required by the “replacement” model 16,17. Finally, and quite surprisingly, hla-e is associated with a good prognosis, at least in certain tumour histotypes 18–20. It will be of considerable interest to find out if and how tumours use nk-decoy tactics. Although there are simpler ways to explain mhc-i–driven tumour evasion from both ctl and nk cells, those explanations have received considerably less attention than the foregoing mechanisms. A straightforward assumption is that, besides mhc-i losses adopted by ctl-sensitive tumours, there are mechanisms of mhc-i gains, and those mechanisms are preferred by another set of tumours that are particularly sensitive to nk lysis. It might be envisaged that the opposing influences of ctl and nk cells prevent any major change in mhc-i expression, making less-aggressive tumours resemble their normal counterparts. By contrast, aggressive tumours may escape by adopting whichever immuno-evasion strategy is the most advantageous in the context of the immune response mounted by an individual host. Indeed, a Gaussian distribution of mhc-i expression around “normal” values was observed in vitro and in vivo in a variety of solid tumours 3,21, mhc-i losses and mhc-i gains both being associated with poor prognosis in colorectal carcinoma 22. Given the opposing effects of mhc-i molecules on ctl and nk cells (Figure 1), an mhc-i phenotype efficiently triggering both effectors is a contradiction in terms. For instance, in the classical paper that pioneered the “missing self” hypothesis, a tap-defective mutant of the murine lymphoma RMA, called RMAS, was shown to be rejected essentially by nk cells 1. Recently, rna interference of the same RMA cells for eraap (just downstream of tap in the antigen-processing machinery pathway 23) similarly resulted in tumour rejection 24, but in addition to nk cells, T cells (CD4 and CD8 alike) were also involved. It appears that poorly folded mhc-i molecules synthesized in the absence of eraap can be “seen” as abnormal by several immune effectors. Quite interestingly, only a few human tumours express low erap1 and erap2 levels 25,26, suggesting that the spontaneous occurrence of this altered, two-edge phenotype is counterselected in vivo. In conclusion, it is fairly clear what tumours look like when they are “out of the hands” of the immune system, but we know much less of “real” tumours under immunologic scrutiny and during immunoediting in vivo. If ctl and nk cells must both be “tuned in” to reject tumours, many more immunoevasive mhc-i (and non-mhc-i 27) phenotypes remain to be discovered.

IRF1 and NF-kB Restore MHC Class I-Restricted Tumor Antigen Processing and Presentation to Cytotoxic T Cells in Aggressive Neuroblastoma

Neuroblastoma (NB), the most common solid extracranial cancer of childhood, displays a remarkable low expression of Major Histocompatibility Complex class I (MHC-I) and Antigen Processing Machinery (APM) molecules, including Endoplasmic Reticulum (ER) Aminopeptidases, and poorly presents tumor antigens to Cytotoxic T Lymphocytes (CTL). We have previously shown that this is due to low expression of the transcription factor NF-kB p65. Herein, we show that not only NF-kB p65, but also the Interferon Regulatory Factor 1 (IRF1) and certain APM components are low in a subset of NB cell lines with aggressive features. Whereas single transfection with either IRF1, or NF-kB p65 is ineffective, co-transfection results in strong synergy and substantial reversion of the MHC-I/APM-low phenotype in all NB cell lines tested. Accordingly, linked immunohistochemistry expression patterns between nuclear IRF1 and p65 on the one hand, and MHC-I on the other hand, were observed in vivo. Absence and presence of the three molecules neatly segregated between high-grade and low-grade NB, respectively. Finally, APM reconstitution by double IRF1/p65 transfection rendered a NB cell line susceptible to killing by anti MAGE-A3 CTLs, lytic efficiency comparable to those seen upon IFN-γ treatment. This is the first demonstration that a complex immune escape phenotype can be rescued by reconstitution of a limited number of master regulatory genes. These findings provide molecular insight into defective MHC-I expression in NB cells and provide the rational for T cell-based immunotherapy in NB variants refractory to conventional therapy.

PD-L1 Is a Therapeutic Target of the Bromodomain Inhibitor JQ1 and, Combined with HLA Class I, a Promising Prognostic Biomarker in Neuroblastoma

Purpose: This study sought to evaluate the expression of programmed cell death-ligand-1 (PD-L1) and HLA class I on neuroblastoma cells and programmed cell death-1 (PD-1) and lymphocyte activation gene 3 (LAG3) on tumor-infiltrating lymphocytes to better define patient risk stratification and understand whether this tumor may benefit from therapies targeting immune checkpoint molecules. Experimental Design: In situ IHC staining for PD-L1, HLA class I, PD-1, and LAG3 was assessed in 77 neuroblastoma specimens, previously characterized for tumor-infiltrating T-cell density and correlated with clinical outcome. Surface expression of PD-L1 was evaluated by flow cytometry and IHC in neuroblastoma cell lines and tumors genetically and/or pharmacologically inhibited for MYC and MYCN. A dataset of 477 human primary neuroblastomas from GEO and ArrayExpress databases was explored for PD-L1, MYC, and MYCN correlation. Results: Multivariate Cox regression analysis demonstrated that the combination of PD-L1 and HLA class I tumor cell density is a prognostic biomarker for predicting overall survival in neuroblastoma patients (P = 0.0448). MYC and MYCN control the expression of PD-L1 in neuroblastoma cells both in vitro and in vivo. Consistently, abundance of PD-L1 transcript correlates with MYC expression in primary neuroblastoma. Conclusions: The combination of PD-L1 and HLA class I represents a novel prognostic biomarker for neuroblastoma. Pharmacologic inhibition of MYCN and MYC may be exploited to target PD-L1 and restore an efficient antitumor immunity in high-risk neuroblastoma. Clin Cancer Res; 23(15); 4462–72. ©2017 AACR.

Epigenetic Deregulation of MicroRNAs in Rhabdomyosarcoma and Neuroblastoma and Translational Perspectives

Gene expression control mediated by microRNAs and epigenetic remodeling of chromatin are interconnected processes often involved in feedback regulatory loops, which strictly guide proper tissue differentiation during embryonal development. Altered expression of microRNAs is one of the mechanisms leading to pathologic conditions, such as cancer. Several lines of evidence pointed to epigenetic alterations as responsible for aberrant microRNA expression in human cancers. Rhabdomyosarcoma and neuroblastoma are pediatric cancers derived from cells presenting features of skeletal muscle and neuronal precursors, respectively, blocked at different stages of differentiation. Consistently, tumor cells express tissue markers of origin but are unable to terminally differentiate. Several microRNAs playing a key role during tissue differentiation are often epigenetically downregulated in rhabdomyosarcoma and neuroblastoma and behave as tumor suppressors when re-expressed. Recently, inhibition of epigenetic modulators in adult tumors has provided encouraging results causing re-expression of anti-tumor master gene pathways. Thus, a similar approach could be used to correct the aberrant epigenetic regulation of microRNAs in rhabdomyosarcoma and neuroblastoma. The present review highlights the current insights on epigenetically deregulated microRNAs in rhabdomyosarcoma and neuroblastoma and their role in tumorigenesis and developmental pathways. The translational clinical implications and challenges regarding modulation of epigenetic chromatin remodeling/microRNAs interconnections are also discussed.

High-Resolution Array CGH Profiling Identifies Na/K Transporting ATPase Interacting 2 (NKAIN2) as a Predisposing Candidate Gene in Neuroblastoma

Neuroblastoma (NB), the most common solid cancer in early childhood, usually occurs sporadically but also its familial occurance is known in 1-2% of NB patients. Germline mutations in the ALK and PHOX2B genes have been found in a subset of familial NBs. However, because some individuals harbouring mutations in these genes do not develop this tumor, additional genetic alterations appear to be required for NB pathogenesis. Herein, we studied an Italian family with three NB patients, two siblings and a first cousin, carrying an ALK germline-activating mutation R1192P, that was inherited from their unaffected mothers and with no mutations in the PHOX2B gene. A comparison between somatic and germline DNA copy number changes in the two affected siblings by a high resolution array-based Comparative Genomic Hybridization (CGH) analysis revealed a germline gain at NKAIN2 (Na/K transporting ATPase interacting 2) locus in one of the sibling, that was inherited from the parent who does not carry the ALK mutation. Surprisingly, NKAIN2 was expressed at high levels also in the affected sibling that lacks the genomic gain at this locus, clearly suggesting the existance of other regulatory mechanisms. High levels of NKAIN2 were detected in the MYCN-amplified NB cell lines and in the most aggressive NB lesions as well as in the peripheral blood of a large cohort of NB patients. Consistent with a role of NKAIN2 in NB development, NKAIN2 was down-regulated during all-trans retinoic acid differentiation in two NB cell lines. Taken together, these data indicate a potential role of NKAIN2 gene in NB growth and differentiation.

MYCN is an immunosuppressive oncogene dampening the expression of ligands for NK-cell-activating receptors in human high-risk neuroblastoma

ABSTRACT Neuroblastoma (NB) is the most common extracranial solid tumor occurring in childhood. Amplification of the MYCN oncogene is associated with poor prognosis. Downregulation on NB cells of ligands recognized by Natural Killer (NK) cell-activating receptors, involved in tumor cell recognition and lysis, may contribute to tumor progression and relapse. Here, we demonstrate that in human NB cell lines MYCN expression inversely correlates with that of ligands recognized by NKG2D and DNAM1 activating receptors in human NB cell lines. In the MYCN-inducible Tet-21/N cell line, downregulation of MYCN resulted in enhanced expression of the activating ligands MICA, ULBPs and PVR, which rendered tumor cells more susceptible to recognition and lysis mediated by NK cells. Conversely, a MYCN non-amplified NB cell line transfected with MYCN showed an opposite behavior compared with control cells. Consistent with these findings, an inverse correlation was detected between the expression of MYCN and that of ligands for NK-cell-activating receptors in 12 NB patient specimens both at mRNA and protein levels. Taken together, these results provide the first demonstration that MYCN acts as an immunosuppressive oncogene in NB cells that negatively regulates the expression of ligands for NKG2D and DNAM-1 NK-cell-activating receptors. Our study provides a clue to exploit MYCN expression levels as a biomarker to predict the efficacy of NK-cell-based immunotherapy in NB patients.

The role of HCMV and HIV-1 MicroRNAs: Processing, and mechanisms of action during viral infection

Viruses infect host cells releasing their genome (DNA or RNA) containing all information needed to replicate themselves. The viral genome takes control of the cells and helps the virus to evade the host immune system. Some viruses alter the functions of infected cells without killing them. In some cases infected cells lose control over normal cell proliferation and becomes cancerous. Viruses, such as HCMV and HIV-1, may leave their viral genome in the host cells for a certain period (latency) and begin to replicate when the cells are stressed causing diseases. HCMV and HIV-1 have developed multiple strategies to avoid recognition and elimination by the host’s immune system. These strategies rely on viral products that mimic specific components of the host cells to prevent immune recognition of virally infected cells. In addition to viral proteins, viruses encode short non-coding RNAs (vmiRNAs) that regulate both viral and host cellular transcripts to favor viral infection and actively curtail the host’s antiviral immune response. In this review, we will give an overview of the general functions of microRNAs generated by HCMV and HIV-1, their processing and interaction with the host’s immune system.