Loredana Cifaldi

Antagomir-17-5p Abolishes the Growth of Therapy- Resistant Neuroblastoma through p21 and BIM

We identified a key oncogenic pathway underlying neuroblastoma progression: specifically, MYCN, expressed at elevated level, transactivates the miRNA 17-5p-92 cluster, which inhibits p21 and BIM translation by interaction with their mRNA 3′ UTRs. Overexpression of miRNA 17-5p-92 cluster in MYCN-not-amplified neuroblastoma cells strongly augments their in vitro and in vivo tumorigenesis. In vitro or in vivo treatment with antagomir-17-5p abolishes the growth of MYCN-amplified and therapy-resistant neuroblastoma through p21 and BIM upmodulation, leading to cell cycling blockade and activation of apoptosis, respectively. In primary neuroblastoma, the majority of cases show a rise of miR-17-5p level leading to p21 downmodulation, which is particularly severe in patients with MYCN amplification and poor prognosis. Altogether, our studies demonstrate for the first time that antagomir treatment can abolish tumor growth in vivo, specifically in therapy-resistant neuroblastoma.

Natural Killer Cells Efficiently Reject Lymphoma Silenced for the Endoplasmic Reticulum Aminopeptidase Associated with Antigen Processing

The endoplasmic reticulum aminopeptidase ERAAP is involved in the final trimming of peptides for presentation by MHC class I (MHC-I) molecules. Herein, we show that ERAAP silencing results in MHC-I peptide-loading defects eliciting rejection of the murine T-cell lymphoma RMA in syngeneic mice. Although CD4 and CD8 T cells are also involved, rejection is mainly due to an immediate natural killer (NK) cell response and depends on the MHC-I-peptide repertoire because replacement of endogenous peptides with correctly trimmed, high-affinity peptides is sufficient to restore an NK-protective effect of MHC-I molecules through the Ly49C/I NK inhibitory receptors. At the crossroad between innate and adaptive immunity, ERAAP is therefore unique in its two-tiered ability to control tumor immunogenicity. Because a large fraction of human tumors express high levels of the homologous ERAP1 and/or ERAP2, the present findings highlight a convenient, novel target for cancer immunotherapy.

Inhibition of Natural Killer Cell Cytotoxicity by Interleukin-6: Implications for the Pathogenesis of Macrophage Activation Syndrome

Systemic juvenile idiopathic arthritis (JIA) is associated with high levels of interleukin‐6 (IL‐6) in the serum and synovial fluid, and impairment of natural killer (NK) cell function is often observed. This study was undertaken to evaluate a possible link between these 2 biologic findings and whether they may be associated with the development of macrophage activation syndrome, a condition frequently observed in systemic JIA.

NF-κB, and not MYCN, Regulates MHC Class I and Endoplasmic Reticulum Aminopeptidases in Human Neuroblastoma Cells

Neuroblastoma (NB) is the most common solid extracranial cancer of childhood. Amplification and overexpression of the MYCN oncogene characterize the most aggressive forms and are believed to severely downregulate MHC class I molecules by transcriptional inhibition of the p50 NF-kappaB subunit. In this study, we found that in human NB cell lines, high MYCN expression is not responsible for low MHC class I expression because neither transfection-mediated overexpression nor small interfering RNA suppression of MYCN affects MHC class I and p50 levels. Furthermore, we identified NF-kappaB as the immediate upstream regulator of MHC class I because the p65 NF-kappaB subunit binds MHC class I promoter in chromatin immunoprecipitation experiments, and MHC class I expression is enhanced by p65 transfection and reduced by (a) the chemical NF-kappaB inhibitor sulfasalazine, (b) a dominant-negative IKBalpha gene, and (c) p65 silencing. Moreover, we showed that the endoplasmic reticulum aminopeptidases ERAP1 and ERAP2, which generate MHC class I binding peptides, are regulated by NF-kappaB, contain functional NF-kappaB-binding elements in their promoters, and mimic MHC class I molecules in the expression pattern. Consistent with these findings, nuclear p65 was detected in NB cells that express MHC class I molecules in human NB specimens. Thus, the coordinated downregulation of MHC class I, ERAP1, and ERAP2 in aggressive NB cells is attributable to a low transcriptional availability of NF-kappaB, possibly due to an unknown suppressor other than MYCN.

Role of Endoplasmic Reticulum Aminopeptidases in Health and Disease: from Infection to Cancer

Endoplasmic reticulum (ER) aminopeptidases ERAP1 and ERAP2 (ERAPs) are essential for the maturation of a wide spectrum of proteins involved in various biological processes. In the ER, these enzymes work in concert to trim peptides for presentation on MHC class I molecules. Loss of ERAPs function substantially alters the repertoire of peptides presented by MHC class I molecules, critically affecting recognition of both NK and CD8+ T cells. In addition, these enzymes are involved in the modulation of inflammatory responses by promoting the shedding of several cytokine receptors, and in the regulation of both blood pressure and angiogenesis. Recent genome-wide association studies have identified common variants of ERAP1 and ERAP2 linked to several human diseases, ranging from viral infections to autoimmunity and cancer. More recently, inhibition of ER peptide trimming has been shown to play a key role in stimulating innate and adaptive anti-tumor immune responses, suggesting that inhibition of ERAPs might be exploited for the establishment of innovative therapeutic approaches against cancer. This review summarizes data currently available for ERAP enzymes in ER peptide trimming and in other immunological and non-immunological functions, paying attention to the emerging role played by these enzymes in human diseases.

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.

ERAP1 regulates natural killer cell function by controlling the engagement of inhibitory receptors

The endoplasmic reticulum aminopeptidase ERAP1 regulates innate and adaptive immune responses by trimming peptides for presentation by MHC class I (MHC-I) molecules. Herein, we demonstrate that genetic or pharmacological inhibition of ERAP1 on human tumor cell lines perturbs their ability to engage several classes of inhibitory receptors by their specific ligands, including killer cell Ig-like receptors (KIR) by classical MHC-I-peptide (pMHC-I) complexes and the lectin-like receptor CD94-NKG2A by nonclassical pMHC-I complexes, in each case leading to natural killer (NK) cell killing. The protective effect of pMHC-I complexes could be restored in ERAP1-deficient settings by the addition of known high-affinity peptides, suggesting that ERAP1 was needed to positively modify the affinity of natural ligands. Notably, ERAP1 inhibition enhanced the ability of NK cells to kill freshly established human lymphoblastoid cell lines from autologous or allogeneic sources, thereby promoting NK cytotoxic activity against target cells that would not be expected because of KIR-KIR ligand matching. Overall, our results identify ERAP1 as a modifier to leverage immune functions that may improve the efficacy of NK cell-based approaches for cancer immunotherapy.

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.

TIM-3/Gal-9 interaction induces IFNγ-dependent IDO1 expression in acute myeloid leukemia blast cells

NK cells expressing TIM-3 show a marked increase in IFNγ production in response to acute myeloid leukemia (AML) blast cells that endogenously express Gal-9. Herein, we demonstrate that NK cell-mediated production of IFNγ, induced by TIM-3/Gal-9 interaction and released in bone marrow microenvironment, is responsible for IDO1 expression in AML blasts. IDO1-expressing AML blasts consequently down-regulate NK cell degranulation activity, by sustaining leukemia immune escape. Furthermore, the blocking of TIM-3/Gal-9 interaction strongly down-regulates IFNγ-dependent IDO1 activity. Thus, the inhibition of TIM-3/Gal-9 immune check point, which affects NK cell-dependent IFNγ production and the consequent IDO1 activation, could usefully integrate current chemotherapeutic approaches.