From the identification of actionable molecular targets to the generation of faithful neuroblastoma patient-derived preclinical models.

BACKGROUND: Neuroblastoma (NB) represents the most frequent and aggressive form of extracranial solid tumor of infants. Although the overall survival of patients with NB has improved in the last years, more than 50% of high-risk patients still undergo a relapse. Thus, in the era of precision/personalized medicine, the need for high-risk NB patient-specific therapies is urgent. METHODS: Within the PeRsonalizEd Medicine (PREME) program, patient-derived NB tumors and bone marrow (BM)-infiltrating NB cells, derived from either iliac crests or tumor bone lesions, underwent to histological and to flow cytometry immunophenotyping, respectively. BM samples containing a NB cells infiltration from 1 to 50 percent, underwent to a subsequent NB cells enrichment using immune-magnetic manipulation. Then, NB samples were used for the identification of actionable targets and for the generation of 3D/tumor-spheres and Patient-Derived Xenografts (PDX) and Cell PDX (CPDX) preclinical models. RESULTS: Eighty-four percent of NB-patients showed potentially therapeutically targetable somatic alterations (including point mutations, copy number variations and mRNA over-expression). Sixty-six percent of samples showed alterations, graded as "very high priority", that are validated to be directly targetable by an approved drug or an investigational agent. A molecular targeted therapy was applied for four patients, while a genetic counseling was suggested to two patients having one pathogenic germline variant in known cancer predisposition genes. Out of eleven samples implanted in mice, five gave rise to (C)PDX, all preserved in a local PDX Bio-bank. Interestingly, comparing all molecular alterations and histological and immunophenotypic features among the original patient's tumors and PDX/CPDX up to second generation, a high grade of similarity was observed. Notably, also 3D models conserved immunophenotypic features and molecular alterations of the original tumors. CONCLUSIONS: PREME confirms the possibility of identifying targetable genomic alterations in NB, indeed, a molecular targeted therapy was applied to four NB patients. PREME paves the way to the creation of clinically relevant repositories of faithful patient-derived (C)PDX and 3D models, on which testing precision, NB standard-of-care and experimental medicines.

News on immune checkpoint inhibitors as immunotherapy strategies in adult and pediatric solid tumors.

Immune checkpoint inhibitors (ICIs) have shown unprecedented benefits in various adult cancers, and this success has prompted the exploration of ICI therapy even in childhood malignances. Although the use of ICIs as individual agents has achieved disappointing response rates, combinational therapies are likely to promise better results. However, only a subset of patients experienced prolonged clinical effects, thus suggesting the need to identify robust bio-markers that predict individual clinical response or resistance to ICI therapy as the main challenge. In this review, we focus on how the use of ICIs in adult cancers can be translated into pediatric malignances. We discuss the physiological mechanism of action of each IC, including PD-1, PD-L1 and CTLA-4 and the new emerging ones, LAG-3, TIM-3, TIGIT, B7-H3, BTLA and IDO-1, and evaluate their prognostic value in both adult and childhood tumors. Furthermore, we offer an overview of preclinical models and clinical trials currently under investigation to improve the effectiveness of cancer immunotherapies in these patients. Finally, we outline the main predictive factors that influence the efficacy of ICIs, in order to lay the basis for the development of a pan-cancer immunogenomic model, able to direct young patients towards more specific immunotherapy.

Redundancy and Complementarity between ERAP1 and ERAP2 Revealed by their Effects on the Behcet's Disease-associated HLA-B*51 Peptidome.

The endoplasmic reticulum aminopeptidases ERAP1 and ERAP2 trim peptides to be loaded onto HLA molecules, including the main risk factor for Behçet's disease HLA-B*51. ERAP1 is also a risk factor among HLA-B*51-positive individuals, whereas no association is known with ERAP2. This study addressed the mutual relationships between both enzymes in the processing of an HLA-bound peptidome, interrogating their differential association with Behçet's disease. CRISPR/Cas9 was used to generate knock outs of ERAP1, ERAP2 or both from transfectant 721.221-HLA-B*51:01 cells. The surface expression of HLA-B*51 was reduced in all cases. The effects of depleting each or both enzymes on the B*51:01 peptidome were analyzed by quantitative label-free mass spectrometry. Substantial quantitative alterations of peptide length, subpeptidome balance, N-terminal residue usage, affinity and presentation of noncanonical ligands were observed. These effects were often different in the presence or absence of the other enzyme, revealing their mutual dependence. In the absence of ERAP1, ERAP2 showed similar and significant processing of B*51:01 ligands, indicating functional redundancy. The high overlap between the peptidomes of wildtype and double KO cells indicates that a large majority of B*51:01 ligands are present in the ER even in the absence of ERAP1/ERAP2. These results indicate that both enzymes have distinct, but complementary and partially redundant effects on the B*51:01 peptidome, leading to its optimization and maximal surface expression. The distinct effects of both enzymes on the HLA-B*51 peptidome provide a basis for their differential association with Behçet's disease and suggest a pathogenetic role of the B*51:01 peptidome.

Quantification of the Immune Content in Neuroblastoma: Deep Learning and Topological Data Analysis in Digital Pathology.

We introduce here a novel machine learning (ML) framework to address the issue of the quantitative assessment of the immune content in neuroblastoma (NB) specimens. First, the EUNet, a U-Net with an EfficientNet encoder, is trained to detect lymphocytes on tissue digital slides stained with the CD3 T-cell marker. The training set consists of 3782 images extracted from an original collection of 54 whole slide images (WSIs), manually annotated for a total of 73,751 lymphocytes. Resampling strategies, data augmentation, and transfer learning approaches are adopted to warrant reproducibility and to reduce the risk of overfitting and selection bias. Topological data analysis (TDA) is then used to define activation maps from different layers of the neural network at different stages of the training process, described by persistence diagrams (PD) and Betti curves. TDA is further integrated with the uniform manifold approximation and projection (UMAP) dimensionality reduction and the hierarchical density-based spatial clustering of applications with noise (HDBSCAN) algorithm for clustering, by the deep features, the relevant subgroups and structures, across different levels of the neural network. Finally, the recent TwoNN approach is leveraged to study the variation of the intrinsic dimensionality of the U-Net model. As the main task, the proposed pipeline is employed to evaluate the density of lymphocytes over the whole tissue area of the WSIs. The model achieves good results with mean absolute error 3.1 on test set, showing significant agreement between densities estimated by our EUNet model and by trained pathologists, thus indicating the potentialities of a promising new strategy in the quantification of the immune content in NB specimens. Moreover, the UMAP algorithm unveiled interesting patterns compatible with pathological characteristics, also highlighting novel insights into the dynamics of the intrinsic dataset dimensionality at different stages of the training process. All the experiments were run on the Microsoft Azure cloud platform.

ERAP1 and ERAP2 Enzymes: A Protective Shield for RAS against COVID-19?

Patients with coronavirus disease 2019 (COVID-19) have a wide variety of clinical outcomes ranging from asymptomatic to severe respiratory syndrome that can progress to life-threatening lung lesions. The identification of prognostic factors can help to improve the risk stratification of patients by promptly defining for each the most effective therapy to resolve the disease. The etiological agent causing COVID-19 is a new coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that enters cells via the ACE2 receptor. SARS-CoV-2 infection causes a reduction in ACE2 levels, leading to an imbalance in the renin-angiotensin system (RAS), and consequently, in blood pressure and systemic vascular resistance. ERAP1 and ERAP2 are two RAS regulators and key components of MHC class I antigen processing. Their polymorphisms have been associated with autoimmune and inflammatory conditions, hypertension, and cancer. Based on their involvement in the RAS, we believe that the dysfunctional status of ERAP1 and ERAP2 enzymes may exacerbate the effect of SARS-CoV-2 infection, aggravating the symptomatology and clinical outcome of the disease. In this review, we discuss this hypothesis.

Genetically driven CD39 expression shapes human tumor-infiltrating CD8+ T-cell functions.

In our study, we investigated the role of CD39 on tumor-infiltrating CD8+ T lymphocytes (CD8+ TILs) in colorectal, head and neck and pancreatic cancers. Partially confirming recent observations correlating the CD39 expression with T-cell exhaustion, we demonstrated a divergent functional activity in CD39+ CD8+ TILs. On the one hand, CD39+ CD8+ TILs (as compared to their CD39- counterparts) produced significantly lower IFN-γ and IL-2 amounts, expressed higher PD-1, and inversely correlated with perforin and granzyme B expression. On the other, they displayed a significantly higher proliferative capacity ex vivo that was inversely correlated with the PD-1 expression. Therefore, CD39+ CD8+ TILs, including those co-expressing the CD103 (a marker of T resident memory [TRM] cells), were defined as partially dysfunctional T cells that correlate with tumor patients with initial progression stages. Interestingly, our results identified for the first time a single nucleotide polymorphism (SNP rs10748643 A>G), as a genetic factor associated with CD39 expression in CD8+ TILs. Finally, we demonstrated that compounds inhibiting CD39-related ATPases improved CD39+ CD8+ T-cell effector function ex vivo, and that CD39+ CD8+ TILs displayed effective suppression function in vitro. Overall these data suggest that the SNP analysis may represent a suitable predictor of CD39+ CD8+ T-cell expression in cancer patients, and propose the modulation of CD39 as a new strategy to restore partially exhausted CD8+ TILs.

Impact of Natural Occurring ERAP1 Single Nucleotide Polymorphisms within miRNA-Binding Sites on HCMV Infection.

Human cytomegalovirus (HCMV) is a ß-herpesvirus that causes serious problems in people with a compromised immune system, whereas it coexists asymptomatically within the host with a healthy immune system. Like other viruses, HCMV has adopted multiples strategies to manipulate the host's immune responses. Among them, expression of viral microRNAs (miRNAs) is one of the most intriguing. HCMV miR-UL112-5p and miR-US4-1 have been found to contribute to immune evasion by targeting the endoplasmic reticulum aminopeptidase 1 (ERAP1), a highly polymorphic key component of antigen processing. The current incomplete picture on the interplay between viral miRNAs and host immunity implies the need to better characterize the host genetic determinants. Naturally occurring single nucleotide polymorphisms (SNPs) within the miRNA binding sites of target genes may affect miRNA-target interactions. In this review, we focus on the relevance of 3' untranslated region (3'UTR) ERAP1 SNPs within miRNA binding sites in modulating miRNA-mRNA interactions and the possible consequent individual susceptibility to HCMV infection. Moreover, we performed an in silico analysis using different bioinformatic algorithms to predict ERAP1 variants with a putative powerful biological function. This evidence provides a basis to deepen the knowledge on how 3'UTR ERAP1 variants may alter the mechanism of action of HCMV miRNAs, in order to develop targeted antiviral therapies.

Counter-regulation of regulatory T cells by autoreactive CD8+ T cells in rheumatoid arthritis.

The mechanisms whereby autoreactive T cells escape peripheral tolerance establishing thus autoimmune diseases in humans remain an unresolved question. Here, we demonstrate that autoreactive polyfunctional CD8+ T cells recognizing self-antigens (i.e., vimentin, actin cytoplasmic 1, or non-muscle myosin heavy chain 9 epitopes) with high avidity, counter-regulate Tregs by killing them, in a consistent percentage of rheumatoid arthritis (RA) patients. Indeed, these CD8+ T cells express a phenotype and gene profile of effector (eff) cells and, upon antigen-specific activation, kill Tregs indirectly in an NKG2D-dependent bystander fashion in vitro. This data provides a mechanistic basis for the finding showing that AE-specific (CD107a+) CD8+ T killer cells correlate, directly with the disease activity score, and inversely with the percentage of activated Tregs, in both steady state and follow-up studies in vivo. In addition, multiplex immunofluorescence imaging analyses of inflamed synovial tissues in vivo show that a remarkable number of CD8+ T cells express granzyme-B and selectively contact FOXP3+ Tregs, some of which are in an apoptotic state, validating hence the possibility that CD8+ Teff cells can counteract neighboring Tregs within inflamed tissues, by killing them. Alternatively, the disease activity score of a different subset of patients is correlated with the expansion of a peculiar subpopulation of autoreactive low avidity, partially-activated (pa)CD8+ T cells that, despite they conserve the conventional naïve (N) phenotype, produce high levels of tumor necrosis factor (TNF)-α and exhibit a gene expression signature of a progressive activation state. Tregs directly correlate with the expansion of this autoreactive (low avidity) paCD8+ TN cell subset in vivo, and efficiently control their differentiation rather their proliferation in vitro. Interestingly, autoreactive high avidity CD8+ Teff cells or low avidity paCD8+ TN cells are significantly expanded in RA patients who would become non-responders or patients who would become responders to TNF-α inhibitor therapy, respectively. These data provide evidence of a previously undescribed role of such mechanisms in the progression and therapy of RA.

Manipulating the tumor immune microenvironment to improve cancer immunotherapy: IGF1R, a promising target.

Cancer immunotherapy has made impressive advances in improving the outcome of patients affected by malignant diseases. Nonetheless, some limitations still need to be tackled to more efficiently and safely treat patients, in particular for those affected by solid tumors. One of the limitations is related to the immunosuppressive tumor microenvironment (TME), which impairs anti-tumor immunity. Efforts to identify targets able to turn the TME into a milieu more auspicious to current immuno-oncotherapy is a real challenge due to the high redundancy of the mechanisms involved. However, the insulin-like growth factor 1 receptor (IGF1R), an attractive drug target for cancer therapy, is emerging as an important immunomodulator and regulator of key immune cell functions. Here, after briefly summarizing the IGF1R signaling pathway in cancer, we review its role in regulating immune cells function and activity, and discuss IGF1R as a promising target to improve anti-cancer immunotherapy.

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.

Multidrug resistance and cancer stem cells in neuroblastoma and hepatoblastoma.

Chemotherapy is one of the major modalities in treating cancers. However, its effectiveness is limited by the acquisition of multidrug resistance (MDR). Several mechanisms could explain the up-regulation of MDR genes/proteins in cancer after chemotherapy. It is known that cancer stem cells (CSCs) play a role as master regulators. Therefore, understanding the mechanisms that regulate some traits of CSCs may help design efficient strategies to overcome chemoresistance. Different CSC phenotypes have been identified, including those found in some pediatric malignancies. As solid tumors in children significantly differ from those observed in adults, this review aims at providing an overview of the mechanistic relationship between MDR and CSCs in common solid tumors, and, in particular, focuses on clinical as well as experimental evidence of the relations between CSCs and MDR in neuroblastoma and hepatoblastoma. Finally, some novel approaches, such as concomitant targeting of multiple key transcription factors governing the stemness of CSCs, as well as nanoparticle-based approaches will also be briefly addressed.

Two bullets in the gun: combining immunotherapy with chemotherapy to defeat neuroblastoma by targeting adrenergic-mesenchymal plasticity.

Neuroblastoma (NB) is a childhood tumor that originates in the peripheral sympathetic nervous system and is responsible for 15% of cancer-related deaths in the pediatric population. Despite intensive multimodal treatment, many patients with high-risk NB relapse and develop a therapy-resistant tumor. One of the phenomena related to therapeutic resistance is intratumor heterogeneity resulting from the adaptation of tumor cells in response to different selective environmental pressures. The transcriptional and epigenetic profiling of NB tissue has recently revealed the existence of two distinct cellular identities in the NB, termed adrenergic (ADRN) and mesenchymal (MES), which can spontaneously interconvert through epigenetic regulation. This phenomenon, known as tumor plasticity, has a major impact on cancer pathogenesis. The aim of this review is to describe the peculiarities of these two cell states, and how their plasticity affects the response to current therapeutic treatments, with special focus on the immunogenic potential of MES cells. Furthermore, we will discuss the opportunity to combine immunotherapy with chemotherapy to counteract NB phenotypic interconversion.

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.

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.

ERAP1 as an emerging therapeutic target for medulloblastoma.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme that shapes the peptide repertoire presented by major histocompatibility complex class I (MHC-I) molecules, thereby affecting tumor immunogenicity. ERAP1 is altered in many tumors, including medulloblastoma (MB). We review the role of ERAP1 in MB development and the possibility of targeting this enzyme for MB treatment.

Targeting the antigen processing and presentation pathway to overcome resistance to immune checkpoint therapy.

Despite the significant clinical advances with the use of immune checkpoint inhibitors (ICIs) in a wide range of cancer patients, response rates to the therapy are variable and do not always result in long-term tumor regression. The development of ICI-resistant disease is one of the pressing issue in clinical oncology, and the identification of new targets and combination therapies is a crucial point to improve response rates and duration. Antigen processing and presentation (APP) pathway is a key element for an efficient response to ICI therapy. Indeed, malignancies that do not express tumor antigens are typically poor infiltrated by T cells and unresponsive to ICIs. Therefore, improving tumor immunogenicity potentially increases the success rate of ICI therapy. In this review, we provide an overview of the key elements of the APP machinery that can be exploited to enhance tumor immunogenicity and increase the efficacy of ICI-based immunotherapy.

DNAM-1-chimeric receptor-engineered NK cells, combined with Nutlin-3a, more effectively fight neuroblastoma cells in vitro: a proof-of-concept study.

Adoptive transfer of engineered NK cells, one of clinical approaches to fight cancer, is gaining great interest in the last decade. However, the development of new strategies is needed to improve clinical efficacy and safety of NK cell-based immunotherapy. NK cell-mediated recognition and lysis of tumor cells are strictly dependent on the expression of ligands for NK cell-activating receptors NKG2D and DNAM-1 on tumor cells. Of note, the PVR/CD155 and Nectin-2/CD112 ligands for DNAM-1 are expressed primarily on solid tumor cells and poorly expressed in normal tissue cells. Here, we generated human NK cells expressing either the full length DNAM-1 receptor or three different DNAM-1-based chimeric receptor that provide the expression of DNAM-1 fused to a costimulatory molecule such as 2B4 and CD3ζ chain. Upon transfection into primary human NK cells isolated from healthy donors, we evaluated the surface expression of DNAM-1 and, as a functional readout, we assessed the extent of degranulation, cytotoxicity and the production of IFNγ and TNFα in response to human leukemic K562 cell line. In addition, we explored the effect of Nutlin-3a, a MDM2-targeting drug able of restoring p53 functions and known to have an immunomodulatory effect, on the degranulation of DNAM-1-engineered NK cells in response to human neuroblastoma (NB) LA-N-5 and SMS-KCNR cell lines. By comparing NK cells transfected with four different plasmid vectors and through blocking experiments, DNAM-1-CD3ζ-engineered NK cells showed the strongest response. Furthermore, both LA-N-5 and SMS-KCNR cells pretreated with Nutlin-3a were significantly more susceptible to DNAM-1-engineered NK cells than NK cells transfected with the empty vector. Our results provide a proof-of-concept suggesting that the combined use of DNAM-1-chimeric receptor-engineered NK cells and Nutlin-3a may represent a novel therapeutic approach for the treatment of solid tumors, such as NB, carrying dysfunctional p53.

Combined mitoxantrone and anti-TGFß treatment with PD-1 blockade enhances antitumor immunity by remodelling the tumor immune landscape in neuroblastoma.

BACKGROUND: Poor infiltration of functioning T cells renders tumors unresponsive to checkpoint-blocking immunotherapies. Here, we identified a combinatorial in situ immunomodulation strategy based on the administration of selected immunogenic drugs and immunotherapy to sensitize poorly T-cell-infiltrated neuroblastoma (NB) to the host antitumor immune response. METHODS: 975A2 and 9464D NB cell lines derived from spontaneous tumors of TH-MYCN transgenic mice were employed to study drug combinations able of enhancing the antitumor immune response using in vivo and ex vivo approaches. Migration of immune cells towards drug-treated murine-derived organotypic tumor spheroids (MDOTS) were assessed by microfluidic devices. Activation status of immune cells co-cultured with drug-treated MDOTS was evaluated by flow cytometry analysis. The effect of drug treatment on the immune content of subcutaneous or orthotopic tumors was comprehensively analyzed by flow-cytometry, immunohistochemistry and multiplex immunofluorescence. The chemokine array assay was used to detect soluble factors released into the tumor microenvironment. Patient-derived organotypic tumor spheroids (PDOTS) were generated from human NB specimens. Migration and activation status of autologous immune cells to drug-treated PDOTS were performed. RESULTS: We found that treatment with low-doses of mitoxantrone (MTX) recalled immune cells and promoted CD8+ T and NK cell activation in MDOTS when combined with TGFß and PD-1 blockade. This combined immunotherapy strategy curbed NB growth resulting in the enrichment of a variety of both lymphoid and myeloid immune cells, especially intratumoral dendritic cells (DC) and IFNγ- and granzyme B-expressing CD8+ T cells and NK cells. A concomitant production of inflammatory chemokines involved in remodelling the tumor immune landscape was also detected. Interestingly, this treatment induced immune cell recruitment against PDOTS and activation of CD8+ T cells and NK cells. CONCLUSIONS: Combined treatment with low-dose of MTX and anti-TGFß treatment with PD-1 blockade improves antitumor immunity by remodelling the tumor immune landscape and overcoming the immunosuppressive microenvironment of aggressive NB.

Class I HLA folding and antigen presentation in beta 2-microglobulin-defective Daudi cells.

To present virus and tumor Ags, HLA class I molecules undergo a complex multistep assembly involving discrete but transient folding intermediates. The most extensive folding abnormalities occur in cells lacking the class I L chain subunit, called beta(2)-microglobulin (beta(2)m). Herein, this issue was investigated taking advantage of eight conformational murine mAbs (including the prototypic W6/32 mAb) to mapped H chain epitopes of class I molecules, four human mAbs to class I alloantigens, as well as radioimmunoprecipitation, in vitro assembly, pulse-chase, flow cytometry, and peptide-pulse/ELISPOT experiments. We show that endogenous (HLA-A1, -A66, and -B58) as well as transfected (HLA-A2) heavy chains in beta(2)m-defective Burkitt lymphoma Daudi cells are capable of being expressed on the cell surface, although at low levels, and exclusively as immature glycoforms. In addition, HLA-A2 is: 1) partially folded at crucial interfaces with beta(2)m, peptide Ag, and CD8; 2) receptive to exogenous peptide; and 3) capable of presenting exogenous peptide epitopes (from virus and tumor Ags) to cytotoxic T lymphocytes (bulk populations as well as clones) educated in a beta(2)m-positive environment. These experiments demonstrate a precursor-product relationship between novel HLA class I folding intermediates, and define a stepwise mechanism whereby distinct interfaces of the class I H chain undergo successive, ligand-induced folding adjustments in vitro as well as in vivo. Due to this unprecedented class I plasticity, Daudi is the first human cell line in which folding and function of class I HLA molecules are observed in the absence of beta(2)m. These findings bear potential implications for tumor immunotherapy.

Dendritic Cells: Behind the Scenes of T-Cell Infiltration into the Tumor Microenvironment.

Tumor-infiltrating CD8+ T cells have been shown to play a crucial role in controlling tumor progression. However, the recruitment and activation of these immune cells at the tumor site are strictly dependent on several factors, including the presence of dendritic cells (DCs), the main orchestrators of the antitumor immune responses. Among the various DC subsets, the role of cDC1s has been demonstrated in several preclinical experimental mouse models. In addition, the high density of tumor-infiltrating cDC1s has been associated with improved survival in many cancer patients. The ability of cDC1s to modulate antitumor activity depends on their interaction with other immune populations, such as NK cells. This evidence has led to the development of new strategies aimed at increasing the abundance and activity of cDC1s in tumors, thus providing attractive new avenues to enhance antitumor immunity for both established and novel anticancer immunotherapies. In this review, we provide an overview of the various subsets of DCs, focusing in particular on the role of cDC1s, their ability to interact with other intratumoral immune cells, and their prognostic significance on solid tumors. Finally, we outline key therapeutic strategies that promote the immunogenic functions of DCs in cancer immunotherapy.