The complex pathophysiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) involves a hyperinflammatory state with excessive cytokine production, leading to an influenza-like syndrome that may need emergency care. The severity of SARS-CoV-2 varies widely, and collective serum immune factors, evaluated in emergency care patients, have not been shown to correlate with disease progression. We applied a machine learning approach to reassess and define serum immune profiles that could align with clinical laboratory parameters and predict disease outcomes in patients with respiratory virus infections, including those with SARS-CoV-2, seeking emergency care. Sixty-two plasma immune molecules, in a cohort of 67 symptomatic SARS-CoV-2, were analyzed for correlation with antibodies (Abs) to spike (S) and nucleocapsid (N) proteins, as well as with clinical laboratory parameters, to identify early indicators of disease prognosis at hospital admission. This approach allowed us to analyze and cluster unlabeled datasets, delineating three distinct serum immune signatures. Two showed significant and opposite modulations, correlating with poorer disease outcomes, while most patients with moderate disease displayed modest immune factor dysregulation. This highlights the complexity of immune responses in the severity of diseases caused by highly respiratory pathogenic virus like SARS-CoV-2, emphasizing the importance of evaluating overall immune imbalance rather than focusing on a few dysregulated factors.

ABSTRACT Background We investigated the role of Nuclear Receptor Binding SET Domain Protein 2 (NSD2) and microRNAs (miRNAs) in melanoma de‐differentiation following Romidepsin and Interferon‐α2b (RI) treatment. Melanoma is the most lethal form of skin cancer, and despite advancements in therapy, treatment resistance remains a major challenge. De‐differentiation has been widely recognized as a key factor contributing to therapy resistance. Methods RNA‐seq and TCGA transcriptomic data were re‐analyzed to identify miRNAs and NSD2 expressions. The functional impact of selected miRNAs was then investigated at the molecular and phenotypic levels using primary and immortalized cell lines. Results Our findings demonstrate that RI treatment induces a de‐differentiation process in primary melanoma cells, resembling that observed in therapy‐resistant melanoma. This effect is particularly pronounced in cells with an intrinsic proliferative phenotype, where we observed significant downregulation of NSD2, a key epigenetic regulator implicated in multiple cancers. Additionally, we identified specific miRNAs as mediators of NSD2 downregulation, influencing melanoma cell viability and fitness. Conclusions These findings provide new insights into the molecular mechanisms driving melanoma progression and highlight potential therapeutic targets to counteract treatment resistance.

Abstract Glioblastoma (GB), IDH-wildtype, is the most common and aggressive high-grade primary malignant brain tumor with extremely poor outcome. Although the use of immune checkpoint inhibitors (ICIs) in GB has shown disappointing results, the identification of a small subgroup of responders underpins the role of the intratumoral immune contexture, in particular tumour-infiltrating lymphocytes (TILs), in determining disease outcome. This study aims to define the features of TIL subpopulations correlating with GB prognosis, and to establish in parallel the predictive roles of peripheral T cell subsets. A single cohort observational study, including patients undergoing GB resection and standard therapeutic and follow-up approaches in Neuro-Oncology, was conducted. Of the 45 patients, histologically confirmed WHO grade 4, 26 had MGMT promoter methylation (60.0%), 24 were male (53.3%) and the median age of young patients (≤63 years) vs elderly patients (>63 years) was 57 vs 71, respectively. Predictors of OS were KPS (≥70 vs <70: 14.7 vs 7.5 months, p=0.0007) and GTR (GTR vs non-GRT: 15 vs 9 months, p=0.004) whereas patients with methylated MGMT promoter had non-significant longer median OS compared to those with unmethylated promoter (14.5 vs 10.5 months). Patients were characterized for the expression of lineage, differentiation, memory, activation, and inhibition markers on intratumoral and peripheral CD4+ and CD8+ T cell subpopulations, and the correlation of an array of CD4+ and CD8+ T cell subsets with OS or PFS was evaluated by multiparametric flow cytometry and uni/multivariate analyses. Intratumoral immune infiltrate was positively associated with CD8+CD103+ tissue-resident memory T cells (Trm), and in particular with CD8+CD103+ Trm expressing PD1 and TIM3 rather than CD4+ Trm. Low CD8+CD103+PD1+ Trm resulted significantly associated with better OS and PFS (p=0.02 and 0.0031, respectively) and even higher significant was the correlation with better OS and PFS of low CD8+CD103+TIM3+ Trm (p=0.0002 and 0.0033, respectively) and CD8+CD103+PD1+TIM3+ Trm (p<0.0001, both). On multivariate analysis, low CD8+CD103+PD1+TIM3+ Trm and KPS ≥70 were confirmed to be the most predictive independent factors associated with longer OS (hazard ratios - HR [95%CI]: 0.14 [0.04 - 0.52] p˂0.001, 0.39 [0.16 - 0.96] p=0.04, respectively). Moreover, CD8+CD103+ Trm subpopulations resulted age-related predictors for GB survival. Consistent with this finding, in vitro PD1 and TIM3 blockade released the capability of intratumoral T cells to produce the effector cytokines IFN-γ and TNF-α, and the cytotoxic factor GrzB. Conversely, T cells expressing PD1 and TIM3 of peripheral blood did not show any prognostic significance. In conclusion, low frequency of Trm expressing PD1 or TIM3 or both markers defined TIL subsets as independent positive prognostic factors for patient survival. Citation Format: Lucia Gabriele, Giulia Romagnoli, Quintino Giorgio D'Alessandris, Imerio Capone, Andrea Tavilla, Irene Canini, Caterina Lapenta, Mariachiara Buccarelli, Martina Giordano, Valentina Tirelli, Massimo Sanchez, Alessandra Fragale, Stefano Giannetti, Rina Di Bonaventura, Liverana Lauretti, Mauro Biffoni, Lucia Ricci-Vitiani, Roberto Pallini. The Influence of PD1 and TIM3 Expression on CD4+CD103+ and CD8+CD103+ Cells in Glioblastoma Progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1053.

Abstract BACKGROUND Glioblastoma, IDH-wildtype (GBM), is the most prevalent and aggressive primary brain cancer with a poor prognosis. Immune checkpoint inhibitors (ICIs) have been explored in GBM with limited success, highlighting the need for a better understanding of the tumor microenvironment (TME). This study investigated whether the expression of specific immune checkpoints on tissue-resident memory T cells (Trm) predicts patient outcomes. METHODS In a single cohort observational study, tumor samples from 45 histologically confirmed GBM patients were processed to obtain single-cell suspensions. Multiparametric flow cytometry was used to analyze the correlation of Trm phenotypes with overall survival (OS) and progression-free survival (PFS). Univariate and multivariate analyses were conducted to assess the prognostic significance of Trm subsets. RESULTS Low frequency of exausted Trm expressing programmed cell death protein 1 (PD1) and T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) was associated with better clinical outcomes. Multivariate analysis identified low CD8+CD103+PD1+TIM3+ Trm and Karnofsky performance status (KPS) ≥70 as the most significant independent prognostic factors for longer OS (hazard ratios—HR [95%CI]: 0.14 [0.04–0.52] p < 0.001, 0.39 [0.16–0.96] p = 0.04, respectively). Additionally, younger patients (<63 years) and males had distinct immune profiles affecting prognosis. Notably, the proportion of exausted CD8+CD103+PD1+ Trms had a prognostic correlate only in male patients, consistent with findings by other groups on the existance of gender-specific immune TME in GBM. CONCLUSIONS Further exploration of Trm-based immune profiling has the potential to shed light on GBM resistance to ICIs and to help in personalizing treatment strategies.

Supplementary Figure 4 shows Viability graphs and Sytox staining of IL4-ARI DC and IFN-lambda1, IFNLR1, IFN-betaand IFN-panalpha expression graphs by RT-qPCR in IL4-ARI DC.

Supplementary Figure 1 contains ten panels of RT-qPCR analisis of CRC

Supplementary Table 3 is an Excell file containig a list of genes that shown ARI-induced modifications referred with ID, symbol, gene name, fold, p.value, by ChIP-seq with H3K9me2 antibody.

Supplementary Figure 2 contains two panels with graphs of IRF8 and IRF7 overexpression cell viability in CRC cell lines transfetced with EV, WT IRF8 or 12Y IRF8 (A), or with EV and IRF7(B) and IRF8 silencing Exps (C)

Supplementary Figure 3 Biological processes categories enriched in the list of genes showing H3K9me2 peaks in the promoter region.

CT-PET scan showing clinical response of Pt#3

Supplementary Figure 1 from Immunization of Stage IV Melanoma Patients with Melan-A/MART-1 and gp100 Peptides plus IFN-α Results in the Activation of Specific CD8⁺ T Cells and Monocyte/Dendritic Cell Precursors

BRAFV600 mutations are the most common oncogenic alterations in melanoma cells, supporting proliferation, invasion, metastasis and immune evasion. In patients, these aberrantly activated cellular pathways are inhibited by BRAFi whose potent antitumor effect and therapeutic potential are dampened by the development of resistance. Here, by using primary melanoma cell lines, generated from lymph node lesions of metastatic patients, we show that the combination of two FDA-approved drugs, the histone deacetylate inhibitor (HDCAi) romidepsin and the immunomodulatory agent IFN-α2b, reduces melanoma proliferation, long-term survival and invasiveness and overcomes acquired resistance to the BRAFi vemurafenib (VEM). Targeted resequencing revealed that each VEM-resistant melanoma cell line and the parental counterpart are characterized by a distinctive and similar genetic fingerprint, shaping the differential and specific antitumor modulation of MAPK/AKT pathways by combined drug treatment. By using RNA-sequencing and functional in vitro assays, we further report that romidepsin-IFN-α2b treatment restores epigenetically silenced immune signals, modulates MITF and AXL expression and induces both apoptosis and necroptosis in sensitive and VEM-resistant primary melanoma cells. Moreover, the immunogenic potential of drug-treated VEM-resistant melanoma cells results significantly enhanced, given the increased phagocytosis rate of these cells by dendritic cells, which in turn exhibit also a selective down-modulation of the immune checkpoint TIM-3. Overall, our results provide evidence that combined epigenetic-immune drugs can overcome VEM resistance of primary melanoma cells by oncogenic and immune pathways reprogramming, and pave the way for rapidly exploiting this combination to improve BRAFi-resistant metastatic melanoma treatment, also via reinforcement of immune checkpoint inhibitor therapy.

Supplementary Table 1. Sequences of oligonucleotides used in RT-qPCR are listed. Supplementary Table 2. Sequences of oligonucleotides used in ChIP assays in qPCR.

Supplementary Table I: IFN-DC release criteria and median values registered among patient-derived IFN-DC (min-max range); Supplementary Table 2: Patients MHC-I and MHC-II alleles and IGHV epitope sequences

Supplementary Figure 1 from Immunization of Stage IV Melanoma Patients with Melan-A/MART-1 and gp100 Peptides plus IFN-α Results in the Activation of Specific CD8+ T Cells and Monocyte/Dendritic Cell Precursors

Glioblastoma (GBM) is the most common and aggressive brain tumor with poor outcome. Immune checkpoint inhibitors (ICIs) have been tested in GBM and, despite disappointing results, the identification of a small subgroup of responders underlies the need to improve our understanding of the tumor microenvironment (TME) immunity. The aim of this study was to determine whether the expression of selected immune checkpoints on tissue-resident memory T cells (Trm) may predict patient outcome. We conducted a single cohort observational study. Tumor samples were collected from 45 patients with histologically confirmed, IDH wild type, GBM (WHO grade IV) and processed to obtain single cell suspensions. Using multiparametric flow cytometry and uni/multivariate analyses, patients were assessed for the correlation of Trm with overall survival (OS) and progression-free survival (PFS). High and low frequency of Trm expressing PD1 and TIM3 was found to be linked to clinical outcome. In fact, low frequency of Trm expressing PD1 or TIM3 or both markers defined subgroups as independent positive prognostic factors for patient survival. On multivariate analysis, low CD8+CD103+PD1+TIM3+ Trm and KPS ≥70 were confirmed to be the most predictive independent factors associated with longer OS (HR [95%CI]: 0.14 [0.04 - 0.52] p˂0.001, 0.39 [0.16 - 0.96] p=0.04, respectively). The CD8+CD103+ Trm subgroups also resulted age-linked predictors for survival in GBM.

CT-PET scan showing clinical response of Pt#2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, induces severe pneumonia mainly in elderly males. Epidemiological data clearly indicate sex-based differences in disease outcomes, with men accounting for about 70 % of deaths, despite similar susceptibility to infection. It is well known that females are endowed with higher capacity to produce antibodies, which correlates with viral clearance and disease resolution in the context of SARS-Cov-2 infection. Many X-linked immune genes escape X inactivation showing biallelic expression in female immune cells, particularly in plasmacytoid dendritic cells (pDCs). PDCs are more active in females and endowed with high capability to induce IFN-α-mediated B cell activation and differentiation into antibody-producing plasma cells throughout epigenetic mechanisms linked to trained immunity. Thus, we hypothesize that following SARS-CoV-2 infection, epigenetic modifications of X-linked genes involved in pDC-mediated type I IFN (IFN-I) signaling occurs more effectively in females, for inducing neutralizing antibody response as an immune correlate driving sex-biased disease outcome.

Clinical studies based on novel rationales and mechanisms of action of chemotherapy agents and cytokines can contribute to the development of new concepts and strategies of antitumor combination therapies. In previous studies, we investigated the paradoxical immunostimulating effects of some chemotherapeutics and the immunoadjuvant activity of interferon alpha (IFN-α) in preclinical and clinical models, thus unraveling novel rationales and mechanisms of action of chemotherapy agents and cytokines for cancer immunotherapy. Here, we carried out a randomized, phase II clinical trial, in which we analyzed the relapse-free (RFS) and overall survival (OS) of 34 completely resected stage III–IV melanoma patients, treated with peptide-based vaccination (Melan-A/MART-1 and NY-ESO-1) in combination with IFN-α2b, with (arm 2) or without (arm 1) dacarbazine preconditioning. All patients were included in the intention-to-treat analysis. At a median follow-up of 4.5 years (interquartile range, 15.4–81.0 months), the rates of RFS were 52.9 and 35.3% in arms 1 and 2, respectively. The 4.5-year OS rates were 68.8% in arm 1 and 62.7% in arm 2. No significant differences were observed between the two arms for both RFS and OS. Interestingly, the RFS and OS curves remained stable starting from 18 and 42 months, respectively. Grade 3 adverse events occurred in 5.9% of patients, whereas grade 4 events were not observed. Both treatments induced a significant expansion of vaccine-specific CD8+ T cells, with no correlation with the clinical outcome. However, treatment-induced increase of polyfunctionality and of interleukin 2 production by Melan-A–specific CD8+ T cells and expansion/activation of natural killer cells correlated with RFS, being observed only in nonrelapsing patients. Despite the recent availability of different therapeutic options, low-cost, low-toxic therapies with long-lasting clinical effects are still needed in patients with high-risk resected stage III/IV melanoma. The combination of peptide vaccination with IFN-α2b showed a minimal toxicity profile and resulted in encouraging RFS and OS rates, justifying further evaluation in clinical trials, which may include the use of checkpoint inhibitors to further expand the antitumor immune response and the clinical outcome.Clinical Trial Registration:https://www.clinicaltrialsregister.eu/ctr-search/search, identifier: 2008-008211-26