Human γδ T-Cells: from surface receptors to the therapy of high-risk leukemias

γδ T lymphocytes are potent effector cells, capable of efficiently killing tumor and leukemia cells. Their activation is mediated by γδ T-cell receptor (TCR) and by activating receptors shared with NK cells (e.g., NKG2D and DNAM-1). γδ T-cell triggering occurs upon interaction with specific ligands, including phosphoantigens (for Vγ9Vδ2 TCR), MICA-B and UL16 binding protein (for NKG2D), and PVR and Nectin-2 (for DNAM-1). They also respond to cytokines undergoing proliferation and release of cytokines/chemokines. Although at the genomic level γδ T-cells have the potential of an extraordinary TCR diversification, in tissues they display a restricted repertoire. Recent studies have identified various γδ TCR rearrangements following either hematopoietic stem cell transplantation (HSCT) or cytomegalovirus infection, accounting for their "adaptive" potential. In humans, peripheral blood γδ T-cells are primarily composed of Vγ9Vδ2 chains, while a minor proportion express Vδ1. They do not recognize antigens in the context of MHC molecules, thus bypassing tumor escape based on MHC class I downregulation. In view of their potent antileukemia activity and absence of any relevant graft-versus-host disease-inducing effect, γδ T-cells may play an important role in the successful clinical outcome of patients undergoing HLA-haploidentical HSCT depleted of TCR αβ T/CD19+ B lymphocytes to cure high-risk acute leukemias. In this setting, high numbers of both γδ T-cells (Vδ1 and Vδ2) and NK cells are infused together with CD34+ HSC and may contribute to rapid control of infections and leukemia relapse. Notably, zoledronic acid potentiates the cytolytic activity of γδ T-cells in vitro and its infusion in patients strongly promotes γδ T-cell differentiation and cytolytic activity; thus, treatment with this agent may contribute to further improve the patient clinical outcome after HLA-haploidentical HSCT depleted of TCR αβ T/CD19+ B lymphocytes

Conceptual Design of Digital Twin for Bio-Methanol Production from Microalgae

In the last decades, microalgae have gained a lot of interest in the energy and chemical industry thanks to their higher biofuel productivity potential rather than other land plants. To better exploit their green nature and renewable power, anaerobic digestion (AD) fits perfectly for the scope. AD is a metabolic process that generates a methane-rich gas, the biogas, which can then be used for clean electricity and chemicals production. High interest has arisen in the field of AD in industrial practice, and a lot of experiments were done to produce biogas from different types of feedstocks. In this manner, microalgae represent a promising opportunity to produce biogas from renewable and self-sustainable organisms. Biogas is mostly used to produce electrical energy and heat through cogeneration cycles or is upgraded to biomethane through the removal of CO2 and impurities, reaching a CH4 purity above 95-97% vol. On the other hand, an interesting perspective of biogas exploitation is its conversion in biofuels such as methanol or dimethyl-ether. This new concept of bio-refining lays the ground for two aspects: The economical valorisation of the biomass with a more valuable product as bio-methanol and the conversion of biogas to biofuel to fix part of the carbon in a chemical molecule, avoiding the re-emission in the atmosphere of CO2. The scope of this work is to present and technically analyse a conceptual design of a circular bio-refinery based on microalgae biomass feedstock with the final output of methanol production. Biogas production from microalgae is modelled with PythonTM (v3.9) while process simulations are computed using state of the art industrial simulation packages like Aspen HYSIS® v11. Interesting factors to analyse are carbon emission, the field of use for functional production, the global process yield and preliminary feasibility analysis

NK cells and other innate lymphoid cells in hematopoietic stem cell transplantation

Natural killer (NK) cells play a major role in the T-cell depleted haploidentical hematopoietic stem cell transplantation (haplo-HSCT) to cure high-risk leukemias. NK cells belong to the expanding family of innate lymphoid cells (ILCs). At variance with NK cells, the other ILC populations (ILC1/2/3) are non-cytolytic, while they secrete different patterns of cytokines. ILCs provide host defenses against viruses, bacteria, and parasites, drive lymphoid organogenesis, and contribute to tissue remodeling. In haplo-HSCT patients, the extensive T-cell depletion is required to prevent graft-versus-host disease (GvHD) but increases risks of developing a wide range of life-threatening infections. However, these patients may rely on innate defenses that are reconstituted more rapidly than the adaptive ones. In this context, ILCs may represent important players in the early phases following transplantation. They may contribute to tissue homeostasis/remodeling and lymphoid tissue reconstitution. While the reconstitution of NK cell repertoire and its role in haplo-HSCT have been largely investigated, little information is available on ILCs. Of note, CD34+ cells isolated from different sources of HSC may differentiate in vitro toward various ILC subsets. Moreover, cytokines released from leukemia blasts (e.g., IL-1β) may alter the proportions of NK cells and ILC3, suggesting the possibility that leukemia may skew the ILC repertoire. Further studies are required to define the timing of ILC development and their potential protective role after HSCT

Predicting FTS products through artificial neural network modelling

Fischer-Tropsch synthesis is essential for converting CO2 into hydrocarbons, creating sustainable fuels and olefins. However, challenges in production yield and reaction kinetics remain. This study introduces an artificial neural network (ANN) to predict FT synthesis products from specific inputs, including temperature, pressure, GHSV, H2/CO2 ratio, and catalyst composition (Fe weight and K as a promoter). The ANN's ability to predict outputs like CH4, C2-4, C5+, CO2 conversion, and CO selectivity, without detailed reaction mechanisms, is a key innovation. This approach circumvents complex kinetic models. The network architecture is optimized for minimal error, and results are validated against a comprehensive database

PD-L1 expression in metastatic neuroblastoma as an additional mechanism for limiting immune surveillance

The prognosis of high-risk neuroblastoma (NB) remains poor, although immunotherapies with anti-GD2 antibodies have been reported to provide some benefit. Immunotherapies can be associated with an IFNγ storm that induces in tumor cells the “adaptive immune resistance” characterized by the de-novo expression of Programmed Death Ligands (PD-Ls). Tumor cells can also constitutively express PD-Ls in response to oncogenic signaling. Here, we analyze the constitutive and the inducible surface expression of PD-Ls in NB cells. We show that virtually all HLA class Ipos NB cell lines constitutively express PD-L1, whereas PD-L2 is rarely detected. IFNγ upregulates or induces PD-L1 both in NB cell lines in vitro and in NB engrafted nude/nude mice. Importantly, after IFNγ stimulation PD-L1 can be acquired by NB cell lines, as well as by metastatic neuroblasts isolated from bone marrow aspirates of high-risk NB patients, characterized by different MYCN amplification status. Interestingly, in one patient NB cells were poorly responsive to IFNγ stimulation, pointing out that responsiveness to IFNγ might represent a further element of heterogeneity in metastatic neuroblasts. Finally, we document the presence of lymphocytes expressing the PD-1 receptor in NB-infiltrated bone marrow of patients. PD-1pos cells are mainly represented by αβ T cells, but also include small populations of γδ T cells and NK cells. Moreover, PD-1pos T cells have a higher expression of activation markers. Overall, our data show that a PD-L1-mediated immune resistance mechanism occurs in metastatic neuroblasts and provide a biological rationale for blocking the PD-1/PD-Ls axis in future combined immunotherapeutic approaches

Inhibitory 2B4 contributes to NK cell education and immunological derangements in XLP1 patients

X-linked lymphoproliferative disease 1 (XLP1) is an inherited immunodeficiency, caused by mutations in SH2D1A encoding Signaling Lymphocyte Activation Molecule (SLAM)-associated protein (SAP). In XLP1, 2B4, upon engagement with CD48, has inhibitory instead of activating function. This causes a selective inability of cytotoxic effectors to kill EBV-infected cells, with dramatic clinical sequelae. Here, we investigated the NK cell education in XLP1, upon characterization of killer Ig-like receptor (KIR)/KIR-L genotype and phenotypic repertoire of self-HLA class I specific inhibitory NK receptors (self-iNKRs). We also analyzed NK-cell cytotoxicity against CD48+ or CD48− KIR-ligand matched or autologous hematopoietic cells in XLP1 patients and healthy controls. XLP1 NK cells may show a defective phenotypic repertoire with substantial proportion of cells lacking self-iNKR. These NK cells are cytotoxic and the inhibitory 2B4/CD48 pathway plays a major role to prevent killing of CD48+ EBV-transformed B cells and M1 macrophages. Importantly, self-iNKR defective NK cells kill CD48− targets, such as mature DCs. Self-iNKR− NK cells in XLP1 patients are functional even in resting conditions, suggesting a role of the inhibitory 2B4/CD48 pathway in the education process during NK-cell maturation. Killing of autologous mature DC by self-iNKR defective XLP1 NK cells may impair adaptive responses, further exacerbating the patients’ immune defect

Killer Ig-like receptors (kirs). their role in nk cell modulation and developments leading to their clinical exploitation

Natural killer (NK) cells contribute to the first line of defense against viruses and to the control of tumor growth and metastasis spread. The discovery of HLA class I specific inhibitory receptors, primarily of killer Ig-like receptors (KIRs), and of activating receptors has been fundamental to unravel NK cell function and the molecular mechanisms of tumor cell killing. Stemmed from the seminal discoveries in early ‘90s, in which Alessandro Moretta was the major actor, an extraordinary amount of research on KIR specificity, genetics, polymorphism, and repertoire has followed. These basic notions on NK cells and their receptors have been successfully translated to clinical applications, primarily to the haploidentical hematopoietic stem cell transplantation to cure otherwise fatal leukemia in patients with no HLA compatible donors. The finding that NK cells may express the PD-1 inhibitory checkpoint, particularly in cancer patients, may allow understanding how anti-PD-1 therapy could function also in case of HLA class Ineg tumors, usually susceptible to NK-mediated killing. This, together with the synergy of therapeutic anti-checkpoint monoclonal antibodies, including those directed against NKG2A or KIRs, emerging in recent or ongoing studies, opened new solid perspectives in cancer therapy

Characterization of the proteins involved in the DNA repair mechanism in M. smegmatis.

: Several alkylating agents that either occur in the environment or are self-produced can cause DNA-damaging injuries in bacterial cells. Therefore, all microorganisms have developed repair systems that are able to counteract DNA alkylation damage. The adaptive response to alkylation stress in Escherichia coli consists of the Ada operon, which has been widely described; however, the homologous system in Mycobacterium tuberculosis (MTB) has been shown to have a different genetic organization but it is still largely unknown. In order to describe the defense system of MTB, we first investigated the proteins involved in the repair mechanism in the homologous non-pathogenic mycobacterium M. smegmatis. Ogt, Ada-AlkA and FadE8 proteins were recombinantly produced, purified and characterized. The biological role of Ogt was examined using proteomic experiments to identify its protein partners in vivo under stress conditions. Our results suggested the formation of a functional complex between Ogt and Ada-AlkA, which was confirmed both in silico by docking calculations and by gel filtration chromatography. We propose that this stable association allows the complex to fulfill the biological roles exerted by Ada in the homologous E. coli system. Finally, FadE8 was demonstrated to be structurally and functionally related to its E. coli homologous, Aid