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T cell-mediated immune responses are compromised in aged individuals, leading to increased morbidity and reduced response to vaccination. While cellular metabolism tightly regulates T cell activation and function, metabolic reprogramming in aged T cells has not been thoroughly studied. Here, we report a systematic analysis of metabolism during young versus aged naïve T cell activation. We observed a decrease in the number and activation of naïve T cells isolated from aged mice. While young T cells demonstrated robust mitochondrial biogenesis and respiration upon activation, aged T cells generated smaller mitochondria with lower respiratory capacity. Using quantitative proteomics, we defined the aged T cell proteome and discovered a specific deficit in the induction of enzymes of one-carbon metabolism. The activation of aged naïve T cells was enhanced by addition of products of one-carbon metabolism (formate and glycine). These studies define mechanisms of skewed metabolic remodeling in aged T cells and provide evidence that modulation of metabolism has the potential to promote immune function in aged individuals.

Immunometabolism describes the interface between immune cell function and metabolism. This interplay is governed by both cell-intrinsic metabolic programs that shape immune cell function, as well as environmental metabolites that, when scarce, can limit fuel choice and, when overabundant, can act as signaling molecules and alter cell fate and function. Here, we present two stories of how metabolites in the extracellular environment can directly impact T cell function.Fluctuations in environmental amino acid concentrations can dramatically alter T cell activity. Building on an observation made nearly 40 years ago showing that T cells fail to activate in the presence of dialyzed FBS, we demonstrate that the addition of a single amino acid, alanine, to dialyzed FBS is sufficient to restore T cell function. Therefore, we sought to investigate why T cells rely on extracellular alanine for their activation. We found that, once inside a T cell, alanine is not metabolized towards pyruvate but is instead directly shunted towards protein synthesis. In the absence of alanine, T cells fail to activate and proliferate due to a block in activation-induced protein synthesis. Thus, T cell activation may be influenced by local concentrations of alanine during physiological or disease states.In our second study, we investigate how metabolites in the tumor microenvironment can act as signaling molecules and suppress T cell function. Mutations in the metabolic gene isocitrate dehydrogenase result in the production of the oncometabolite D-2-hydroxyglutarete (D-2HG), which accumulates at high levels in the surrounding microenvironment. We demonstrate that D-2HG is taken up by CD8+ T cells and directly inhibits the activity of the glycolytic enzyme lactate dehydrogenase. This inhibition drives a metabolic program and a CD8+ T cell signature marked by decreased cytotoxicity and altered interferon gamma signaling that is recapitulated in clinical samples. Thus, we describe a novel role for the oncometabolite D-2HG in shaping anti-tumor immunity in the tumor microenvironment of IDH mutant cancers.Together, our studies highlight the role of metabolism in regulating T cell function and how alterations in metabolite levels in the surrounding environment can impact such regulation.

X-linked immunodeficiency with magnesium defect, EBV infection, and neoplasia (XMEN) disease are caused by deficiency of the magnesium transporter 1 (MAGT1) gene. We studied 23 patients with XMEN, 8 of whom were EBV naive. We observed lymphadenopathy (LAD), cytopenias, liver disease, cavum septum pellucidum (CSP), and increased CD4-CD8-B220-TCRαβ+ T cells (αβDNTs), in addition to the previously described features of an inverted CD4/CD8 ratio, CD4+ T lymphocytopenia, increased B cells, dysgammaglobulinemia, and decreased expression of the natural killer group 2, member D (NKG2D) receptor. EBV-associated B cell malignancies occurred frequently in EBV-infected patients. We studied patients with XMEN and patients with autoimmune lymphoproliferative syndrome (ALPS) by deep immunophenotyping (32 immune markers) using time-of-flight mass cytometry (CyTOF). Our analysis revealed that the abundance of 2 populations of naive B cells (CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4++CD10+CD38+ and CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4+CD10-CD38-) could differentially classify XMEN, ALPS, and healthy individuals. We also performed glycoproteomics analysis on T lymphocytes and show that XMEN disease is a congenital disorder of glycosylation that affects a restricted subset of glycoproteins. Transfection of MAGT1 mRNA enabled us to rescue proteins with defective glycosylation. Together, these data provide new clinical and pathophysiological foundations with important ramifications for the diagnosis and treatment of XMEN disease.

PurposeTo determine the impact of BRCA1 and BRCA2 mutations on ovarian reserve and fertility preservation outcome. The main purpose and research question of the study is to determine the impact of BRCA1 and BRCA2 mutations on ovarian reserve and fertility preservation outcomes.MethodsProspective study: 67 breast cancer patients between 18 and 40 years old, undergoing a fertility preservation by means of oocyte storage were considered. Inclusions criteria for the study were age between 18 and 40 years old, BMI between 18 and 28, breast cancer neoplasm stage I and II according to American Joint Committee on Cancer classification (2017) and no metastasis. Exclusion criteria: age over 40 years old, BMI < 18 and > 28, breast cancer neoplasm stage III and IV and do not performed the BRCA test. A total of 21 patients had not performed the test and were excluded. Patients were divided into four groups: Group A was composed by 11 breast cancer patients with BRCA 1 mutations, Group B was composed by 11 breast cancer patients with BRCA 2 mutations, Group C was composed by 24 women with breast cancer without BRCA mutations, and Group D (control) was composed by 181 normal women.ResultsGroup A showed significant lower AMH levels compared to Group C and D (1.2 ± 1.1 vs 4.5 ± 4.1 p < 0.05 and 1.2 ± 1.1 vs 3.8 ± 2.5 p < 0.05). BRCA1 mutated patients showed a significant lower rate of mature oocytes (MII) compared to Group C (3.1 ± 2.3 vs 7.2 ± 4.4 p < 0,05) and Group D (3.1 ± 2.3 vs 7.3 ± 3.4; p < 0,05). Breast cancer patients needed a higher dose of gonadotropins compared to controls (Group A 2206 ± 1392 Group B2047.5 ± 829.9 Group C 2106 ± 1336 Group D 1597 ± 709 p < 0,05). No significant differences were found among the groups considering basal FSH levels, duration of stimulation, number of developed follicles, and number of total retrieved oocytes. Regarding BRCA2 mutation, no effect on fertility was shown in this study.ConclusionsThe study showed that BRCA1 patients had a higher risk of premature ovarian insufficiency (POI) confirmed by a diminished ovarian reserve and a lower number of mature oocytes suitable for cryopreservation.

The base excision repair (BER) Apurinic/apyrimidinic endonuclease 1 (APE1) enzyme is endowed with several non-repair activities including miRNAs processing. APE1 is overexpressed in many cancers but its causal role in the tumorigenic processes is largely unknown. We recently described that APE1 can be actively secreted by mammalian cells through exosomes. However, APE1 role in EVs or exosomes is still unknown, especially regarding a putative regulatory function on vesicular small non-coding RNAs. Through dedicated transcriptomic analysis on cellular and vesicular small RNAs of different APE1-depleted cancer cell lines, we found that miRNAs loading into EVs is a regulated process, dependent on APE1, distinctly conveying RNA subsets into vesicles. We identified APE1-dependent secreted miRNAs characterized by enriched sequence motifs and possible binding sites for APE1. In 33 out of 34 APE1-dependent-miRNA precursors, we surprisingly found EXO-motifs and proved that APE1 cooperates with hnRNPA2B1 for the EV-sorting of a subset of miRNAs, including miR-1246, through direct binding to GGAG stretches. Using TCGA-datasets, we showed that these miRNAs identify a signature with high prognostic significance in cancer. In summary, we provided evidence that the ubiquitous DNA-repair enzyme APE1 is part of the EV protein cargo with a novel post-transcriptional role for this ubiquitous DNA-repair enzyme that could explain its role in cancer progression. These findings could open new translational perspectives in cancer biology.

Cooks syndrome (CS) is an ultrarare limb malformation due to in tandem microduplications involving KCNJ2 and extending to the 5′ regulatory element of SOX9. To date, six CS families were resolved at the molecular level. Subsequent studies explored the evolutionary and pathological complexities of the SOX9-KCNJ2/Sox9-Kcnj2 locus, and suggested a key role for the formation of novel topologically associating domain (TAD) by inter-TAD duplications in causing CS. Here, we report a unique case of CS associated with a de novo 1;17 translocation affecting the KCNJ2 locus. On chromosome 17, the breakpoint mapped between KCNJ16 and KCNJ2, and combined with a ~ 5 kb deletion in the 5′ of KCNJ2. Based on available capture Hi-C data, the breakpoint on chromosome 17 separated KCNJ2 from a putative enhancer. Gene expression analysis demonstrated downregulation of KCNJ2 in both patient’s blood cells and cultured skin fibroblasts. Our findings suggest that a complex rearrangement falling in the 5′ of KCNJ2 may mimic the developmental consequences of in tandem duplications affecting the SOX9-KCNJ2/Sox9-Kcnj2 locus. This finding adds weight to the notion of an intricate role of gene regulatory regions and, presumably, the related three-dimensional chromatin structure in normal and abnormal human morphology.

Background Extracellular vesicles (EVs) are cell-secreted particles conceived as natural vehicles for intercellular communication. The capacity to entrap heterogeneous molecular cargoes and target specific cell populations through EV functionalization promises advancements in biomedical applications. However, the efficiency of the obtained EVs, the contribution of cell-exposed receptors to EV interactions, and the predictability of functional cargo release with potential sharing of high molecular weight recombinant mRNAs are crucial for advancing heterologous EVs in targeted therapy applications. Methods In this work, we selected the popular EV marker CD81 as a transmembrane guide for fusion proteins with a C-terminal GFP reporter encompassing or not Trastuzumab light chains targeting the HER2 receptor. We performed high-content imaging analyses to track EV-cell interactions, including isogenic breast cancer cells with manipulated HER2 expression. We validated the functional cargo delivery of recombinant EVs carrying doxorubicin upon EV-donor cell treatment. Then, we performed an in vivo study using JIMT-1 cells commonly used as HER2-refractory, trastuzumab-resistant model to detect a more than 2000 nt length recombinant mRNA in engrafted tumors. Results Fusion proteins participated in vesicular trafficking dynamics and accumulated on secreted EVs according to their expression levels in HEK293T cells. Despite the presence of GFP, secreted EV populations retained a HER2 receptor-binding capacity and were used to track EV-cell interactions. In time-frames where the global EV distribution did not change between HER2-positive (SK-BR-3) or -negative (MDA-MB-231) breast cancer cell lines, the HER2 exposure in isogenic cells remarkably affected the tropism of heterologous EVs, demonstrating the specificity of antiHER2 EVs representing about 20% of secreted bulk vesicles. The specific interaction strongly correlated with improved cell-killing activity of doxorubicin-EVs in MDA-MB-231 ectopically expressing HER2 and reduced toxicity in SK-BR-3 with a knocked-out HER2 receptor, overcoming the effects of the free drug. Interestingly, the fusion protein-corresponding transcripts present as full-length mRNAs in recombinant EVs could reach orthotopic breast tumors in JIMT-1-xenografted mice, improving our sensitivity in detecting penetrant cargoes in tissue biopsies. Conclusions This study highlights the quantitative aspects underlying the creation of a platform for secreted heterologous EVs and shows the limits of single receptor-ligand interactions behind EV-cell engagement mechanisms, which now become the pivotal step to predict functional tropism and design new generations of EV-based nanovehicles.

Adenosine deaminase (ADA) deficiency results in severe combined immunodeficiency, the first genetic disorder treated by gene therapy. Two different retroviral vectors were used to transfer ex vivo the human ADA minigene into bone marrow cells and peripheral blood lymphocytes from two patients undergoing exogenous enzyme replacement therapy. After 2 years of treatment, long-term survival of T and B lymphocytes, marrow cells, and granulocytes expressing the transferred ADA gene was demonstrated and resulted in normalization of the immune repertoire and restoration of cellular and humoral immunity. After discontinuation of treatment, T lymphocytes, derived from transduced peripheral blood lymphocytes, were progressively replaced by marrow-derived T cells in both patients. These results indicate successful gene transfer into long-lasting progenitor cells, producing a functional multilineage progeny.

Adenosine deaminase (ADA) deficiency leads to severe combined immunodeficiency (SCID). Previous clinical trials showed that autologous CD34 + cell gene therapy (GT) following busulfan reduced-intensity conditioning is a promising therapeutic approach for ADA-SCID, but long-term data are warranted. Here we report an analysis on long-term safety and efficacy data of 43 patients with ADA-SCID who received retroviral ex vivo bone marrow-derived hematopoietic stem cell GT. Twenty-two individuals (median follow-up 15.4 years) were treated in the context of clinical development or named patient program. Nineteen patients were treated post-marketing authorization (median follow-up 3.2 years), and two additional patients received mobilized peripheral blood CD34 + cell GT. At data cutoff, all 43 patients were alive, with a median follow-up of 5.0 years (interquartile range 2.4–15.4) and 2 years intervention-free survival (no need for long-term enzyme replacement therapy or allogeneic hematopoietic stem cell transplantation) of 88% (95% confidence interval 78.7–98.4%). Most adverse events/reactions were related to disease background, busulfan conditioning or immune reconstitution; the safety profile of the real world experience was in line with premarketing cohort. One patient from the named patient program developed a T cell leukemia related to treatment 4.7 years after GT and is currently in remission. Long-term persistence of multilineage gene-corrected cells, metabolic detoxification, immune reconstitution and decreased infection rates were observed. Estimated mixed-effects models showed that higher dose of CD34 + cells infused and younger age at GT affected positively the plateau of CD3 + transduced cells, lymphocytes and CD4 + CD45RA + naive T cells, whereas the cell dose positively influenced the final plateau of CD15 + transduced cells. These long-term data suggest that the risk–benefit of GT in ADA remains favorable and warrant for continuing long-term safety monitoring. Clinical trial registration: NCT00598481 , NCT03478670 . Fifteen years’ follow-up of clinical development and real-world data from 43 patients show that gammaretroviral gene therapy for adenosine deaminase deficiency has a positive long-term efficacy profile, warranting continued safety monitoring of patients receiving gene therapy.