Cytotoxic T lymphocytes (CTLs) used in immunotherapy are typically cultured under atmospheric O pressure but encounter hypoxic conditions inside tumors. Activating CTLs under hypoxic conditions has been shown to improve their cytotoxicity in vitro, but the mechanism employed and the implications for immunotherapy remain unknown. We activated and cultured OT-I CD8 T cells at either 1% or 20% O. Hypoxic CTLs survived, as well as normoxic ones, in vitro but killed OVA-expressing B16 melanoma cells more efficiently. Hypoxic CTLs contained similar numbers of cytolytic granules and released them as efficiently but packaged more granzyme-B in each granule without producing more perforin. We imaged CTL distribution and motility inside B16-OVA tumors using confocal and intravital 2-photon microscopy and observed no obvious differences. However, mice treated with hypoxic CTLs exhibited better tumor regression and survived longer. Thus, hypoxic CTLs may perform better in tumor immunotherapy because of higher intrinsic cytotoxicity rather than improved migration inside tumors.

A point mutation, Gly380Arg, in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3) leads to achondroplasia, the most common form of genetic dwarfism in humans. This substitution was suggested to enhance mutant receptor dimerization, leading to constitutive, ligand-independent activation. We found that dimerization and activation of the G380R mutant receptor are predominantly ligand dependent. However, using both transient and stable transfections, we found significant overexpression only of the mutant receptor protein. Metabolic pulse-chase experiments, cell surface labeling, and kinetics of uptake of radiolabeled ligand demonstrated a selective delay in the down-regulation of the mutant receptor. Moreover, this receptor was now resistant to ligand-mediated internalization, even at saturating ligand concentrations. Finally, transgenic mice expressing the human G380R mutant receptor under the mouse receptor transcriptional control demonstrated a markedly expanded area of FGFR3 immunoreactivity within their epiphyseal growth plates, compatible with an in vivo defect in receptor down-regulation. We propose that the achondroplasia mutation G380R uncouples ligand-mediated receptor activation from down-regulation at a site where the levels and kinetics of FGFR3 signals are crucial for chondrocyte maturation and bone formation.Fibroblast growth factor (FGF) receptors (FGFR) constitute a family of four genes that encode multiple receptor isoforms, all of which have intrinsic tyrosine kinase activity (8,12). Upon binding of a ligand, receptor dimerization is induced, leading to auto-and transphosphorylation followed by receptor internalization and down-regulation. These lead to the controlled activation of specific signal transduction pathways and the expression of FGF target genes, critically required during embryogenesis, tissue repair, and angiogenesis (1).Multiple mutations in FGFR 1, 2, and 3 (FGFR1, FGFR2, and FGFR3, respectively) give rise to a variety of inherited skeletal malformations (40). Mutations in FGFR3 are responsible for disorders predominantly of the long bones, including achondroplasia, the most common form of human genetic dwarfism (27,29). Over 97% of cases of achondroplasia result from either a G-to-A transition or a G-to-C transversion, changing the codon for Gly380 (GGG) to Arg (AGG or CGG) in the transmembrane domain of FGFR3. An Asn540Lys mutation in the proximal tyrosine kinase domain of FGFR3 is found in the milder disorder of hypochondroplasia (2), while substitution to a cysteine of residue 248, 249, 370, or 371 in the extracellular domain or a Lys650Glu mutation in the kinase activation loop gives rise to the most severe and neonatal lethal thanatophoric dysplasia (TD) types, I and II, respectively (28, 33). All of these skeletal malformations represent autosomal dominant disorders characterized by disproportionately short limbs and relative macrocephaly (23).The cellular basis underlying the clinical features of achondroplasia is a defect in chondrocyte function durin...

IntroductionAnti-acetylcholine receptor (AChR) autoantibodies target muscles in spontaneous human myasthenia gravis (MG) and its induced experimental autoimmune model MG (EAMG). The aim of this study was to identify novel functional mechanisms occurring in the muscle pathology of myasthenia.ResultsA transcriptome analysis performed on muscle tissue from MG patients (compared with healthy controls) and from EAMG rats (compared with control rats) revealed a deregulation of genes associated with the Interleukin-6 (IL-6) and Insulin-Like Growth Factor 1 (IGF-1) pathways in both humans and rats. The expression of IL-6 and its receptor IL-6R transcripts was found to be altered in muscles of EAMG rats and mice compared with control animals. In muscle biopsies from MG patients, IL-6 protein level was higher than in control muscles. Using cultures of human muscle cells, we evaluated the effects of anti-AChR antibodies on IL-6 production and on the phosphorylation of Protein Kinase B (PKB/Akt). Most MG sera and some monoclonal anti-AChR antibodies induced a significant increase in IL-6 production by human muscle cells. Furthermore, Akt phosphorylation in response to insulin was decreased in the presence of monoclonal anti-AChR antibodies.ConclusionsAnti-AChR antibodies alter IL-6 production by muscle cells, suggesting a putative novel functional mechanism of action for the anti-AChR antibodies. IL-6 is a myokine with known effects on signaling pathways such as Akt/mTOR (mammalian Target of Rapamycin). Since Akt plays a key role in multiple cellular processes, the reduced phosphorylation of Akt by the anti-AChR antibodies may have a significant impact on the muscle fatigability observed in MG patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-014-0179-6) contains supplementary material, which is available to authorized users.

The quest for tumor-associated antigens (TAA) and neoantigens is a major focus of cancer immunotherapy. Here, we combine a neoantigen prediction pipeline and human leukocyte antigen (HLA) peptidomics to identify TAAs and neoantigens in 16 tumors derived from seven patients with melanoma and characterize their interactions with their tumor-infiltrating lymphocytes (TIL). Our investigation of the antigenic and T-cell landscapes encompassing the TAA and neoantigen signatures, their immune reactivity, and their corresponding T-cell identities provides the first comprehensive analysis of cancer cell T-cell cosignatures, allowing us to discover remarkable antigenic and TIL similarities between metastases from the same patient. Furthermore, we reveal that two neoantigen-specific clonotypes killed 90% of autologous melanoma cells, both and, showing that a limited set of neoantigen-specific T cells may play a central role in melanoma tumor rejection. Our findings indicate that combining HLA peptidomics with neoantigen predictions allows robust identification of targetable neoantigens, which could successfully guide personalized cancer immunotherapies. As neoantigen targeting is becoming more established as a powerful therapeutic approach, investigating these molecules has taken center stage. Here, we show that a limited set of neoantigen-specific T cells mediates tumor rejection, suggesting that identifying just a few antigens and their corresponding T-cell clones could guide personalized immunotherapy. .

Naturally occurring CD4+CD25+ regulatory T (Treg) cells are key players in immune tolerance and have therefore been suggested as potential therapeutic tools for autoimmune diseases. In myasthenia gravis (MG), reduced numbers or functionally impaired Treg cells have been reported. We have observed that PBL from myasthenic rats contain decreased numbers of CD4+CD25highFoxp3+ cells as compared with PBL from healthy controls, and we have tested whether Treg cells from healthy donors can suppress experimental autoimmune MG in rats. Because the number of naturally occurring Treg cells is low, we used an approach for a large-scale ex vivo generation of functional Treg cells from CD4+ splenocytes of healthy donor rats. Treg cells were generated ex vivo from CD4+ cells by stimulation with anti-CD3 and anti-CD28 Abs in the presence of TGF-β and IL-2. The obtained cells expressed high levels of CD25, CTLA-4, and Foxp3, and they were capable of suppressing in vitro proliferation of T cells from myasthenic rats in response to acetylcholine receptor, the major autoantigen in myasthenia. Administration of ex vivo-generated Treg cells to myasthenic rats inhibited the progression of experimental autoimmune MG and led to down-regulation of humoral acetylcholine receptor-specific responses, and to decreased IL-18 and IL-10 expression. The number of CD4+CD25+ cells in the spleen of treated rats remained unchanged, but the subpopulation of CD4+CD25+ cells expressing Foxp3 was significantly elevated. Our findings imply that Treg cells play a critical role in the control of myasthenia and could thus be considered as potential agents for the treatment of MG patients.

Myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are autoimmune disorders in which the acetylcholine receptor (AChR) is the major autoantigen. Microarray technology was used to identify new potential drug targets for treatment of myasthenia that would reduce the need for the currently used nonspecific immunosuppression. The chemokine IFN-γ-inducible protein 10 (IP-10; CXCL10), a CXC chemokine, and its receptor, CXCR3, were found to be overexpressed in lymph node cells of EAMG rats. Quantitative real-time PCR confirmed these findings and revealed up-regulated mRNA levels of another chemoattractant that activates CXCR3, monokine induced by IFN-γ (Mig; CXCL9). TNF-α and IL-1β, which act synergistically with IFN-γ to induce IP-10, were also up-regulated. These up-regulations were observed in immune response effector cells, namely, lymph node cells, and in the target organ of the autoimmune attack, the muscle of myasthenic rats, and were significantly reduced after suppression of EAMG by mucosal tolerance induction with an AChR fragment. The relevance of IP-10/CXCR3 signaling in myasthenia was validated by similar observations in MG patients. A significant increase in IP-10 and CXCR3 mRNA levels in both thymus and muscle was observed in myasthenic patients compared with age-matched controls. CXCR3 expression in PBMC of MG patients was markedly increased in CD4+, but not in CD8+, T cells or in CD19+ B cells. Our results demonstrate a positive association of IP-10/CXCR3 signaling with the pathogenesis of EAMG in rats as well as in human MG patients.

Messenger RNA-based vaccines against COVID-19 induce a robust anti-SARS-CoV-2 antibody response with potent viral neutralization activity. Antibody effector functions are determined by their constant region subclasses and by their glycosylation patterns, but their role in vaccine efficacy is unclear. Moreover, whether vaccination induces antibodies similar to those in patients with COVID-19 remains unknown. We analyze BNT162b2 vaccine-induced IgG subclass distribution and Fc glycosylation patterns and their potential to drive effector function via Fcg receptors and complement pathways. We identify unique and dynamic pro-inflammatory Fc compositions that are distinct from those in patients with COVID-19 and convalescents. Vaccine-induced anti-Spike IgG is characterized by distinct Fab-and Fc-mediated functions between different age groups and in comparison to antibodies generated during natural viral infection. These data highlight the heterogeneity of Fc responses to SARS-CoV-2 infection and vaccination and suggest that they support long-lasting protection differently.

The COVID-19 pandemic and the fast global spread of the disease resulted in unprecedented decline in world trade and travel. A critical priority is, therefore, to quickly develop serological diagnostic capacity and identify individuals with past exposure to SARS-CoV-2. In this study serum samples obtained from 309 persons infected by SARS-CoV-2 and 324 of healthy, uninfected individuals as well as serum from 7 COVID-19 patients with 4–7 samples each ranging between 1–92 days post first positive PCR were tested by an “in house” ELISA which detects IgM, IgA and IgG antibodies against the receptor binding domain (RBD) of SARS-CoV-2. Sensitivity of 47%, 80% and 88% and specificity of 100%, 98% and 98% in detection of IgM, IgA and IgG antibodies, respectively, were observed. IgG antibody levels against the RBD were demonstrated to be up regulated between 1–7 days after COVID-19 detection, earlier than both IgM and IgA antibodies. Study of the antibody kinetics of seven COVID 19 patients revealed that while IgG levels are high and maintained for at least 3 months, IgM and IgA levels decline after a 35–50 days following infection. Altogether, these results highlight the usefulness of the RBD based ELISA, which is both easy and cheap to prepare, to identify COVID-19 patients even at the acute phase. Most importantly our results demonstrate that measuring IgG levels alone is both sufficient and necessary to diagnose past exposure to SARS-CoV-2.