Efficient targeting of NY-ESO-1 tumor antigen to human cDC1s by lymphotactin results in cross-presentation and antigen-specific T cell expansion.

BACKGROUND: Type 1 conventional dendritic cells (cDC1s) are characterized by their ability to induce potent CD8+ T cell responses. In efforts to generate novel vaccination strategies, notably against cancer, human cDC1s emerge as an ideal target to deliver antigens. cDC1s uniquely express XCR1, a seven transmembrane G protein-coupled receptor. Due to its restricted expression and endocytic nature, XCR1 represents an attractive receptor to mediate antigen-delivery to human cDC1s. METHODS: To explore tumor antigen delivery to human cDC1s, we used an engineered version of XCR1-binding lymphotactin (XCL1), XCL1(CC3). Site-specific sortase-mediated transpeptidation was performed to conjugate XCL1(CC3) to an analog of the HLA-A*02:01 epitope of the cancer testis antigen New York Esophageal Squamous Cell Carcinoma-1 (NY-ESO-1). While poor epitope solubility prevented isolation of stable XCL1-antigen conjugates, incorporation of a single polyethylene glycol (PEG) chain upstream of the epitope-containing peptide enabled generation of soluble XCL1(CC3)-antigen fusion constructs. Binding and chemotactic characteristics of the XCL1-antigen conjugate, as well as its ability to induce antigen-specific CD8+ T cell activation by cDC1s, was assessed. RESULTS: PEGylated XCL1(CC3)-antigen conjugates retained binding to XCR1, and induced cDC1 chemoattraction in vitro. The model epitope was efficiently cross-presented by human cDC1s to activate NY-ESO-1-specific CD8+ T cells. Importantly, vaccine activity was increased by targeting XCR1 at the surface of cDC1s. CONCLUSION: Our results present a novel strategy for the generation of targeted vaccines fused to insoluble antigens. Moreover, our data emphasize the potential of targeting XCR1 at the surface of primary human cDC1s to induce potent CD8+ T cell responses.

Selective BET-bromodomain inhibition by JQ1 suppresses dendritic cell maturation and antigen-specific T-cell responses.

Bromo- and extra-terminal domain (BET) inhibitors represent potential therapeutic approaches in solid and hematological malignancies that are currently analyzed in several clinical trials. Additionally, BET are involved in the epigenetic regulation of immune responses by macrophages and dendritic cells (DCs), that play a central role in the regulation of immune responses, indicating that cancer treatment with BET inhibitors can promote immunosuppressive effects. The aim of this study was to further characterize the effects of selective BET inhibition by JQ1 on DC maturation and DC-mediated antigen-specific T-cell responses. Selective BET inhibition by JQ1 impairs LPS-induced DC maturation and inhibits the migrational activity of DCs, while antigen uptake is not affected. JQ1-treated DCs show reduced ability to induce antigen-specific T-cell proliferation. Moreover, antigen-specific T cells co-cultured with JQ1-treated DCs exhibit an inactive phenotype and reduced cytokine production. JQ1-treated mice show reduced immune responses in vivo to sublethal doses of LPS, characterized by a reduced white blood cell count, an immature phenotype of splenic DCs and T cells and lower blood levels of IL-6. In our study, we demonstrate that selective BET inhibition by JQ1, a drug currently tested in clinical trials for malignant diseases, has profound effects on DC maturation and DC-mediated antigen-specific T-cell responses. These immunosuppressive effects can result in the induction of possible infectious side effects in cancer treatments. In addition, based on our results, these compounds should not be used in combinatorial regimes using immunotherapeutic approaches such as check point inhibitors, T-cell therapies, or vaccines.

Blocking PDGF-CC signaling ameliorates multiple sclerosis-like neuroinflammation by inhibiting disruption of the blood-brain barrier.

Disruption of blood-brain barrier (BBB) integrity is a feature of various neurological disorders. Here we found that the BBB is differently affected during the preclinical, progression and remission phase of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We have identified an upregulation of pro-inflammatory and pro-angiogenic factors in the BBB transcriptome and down-regulation of endothelial tight junction members coinciding with elevated BBB leakage specifically during the progression phase. These changes were antagonized by blocking PDGFRα signaling with the small tyrosine kinase inhibitor imatinib. Moreover, targeting the PDGFRα ligand PDGF-CC using a neutralizing antibody, facilitated recovery of BBB integrity and improvement of EAE symptoms. Intracerebroventricular injection of PDGF-CC induced upregulation, whereas blocking PDGF-CC during EAE led to downregulation of Tnfa and Il1a at the BBB. Our findings suggest that blocking PDGF-CC counteracts fundamental aspects of endothelial cell activation and disruption of the BBB by decreasing Tnfa and Il1a expression. We also demonstrate that both PDGF-CC and its receptor PDGFRα were upregulated in MS lesions indicating that blocking PDGF-CC may be considered a novel treatment for MS.

Identification of a four immune-related genes signature based on an immunogenomic landscape analysis of clear cell renal cell carcinoma.

Renal clear cell carcinoma (ccRCC) is the most common type of renal cell carcinoma, which has strong immunogenicity. A comprehensive study of the role of immune-related genes (IRGs) in ccRCC is of great significance in finding ccRCC treatment targets and improving patient prognosis. In this study, we comprehensively analyzed the expression of IRGs in ccRCC based on The Cancer Genome Atlas datasets. The mechanism of differentially expressed IRGs in ccRCC was analyzed by bioinformatics. In addition, Cox regression analysis was used to screen prognostic related IRGs from differentially expressed IRGs. We also identified a four IRGs signature consisting of four IRGs (CXCL2, SEMA3G, PDGFD, and UCN) through lasso regression and multivariate Cox regression analysis. Further analysis results showed that the four IRGs signature could effectively predict the prognosis of patients with ccRCC, and its predictive power is independent of other clinical factors. In addition, the correlation analysis of immune cell infiltration showed that this four IRGs signature could effectively reflect the level of immune cell infiltration of ccRCC. We also found that the expression of immune checkpoint genes CTLA-4, LAG3, and PD-1 in the high-risk group was higher than that in the low-risk group. Our research revealed the role of IRGs in ccRCC, and developed a four IRGs signature that could be used to evaluate the prognosis of patients with ccRCC, which will help to develop personalized treatment strategies for patients with ccRCC and improve their prognosis. In addition, these four IRGs may be effective therapeutic targets for ccRCC.

[Probiotics in the treatment of atopic dermatitis: the induction of tolerogenic dendritic cells]. / Traiter la dermatite atopique par les probiotiques - Induction de cellules dendritiques tolérogènes.

TITLE: Traiter la dermatite atopique par les probiotiques - Induction de cellules dendritiques tolérogènes. ABSTRACT: Dans le cadre du module d'enseignement Communication Scientifique et Littérature du Master Biologie Moléculaire et Cellulaire de Lyon, les étudiants des parcours M2 Génopath et Biologie de la Peau se sont formés à l'écriture scientifique sur un sujet libre. Suite à un travail préparatoire avec l'équipe pédagogique, chaque étudiant a rédigé, conseillé par un chercheur, une Nouvelle. Le parcours M2 Génopath s'adresse aux étudiants scientifiques et médecins et les forme à la recherche fondamentale dans les domaines de la génétique, de la biologie cellulaire et de leurs applications biomédicales. Le parcours M2 Biologie de la Peau est une formation unique en France, et forme des spécialistes de la recherche en biologie cutanée qui s'inséreront dans les services de recherche et développement hospitalier ou de l'industrie dermo-cosmétique et dermo-pharmaceutique.

Allogeneic murine pregnancy models for assessing the developmental effects of immune-stimulating antibodies: Challenges in reproducibility.

Literature suggests that murine allogeneic pregnancy models are an alternative approach for evaluating the developmental toxicity of immune-stimulating agents. In this study, multiple syngeneic and allogeneic murine pregnancy models were used to assess the potential embryo-fetal effects of four different murine antibodies (IgG1 or IgG2 ) that activate the immune system by binding to T-cell receptors (PD-L1, LAG-3, and GITR). The pregnancy models were generated by within and between matings of five different inbred strains of mice (CBA/CaJ, DBA/2J, BALB/c, C57BL/6, and CBA/J). The antibodies were administered every 2-3 days by intraperitoneal injection (n = 12-29/group) during gestation days 6 to 14. There were no differences in embryo-fetal endpoints between the allogeneic and syngeneic pregnancies. Additionally, treatment with the antibodies had no effect on mean postimplantation loss in either the syngeneic or allogeneic pregnancies despite confirmation of pharmacologically-relevant systemic exposures. These results suggest that allogeneic murine pregnancy models need further validation and testing before they can be reliably used as an alternative approach for assessing the developmental effects of agents that stimulate the immune system.

The mouse intron-nested gene, Israa, is expressed in the lymphoid organs and involved in T-cell activation and signaling.

We have previously reported Israa, immune-system-released activating agent, as a novel gene nested in intron 8 of the mouse Zmiz1 gene. We have also shown that Israa encodes for a novel FYN-binding protein and might be involved in the regulation of T-cell activation. In this report, we demonstrate that Israa gene product regulates the expression of a pool of genes involved in T-cell activation and signaling. Real time PCR and GFP knock-in expression analysis showed that Israa is transcribed and expressed in the spleen mainly by CD3+CD8+ cells as well as in the thymus by CD3+ (DP and DN), CD4+SP and CD8+SP cells at different developmental stages. We also showed that Israa is downregulated in T-cells following activation of T-cell receptor. Using yeast two-hybrid analysis, we identified ELF1, a transcription factor involved in T-cell regulation, as an ISRAA-binding partner. Transcriptomic analysis of an EL4 cell line overexpressing ISRAA revealed differential expression of several genes involved in T-cell signaling, activation and development. Among these genes, Prkcb, Mib2, Fos, Ndfip2, Cxxc5, B2m, Gata3 and Cd247 were upregulated whereas Itk, Socs3, Tigit, Ifng, Il2ra and FoxJ1 were downregulated. Our findings support the existence in mouse of a novel FYN-related T-cell regulation pathway involving the product of an intron-nested gene.

A Highly Active Form of XCL1/Lymphotactin Functions as an Effective Adjuvant to Recruit Cross-Presenting Dendritic Cells for Induction of Effector and Memory CD8+ T Cells.

The chemokine receptor XCR1 is known to be selectively expressed by cross-presenting dendritic cells (DCs), while its ligand XCL1/lymphotactin is mainly produced by activated CD8+ T cells and natural killer cells. Recent studies have shown that XCL1-antigen fusion proteins efficiently induce CD8+ T cell responses by preferentially delivering antigens to XCR1+ DCs. However, XCL1 per se was found to be a poor adjuvant for induction of CD8+ T cell responses. XCL1 is unique because of its lack of one of the two disulfide bonds commonly conserved in all other chemokines and thus has an unstable structure with a relatively weak chemokine activity. In the present study, we generated a variant form of murine XCL1 termed mXCL1-V21C/A59C that contained a second disulfide bond to stabilize its chemokine structure. We confirmed that mXCL1-V21C/A59C had much more potent chemotactic and calcium mobilization activities than the wild type XCL1 (mXCL1-WT). Intradermal injection of mXCL1-V21C/A59C, but not that of mXCL1-WT, significantly increased the accumulation of XCR1+CD103+ DCs in the injection site, and most of the accumulated XCR1+CD103+ DCs were found to take up co-injected ovalbumin (OVA). Furthermore, recruited XCR1+CD103+ DCs efficiently migrated to the draining lymph nodes and stayed for a prolonged period of time. Consequently, mXCL1-V21C/A59C strongly induced OVA-specific CD8+ T cells. The combination of OVA and mXCL1-V21C/A59C well protected mice from E.G7-OVA tumor growth in both prophylactic and therapeutic protocols. Finally, memory CTL responses were efficiently induced in mice immunized with OVA and mXCL1-V21C/A59C. Although intradermal injection of OVA and polyinosinic-polycytidylic acid (poly(I:C)) as an adjuvant also induced CD8+ T cell responses to OVA, poly (I:C) poorly recruited XCR1+CD103+ DCs in the injection site and failed to induce significant memory CTL responses to OVA. Collectively, our findings demonstrate that a highly active form of XCL1 is a promising vaccine adjuvant for cross-presenting DCs to induce antigen-specific effector and memory CD8+ T cells.

Development of monoclonal anti-PDGF-CC antibodies as tools for investigating human tissue expression and for blocking PDGF-CC induced PDGFRα signalling in vivo.

PDGF-CC is a member of the platelet-derived growth factor (PDGF) family that stimulates PDGFRα phosphorylation and thereby activates intracellular signalling events essential for development but also in cancer, fibrosis and neuropathologies involving blood-brain barrier (BBB) disruption. In order to elucidate the biological and pathological role(s) of PDGF-CC signalling, we have generated high affinity neutralizing monoclonal antibodies (mAbs) recognizing human PDGF-CC. We determined the complementarity determining regions (CDRs) of the selected clones, and mapped the binding epitope for clone 6B3. Using the monoclonal 6B3, we determined the expression pattern for PDGF-CC in different human primary tumours and control tissues, and explored its ability to neutralize PDGF-CC-induced phosphorylation of PDGFRα. In addition, we showed that PDGF-CC induced disruption of the blood-retinal barrier (BRB) was significantly reduced upon intraperitoneal administration of a chimeric anti-PDGF-CC antibody. In summary, we report on high affinity monoclonal antibodies against PDGF-CC that have therapeutic efficacy in vivo.

Preclinical toxicological assessment of a novel monoclonal antibody targeting human platelet-derived growth factor CC (PDGF-CC) in PDGF-CChum mice.

Platelet-derived growth factor CC (PDGF-CC) is important during foetal development but also in pathogenesis of neurologic diseases, cancer and fibrosis. We have previously demonstrated that blocking the PDGF-CC/PDGF receptor alpha (PDGFRα) axis resulted in reduction of stroke volume and cerebrovascular permeability after experimentally induced stroke. Recently, we could translate these findings into the clinic showing that imatinib, a small tyrosine kinase inhibitor targeting PDGF receptors, can significantly improve neurological outcome after ischemic stroke in human. Herein we report preclinical toxicological analyses of our newly generated monoclonal anti-human PDGF-CC antibody 6B3 (mAb 6B3) in PDGF-CC humanized mice. Beside histological organ assessment, we also analysed serum, urine, haematological parameters and the general health status of the treated mice. We could not find any indications that mAb 6B3 is toxic or has other significant side effects neither in short, nor in long treatment regimens. Our results indicate that mAb 6B3 can be further developed for clinical use. This opens up the possibility to assess the therapeutic potential of blocking PDGF-CC in diverse pathological conditions such as neurologic diseases, cancer and fibrosis.

Effect of leukocyte inhibitory factor on neuron differentiation from human induced pluripotent stem cell-derived neural precursor cells.

Direct derivation of human induced pluripotent stem cells into neural precursor cells and differentiation of these into neurons holds great promise in the cell therapy of neurodegenerative diseases. However, the availability and survival rate of neurons requires improvement. In the present study, it was found that the addition of 5 ng/ml leukocyte inhibitory factor (LIF) during the process of differentiation significantly improved the expression of neuron­specific class III ß­tubulin (TUJ1) and microtubule­associated protein 2 (MAP2), as detected by immunofluorescence and western blotting. In addition, LIF improved the cell viability, increased the expression of phosphorylated­protein kinase B (AKT), downregulated the expression of proinflammatory cytokines, including interleukin­1α (IL­1α) and tumor necrosis factor­α (TNF-α), and upregulated the expression of anti­inflammatory cytokines, including interleukin­10 (IL­10) and transforming growth factor­ß (TGF-ß). After adding the phosphatidylinositol 3-kinase (PI3K)/AKT signaling inhibitor LY294002 or wortmannin to the LIF differentiation group, LIF-induced changes in the protein expression of TUJ1 and MAP2 were reversed, but this effect could not be prevented by rapamycin, a mechanistic target of rapamycin signaling inhibitor. The expression of cytokines associated with inflammation and cell viability was reversed by LY294002 and wortmannin, but not by rapamycin. In conclusion, LIF could improve neuronal differentiation and survival through the activation of PI3K/AKT signaling and the anti­inflammatory effect. The anti­inflammatory effect may be mediated by the activation of PI3K/AKT.

Human neonatal Fc receptor as a new potential antibody binding protein for antibody immobilization.

A critical challenge in producing an antibody-based assay with the highest reproducibility and sensitivity is the strategy to immobilize antibodies to solid phase. To date, numerous methods of antibody immobilization were reported but each was subjected to its advantages and limitations. The current study proposes a new potential antibody binding protein, the human neonatal fragment crystallizable (Fc) receptor. This protein has shown its high affinity to the Fc of antibody either in vivo or in vitro. Human neonatal Fc receptor is a heterodimer constructed by p51 α-heavy chain and ß2-microglobulin light chain; however, the binding sites toward the antibody are located in the p51 α-heavy chain. Hence, vector cloning and recombinant protein expression were carried out to express the p51 α-heavy chain of the human neonatal Fc receptor (hFcRn-α). The recombinant protein expressed, hFcRn-α, was adopted to pin rabbit IgG against hepatitis B virus surface antigen to a solid phase. A sandwich enzyme-linked immunosorbent assay was further developed to evaluate the efficiency of hFcRn-α-directed immobilization in antigen detection. The result was compared with the conventional physical adsorption method. The findings demonstrated that human neonatal Fc receptor was efficient in pinning antibodies and generating higher signals compared with the physical adsorption of antibody.

Immodin and its immune system supportive role in paclitaxel therapy of 4T1 mouse breast cancer.

It is evident that standard chemotherapy agents may have an impact on both tumor and host immune system. Paclitaxel (PTX), a very potent anticancer drug from a taxane family, has achieved prominence in clinical oncology for its efficacy against a wide range of tumors including breast cancer. However, significant toxicity, such as myelosuppression, limit the effectiveness of Paclitaxel-based treatment regimens. Immodin (IM) is low molecular dialysate fraction of homogenate made from human leukocytes. It contains a mixture of substances from which so far have been described e.g. Imreg 1 and Imreg 2 formed by the dipeptide tyrosine-glycine and the tripeptide tyrosine-glycine-glycine, respectively. The aim of this study was to explore immunopharmacological activities of IM, using the strongly immunogenic 4T1 mouse breast cancer model, and evaluate its effect on the reactivity and the efficiency of PTX cancer therapy. The results highlight a potentially beneficial role for IM in alleviating PTX-induced toxicity, especially on the nonspecific immunity, during breast cancer therapy. Co-treatment exhibited an antitumor effect including reduced tumor growth, prolonged survival of tumor bearing mice, increased number of monocytes and lymphocytes in peripheral blood. In spleens, IM+PTX therapy elevated proportion of whole lymphocytes in the account of myelo-monocytic cells characteristic with low expression of CD11c+ and bearing Fc receptor (CD16/32) as well as T-lymphocytes, NK cells and dendritic cells. Accumulation of tumor-associated granulocytes in stroma of PTX-treated group and intensive 4T1-necrosis/apoptosis in tumors after co-treatment were also recorded. These findings suggest the possibility of using IM alongside PTX treatment for maintaining the immune system functions and increasing patient survival.

Autologous and Allogeneic Equine Mesenchymal Stem Cells Exhibit Equivalent Immunomodulatory Properties In Vitro.

The use of allogeneic bone marrow-derived mesenchymal stem cells (BMDMSCs) may provide an effective alternative to autologous BMDMSCs for treatment of equine musculoskeletal injuries. However, concerns have been raised regarding the potential safety and effectiveness of allogeneic BMDMSCs. We conducted studies to assess the immunological properties of equine allogeneic BMDMSCs compared with those of autologous BMDMSCs. For assessment of inherent immunogenicity, the relative ability of allogeneic and autologous BMDMSCs to stimulate spontaneous proliferation of equine lymphocytes was compared. The immunosuppressive activity of the two cell types was evaluated by adding autologous or allogeneic BMDMSCs to activated lymphocytes and assessing suppression of lymphocyte proliferation and IFNγ production. Fifty-six allogeneic and 12 autologous combinations were evaluated. Studies were also done to elucidate mechanisms by which equine mesenchymal stem cells (MSCs) suppress lymphocyte function. Potential mechanisms evaluated included production of prostaglandin E2 (PGE2), nitric oxide, transforming growth factor-beta, and indoleamine 2,3-dioxygenase. We found that autologous and allogeneic BMDMSCs both induced mild but equivalent levels of spontaneous lymphocyte activation in vitro. In in vitro assays assessing the ability of BMDMSCs to suppress activated lymphocytes, both allogeneic and autologous BMDMSCs suppressed T cell proliferation and IFNγ production to an equal degree. The primary mechanism of equine BMDMSC suppression of T cells was mediated by PGE2. We concluded that allogeneic and autologous BMDMSCs are equivalent in terms of their immunomodulatory properties, and stimulated peripheral blood mononuclear cells appear to trigger the immunosuppressive properties of MSCs. Therefore, both cell types appear to have equal potency in modulating inflammatory processes related to acute or chronic musculoskeletal injuries in the horse.

Fully synthetic self-adjuvanting MUC1-fibroblast stimulating lipopeptide 1 conjugates as potential cancer vaccines.

We have designed and synthesized MUC1-fibroblast stimulating lipopeptide 1 conjugates as potential self-adjuvanting cancer vaccines using a linear solid phase peptide synthesis strategy. These vaccine candidates have elicited T cell dependent immune response and the induced antibodies showed intense specific binding affinity to human MCF-7 cells.

A novel, rapid and efficient method of cloning functional antigen-specific T-cell receptors from single human and mouse T-cells.

T-cell receptor (TCR) gene therapy is a promising approach for the treatment of infectious diseases and cancers. However, the paired cloning and functional assays of antigen-specific TCRα and TCRß is time-consuming and laborious. In this study, we developed a novel, rapid and efficient antigen-specific TCR-cloning system by combining three technologies: multiplex one-step RT-PCR, transcriptionally active PCR (TAP) and luciferase reporter assays. Multiplex one-step RT-PCR with leader primers designed from leader peptide sequences of TCRs enabled us to amplify cDNAs of TCRα and ß pairs from single T-cells with remarkably high efficiency. The combination of TAP fragments and HEK293T-based NFAT-luciferase reporter cells allowed for a rapid functional assay without the need to construct expression vectors. Using this system, we cloned human TCRs specific for Epstein-Barr virus BRLF-1-derived peptide as well as mouse TCRs specific for melanoma-associated antigen tyrosinase-related protein 2 (TRP-2) within four days. These results suggest that our system provides rapid and efficient cloning of functional antigen-specific human and mouse TCRs and contributes to TCR-based immunotherapy for cancers and infectious diseases.

Immune responses to Mycobacterial heat shock protein 70 accompany self-reactivity to human BiP in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease, and a member of human heat shock protein (HSP) 70 protein family, Binding Immunoglobulin Protein (BiP), has been identified as an important autoantigen for T and B cells. We herein focused on Mycobacterial (Myc) HSPs and immune responses to MycHSPs in RA patients. Serum titers of antibodies against MycHSP70 were significantly elevated in RA patients and correlated with serum anti-BiP antibody titers. A MycHSP70-derived HLA-DR4 major epitope was identified using the proliferative capacity of RA PBMCs as an indicator. The major epitope, MycHSP70287-306, was located at the corresponding position in the major epitope for human BiP336-355, and a strong correlation was found between the proliferation of PBMCs in response to MycHSP70287-306 and BiP336-355. The immunization of HLA-DR4 transgenic mice with MycHSP70 induced the proliferation of T cells and development of anti-BiP antibodies. In contrast, the oral administration of MycHSP70287-306 resulted in the amelioration of collagen-induced arthritis, serum antibody responses, and T cell proliferation. In conclusion, immune responses to MycHSP70 were associated with adaptive immunity against BiP in RA, and could be an important mechanism underlying the development of autoimmunity.

Development of Phage-Based Antibody Fragment Reagents for Affinity Enrichment of Bacterial Immunoglobulin G Binding Proteins.

Disease and death caused by bacterial infections are global health problems. Effective bacterial strategies are required to promote survival and proliferation within a human host, and it is important to explore how this adaption occurs. However, the detection and quantification of bacterial virulence factors in complex biological samples are technically demanding challenges. These can be addressed by combining targeted affinity enrichment of antibodies with the sensitivity of liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS). However, many virulence factors have evolved properties that make specific detection by conventional antibodies difficult. We here present an antibody format that is particularly well suited for detection and analysis of immunoglobulin G (IgG)-binding virulence factors. As proof of concept, we have generated single chain fragment variable (scFv) antibodies that specifically target the IgG-binding surface proteins M1 and H of Streptococcus pyogenes. The binding ability of the developed scFv is demonstrated against both recombinant soluble protein M1 and H as well as the intact surface proteins on a wild-type S. pyogenes strain. Additionally, the capacity of the developed scFv antibodies to enrich their target proteins from both simple and complex backgrounds, thereby allowing for detection and quantification with LC-SRM MS, was demonstrated. We have established a workflow that allows for affinity enrichment of bacterial virulence factors.

New technologies for monitoring human antigen-specific T cells and regulatory T cells by flow-cytometry.

T cells orchestrate and execute immune responses against certain antigens recognized by their antigen receptor. They can acquire a highly divers set of functional properties, which provide the basis for immune protection, but also for immune-pathologies and thus represent highly specific diagnostic and therapeutic targets. New cytometric technologies now allow identification and precise characterization of human conventional and regulatory T cells against basically any antigen and even within naive donors. These provide the basis for thorough analyses of immune protection against infections and to tackle unmet challenges such as T cell responses involved in tolerance and/or directed against undefined or complex antigens, that is, in autoimmunity or allergy. Together with the parallel evolution of single cell multi-parameter approaches this has revolutionized the quantitative and qualitative characterization of human T cells, bearing important diagnostic, prognostic or therapeutic potential.

Modulation of the spleen transcriptome in domestic turkey (Meleagris gallopavo) in response to aflatoxin B1 and probiotics.

Poultry are highly susceptible to the immunotoxic effects of the food-borne mycotoxin aflatoxin B1 (AFB1). Exposure impairs cell-mediated and humoral immunity, limits vaccine efficacy, and increases the incidence of costly secondary infections. We investigated the molecular mechanisms of AFB1 immunotoxicity and the ability of a Lactobacillus-based probiotic to protect against aflatoxicosis in the domestic turkey (Meleagris gallopavo). The spleen transcriptome was examined by RNA sequencing (RNA-seq) of 12 individuals representing four treatment groups. Sequences (6.9 Gb) were de novo assembled to produce over 270,000 predicted transcripts and transcript fragments. Differential expression analysis identified 982 transcripts with statistical significance in at least one comparison between treatment groups. Transcripts with known immune functions comprised 27.6 % of significant expression changes in the AFB1-exposed group. Short exposure to AFB1 suppressed innate immune transcripts, especially from antimicrobial genes, but increased the expression of transcripts from E3 ubiquitin-protein ligase CBL-B and multiple interleukin-2 response genes. Up-regulation of transcripts from lymphotactin, granzyme A, and perforin 1 could indicate either increased cytotoxic potential or activation-induced cell death in the spleen during aflatoxicosis. Supplementation with probiotics was found to ameliorate AFB1-induced expression changes for multiple transcripts from antimicrobial and IL-2-response genes. However, probiotics had an overall suppressive effect on immune-related transcripts.