Human Repertoire Research Articles

Referencing cross-reactivity of detection antibodies for protein array experiments.

Protein arrays are frequently used to profile antibody repertoires in humans and animals. High-throughput protein array characterisation of complex antibody repertoires necessitates the use of extensively validated secondary detection antibodies. This article details the validation of an affinity-isolated anti-chicken IgY antibody produced in rabbit and a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase using protein arrays consisting of 7,390 distinct human proteins. Probing protein arrays with secondary antibodies in absence of chicken serum revealed non-specific binding to 61 distinct human proteins. Despite the identified non-specific binding, the tested antibodies are well suited for use in protein array experiments as the cross-reactive binding partners can be readily excluded from further analysis. The evident cross-reactivity of the tested secondary detection antibodies points towards the necessity of platform-specific antibody characterisation studies for all secondary immunoreagents. Furthermore, secondary antibody characterisation using protein arrays enables the generation of reference lists of cross-reactive proteins, which can be then marked as potential false positives in follow-up experiments. Providing such cross-reactivity reference lists accessible to the wider research community may help to interpret data generated with the same antibodies in applications not only related to protein arrays such as immunoprecipitation, Western blots or other immunoassays.

Affective and Behavioral Responses to Robot-Initiated Social Touch: Toward Understanding the Opportunities and Limitations of Physical Contact in Human–Robot Interaction

Social touch forms an important aspect of the human non-verbal communication repertoire, but is often overlooked in human-robot interaction. In this study, we investigated whether robot-initiated touches can induce physiological, emotional, and behavioral responses similar to those reported for human touches. 39 Participants were invited to watch a scary movie together with a robot that spoke soothing words. In the Touch condition, these words were accompanied by a touch on the shoulder. We hypothesized that this touch – as compared with no touch – could (H1) attenuate physiological (heart rate (variability), skin conductance, cortisol, and respiration rate) and subjective stress responses that were caused by the movie. Moreover, we expected that a touch could (H2) decrease aversion towards the movie, (H3) increase positive perceptions of the robot (e.g., its appearance and one’s attitude towards it), and (H4) increase compliance to the robot’s request to make a monetary donation. Although the movie did increase arousal as intended, none of the hypotheses could be confirmed. Our findings suggest that merely simulating a human touching action with the robot’s limbs is insufficient to elicit physiological, emotional, and behavioral responses in this specific context and with this amount of participants. To inform future research on the opportunities and limitations of robot-initiated touch, we reflect on our methodology and identify dimensions that may play a role in physical human-robot interactions: e.g., the robot’s touching behavior, its appearance and behavior, the user’s personality, the body location where the touch is applied, and the (social) context of the interaction. Social touch can only become an integral and effective part of a robot’s non-verbal communication repertoire, when we better understand if, and under which boundary conditions such touches can elicit responses in humans.

Aging-related changes in human T-cell repertoire over 20 years delineated by deep sequencing of peripheral T-cell receptors

Recent deep sequencing studies on T-cell receptor (TCR) repertoire have provided robust data to characterize diversity of T-cell immune responsiveness to a wide variety of peptide antigens, including viral and tumor antigens. The human TCR repertoire declines with age, but this decline has not been fully investigated longitudinally in individuals. Using a deep sequencing approach, we analyzed TCRβ repertoires longitudinally over approximately 20years, with ages ranging from 23 to 50years at the start (23 to 65years overall), in peripheral-blood CD4 and CD8 T-cell populations that were collected and cryopreserved 3 times at intervals of approximately 10years from each of 6 healthy adults (3 men and 3 women). Sequence data at the hypervariable complementarity determining region 3 (CDR3) in the TCRB gene locus were evaluated by applying a random-coefficient statistical regression model. Two outcomes were analyzed: total number of distinct TCRB CDR3 sequences as a TCR diversity metric, and clonality of the T-cell populations. TCR repertoire diversity decreased (p<0.001) and frequencies of clonal populations increased (p=0.003) with age in CD8 T cells, whereas CD4 T cells retained fairly diverse TCR repertoires along with relatively low clonality. We also found that approximately 10–30% and 30–80% of read sequences in CD4 and CD8 T cells, respectively, overlapped at different ages within each individual, indicating long-term stable maintenance of T-cell clonal composition. Moreover, many of the most frequent TCRB CDR3 sequences (i.e., top T-cell clones) persisted over 20years, and some of them expanded and exerted a dominating influence on clonality of peripheral T-cell populations. It is thus possible that persistence or expansion of top T-cell clones is a driver of T-cell immunity aging, and therefore represents a potential interventional target.

Research on the diversity of T cell receptor repertoire in the process of malignant transformation of HBV chronic infection.

e23086 Background: The progression of liver inflammation resulted from HBV infection is the main cause of HBV-related liver cancer. T cells play a central role in anti-HBV and anti-tumor specific immune response during disease progression, and the T cell receptor (TCR) repertoire can quantitatively reflect the immune function of T cells. The accurate information of TCR repertoire change in the progression from chronic hepatitis B to tumor was obtained in this study. Methods: CD4+ T cells from the spleens of mice and CD8+ T cells from 50ml peripheral blood of the patients with chronic hepatitis B and liver cancer were sorted by flow cytometry for establishing next generation sequencing (NGS) and the related bioinformatics research system of TCR repertoire. The sequence in V region of TCR was amplified bu using 5 'RACE technology with the mRNA as template. The sequences of TCRα and TCRβ were determined by the NGS technology, and the sequence data with 6Gb were obtained for each chain of each individual. Results: Acoording to analysis of TCR repertoire in mouse CD4+ T cells, the V-J pairing and diversity of CDR3 sequence in TCRβ were higher than TCRα; With the increasing of the recombination number in D gene fragments in the TCRβ chain, a higher diversity of V-J pairing could be introduced in TCRβ.Acoording to analysis of TCR repertoire in human CD8+ T cells, the effects of chronic hepatitis and HCC condition on the overall diversity of TCR receptor repertoire were not significant; The level of receptor repertoire in patients with chronic hepatitis and HCC showed major difference only at the level of low frequency ratio compared with healthy volunteers; the distinctive V-J paring and CDR3 could be detected in the group of patients with chronic hepatitis and HCC based on the above TCR clone at the level of low frequency. Conclusions: NGS sequencing and the related bioinformatics research system of mouse and human TCR repertoire were established. The level of receptor repertoire in patients with chronic hepatitis and HCC showed major difference only at the level of low frequency ratio compared with healthy volunteers.

Referencing cross-reactivity of detection antibodies for protein array experiments

Protein arrays are frequently used to profile antibody repertoires in humans and animals. High-throughput protein array characterisation of complex antibody repertoires necessitates the use of extensively validated secondary detection antibodies. This article details the validation of an affinity-isolated anti-chicken IgY antibody produced in rabbit and a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase using protein arrays consisting of 7,390 distinct human proteins. Probing protein arrays with secondary antibodies in absence of chicken serum revealed non-specific binding to 61 distinct human proteins. Despite the identified non-specific binding, the tested antibodies are well suited for use in protein array experiments as the cross-reactive binding partners can be readily excluded from further analysis. The evident cross-reactivity of the tested secondary detection antibodies points towards the necessity of platform-specific antibody characterisation studies for all secondary immunoreagents. Furthermore, secondary antibody characterisation using protein arrays enables the generation of reference lists of cross-reactive proteins, which can be then marked as potential false positives in follow-up experiments. Providing such cross-reactivity reference lists accessible to the wider research community may help to interpret data generated with the same antibodies in applications not only related to protein arrays such as immunoprecipitation, Western blots or other immunoassays.

Lust for violence: Appetitive aggression as a fundamental part of human nature

AbstractAppetitive aggression describes a biologically-driven form of aggressive behaviour and violence characterized by positive affect. In contrast to reactive aggression, which has the function of resisting a threat, and reducing concomitant negative emotional arousal and anger, appetitive aggression underlies the pleasure of violence. A prototypical example is hunting, which can in turn transfer to the hunting of humans and can even result in bloodlust, and killing for its own sake. At the physiological level, this morally illicit pleasure is accompanied by an adrenalin surge, the release of cortisol and endorphins. In order to activate reward systems via appetitive aggression, their moral and cultural restraints need to be overridden. For example, armed groups work to dehumanize the enemy. Once initiated, a positive feedback loop is generated: As the individual commits more acts of violence with elements of positive affect, the tendency to commit them grows, and they begin to be perceived more positively. A latent passion for fighting and dominance can probably be evoked in almost all men and in some women. The cumulative outcome of whole groups, tribes, or communities enacting this aggression is war and destruction, to the point of trying to extinguish entire ethnic groups:“… and yes, human beings, hundreds of thousands of otherwise normal people, not professional killers, did it.”(from “The Killers in Rwanda Speak” by Jean Hatzfeld, 2005). Thus, appetitive aggression, the disposition towards a lust for violence, is by no means a psychopathological anomaly but an intrinsic part of the human behavioural repertoire. Morality, culture and the state monopoly on violence constitute the guards that regulate aggression potential and to channel it into socially useful forms.

Mechanisms That Shape Human Antibody Repertoire Development in Mice Transgenic for Human Ig H and L Chain Loci.

To determine the impact of the milieu on the development of the human B cell repertoire, we carried out a comprehensive analysis of productive and nonproductive Ig gene rearrangements from transgenic mice engineered to express single copies of the unrearranged human H chain and L chain Ig gene loci. By examining the nonproductive repertoire as an indication of the immediate product of the rearrangement machinery without an impact of selection, we discovered that the distribution of human rearrangements arising in the mouse was generally comparable to that seen in humans. However, differences between the distribution of nonproductive and productive rearrangements that reflect the impact of selection suggested species-specific selection played a role in shaping the respective repertoires. Although expression of some VH genes was similar in mouse and human (IGHV3-23, IGHV3-30, and IGHV4-59), other genes behaved differently (IGHV3-33, IGHV3-48, IGHV4-31, IGHV4-34, and IGHV1-18). Gene selection differences were also noted in L chains. Notably, nonproductive human VH rearrangements in the transgenic mice expressed shorter CDRH3 with less N addition. Even the CDRH3s in the productive rearrangements were shorter in length than those of the normal human productive repertoire. Amino acids in the CDRH3s in both species showed positive selection of tyrosines and glycines, and negative selection of leucines. The data indicate that the environment in which B cells develop can affect the expressed Ig repertoire by exerting influences on the distribution of expressed VH and VL genes and by influencing the amino acid composition of the Ag binding site.

What We're Reading

Prostate adenocarcinoma (by Nephron, Wikimedia Commons).A chimeric mouse model with reliable formation of prostate tumors was used to test therapies for advanced prostate cancer. Checkpoint blockade alone was ineffective, but, by also targeting MDSCs (by inhibiting tyrosine kinases and the PI3K pathway), IL1 receptor antagonist was upregulated, suppressive Gr-MDSCs and the supporting cytokines were reduced, infiltration of CD8+ T cells into tumors increased, and growth of tumors and metastases were inhibited.Lu X, …, DePinho RA. Nature 2017 Mar 20; doi: 10.1038/nature21676.DCs and peacock flounder have signatures responsive to their locales (by B. Inaglory, Wikimedia).Organ donor tissue from 78 people of different ages was used to assess DC populations in mucosal tissues and lymph nodes during the human lifespan. cDC2s were the dominant cDCs in lymph nodes from an early age. DC subset composition varied by location, with DCs expressing tissue-specific signatures. cDC2s also appear to function as the primary guardians at mucosal surfaces.Granot T, …, Farber DL. Immunity 2017;46:504–15.Strengthening macrophage antitumor responses (from PublicDomainPictures.net).Tumor-associated macrophages are often immunosuppressive. The class IIa HDAC inhibitor TMP195 was found to activate macrophages at the tumor site and normalize the vasculature without directly altering lymphocytes. Administering the drug reduced tumor burdens and decreased metastasis. When combined with checkpoint blockade, TMP195 induced a durable antitumor response in a mouse MMTV-PyMT breast cancer model.Guerriero JL., …, Letai A. Nature 2016;543:428–32.SPADE tree of V δ2 subsets (Lo Presti et al. Cancer Immunol Res 2017. DOI: 10.1158/2326-6066.CIR-16-0348).Knowledge of the human γδ TCR repertoire during development or after infection is scant. Next-gen sequencing revealed that repertoires of rearranged genes encoding γδ TCRs in the peripheral blood of healthy adults were stable over time. Although γδ T cells were quickly reconstituted after hematopoietic stem cell transplantation, the TCR repertoires were profoundly altered. CMV infection resulted in clonal expansion of distinct γδ T cell responses, underscoring the clinical association between increased γδ T cells and improved long-term disease-free survival, and providing further evidence for adaptive anti-viral γδ T cell immunity.Ravens S, …, Prinz I. Nat Immunol 2017 Apr; 18:393–401.Mantle cell lymphoma of the terminal ileum (by Nephron, Wikimedia Commons).Analysis by liquid chromatography and tandem mass spectrometry of 17 human mantle cell lymphomas revealed that all presented neoepitopes were derived from the lymphoma's unique immunoglobulin. The neoantigenic peptides were exclusively derived from the Ig heavy and light chain variable regions, and presented by MHC class II. Circulating CD4+ T cells specific for these neoantigens were isolated from patients and could kill autologous lymphoma cells. Peptides from other genes were not recovered from MHC, supporting a focus for immunotherapy on tumor-expressed immunoglobulins.Khodadoust MS, …, Alizadeh AA. Nature 2017;543:723–7.PD-1 blocks signal flow from CD28 (from Siena College on Flickr)..Two papers in SCIENCE looked at the interplay between PD-1 and CD28 signaling, in different systems. Kamphorst and colleagues found that blockade of PD-1 will not restore T cell responsiveness unless CD28 can signal. A biochemical analysis by Hui and colleagues surprisingly revealed that CD28 is more sensitive than the TCR to PD-1 and SHP-2 dephosphorylation. These studies suggest costimulatory pathways play key roles in regulating responses to anti-PD-L1/PD-1 therapy, and imply a distinct early memory population may be the target of rescue.Kamphorst AO, …, Ahmed R. Science 2017 Mar 31;355:1423–7.Hui E, …, Vale RD. Science 2017 Mar 31;355:1428–33.

Exploring Human Antimicrobial Antibody Responses on a Single B Cell Level.

Analysis of monoclonal antibodies (MAbs) derived from single B cell cloning has been highly beneficial for antimicrobial immunotherapy, vaccine design, and advancing our understanding of pathogen-triggered effects on the human immunoglobulin repertoire. Sequencing of variable domains of single B cells, and characterization of binding and functional activities of MAbs derived from those sequences, provides in-depth insight not only into sites of susceptibility for antibody-mediated neutralization or opsonization of the pathogen but also into the dynamics of protective antibody evolution during infection. This information can be utilized to rapidly develop novel immunotherapies of completely human origin and provides a roadmap for structure-based vaccine design that aims to elicit similar protective antibody responses. Here, we summarize recent aspects of the single B cell cloning approach.

Gene conversion contributes significantly to IgHV somatic hypermutation in mice and humans

Abstract During antigen-driven affinity maturation germinal center B lymphocytes undergo multiple rounds of somatic mutagenesis in the rearranged IgHV gene segment. A direct consequence of this process is the generation of multiple antibody mutants that are, in general, specific for antigen. Survival of these mutants is contingent on preserving or improving antigen-specificity in the complementarity-determining regions (CDRs) as well as retaining a functional framework region (FWR). Broadly, somatic mutations in germinal center B cells can be classified as templated or untemplated. In animals such as rabbits and chickens, somatic mutations are mostly templated. Conversely, in mice and humans it is widely accepted that mutations are untemplated. Here, we argue in favor of the alternative, that a predominant number of somatic mutations in IgHV gene segments in mice and humans can be traced to germline IgHV genes and occur through gene conversion. Using the NP hapten model, we demonstrate that mutations in germinal center B cells and plasma cells are traceable to other germline IgHV genes. Subsequently we also demonstrate using the novel V-LAIR1-DJ antibodies described by Tan et al (Nature 2016) that mutations in human antibody IgHV rearrangements, including those in non-immunoglobulin sequences (LAIR1), are traceable to the human IgHV germline repertoire. Together, this suggests that the plethora of diversity generated in antigen specific B cells is primarily directed by the germline repertoire of IgHV genes.

Btk-specific inhibition blocks pathogenic plasma cell signatures and myeloid cell-associated damage in IFNα-driven lupus nephritis.

Systemic lupus erythematosus (SLE) is often associated with exaggerated B cell activation promoting plasma cell generation, immune-complex deposition in the kidney, renal infiltration of myeloid cells, and glomerular nephritis. Type-I IFNs amplify these autoimmune processes and promote severe disease. Bruton's tyrosine kinase (Btk) inhibitors are considered novel therapies for SLE. We describe the characterization of a highly selective reversible Btk inhibitor, G-744. G-744 is efficacious, and superior to blocking BAFF and Syk, in ameliorating severe lupus nephritis in both spontaneous and IFNα-accelerated lupus in NZB/W_F1 mice in therapeutic regimens. Selective Btk inhibition ablated plasmablast generation, reduced autoantibodies, and - similar to cyclophosphamide - improved renal pathology in IFNα-accelerated lupus. Employing global transcriptional profiling of spleen and kidney coupled with cross-species human modular repertoire analyses, we identify similarities in the inflammatory process between mice and humans, and we demonstrate that G-744 reduced gene expression signatures essential for splenic B cell terminal differentiation, particularly the secretory pathway, as well as renal transcriptional profiles coupled with myeloid cell-mediated pathology and glomerular plus tubulointerstitial disease in human glomerulonephritis patients. These findings reveal the mechanism through which a selective Btk inhibitor blocks murine autoimmune kidney disease, highlighting pathway activity that may translate to human SLE.

Immunological memory to hyperphosphorylated tau in asymptomatic individuals

Several reports have described the presence of antibodies against Alzheimer’s disease-associated hyperphosphorylated forms of tau in serum of healthy individuals. To characterize the specificities that can be found, we interrogated peripheral IgG+ memory B cells from asymptomatic blood donors for reactivity to a panel of phosphorylated tau peptides using a single-cell screening assay. Antibody sequences were recovered, cloned, and expressed as full-length IgGs. In total, 52 somatically mutated tau-binding antibodies were identified, corresponding to 35 unique clonal families. Forty-one of these antibodies recognize epitopes in the proline-rich and C-terminal domains, and binding of 26 of these antibodies is strictly phosphorylation dependent. Thirteen antibodies showed inhibitory activity in a P301S lysate seeded in vitro tau aggregation assay. Two such antibodies, CBTAU-7.1 and CBTAU-22.1, which bind to the proline-rich and C-terminal regions of tau, respectively, were characterized in more detail. CBTAU-7.1 recognizes an epitope that is similar to that of murine anti-PHF antibody AT8, but has different phospho requirements. Both CBTAU-7.1 and CBTAU-22.1 detect pathological tau deposits in post-mortem brain tissue. CBTAU-7.1 reveals a similar IHC distribution pattern as AT8, immunostaining (pre)tangles, threads, and neuritic plaques. CBTAU-22.1 shows selective detection of neurofibrillary changes by IHC. Taken together, these results suggest the presence of an ongoing antigen-driven immune response against tau in healthy individuals. The wide range of specificities to tau suggests that the human immune repertoire may contain antibodies that can serve as biomarkers or be exploited for therapy.

Human γδ T cells are quickly reconstituted after stem-cell transplantation and show adaptive clonal expansion in response to viral infection.

To investigate how the human γδ T cell pool is shaped during ontogeny and how it is regenerated after transplantation of hematopoietic stem cells (HSCs), we applied an RNA-based next-generation sequencing approach to monitor the dynamics of the repertoires of γδ T cell antigen receptors (TCRs) before and after transplantation in a prospective cohort study. We found that repertoires of rearranged genes encoding γδ TCRs (TRG and TRD) in the peripheral blood of healthy adults were stable over time. Although a large fraction of human TRG repertoires consisted of public sequences, the TRD repertoires were private. In patients undergoing HSC transplantation, γδ T cells were quickly reconstituted; however, they had profoundly altered TCR repertoires. Notably, the clonal proliferation of individual virus-reactive γδ TCR sequences in patients with reactivation of cytomegalovirus revealed strong evidence for adaptive anti-viral γδ T cell immune responses.

BRILIA: Integrated Tool for High-Throughput Annotation and Lineage Tree Assembly of B-Cell Repertoires.

The somatic diversity of antigen-recognizing B-cell receptors (BCRs) arises from Variable (V), Diversity (D), and Joining (J) (VDJ) recombination and somatic hypermutation (SHM) during B-cell development and affinity maturation. The VDJ junction of the BCR heavy chain forms the highly variable complementarity determining region 3 (CDR3), which plays a critical role in antigen specificity and binding affinity. Tracking the selection and mutation of the CDR3 can be useful in characterizing humoral responses to infection and vaccination. Although tens to hundreds of thousands of unique BCR genes within an expressed B-cell repertoire can now be resolved with high-throughput sequencing, tracking SHMs is still challenging because existing annotation methods are often limited by poor annotation coverage, inconsistent SHM identification across the VDJ junction, or lack of B-cell lineage data. Here, we present B-cell repertoire inductive lineage and immunosequence annotator (BRILIA), an algorithm that leverages repertoire-wide sequencing data to globally improve the VDJ annotation coverage, lineage tree assembly, and SHM identification. On benchmark tests against simulated human and mouse BCR repertoires, BRILIA correctly annotated germline and clonally expanded sequences with 94 and 70% accuracy, respectively, and it has a 90% SHM-positive prediction rate in the CDR3 of heavily mutated sequences; these are substantial improvements over existing methods. We used BRILIA to process BCR sequences obtained from splenic germinal center B cells extracted from C57BL/6 mice. BRILIA returned robust B-cell lineage trees and yielded SHM patterns that are consistent across the VDJ junction and agree with known biological mechanisms of SHM. By contrast, existing BCR annotation tools, which do not account for repertoire-wide clonal relationships, systematically underestimated both the size of clonally related B-cell clusters and yielded inconsistent SHM frequencies. We demonstrate BRILIA’s utility in B-cell repertoire studies related to VDJ gene usage, mechanisms for adenosine mutations, and SHM hot spot motifs. Furthermore, we show that the complete gene usage annotation and SHM identification across the entire CDR3 are essential for studying the B-cell affinity maturation process through immunosequencing methods.

Rhetorical Functions of Silence among Speakers of Some Nigerian Languages: Cultural and Individual Perspectives

Rhetorical Functions of Silence among Speakers of Some Nigerian Languages: Cultural and Individual Perspectives Bashir Ibrahim, Ain Nadzimah Abdullah, Shameem Rafik-Galea1, Zalina Mohd Kasim Abstract Recently, there has been renewed interest in the study of silence as a linguistic tool used by humans for communication purposes. Since the work of Nwoye (1985) on eloquent silence among the Igbo of Nigeria, little study has been conducted on the use of silence among other myriad Nigerian tribes. Yet, there has been a general claim that Africans use silence in formal and social situations more frequently than their European and North American counterparts. This study seeks to understand and explain rhetorical functions of silence among speakers of some Nigerian languages. Focus group discussions were held to elicit data from some undergraduate students of two public universities in Northern Nigeria involving speakers of Hausa/Fulani, Igala, Yoruba, Idoma, Igbo, Tiv,Meroeh, Lunguda, and Nupe. Two discussions were held with 300 level students of the Department of English, 400 level students of the Department of Physics and 300 level students of the Department of Biological Sciences. The discussion lasted about twenty minutes with each of the group.The results indicated that silence in conversation performs at both illocutionary and perlocutionary levels, having both negative and positive connotations depending on the context, situation, and the participants involved. The findings indicated that silence is ubiquitous in human communication repertoire, irrespective of culture. Intra- and intercultural variation, however, exist on the perception of various cultures around the world on the use of silence in conversation. Full Text: PDF DOI: 10.15640/ijlc.v5n1a6

The long non-coding RNA ROCR contributes to SOX9 expression and chondrogenic differentiation of human mesenchymal stem cells.

Long non-coding RNAs (lncRNAs) are expressed in a highly tissue-specific manner and function in various aspects of cell biology, often as key regulators of gene expression. In this study, we established a role for lncRNAs in chondrocyte differentiation. Using RNA sequencing we identified a human articular chondrocyte repertoire of lncRNAs from normal hip cartilage donated by neck of femur fracture patients. Of particular interest are lncRNAs upstream of the master chondrocyte transcription factor SOX9 locus. SOX9 is an HMG-box transcription factor that plays an essential role in chondrocyte development by directing the expression of chondrocyte-specific genes. Two of these lncRNAs are upregulated during chondrogenic differentiation of mesenchymal stem cells (MSCs). Depletion of one of these lncRNAs, LOC102723505, which we termed ROCR (regulator of chondrogenesis RNA), by RNA interference disrupted MSC chondrogenesis, concomitant with reduced cartilage-specific gene expression and incomplete matrix component production, indicating an important role in chondrocyte biology. Specifically, SOX9 induction was significantly ablated in the absence of ROCR, and overexpression of SOX9 rescued the differentiation of MSCs into chondrocytes. Our work sheds further light on chondrocyte-specific SOX9 expression and highlights a novel method of chondrocyte gene regulation involving a lncRNA.

Antibodies from multiple sclerosis patients preferentially recognize hyperglucosylated adhesin of non-typeable Haemophilus influenzae.

In autoimmune diseases, there have been proposals that exogenous “molecular triggers”, i.e., specific this should be ‘non-self antigens’ accompanying infectious agents, might disrupt control of the adaptive immune system resulting in serious pathologies. The etiology of the multiple sclerosis (MS) remains unclear. However, epidemiologic data suggest that exposure to infectious agents may be associated with increased MS risk and progression may be linked to exogenous, bacterially-derived, antigenic molecules, mimicking mammalian cell surface glycoconjugates triggering autoimmune responses. Previously, antibodies specific to a gluco-asparagine (N-Glc) glycopeptide, CSF114(N-Glc), were identified in sera of an MS patient subpopulation. Since the human glycoproteome repertoire lacks this uniquely modified amino acid, we turned our attention to bacteria, i.e., Haemophilus influenzae, expressing cell-surface adhesins including N-Glc, to establish a connection between H. influenzae infection and MS. We exploited the biosynthetic machinery from the opportunistic pathogen H. influenzae (and the homologous enzymes from A. pleuropneumoniae) to produce a unique set of defined glucosylated adhesin proteins. Interestingly we revealed that a hyperglucosylated protein domain, based on the cell-surface adhesin HMW1A, is preferentially recognized by antibodies from sera of an MS patient subpopulation. In conclusion the hyperglucosylated adhesin is the first example of an N-glucosylated native antigen that can be considered a relevant candidate for triggering pathogenic antibodies in MS.

The newborn human NK cell repertoire is phenotypically formed but functionally reduced.

Infection is a leading cause of death worldwide in babies under 1 month of age. Better vaccines and therapeutics are desperately needed for this vulnerable population. Because newborns rely heavily on the innate immune system, we evaluated cell phenotype and function of some of the earliest cellular responders during infection, natural killer (NK) cells. We used mass cytometry to provide a comprehensive comparison of NK cells from umbilical cord blood and adult peripheral blood. In unsupervised analyses, including viSNE and principal component analysis, the structure of the cord blood and adult NK cell repertoires are highly similar, distinguishable mainly by maturity-related markers expressed on rare subpopulations of cells. However, in functional analyses, cord blood NK cells show reduced degranulation and cytokine production following target recognition, as well as antibody-dependent cell-mediated cytotoxicity and apoptosis induction in targets. These findings show that the structure of the NK cell repertoire is intact at birth, suggesting great potential for vaccine and therapeutic strategies targeting this cell population. © 2016 International Clinical Cytometry Society.

Culture of previously uncultured members of the human gut microbiota by culturomics.

Metagenomics revolutionized the understanding of the relations among the human microbiome, health and diseases, but generated a countless number of sequences that have not been assigned to a known microorganism1. The pure culture of prokaryotes, neglected in recent decades, remains essential to elucidating the role of these organisms2. We recently introduced microbial culturomics, a culturing approach that uses multiple culture conditions and matrix-assisted laser desorption/ionization-time of flight and 16S rRNA for identification2. Here, we have selected the best culture conditions to increase the number of studied samples and have applied new protocols (fresh-sample inoculation; detection of microcolonies and specific cultures of Proteobacteria and microaerophilic and halophilic prokaryotes) to address the weaknesses of the previous studies3-5. We identified 1,057 prokaryotic species, thereby adding 531 species to the human gut repertoire: 146 bacteria known in humans but not in the gut, 187 bacteria and 1 archaea not previously isolated in humans, and 197 potentially new species. Genome sequencing was performed on the new species. By comparing the results of the metagenomic and culturomic analyses, we show that the use of culturomics allows the culture of organisms corresponding to sequences previously not assigned. Altogether, culturomics doubles the number of species isolated at least once from the human gut.

12PD - MemoMAB, a novel technology for the banking and screening of cognate human immunoreceptor repertoires

Aim/Background: Providing a platform for the comprehensive analysis of the repertoires of human T and B cells in recombinant form while preserving the original heavy and light chain pairing of immunoreceptors (cognate, non-combinatorial libraries). Discovery of antibodies and TCRs generated in response to immune-stimulation such as administration of checkpoint inhibitors in cancer immunotherapy. Methods: A high throughput microfluidics-based single-cell molecular cloning and recombinant expression platform was developed that enables the generation of cognate human immunoreceptor repertoire libraries from human B and T cells (MemoMAB platform). Screening of this library can be performed by high-throughput single-cell-based mammalian display methods such as cell surface expression followed by cytometric sorting or encapsulation and culture of single recombinant cells in nanoliter reactors followed by functional screenings. Results: Using the MemoMAB platform technology, we have generated and screened antibody repertoire libraries from more than 100’000 human memory B cells from several human donors. Initial proof of concept of the platform has been performed in rabbits where monoclonal antibodies to several targets have been identified using cell surface expression or single cell encapsulation in nanoliter reactors. Conclusions: The generation of human cognate antibody repertoire analysis comprising 100’000 and more human memory B cells is feasible. Screening of these libraries is a straightforward process directly leading to the identification of clonal cell lines producing the immunoreceptor of interest in recombinant form. This tool can be expected to aid in the discovery of novel antibodies and their targets as candidate agents for cancer immuno-therapy. A particular area of interest for the application of the MemoMAB technology would be the analysis of antibody and T cell repertoires of patients undergoing immune-checkpoint-inhibitor therapy with the goal to identify novel immunogenic determinants for cancer immunotherapy. Legal entity responsible for the study: Klonale Analyse von menschlichen Antikörperrepertoires (2016-01260) Kantonale Ethikkommission Zürich Funding: Memo Therapeutics AG Disclosure: C. Esslinger: Shareholder and employee of Memo Therapeutics. S. Schmitt, M. Weber, A. Guimaraes: Employee of Memo Therapeutics.