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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2018

HLA-B*39:06 Efficiently Mediates Type 1 Diabetes in a Mouse Model Incorporating Reduced Thymic Insulin Expression

Type 1 diabetes (T1D) is characterized by T cell-mediated destruction of the insulin-producing {beta}cells of the pancreatic islets. Among the loci associated with T1D risk, those most predisposing are found in the MHC region. HLA-B*39:06 is the most predisposing class I MHC allele and is associated with an early age of onset. To establish an NOD mouse model for the study of HLA-B*39:06, we expressed it in the absence of murine class I MHC. HLA-B*39:06 was able to mediate the development of CD8 T cells, support lymphocytic infiltration of the islets, and confer T1D susceptibility. Because reduced thymic insulin expression is associated with increased T1D risk in patients, we incorporated this in our model as well, finding that HLA-B*39:06-transgenic NOD mice with reduced thymic insulin expression have an earlier age of disease onset and a higher overall prevalence as compared to littermates with typical thymic insulin expression. This was despite virtually indistinguishable blood insulin levels, T cell subset percentages, and TCR V{beta} family usage, indicating that reduced thymic insulin expression does not impact T cell development on a global scale. Rather, we propose that it allows the thymic escape of insulin-reactive HLA-B*39:06-restricted T cells which participate in {beta} cell destruction. We also found that in mice expressing either HLA-B*39:06 or HLA-A*02:01 in the absence of murine class I MHC, HLA transgene identity alters TCR V{beta} usage, which may contribute to varying diabetogenic CD8 T cell repertoires in the presence of different HLA class I alleles.

immunology↗

bioRxiv2018

A long-chain fatty acid elongase Elovl6 regulates mechanical damage-induced keratinocyte death and skin inflammation

Mechanical damage on the skin not only affect the barrier function but also induce various immune responses, which trigger or exacerbate the inflammation in healthy individuals and patients with inflammatory skin diseases. However, how mechanical damage-induced skin inflammation is regulated remains largely unknown. Here, we show that mechanical damage due to tape stripping triggered keratinocyte death and release of danger-associated molecular patterns (DAMPs) such as high-mobility group box 1 protein (HMGB-1) and IL-1, which induced production of proinflammatory cytokines and chemokines IL-1{beta} and CXCL-1 by keratinocytes in mice. We also show that a long-chain fatty acid elongase Elovl6 is expressed in keratinocytes. Mice deficient in Elovl6 had increased epidermal levels of cis-vaccenic acid (CVA); this accelerated keratinocyte death triggered by tape stripping and release of DAMPs and exacerbated skin inflammation. Our results demonstrate that Elovl6 regulates mechanical damage-triggered keratinocyte death and skin inflammation.

immunology↗

bioRxiv2018

Primate MHC class I from Genomes

The major histocompatibility complex (MHC) molecule plays a central role in the adaptive immunity of jawed vertebrates. Allelic variations have been studied extensively in some primate species, however a comprehensive description of the number of genes remains incomplete. Here, a bioinformatics program was developed to identify three MHC Class I exons (EX2, EX3 and EX4) from Whole Genome Sequencing (WGS) datasets. With this algorithm, MHC Class I exons sequences were extracted from 30 WGS datasets of primates, representatives of Apes, Old World and New World monkeys and prosimians. There is a high variability in the number of genes between species. From human WGS, six viable genes (HLA-A, -B, -C, -E, -F, and -G) and four pseudogene sequences (HLA-H, -J, -L, -V) are obtained. These genes serve to identify the phylogenetic clades of MHC-I in primates. The results indicate that human clades of HLA-A -B and -C were generated shortly after the separation of Old World monkeys. The clades pertaining to HLA-E, -H and -F are found in all primate families, except in Prosimians. In the clades defined by HLA-G, -L and -J, there are sequences from Old world monkeys. Specific clades are found in the four primate families. The evolution of these genes is consistent with birth and death processes having a high turnover rates.

immunology↗

bioRxiv2018

Nanoscale colocalization of NK cell activating and inhibitory receptors controls signal integration

NK cell responses depend on the balance of signals from inhibitory and activating receptors. However, how the integration of antagonistic signals occurs upon NK cell-target cell interaction is not fully understood. Here, we provide evidence that NK cell inhibition via the inhibitory receptor Ly49A is dependent on its relative colocalization at the nanometer scale with the activating receptor NKG2D upon immune synapse (IS) formation. NKG2D and Ly49A signal integration and colocalization was studied using NKG2D-GFP and Ly49A-RFP-expressing primary NK cells, forming ISs with NIH3T3 target cells, with or without expression of single chain trimer (SCT) H2-Dd and an extended form of SCT H2-Dd-CD4 MHC-I molecules. Nanoscale colocalization was assessed by Forster resonance energy transfer (FRET) between NKG2D-GFP and Ly49A-RFP and measured for each synapse. In the presence of their respective cognate ligands, NKG2D and Ly49A colocalize at a nanometer scale leading to NK cell inhibition. However, increasing the size of the Ly49A ligand reduced the nanoscale colocalization with NKG2D consequently impairing Ly49A-mediated inhibition. Thus, our data shows NK cell signal integration is critically dependent on the dimensions of NK cell ligand-receptor pairs by affecting their relative nanometer-scale colocalization at the IS. Together, our results suggest the balance of NK cell signals, and NK cell responses, are determined by the relative nanoscale colocalization of activating and inhibitory receptors in the immune synapse.

immunology↗

bioRxiv2018

The plasma biomarker soluble SIGLEC-1 is associated with the type I interferon transcriptional signature, ethnic background and renal disease in systemic lupus erythematosus

The molecular heterogeneity of autoimmune and inflammatory diseases has been one of the main obstacles to the development of safe and specific therapeutic options. Here we have evaluated the diagnostic and clinical value of a robust, inexpensive, immunoassay detecting the circulating soluble form of the monocyte-specific surface receptor sialic acid binding Ig-like lectin 1 (sSIGLEC-1).\n\nWe developed an immunoassay to measure sSIGLEC-1 in small volumes of plasma/serum from systemic lupus erythematosus (SLE) patients and healthy donors. Plasma concentrations of sSIGLEC-1 strongly correlated with expression of SIGLEC-1 on the surface of blood monocytes and with type I interferon (IFN)-regulated gene (IRG) expression in SLE patients. In addition, we identified marked ancestry-related differences in sSIGLEC-1 concentrations in SLE patients, with patients of non-European ancestry showing higher levels compared to patients of European ancestry. Higher sSIGLEC-1 concentrations were associated with lower serum complement component 3 and increased frequency of renal complications in European patients, but not with the SLEDAI clinical score.\n\nOur sSIGLEC-1 immunoassay provides a specific and easily-assayed marker for monocyte-macrophage activation, and interferonopathy in SLE and other diseases. Further studies can extend its clinical associations and its potential use to stratify patients and as a secondary endpoint in trials.

immunology↗

bioRxiv2018

Experimental and mathematical approaches to quantify recirculation kinetics of lymphocytes

One of the properties of the immune system that makes it different from nervous and en-docrine systems of mammals is the ability of immune cells to migrate between different tissues. Lymphocytes such as T and B cells have the ability to migrate from the blood to secondary lymphoid tissues such as spleen, lymph nodes, and Peyers patches, and then migrate back to the blood, i.e., they can recirculate. Recirculation of lymphocytes has been a subject of intensive investigation decades ago with wealth of data on the kinetics of lymphocyte recirculation available. However, these data have not been widely used to estimate the kinetics of recirculation of different lymphocyte subsets in naive and immunized animals. In this paper we review pioneering studies addressing the question of lymphocyte recirculation, overview quantitative approaches that have been used to estimate the kinetics of lymphocyte recirculation, and provide currently published estimates of the residence times of resting lymphocytes in secondary lymphoid tissues of mammals.

immunology↗

bioRxiv2018

Trends in CRP, D-dimer and fibrinogen during therapy for HIV associated multidrug resistant tuberculosis

BackgroundHIV positive adults on treatment for multidrug-resistant tuberculosis (MDR-TB) experience high mortality. Biomarkers of HIV/MDR-TB treatment response may enable earlier treatment modifications that improve outcomes.\n\nMethodsTo determine whether trends in C-reactive protein (CRP), D-dimer and fibrinogen predict treatment outcome among those with HIV/MDR-TB co-infection we studied 20 HIV positive participants initiating therapy for MDR-TB. Serum CRP, fibrinogen, and D-dimer were measured at baseline and serially while on treatment. Results: At baseline, all biomarkers were elevated with median CRP 86.15 mg/L (IQR 29.25-149.32), D-dimer 0.85 g/mL (IQR 0.34-1.80) and fibrinogen 4.11 g/L (IQR 3.75-6.31). CRP decreased significantly within 10 days of treatment initiation and fibrinogen within 28 days; D-dimer did not change significantly. 5 (25%) participants died. Older age (median age of 38y among survivors and 54y among deceased, p=0.008) and higher baseline fibrinogen (3.86 g/L among survivors and 6.37 g/L among deceased, p=0.02) were significantly associated with death. Higher CRP concentrations at the beginning of each measurement interval were significantly associated with a higher risk of death during that interval.\n\nConclusionTrends in fibrinogen and CRP may be useful for evaluating early response to treatment among individuals with HIV/MDR-TB co-infection.

immunology↗

bioRxiv2018

Clonal competition in B-cell repertoires during chronic HIV-1 infection

During chronic infection, HIV-1 engages in a rapid coevolutionary arms race with the hosts adaptive immune system. While it is clear that HIV exerts strong selection on the adaptive immune system, the characteristics of the somatic evolution that shape the immune response are still unknown. Traditional population genetics methods fail to distinguish chronic immune response from healthy repertoire evolution. Here, we infer the evolutionary modes of B-cell repertoires and identify complex dynamics with a constant production of better B-cell receptor mutants that compete, maintaining large clonal diversity and potentially slowing down adaptation. A substantial fraction of mutations that rise to high frequencies in pathogen engaging CDRs of B-cell receptors (BCRs) are beneficial, in contrast to many such changes in structurally relevant frameworks that are deleterious and circulate by hitchhiking. We identify a pattern where BCRs in patients who experience larger viral expansions undergo stronger selection with a rapid turnover of beneficial mutations due to clonal interference in their CDR3 regions. Using population genetics modeling, we show that the extinction of these beneficial mutations can be attributed to the rise of competing beneficial alleles and clonal interference. The picture is of a dynamic repertoire, where better clones may be outcompeted by new mutants before they fix.

immunology↗

bioRxiv2018

Differential expression of an alternative splice variant of IL-12Rβ1 impacts early dissemination in the mouse and associates with disease outcome in both mouse and humans exposed to tuberculosis

Experimental mouse models of TB suggest that early events in the lung impact immunity. Early events in the human lung in response to TB are difficult to probe and their impact on disease outcome is unknown. We have shown in mouse that a secreted alternatively-spliced variant of IL-12R{beta}1, lacking the transmembrane domain and termed {Delta}TM-IL-12R{beta}1, promotes dendritic cell migration to the draining lymph node, augments T cell activation and limits dissemination of M. tuberculosis (Mtb). We show here that CBA/J and C3H/HeJ mice (both highly susceptible to Mtb) express higher levels of {Delta}TM-IL-12R{beta}1 than resistant C57BL6 mice and limit early dissemination of Mtb from the lungs. Both CD11c+ cells and T cells express {Delta}TM-IL-12R{beta}1 in humans, and mice unable to make {Delta}TM-IL-12R{beta}1 in either CD4 or CD11c expressing cells permit early dissemination from the lung. Analysis of publically available blood transcriptomes indicates that pulmonary TB is associated with high {Delta}TM-IL-12R{beta}1 expression and that of all IL-12 related signals, the {Delta}TM-IL-12R{beta}1 signal best predicts active disease. {Delta}TM-IL-12R{beta}1 expression reflects the heterogeneity of latent TB infection and has the capacity to discriminate between latent and active disease. In a new Chinese TB patient cohort, {Delta}TM-IL-12R{beta}1 effectively differentiates TB from latent TB, healthy controls and pneumonia patients. Finally, {Delta}TM-IL-12R{beta}1 expression drops in drug-treated individuals in the UK and China where infection pressure is low. We propose that {Delta}TM-IL-12R{beta}1 regulates early dissemination from the lung and that it has diagnostic potential and provides mechanistic insights into human TB.

immunology↗

bioRxiv2018

RNASeq analysis identifies non-canonical role of STAT2 and IRF9 in the regulation of a STAT1-independent antiviral and immunoregulatory transcriptional program induced by IFNβ and TNFα

IFN{beta} typically induces an antiviral and immunoregulatory transcriptional program through the activation of ISGF3 (STAT1, STAT2 and IRF9) transcriptional complexes. The response to IFN{beta} is context-dependent and is prone to crosstalk with other cytokines, such as TNF IFN{beta} and TNF synergize to drive a specific delayed transcriptional program. Previous observation led to the hypothesis that an alternative STAT1-independent pathway involving STAT2 and IRF9 might be involved in gene induction by the combination of IFN{beta} and TNF. Using genome wide transcriptional profiling by RNASeq, we found that the costimulation with IFN{beta} and TNF induces a broad antiviral and immunoregulatory transcriptional program independently of STAT1. Additionally, STAT2 and IRF9 are involved in the regulation of only a subset of these STAT1-independent genes. Consistent with the growing literature, STAT2 and IRF9 act in concert to regulate a subgroup of these genes. Unexpectedly, STAT2 and IRF9 were also engaged in specific independent pathways to regulate distinct sets of IFN{beta} and TNF-induced genes. Altogether these observations highlight the existence of distinct previously unrecognized non-canonical STAT1-independent, but STAT2 and/or IRF9-dependent pathways in the establishment of a delayed antiviral and immunoregulatory transcriptional program in conditions where elevated levels of both IFN{beta} and TNF are present.

immunology↗

bioRxiv2018

Perforin-2 Permeabilizes the Envelope of Phagocytosed Bacteria

Perforin-2, the product of the MPEG1 gene, limits the spread and dissemination of bacterial pathogens in vivo. It is highly expressed in murine and human phagocytes, and macrophages lacking Perforin-2 are compromised in their ability to kill phagocytosed bacteria. In this study we used Salmonella typhimurium as a model intracellular pathogen to elucidate the mechanism of Perforin-2 s bactericidal activity. In vitro Perforin-2 was found to facilitate the degradation of antigens contained within the envelope of phagocytosed bacteria. In contrast, degradation of a representative surface antigen was found to be independent of Perforin-2. Consistent with our in vitro results a protease sensitive, periplasmic superoxide disumutase (SodCII) contributed to the virulence of S. typhimurium in Perforin-2 knockout but not wild-type mice. In aggregate our studies indicate that Perforin-2 breaches the envelope of phagocytosed bacteria facilitating the delivery of proteases and other antimicrobial effectors to sites within the bacterial envelope.

immunology↗

bioRxiv2018

Anti-HIV-1 B cell responses are dependent on B cell precursor frequency and antigen binding affinity

The discovery that humans can produce potent broadly neutralizing antibodies (bNAbs) to several different epitopes on the HIV-1 spike has reinvigorated efforts to develop an antibody based HIV-1 vaccine. Antibody cloning from single cells revealed that nearly all bNAbs show unusual features that could help explain why it has not been possible to elicit them by traditional vaccination, and instead that it would require a sequence of different immunogens. This idea is supported by experiments with genetically modified immunoglobulin knock-in mice. Sequential immunization with a series of specifically designed immunogens was required to shepherd the development of bNAbs. However, knock-in mice contain super-physiologic numbers of bNAb precursor expressing B cells and therefore how these results can be translated to a more physiologic setting remains to be determined. Here we make use of adoptive transfer experiments using knock-in B cells that carry a synthetic intermediate in the pathway to anti-HIV-1 bNAb development to examine how the relationship between B cell receptor affinity and precursor frequency affects germinal center B cell recrutiment and clonal expansion. Immunization with soluble HIV-1 antigens can recruit bNAb precursor B cells to the germinal center when there are as few as 10 such cells per mouse. However, at low precursor frequencies the extent of clonal expansion is directly proportional to the affinity of the antigen for the B cell receptor, and recruitment to germinal centers is variable and dependent on re-circulation.\n\nSignificance statementAn essential requirement for an HIV-vaccine is to elicit antibodies to conserved regions of the spike protein (Env) becasue these antibodies can protect against infection. Although broadly neutralizing antibodies develop naturally in rare individuals after prolongued HIV infection, eliciting them by vaccination has only been possible in artificial knock-in mouse models wherein the number of B cells expressing the antibody precursor is super-physiologic. To understand the relationship between precursor frequency, antigen affinity and germinal center recruitment we have performed adoptive transfer experiments in which fixed numbers of precursor cells are engrafted in wild type mice. Our results provide a framework for understanding how precursor frequency and antigen affinity shape humoral immunity to HIV.

immunology↗

bioRxiv2018

A mouse model of maternal obesity leads to uterine natural killer (uNK) cell activation and uterine artery remodeling defects

Maternal obesity associates with multiple adverse reproductive outcomes, negatively affecting the health and survival of both the mother and the fetus. The contributory effects of obesity on adipose-derived immune changes have been well established, however the mechanisms that link obesity to pregnancy complications remain unclear. Proper development of the placenta and establishment of utero-placental vasculature is essential in early pregnancy to allow for optimal fetal growth and survival. Uterine immune cells, particularly uterine natural killer (uNK) cells, play fundamental roles in promoting these events. Using an obesogenic high-fat/high-sucrose (HFD) mouse model of maternal obesity, uNK cells and placenta/uterine vascular remodeling were examined at gestational days (Gd) 10.5 and 14.5 of pregnancy. While mice fed a HFD 13 weeks prior to pregnancy significantly gained more weight than control mice fed a low-fat/no-sucrose diet (LFD), fetal survival was not different in either diet. At Gd 10.5, HFD had no effect on total uNK proportions, nor did it affect proportions of distinct CD122+/CD49a tissue resident or CD122+/DX5+ conventional uNKs. However, HFD resulted in increased proportions of NCR1+ uNK and overall heightened uNK activity. Moreover, HFD resulted in impairments in uterine vascular remodeling at Gd10.5. By Gd14.5, uterine artery defects were no longer observed and placental structure was comparable between HFD and LFD mice. Gene analysis of IFN{gamma}, TNF and VEGFA in Gd10.5 uNK indicated that, while not significant, a HFD leads to subtle alterations in uNK cytokine gene expression. These findings show that HFD in mice results in changes in uNK biology and temporary uterine artery remodeling impairments. Further, this work establishes insight into the cellular changes occurring in early pregnancy as a result of HFD exposure.

immunology↗

bioRxiv2018

Predicting the spectrum of TCR repertoire sharing with a data-driven model of recombination

Despite the extreme diversity of T cell repertoires, many identical T cell receptor (TCR) sequences are found in a large number of individual mice and humans. These widely-shared sequences, often referred to as public , have been suggested to be over-represented due to their potential immune functionality or their ease of generation by V(D)J recombination. Here we show that even for large cohorts the observed degree of sharing of TCR sequences between individuals is well predicted by a model accounting for the known quantitative statistical biases in the generation process, together with a simple model of thymic selection. Whether a sequence is shared by many individuals is predicted to depend on the number of queried individuals and the sampling depth, as well as on the sequence itself, in agreement with the data. We introduce the degree of publicness conditional on the queried cohort size and the size of the sampled repertoires. Based on these observations we propose a public/private sequence classifier, PUBLIC (Public Universal Binary Likelihood Inference Classifier), based on the generation probability, which performs very well even for small cohort sizes.

immunology↗

bioRxiv2018

A protocol for single-cell transcriptomics from cryopreserved renal tissue and urine for the Accelerating Medicine Partnership (AMP) RA/SLE network

OBJECTIVEThere is a critical need to define the cells that mediate tissue damage in lupus nephritis. Here we aimed to establish a protocol to preserve lupus nephritis kidney biopsies and urine cell samples obtained at multiple clinical sites for subsequent isolation and transcriptomic analysis of single cells.\n\nMETHODSFresh and cryopreserved kidney tissue from tumor nephrectomies and lupus nephritis kidney biopsies were disaggregated by enzymatic digestion. Cell yields and cell composition were assessed by flow cytometry. Transcriptomes of leukocytes and epithelial cells were evaluated by low-input and single cell RNA-seq.\n\nRESULTSCryopreserved kidney tissue from tumor nephrectomies and lupus nephritis biopsies can be thawed and dissociated to yield intact, viable leukocytes and epithelial cells. Cryopreservation of intact kidney tissue provides higher epithelial cell yields compared to cryopreservation of single cell suspensions from dissociated kidneys. Cell yields and flow cytometric cell phenotypes are comparable between cryopreserved kidney samples and paired kidney samples shipped overnight on wet ice. High-quality single cell and low-input transcriptomic data were generated from leukocytes from both cryopreserved lupus nephritis kidney biopsies and urine, as well as from a subset of kidney epithelial cells.\n\nCONCLUSIONThe AMP RA/SLE cryopreserved tissue analysis pipeline provides a method for centralized processing of lupus nephritis kidney biopsies and urine samples to generate robust transcriptomic analyses in multi-center studies.

immunology↗

bioRxiv2018

Inflammatory bowel disease microbiotas alter gut CD4 T-cell homeostasis and drive colitis in mice

To examine the functional contribution of Inflammatory Bowel Disease (IBD) microbes to immune homeostasis and colitis, we colonized unchallenged and colitis-susceptible germ-free mice with over twenty human intestinal microbiotas from healthy and IBD donors. Compared to healthy microbiotas, IBD microbiotas led to expanded ROR{gamma}t+Th17 cells and reduced ROR{gamma}t+Treg in the gut of unchallenged gnotobiotic mice and increased disease severity in colitis-susceptible mice. The proportions of ROR{gamma}t+Th17 and ROR{gamma}t+Treg induced by each microbiota were highly predictive of the human disease status and strongly correlated with disease severity in colitis-susceptible mice colonized with the same human microbiotas. The transmittable functional potential of IBD microbes suggests a mechanism for a microbial contribution to IBD pathogenesis and a potential route for its treatment and prevention.

immunology↗

bioRxiv2018

Perturbation of ubiquitin homeostasis promotes macrophage oxidative defenses

Innate immune responses rely on specific pattern recognition receptors that induce downstream signaling cascades and promote inflammatory responses. Emerging evidence suggests that cells may also recognize alterations in cellular processes induced by infection. Protein ubiquitination is a post-translational modification essential for maintaining cellular homeostasis, and infection can cause global alterations in the host ubiquitin proteome. Here we used a chemical biology approach to perturb the cellular ubiquitin proteome as a simplified model to study the direct effect of ubiquitin homeostasis on macrophage responses. We show that perturbation of ubiquitin homeostasis results in a rapid and transient burst of reactive oxygen species (ROS) that promotes macrophage anti-infective capacity. ROS production was dependent on the activity of the phagocyte NADPH oxidase NOX2 and was associated with an increase in intracellular calcium. Our findings suggest that major changes in the host ubiquitin landscape may be a potent signal to rapidly deploy innate immune defenses.

immunology↗

bioRxiv2018

Mitochondria-derived vesicles deliver antimicrobial payload to control phagosomal bacteria

Pathogenic bacteria taken up into the macrophage phagosome are the target of many anti-microbial effector molecules. Although mitochondria-derived antimicrobial effectors such as reactive oxygen species (mROS) are reported to aid in bacterial killing, it is unclear how these effectors reach bacteria within the phagosomal lumen. To examine the crosstalk between mitochondria and phagosomes, we monitored the production and the spatial localization of mROS during methicillin-resistant Staphylococcus aureus (MRSA) infection. We showed here mROS, specifically hydrogen peroxide (mH2O2) can be delivered into phagosomes via infection-induced mitochondria-derived vesicles, which are generated in a Parkin-dependent manner. Accumulation of mH2O2 in phagosomes required TLR signaling and the mitochondrial superoxide dismutase, Sod2, which converts superoxide into mH2O2. These data highlight a novel mechanism by which the mitochondrial redox capacity enhances macrophage antimicrobial function by delivering mitochondria-derived effector molecules into bacteria-containing phagosomes.

immunology↗