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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

Precise tracking of vaccine-responding T-cell clones reveals convergent and personalized response in identical twins

T-cell receptor (TCR) repertoire data contain information about infections that could be used in disease diagnostics and vaccine development, but extracting that information remains a major challenge. Here we developed a statistical framework to detect TCR clone proliferation and contraction from longitudinal repertoire data. We applied this framework to data from three pairs of identical twins immunized with the yellow fever vaccine. We identified 500-1500 responding TCRs in each donor and validated them using three independent assays. While the responding TCRs were mostly private, albeit with higher overlap between twins, they could be well predicted using a classifier based on sequence similarity. Our method can also be applied to samples obtained post-infection, making it suitable for systematic discovery of new infection-specific TCRs in the clinic.

immunology↗

bioRxiv2018

Predicting influenza H3N2 vaccine efficacy from evolution of the dominant epitope

We predict vaccine efficacy with a measure of antigenic distance between influenza A(H3N2) and candidate vaccine viruses based on amino acid substitutions in the dominant epitopes. In 2016-2017, our model predicts 19% efficacy compared to 20% observed. This tool assists candidate vaccine selection by predicting human protection against circulating strains.\n\n40-word summary of main pointOur pepitope model predicts the ability of the influenza vaccine to reduce the A(H3N2) disease attack rate, with an r^2=0.77. This fast, sequence-based method compliments strain-to-strain antigenic comparisons from ferret models and provides antigenic comparisons for all circulating sequences.

immunology↗

bioRxiv2018

B cell receptor crosslinking can augment T cell help-mediated germinal center B cell selection.

Selection of germinal center (GC) B cells with B cell receptors (BCR) possessing high affinity to foreign antigen (Ag) and their differentiation into antibody-secreting long-lived plasma cells is critical for potent long-term humoral immunity. Ag-dependent engagement of GC B cell BCR triggers Ag internalization and loading of antigenic peptides on MHCII molecules for presentation to follicular helper T cells (Tfh) and acquisition of T cell help. However, whether it also provides signals that are critical or synergistic with T cell help for GC B cell selection and differentiation in vivo is not known. Here we show that T cell help is sufficient to induce GC B cell expansion and plasmablast (PB) formation in the absence of recurrent BCR engagement with Ag. Ag-mediated BCR crosslinking on the other hand is not sufficient to promote GC B cell selection, but can synergize with T cell help to enhance the GC B cell and PB responses when T cell help is limiting.

immunology↗

bioRxiv2018

Brucella Peptide Cross-Reactive MHC I Presentation Activates SIINFEKL-Specific TCR Expressing T Cells

Brucella spp are intracellular pathogenic bacteria remarkable in their ability to escape immune surveillance and therefore inflict a state of chronic disease within the host. To enable further immune response studies, Brucella were engineered to express the well characterized chicken ovalbumin (OVA). Surprisingly, we found that CD8 T cells bearing T cell receptors (TCR) nominally specific for the OVA peptide SIINFEKL (OT-1) reacted to parental Brucella-infected targets as well as OVA-expressing Brucella variants in cytotoxicity assays. Furthermore, splenocytes from Brucella immunized mice produced IFN-{gamma} and exhibited cytotoxicity in response to SIINFEKL-pulsed target cells. To determine if the SIINFEKL-reactive OT-1 TCR could be cross-reacting to Brucella peptides, we searched the Brucella proteome using an algorithm to generate a list of near-neighbor nonamer peptides that would bind to H2Kb. Selecting five Brucella peptide candidates, along with controls, we verified that several of these peptides mimicked SIINFEKL resulting in T cell activation through the \"SIINFEKL-specific\" TCR. Activation was dependent on peptide concentration as well as sequence. Our results underscore the complexity and ubiquity of cross-reactivity in T cell recognition. This cross-reactivity may enable microbes such as Brucella to escape immune surveillance by presenting peptides similar to the host, and may also lead to the activation of autoreactive T cells.

immunology↗

bioRxiv2018

Identification of a Novel Neutralizing and Two Non-Neutralizing Epitopes on Epstein-Barr Virus gp350 Protein

Prevention of Epstein-Barr virus (EBV) primary infection has focused on generating neutralizing antibodies (nAbs) targeting the major envelope glycoprotein gp350/220 (gp350). To date, eight gp350 epitopes have been identified, but only one has elicited nAbs. In this study, we generated 23 hybridomas that produced anti-gp350 antibodies. We compared the candidate anti-gp350 antibodies to nAb-72A1 by: (1) testing their ability to detect gp350 using ELISA, flow cytometry, and immunoblot; (2) sequencing their heavy and light chain complementarity-determining regions (CDRs); (3) measuring the ability of each monoclonal antibody (mAb) to neutralize EBV infection in vitro; and (4) mapping the gp350 amino acids bound by the mAbs using RepliTope peptide microarrays. Eight antibodies recognized both denatured and non-denatured gp350, whereas five failed to react with denatured gp350 but recognized native gp350, suggesting they recognized conformational epitope(s). Sequence analysis of the heavy and light chain variable regions of the hybridomas identified 15 as mAbs with novel CDR regions unique from those of nAb-72A1. Seven of the new mAbs neutralized EBV in vitro, with HB20 and HB17 reducing EBV infection by 40% and >60%, and >30% and 80%, at 10 g/ml and 50 g/ml, respectively. Epitope mapping identified nine epitopes and defined their core residues, including two unique immunodominant epitopes, 253TPIPGTGYAYSLRLTPRPVSRFL253 and 875LLLLVMADCAFRRNLSTSHTYTTPPY899, and a novel nAb epitope 381GAFASNRTFDIT392. This study provides comprehensive in vitro mapping of the exact residues defining nine epitopes of EBV gp350. Our findings will inform novel strategies to design optimal EBV vaccines capable of conferring broader protection against the virus.\n\nImportanceNeutralizing antibodies (nAbs) directed against Epstein-Barr virus envelope glycoprotein gp350/220 (gp350) are generated in humans upon infection or immunization, and are thought to prevent neonatal infection. However, clinical use of exogenous nAbs (passive immunization) is limited to a single study using the only well-characterized nAb, 72A1. The gp350 ectodomain contains at least eight unique B-cell binding epitopes; two of these epitopes are recognized by nAb-72A1. The exact amino acid residues of the other six epitopes and their role in generating nAbs has not been elucidated. We used our 15 newly generated and fully characterized monoclonal antibodies and a peptide-overlapping RepliTope array to provide a comprehensive map of the core amino acid residues that define epitopes of gp350 and to understand their role in generating nAbs. These results will inform design of better-targeted gp350 peptide vaccines that contain only protective epitopes, which will focus the B-cell response to produce predominantly nAbs.

immunology↗

bioRxiv2018

B cells transiently exposed to antigen that return to quiescence respond to protein immunization similarly to naive B cells

Transient exposure of B cells to moderately multivalent antigens has been shown to lead to their activation and recruitment into T-dependent B cell response in the presence of T cell help. However, when T cell help is delayed, transiently activated B cells return to a naive-like state. While B cells that briefly encountered cognate antigen and then underwent inactivation are not tolerant, whether they may be hypo-or hyper-responsive to cognate antigen is not known. In this study we show that naive B cells and those previously exposed to antigen and then inactivated generate comparable germinal center and plasma cell responses after immunization with a wide range of antigen concentrations.

immunology↗

bioRxiv2018

Comparative analysis of IgG Responses to recombinant Qβ phage displayed MSP3 and UB05 in Dual HIV-malaria infected adults living in areas differing in Malaria transmission intensities

Immunoglobulin G specific responses against Plasmodium falciparum merozoite antigens such as the merozoite surface protein 3 (MSP3) and UB05 are known to play critical roles in parasitemia control and protection from symptomatic illness. However when there is intense perennial malaria transmission coupled with concurrent infection with the human immunodeficiency virus type 1 (HIV), knowledge of IgG antibody response profiles is limited. In this study we assessed the impact of dual HIV-Malaria infections on IgG subclass responses to MSP3 (Q{beta}MSP3) and UB05 (Q{beta}UB05) in individuals living in two areas of Cameroon differing in transmission intensity. We observed differences in antigen specific IgG and IgG subclass responses which was dependent upon the antigen type, malaria transmission intensity, HIV infection, malaria infection and dual HIV-malaria infections. Individuals living in high malaria transmission areas irrespective of HIV or malaria status had significantly higher IgG responses to both antigens (P=0.0001 for Q{beta}MSP3, P=0.0001 for Q{beta}UB05) than their counterpart from low transmission areas. When dual HIV-Malaria infection is considered significantly higher Q{beta}MSP3 specific IgG1 (P=0.0001) and IgG3 (P=0.04) responses in double negative individuals was associated with protection against malaria in low transmission areas. Superior Q{beta}UBO5 specific IgG1 responses (P=0.0001) in double negative individuals were associated with protection in high transmission areas in contrast to significantly higher IgG3 responses to Q{beta}UB05 (P=0.0001) which were more relevant to protection in low malaria transmission areas in the same population. Thus, understanding immune responses to Q{beta}UB05 and Q{beta}MSP3 could facilitate the development of immunotherapeutic strategies suitable for areas differing in malaria transmission intensity.

immunology↗

bioRxiv2018

TGF-β induced CXCL13 in CD8+ T cells is associated with tertiary lymphoid structures in cancer

Coordinated immune responses against human tumors are frequently characterized by tertiary lymphoid structures (TLS) which predict improved prognosis. The development of TLS is dependent on the chemokine CXCL13, reported to be secreted by dendritic cells and follicular helper T cells only. We report the unexpected finding that CXCL13 is also secreted by activated CD8+ T cells following stimulation by transforming growth factor beta (TGF-{beta}). Using single cell RNA sequencing we found that expression of CXCL13 in CD8+ T cells was restricted to the intraepithelial CD103+ population. Accordingly, CD8+ T cells activated in the presence of TGF-{beta} simultaneously upregulated CD103 and secreted CXCL13. CXCL13 expression was strongly correlated with neo-antigen burden and cytolytic gene signatures in bulk tumors. In line with this, TLS were abundant in neo-antigen-high, CD103+ T cell-enriched tumors. TGF-{beta} thus appears to play a role in coordinating immune responses against human tumors through CD8-dependent CXCL13-associated formation of TLS.

immunology↗

bioRxiv2018

Frequencies of circulating Th1-biased T follicular helper cells in acute HIV-1 infection correlate with the development of HIV-specific antibody responses and lower set point viral load

Despite decades of focused research, the field has yet to develop a prophylactic vaccine. In the RV144 vaccine trial, non-neutralizing antibody responses were identified as a correlate for prevention of HIV acquisition. However, factors that predict the development of such antibodies are not fully elucidated. We sought to define the contribution of circulating T follicular helper (cTfh) cell subsets to the development of non-neutralizing antibodies in HIV-1 clade C infection. Study participants were recruited from an acute HIV-1 clade C infection cohort. Plasma anti-gp41, -gp120, -p24 and -p17 antibodies were screened using a customized multivariate Luminex assay. Phenotypic and functional characterization of cTfh were performed using HLA class II tetramers and intracellular cytokine staining. In this study, we found that acute HIV-1 clade C infection skewed differentiation of functional cTfh subsets towards increased Tfh1 (p=0.02) and Tfh2 (p<0.0001) subsets, with a concomitant decrease in overall Tfh1-17 (that shares both Tfh1 and Tfh17 properties) (p=0.01) and Tfh17 subsets (p<0.0001) compared to HIV negative subjects. Interestingly, the frequencies of Tfh1 during acute infection (5.0-8.0 weeks post-infection) correlated negatively with set point viral load (p=0.03, r=-60) and were predictive of p24-specific plasma IgG titers at one year of infection (p=0.003, r=0.85). Taken together, our results suggest that circulating the Tfh1 subset plays an important role in the development of anti-HIV antibody responses and contributes to HIV suppression during acute HIV-1 infection. These results have implications for vaccine studies aimed at inducing long lasting anti-HIV antibody responses.\n\nImportanceThe HIV epidemic in southern Africa accounts for almost half of the global HIV burden with HIV-1 clade C being the predominant strain. It is therefore important to define immune correlates of clade C HIV control that might have implications for vaccine design in this region. T follicular helper (Tfh) cells are critical for the development of HIV-specific antibody responses and could play a role in viral control. Here we showed that the early induction of circulating Tfh1 cells during acute infection correlated positively with the magnitude of p24-specific IgG and was associated with lower set point viral load. This study highlights a key Tfh cell subset that could limit HIV replication by enhancing antibody generation. This study underscores the importance of circulating Tfh cells in promoting non-neutralizing antibodies during HIV-1 infection.

immunology↗

bioRxiv2018

Regulatory B cells contribute to the alleviation of colitis induced by Dextran Sulphate Sodium after H.pylori infection

BackgroundEpidemiological studies showed that there was an inverse relationship between Helicobacter pylori infection and the incidence of inflammatory bowel disease. Our previous research indicated that the regulatory immune responses induced by H.pylori infection were not limited to gastric mucosa, and the balance of intestinal mucosal immunity was influenced. In this study, we investigated the role of the regulatory B cells in the effects of the H.pylori infection on acute and chronic colitis induced by dextran sulphate sodium.\n\nMethodsMice were infected with H.pylori and then colitis was induced by 3% DSS, the CD19+IL-10+Breg cells and CD4+CD25+Foxp3+Treg cells in blood spleen mesenteric lymph nodes (MLN) Peyers Patches (PP) and gastrointestinal mucosa were measured and examined. The anti- and pro-inflammatory cytokines were also detected at mRNA level.\n\nResultsIn the acute and chronic colitis groups, DAI and colonic histological scores reduced significantly and colon length shorted less; the anti-inflammatory cytokines mRNA expression upregulated while the pro-inflammatory cytokines mRNA expression downregulated in colonic mucosa; and the percentages of CD19+IL-10+Breg cells in blood spleen MLN PP were higher in the H.pylori/DSS co-treated group compared with the DSS treated group.\n\nConclusionH.pylori infection can alleviate the acute and chronic colitis induced by DSS. CD19+IL-10+Breg cells may play a critical role in the alleviation of acute and chronic colitis following H.pylori infection.

immunology↗

bioRxiv2018

Preliminary study on the application of PspA as carrier

The aim of the study is to research the feasibility of pneumococcal surface protein A (PspA) using as carrier protein. Three recombinant pneumococcal surface proteins A (come from family1 and family 2) were expressed by prokaryotic expression system and were conjugated to group A meningococcal polysaccharide (GAMP) to make three polysaccharide-protein conjugates. The conjugates, un-conjugated proteins, GAMP and GAMP-TT vaccine bulk (used as positive control) were immunized to mice and their immune effects were evaluated by the method of ELISA, FCM and SBA. The results showed that the polysaccharide-protein conjugates can produce higher levels of anti-GAMP IgG titers (P < 0.05), higher ratios of Th1/Th2 (P < 0.05) and higher levels of serum bactericidal activity (P < 0.05) compared with the un-conjugated GAMP. The conjugation of PspAs to GAMP also enhanced the anti-PspA responses compared with un-conjugated PspAs except PspA3. In conclusion, all the results indicated that three PspAs were suitable carrier protein as demonstrated by the characteristics of a T-cell dependent response to the GAMP, and would protect against group A of epidemic cerebrospinal meningitis and also have the potential to provide broad protection from Streptococcus pneumonia.

immunology↗

bioRxiv2018

CD161 mediates prenatal immune suppression of IFNγ-producing PLZF+ T cells

While the fetal immune system defaults to a program of tolerance, there is concurrent need for protective immunity to meet the antigenic challenges after birth. Activation of fetal T cells is associated with fetal inflammation and the termination of pregnancy, yet which fetal T cells contribute to this process is poorly understood. Here we show a transcriptionally distinct population of pro-inflammatory T cells that predominates in the human fetal intestine. Activation of PLZF+ T cells results in rapid production of Th1 cytokines and is inhibited upon ligation of surface CD161. This mechanism of fetal immune suppression may inform how immune dysregulation could result in fetal and neonatal inflammatory pathologies such as preterm birth. Our data support that human development of protective adaptive immunity originates in utero within the specialized microenvironment of the fetal intestine.

immunology↗

bioRxiv2018

Possibilities for improvement of humoral innate immunity in turkeys and hens in conditions of thermal stress by the immunomodulator Immunobeta

The experiments for evaluation of the effect of Immunobeta were performed with turkeys and organically reared layer hens. Turkeys received Immunobeta at a dose of 4 g/kg feed during the beginning of lay (cold period), thermoneutral period and hot summer period. In hens, two dietary levels of Immunobeta were tested: 2 g/kg and 4 g/kg. Blood serum and egg lysozyme levels, alternative pathway of complement activation (APCA) and beta-lysins concentrations were determined. It was found out that Immunobeta had a beneficial effect on serum lysozyme, APCA activity and beta-lysins in turkeys and hens. The lysozyme concentration in egg white was statistically significantly higher in eggs produced by birds treated with both doses of the immunomodulator.

immunology↗

bioRxiv2018

Determining the Quantitative Principles of T Cell Response to Antigenic Disparity: The Case of Stem Cell Transplant Donor-Recipient Exome Wide Mismatching and HLA Bound Alloreactive Peptides

Alloreactivity compromising clinical outcomes in stem cell transplantation is observed despite HLA matching of donors and recipients. This has its origin in the variation between the exomes of the two, which provides the basis for minor histocompatibility antigens (mHA). The mHA presented on the HLA class I and II molecules and the ensuing T cell response to these antigens results in graft versus host disease. In this paper, results of a whole exome sequencing study are presented, with resulting alloreactive polymorphic peptides and their HLA class I and HLA class II (DRB1) binding affinity quantified. Large libraries of potentially alloreactive recipient peptides binding both sets of molecules were identified, with HLA-DRB1 presenting an order of magnitude greater number of peptides. These results are used to develop a quantitative framework to understand the immunobiology of transplantation. A tensor-based approach is used to derive the equations needed to determine the alloreactive donor T cell response from the mHA-HLA binding affinity and protein expression data. This approach may be used in future studies to simulate the magnitude of expected donor T cell response and risk for alloreactive complications in HLA matched or mismatched hematopoietic cell and solid organ transplantation.

immunology↗

bioRxiv2018

Elevated O-GlcNAcylation Enhances Pro-Inflammatory Th17 Function by Altering the Lipid Microenvironment

Chronic, low-grade inflammation increases the risk of atherosclerosis, cancer, and autoimmunity in diseases like obesity and diabetes. Here, we show that increased levels of the nutrient-responsive, post-translational protein modification, O-GlcNAc (O-linked {beta}-N-acetylglucosamine) are present in naive CD4+ T cells from a diet-induced obesity murine model, and elevation in O-GlcNAc leads to increased pro-inflammatory IL-17A production. Importantly, CD4+ T helper 17 (Th17) cells, which secrete IL-17A, are increased in obesity and contribute to the inflammatory milieu. We found increased binding of the Th17 master transcription factor, ROR{gamma}t, at the IL-17 locus and significant alterations in the lipid microenvironment, leading to increased ligands capable of increasing ROR{gamma}t transcriptional activity. Importantly, the rate-limiting enzyme of fatty acid biosynthesis, acetyl CoA carboxylase 1 (ACC1), is necessary for production of these ROR{gamma}t activating ligands and is O-GlcNAcylated. Thus, we have identified O-GlcNAc as a critical link between excess nutrients and pathological inflammation.

immunology↗

bioRxiv2018

TRPML1-mediated Modulation of Dense-core Granules Tunes Functional Potential in NK Cells

Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation; however the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. We found that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) show accumulation of granzyme B, localized in dense-core secretory lysosomes, converged close to the centrosome. This discrete morphological phenotype persists in self-KIR+ NK cells independently of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. The granzymeB dense, large secretory lysosomes in self-KIR+ NK cells were efficiently released upon target cell recognition, contributing to their enhanced cytotoxic capacity. Secretory lysosomes are part of the acidic lysosomal compartment, which has been shown to channel calcium and mediate intracellular signalling in several cell types. Interference of signaling from acidic Ca2+ stores in primary NK cells reduced both target-specific Ca2+-flux, degranulation and cytokine production. Furthermore, inhibition of PI(3,5)P2 synthesis or genetic silencing of the PI(3,5)P2-regulated lysosomal Ca2+-channel TRPML1 led to increased levels of granzyme B and enhanced functional potential. These results indicate an intrinsic role for lysosomal homeostasis in NK cell education.

immunology↗

bioRxiv2018

Epitope-based vaccine design yields fusion peptide-directed antibodies that neutralize diverse strains of HIV-1

A central goal of HIV-1-vaccine research is the elicitation of antibodies capable of neutralizing diverse primary isolates of HIV-1. Here we show that focusing the immune response to exposed N-terminal residues of the fusion peptide, a critical component of the viral entry machinery and the epitope of antibodies elicited by HIV-1 infection, through immunization with fusion peptide-coupled carriers and prefusion-stabilized envelope trimers, induces cross-clade neutralizing responses. In mice, these immunogens elicited monoclonal antibodies capable of neutralizing up to 31% of a cross-clade panel of 208 HIV-1 strains. Crystal and cryo-electron microscopy structures of these antibodies revealed fusion peptide-conformational diversity as a molecular explanation for the cross-clade neutralization. Immunization of guinea pigs and rhesus macaques induced similarly broad fusion peptide-directed neutralizing responses suggesting translatability. The N terminus of the HIV-1-fusion peptide is thus a promising target of vaccine efforts aimed at eliciting broadly neutralizing antibodies.

immunology↗

bioRxiv2018

Cognate T and B cell interaction and association of Follicular helper T cells with B cell responses in Vibrio cholerae O1 infected Bangladeshi adults

Vibrio cholerae O1 can cause life threatening diarrheal disease if left untreated. A long lasting immune response, producing 3-5 years of protection from subsequent, symptomatic disease following natural infection, is mediated by B cell mediated humoral immunity. T cells can play critical roles in inducing such immunity. However, the mechanism of T cell dependent B cell maturation and whether a key sub-population of T cells are involved is not well established in cholera. We hypothesized that a specific population of T cells, follicular helper T (Tfh) cells, are involved in B cell maturation following cholera; we used flow cytometry, culture and colorimetric assays to address this question. We found that V. cholerae infection induces significant increase in circulating Tfh cells expressing B cell maturation associated protein CD40L early in disease. The increased Tfh cells expressing CD40L recognize cholera toxin most prominently, with lessened responses to two antigens tested, V. cholerae membrane preparation (MP) and Vibrio cholerae cytolysin (VCC). We further showed that early induction of Tfh cells and CD40L was associated with later memory B cell responses to same antigens. Lastly, we demonstrated in vitro that Tfh cells isolated after cholera can stimulate class switching of cocultured, isolated B cells from patients with cholera, leading to production of the more durable IgG antibody isotype. These studies were conducted on circulating Tfh cells; future studies will be directed at examining role of Tfh cells during cholera directly in the gut mucosa of biopsied samples, at the single cell level if feasible.

immunology↗