10 results found
Pinder CL, McKay PF, Shattock RJ, 2021, Use of chlamydial elementary bodies as probes to isolate pathogen-specific human monoclonal antibodies., Methods in Molecular Biology, Vol: 2183, Pages: 19-28, ISSN: 1064-3745
Chlamydia trachomatis is one of the most prevalent sexually transmitted infectious agents in the world and the leading cause of infectious blindness. The role of antibodies in the prevention and clearance of infection is still not fully understood, but the analysis of the immunoglobulin response to novel vaccine candidates is an important part of many of these studies. In this chapter, we describe a novel method to identify and isolate Chlamydia-specific memory B cells by fluorescence-activated cell sorting (FACS) using fluorescently labeled whole bacteria from cryopreserved human PBMC samples. This method allows for live single cells to be sorted for cell culture, in vitro assays, single-cell RNA sequencing, and cloning of paired heavy and light chains for recombinant monoclonal antibody production.
Siris S, Gladstone CA, Guo Y, et al., 2021, Isolating Pathogen-Specific Human Monoclonal Antibodies (hmAbs) Using Bacterial Whole Cells as Molecular Probes., Methods Mol Biol, Vol: 2183, Pages: 9-18
The immunoglobulin capture assay (ICA) enables the enrichment for pathogen-specific plasmablasts from individuals with a confirmed adaptive immune response to vaccination or disseminated infection. Only single recombinant antigens have been used previously as probes in this ICA and it was unclear whether the method was applicable to complex probes such as whole bacterial cells. Here, we describe the enrichment of plasmablasts specific for polysaccharide and protein antigens of both Streptococcus pneumoniae and Neisseria meningitidis using whole formalin-fixed bacterial cells as probes. The modified ICA protocol described here allowed for a pathogen-specific hmAb cloning efficiency of >80%.
Oliveira JJ, Karrar S, Rainbow DB, et al., 2018, The plasma biomarker soluble SIGLEC-1 is associated with the type I interferon transcriptional signature, ethnic background and renal disease in systemic lupus erythematosus, Arthritis Research and Therapy, Vol: 20, ISSN: 1478-6354
BackgroundThe 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 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).MethodsWe developed an immunoassay to measure sSIGLEC-1 in small volumes of plasma/serum from systemic lupus erythematosus (SLE) patients (n = 75) and healthy donors (n = 504). Samples from systemic sclerosis patients (n = 99) were studied as an autoimmune control. We investigated the correlation between sSIGLEC-1 and both monocyte surface SIGLEC-1 and type I interferon-regulated gene (IRG) expression. Associations of sSIGLEC-1 with clinical features were evaluated in an independent cohort of SLE patients (n = 656).ResultsPlasma concentrations of sSIGLEC-1 strongly correlated with expression of SIGLEC-1 on the surface of blood monocytes and with IRG expression in SLE patients. We found 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 SLE Disease Activity Index clinical score.ConclusionsOur 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 clinical trials.
Muir L, McKay PF, Petrova VN, et al., 2018, Optimisation ofex vivomemory B cell expansion/differentiation for interrogation of rare peripheral memory B cell subset responses [version 2; referees: 2 approved], Wellcome Open Research, Vol: 2, Pages: 97-97, ISSN: 2398-502X
Background: Human memory B cells play a vital role in the long-term protection of the host from pathogenic re-challenge. In recent years the importance of a number of different memory B cell subsets that can be formed in response to vaccination or infection has started to become clear. To study memory B cell responses, cells can be culturedex vivo,allowing for an increase in cell number and activation of these quiescent cells, providing sufficient quantities of each memory subset to enable full investigation of functionality. However, despite numerous papers being published demonstrating bulk memory B cell culture, we could find no literature on optimised conditions for the study of memory B cell subsets, such as IgM+memory B cells. Methods:Following a literature review, we carried out a large screen of memory B cell expansion conditions to identify the combination that induced the highest levels of memory B cell expansion. We subsequently used a novel Design of Experiments approach to finely tune the optimal memory B cell expansion and differentiation conditions for human memory B cell subsets. Finally, we characterised the resultant memory B cell subpopulations by IgH sequencing and flow cytometry. Results:The application of specific optimised conditions induce multiple rounds of memory B cell proliferation equally across Ig isotypes, differentiation of memory B cells to antibody secreting cells, and importantly do not alter the Ig genotype of the stimulated cells. Conclusions:Overall, our data identify a memory B cell culture system that offers a robust platform for investigating the functionality of rare memory B cell subsets to infection and/or vaccination.
Pinder CL, kratochvil S, Cizmeci D, et al., 2017, Isolation and Characterization of Antigen-Speciﬁc Plasmablasts Using a Novel Flow Cytometry–Based Ig Capture Assay, Journal of Immunology, ISSN: 1550-6606
We report the development of a novel flow cytometry–based Ig capture assay (ICA) for the identification and sorting of individual Ab-secreting cells based on their Ag reactivity. The ICA represents a fast and versatile tool for single-cell sorting of peripheral plasmablasts, streamlining subsequent Ab analysis, and cloning. We demonstrate the utility of the assay by isolating Ag-reactive plasmablasts from cryopreserved PBMC obtained from volunteers vaccinated with a recombinant HIV envelope protein. To show the specificity of the ICA, we produced Ag-specific Abs from these cells and subsequently verified their Ag reactivity via ELISA. Furthermore, we used the ICA to track Ag-specific plasmablast responses in HIV-vaccine recipients over a period of 42 d and performed a head-to-head comparison with a conventional B cell ELISpot. Results were highly comparable, highlighting that this assay is a viable alternative for monitoring Ag-specific plasmablast responses at early time points after infection or vaccination. The ICA provides important added benefits in that phenotypic information can be obtained from the identified Ag-specific cells that can then be captured for downstream applications such as B cell sequencing and/or Ab cloning. We envisage the ICA as being a useful tool in Ab repertoire analysis for future clinical trials.
Muir L, McKay P, Petrova V, et al., 2017, Optimisation of ex vivo memory B cell expansion/differentiation for interrogation of rare peripheral memory B cell subset responses [version 2; peer review: 2 approved], Wellcome Open Research, Vol: 2, ISSN: 2398-502X
Background: Human memory B cells play a vital role in the long-term protection of the host from pathogenic re-challenge. In recent years the importance of a number of different memory B cell subsets that can be formed in response to vaccination or infection has started to become clear. To study memory B cell responses, cells can be cultured ex vivo, allowing for an increase in cell number and activation of these quiescent cells, providing sufficient quantities of each memory subset to enable full investigation of functionality. However, despite numerous papers being published demonstrating bulk memory B cell culture, we could find no literature on optimised conditions for the study of memory B cell subsets, such as IgM+ memory B cells.Methods: Following a literature review, we carried out a large screen of memory B cell expansion conditions to identify the combination that induced the highest levels of memory B cell expansion. We subsequently used a novel Design of Experiments approach to finely tune the optimal memory B cell expansion and differentiation conditions for human memory B cell subsets. Finally, we characterised the resultant memory B cell subpopulations by IgH sequencing and flow cytometry.Results: The application of specific optimised conditions induce multiple rounds of memory B cell proliferation equally across Ig isotypes, differentiation of memory B cells to antibody secreting cells, and importantly do not alter the Ig genotype of the stimulated cells. Conclusions: Overall, our data identify a memory B cell culture system that offers a robust platform for investigating the functionality of rare memory B cell subsets to infection and/or vaccination.
Fischetti L, Zhong Z, Pinder CL, et al., 2017, The synergistic effects of combining TLR ligand based adjuvants on the cytokine response are dependent upon p38/JNK signalling., Cytokine, Vol: 99, Pages: 287-296, ISSN: 1043-4666
Toll like receptor (TLR) ligands are important adjuvant candidates, causing antigen presenting cells to release inflammatory mediators, leading to the recruitment and activation of other leukocytes. The aim of this study was to define the response of human blood derived dendritic cells and macrophages to three TLR ligands acting singly or in combination, Poly I:C (TLR3), GLA (TLR4) and R848 (TLR7/8). Combinations of TLR agonists have been shown to have a synergistic effect on individual cytokines, here we look at the global inflammatory response measuring both cytokines and chemokines. Using a custom Luminex assay we saw dose responses in several mediators including CCL3 (MIP1α), IL-1α, IL-1β, IL-12, CXCL10 (IP-10) and IL-6, all of which were significantly increased by the combination of R848 and GLA, even when low dose GLA was added. The synergistic effect was inhibited by specific MAP kinase inhibitors blocking the kinases p38 and JNK but not MEK1. Combining TLR adjuvants also had a synergistic effect on cytokine responses in human mucosal tissue explants. From this we conclude that the combination of R848 and GLA potentiates the inflammatory profile of antigen presenting cells. Since the pattern of inflammatory mediators released can alter the quality and quantity of the adaptive immune response to vaccination, this study informs vaccine adjuvant design.
Ellinghaus U, Cortini A, Pinder CL, et al., 2017, Dysregulated CD46 shedding interferes with Th1-contraction in systemic lupus erythematosus, European Journal of Immunology, Vol: 47, Pages: 1200-1210, ISSN: 0014-2980
IFN‐γ‐producing T helper 1 (Th1) cell responses mediate protection against infections but uncontrolled Th1 activity also contributes to a broad range of autoimmune diseases. Autocrine complement activation has recently emerged as key in the induction and contraction of human Th1 immunity: activation of the complement regulator CD46 and the C3aR expressed by CD4+ T cells via autocrine generated ligands C3b and C3a, respectively, are critical to IFN‐γ production. Further, CD46‐mediated signals also induce co‐expression of immunosuppressive IL‐10 in Th1 cells and transition into a (self)‐regulating and contracting phase. In consequence, C3 or CD46‐deficient patients suffer from recurrent infections while dysregulation of CD46 signaling contributes to Th1 hyperactivity in rheumatoid arthritis and multiple sclerosis. Here, we report a defect in CD46‐regulated Th1 contraction in patients with systemic lupus erythematosus (SLE). We observed that MMP‐9‐mediated increased shedding of soluble CD46 by Th1 cells was associated with this defect and that inhibition of MMP‐9 activity normalized release of soluble CD46 and restored Th1 contraction in patients’ T cells. These data may deliver the first mechanistic explanation for the increased serum CD46 levels observed in SLE patients and indicate that targeting CD46‐cleaving proteases could be a novel avenue to modulate Th1 responses.
Kratochvil S, McKay PF, Kopycinski JT, et al., 2017, A phase 1 human immunodeficiency virus vaccine Trial for cross-profiling the kinetics of serum and mucosal antibody responses to CN54gp140 modulated by two homologous prime-boost vaccine regimens, Frontiers in Immunology, Vol: 8, ISSN: 1664-3224
A key aspect to finding an efficacious human immunodeficiency virus (HIV) vaccine is the optimization of vaccine schedules that can mediate the efficient maturation of protective immune responses. In the present study, we investigated the effect of alternate booster regimens on the immune responses to a candidate HIV-1 clade C CN54gp140 envelope protein, which was coadministered with the TLR4-agonist glucopyranosyl lipid A-aqueous formulation. Twelve study participants received a common three-dose intramuscular priming series followed by a final booster at either 6 or 12 months. The two homologous prime-boost regimens were well tolerated and induced CN54gp140-specific responses that were observed in both the systemic and mucosal compartments. Levels of vaccine-induced IgG-subclass antibodies correlated significantly with FcγR engagement, and both vaccine regimens were associated with strikingly similar patterns in antibody titer and FcγR-binding profiles. In both groups, identical changes in the antigen (Ag)-specific IgG-subclass fingerprint, leading to a decrease in IgG1 and an increase in IgG4 levels, were modulated by booster injections. Here, the dissection of immune profiles further supports the notion that prime-boost strategies are essential for the induction of diverse Ag-specific HIV-1 responses. The results reported here clearly demonstrate that identical responses were effectively and safely induced by both vaccine regimens, indicating that an accelerated 6-month regimen could be employed for the rapid induction of immune responses against CN54gp140 with no apparent impact on the overall quality of the induced immune response. (This study has been registered at http://ClinicalTrials.gov under registration no. NCT01966900.)
Bentham J, Morris DL, Cunninghame Graham DS, et al., 2015, Genetic association analyses implicate aberrant regulation of innate and adaptive immunity genes in the pathogenesis of systemic lupus erythematosus, Nature Genetics, Vol: 47, Pages: 1457-1464, ISSN: 1546-1718
Systemic lupus erythematosus (SLE) is a genetically complex autoimmune disease characterized by loss of immune tolerance to nuclear and cell surface antigens. Previous genome-wide association studies (GWAS) had modest sample sizes, reducing their scope and reliability. Our study comprised 7,219 cases and 15,991 controls of European ancestry, constituting a new GWAS, a meta-analysis with a published GWAS and a replication study. We have mapped 43 susceptibility loci, including ten new associations. Assisted by dense genome coverage, imputation provided evidence for missense variants underpinning associations in eight genes. Other likely causal genes were established by examining associated alleles for cis-acting eQTL effects in a range of ex vivo immune cells. We found an over-representation (n = 16) of transcription factors among SLE susceptibility genes. This finding supports the view that aberrantly regulated gene expression networks in multiple cell types in both the innate and adaptive immune response contribute to the risk of developing SLE.
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