IgG4-related disease (IgG4-RD) is a fibroinflammatory condition involving loss of B-cell tolerance and production of autoantibodies. However, the relevant targets and role of these aberrant humoral ...immune responses are not defined.
Our aim was to identify novel autoantibodies and autoantigen targets that promote pathogenic responses in IgG4-RD.
We sequenced plasmablast antibody repertoires in patients with IgG4-RD. Representative mAbs were expressed and their specificities characterized by using cytokine microarrays. The role of anti–IL-1 receptor antagonist (IL-1RA) autoantibodies was investigated by using in vitro assays.
We identified strong reactivity against human IL-1RA by using a clonally expanded plasmablast-derived mAb from a patient with IgG4-RD. Plasma from patients with IgG4-RD exhibited elevated levels of reactivity against IL-1RA compared with plasma from the controls and neutralized IL-1RA activity, resulting in inflammatory and fibrotic mediator production in vitro. IL-1RA was detected in lesional tissues from patients with IgG4-RD. Patients with anti–IL-1RA autoantibodies of the IgG4 subclass had greater numbers of organs affected than did those without anti–IL-1RA autoantibodies. Peptide analyses identified IL-1RA epitopes targeted by anti–IL-1RA antibodies at sites near the IL-1RA/IL-1R interface. Serum from patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) also had elevated levels of anti–IL-1RA autoantibodies compared with those of the controls.
A subset of patients with IgG4-RD have anti–IL-1RA autoantibodies, which promote proinflammatory and profibrotic meditator production via IL-1RA neutralization. These findings support a novel immunologic mechanism underlying the pathogenesis of IgG4-RD. Anti–IL-1RA autoantibodies are also present in a subset of patients with SLE and RA, suggesting a potential common pathway in multiple autoimmune diseases.
Innate natural killer (NK) cells are diverse at the single-cell level because of variegated expressions of activating and inhibitory receptors, yet the developmental roots and functional consequences ...of this diversity remain unknown. Because NK cells are critical for antiviral and antitumor responses, a better understanding of their diversity could lead to an improved ability to harness them therapeutically. We found that NK diversity is lower at birth than in adults. During an antiviral response to either HIV-1 or West Nile virus, NK diversity increases, resulting in terminal differentiation and cytokine production at the cost of cell division and degranulation. In African women matched for HIV-1 exposure risk, high NK diversity is associated with increased risk of HIV-1 acquisition. Existing diversity may therefore decrease the flexibility of the antiviral response. Collectively, the data reveal that human NK diversity is a previously undefined metric of immune history and function that may be clinically useful in forecasting the outcomes of infection and malignancy.
Protein chips are widely used for high-throughput proteomic analysis, but to date, the low sensitivity and narrow dynamic range have limited their capabilities in diagnostics and proteomics. Here we ...present protein microarrays on a novel nanostructured, plasmonic gold film with near-infrared fluorescence enhancement of up to 100-fold, extending the dynamic range of protein detection by three orders of magnitude towards the fM regime. We employ plasmonic protein microarrays for the early detection of a cancer biomarker, carcinoembryonic antigen, in the sera of mice bearing a xenograft tumour model. Further, we demonstrate a multiplexed autoantigen array for human autoantibodies implicated in a range of autoimmune diseases with superior signal-to-noise ratios and broader dynamic range compared with commercial nitrocellulose and glass substrates. The high sensitivity, broad dynamic range and easy adaptability of plasmonic protein chips presents new opportunities in proteomic research and diagnostics applications.
Autoantibodies against cytokines, chemokines, and growth factors inhibit normal immunity and are implicated in inflammatory autoimmune disease and diseases of immune deficiency. In an effort to ...evaluate serum from autoimmune and immunodeficient patients for Abs against cytokines, chemokines, and growth factors in a high-throughput and unbiased manner, we constructed a multiplex protein microarray for detection of serum factor-binding Abs and used the microarray to detect autoantibody targets in SLE. We designed a nitrocellulose-surface microarray containing human cytokines, chemokines, and other circulating proteins and demonstrated that the array permitted specific detection of serum factor-binding probes. We used the arrays to detect previously described autoantibodies against cytokines in samples from individuals with autoimmune polyendocrine syndrome type 1 and chronic mycobacterial infection. Serum profiling from individuals with SLE revealed that among several targets, elevated IgG autoantibody reactivity to B cell-activating factor (BAFF) was associated with SLE compared with control samples. BAFF reactivity correlated with the severity of disease-associated features, including IFN-α-driven SLE pathology. Our results showed that serum factor protein microarrays facilitate detection of autoantibody reactivity to serum factors in human samples and that BAFF-reactive autoantibodies may be associated with an elevated inflammatory disease state within the spectrum of SLE.
Existing methods to measure influenza vaccine immunogenicity prohibit detailed analysis of epitope determinants recognized by immunoglobulins. The development of highly multiplex proteomics platforms ...capable of capturing a high level of antibody binding information will enable researchers and clinicians to generate rapid and meaningful readouts of influenza-specific antibody reactivity.
We developed influenza hemagglutinin (HA) whole-protein and peptide microarrays and validated that the arrays allow detection of specific antibody reactivity across a broad dynamic range using commercially available antibodies targeted to linear and conformational HA epitopes. We derived serum from blood draws taken from 76 young and elderly subjects immediately before and 28±7 days post-vaccination with the 2008/2009 trivalent influenza vaccine and determined the antibody reactivity of these sera to influenza array antigens.
Using linear regression and correcting for multiple hypothesis testing by the Benjamini and Hochberg method of permutations over 1000 resamplings, we identified antibody reactivity to influenza whole-protein and peptide array features that correlated significantly with age, H1N1, and B-strain post-vaccine titer as assessed through a standard microneutralization assay (p<0.05, q <0.2). Notably, we identified several peptide epitopes that were inversely correlated with regard to age and seasonal H1N1 and B-strain neutralization titer (p<0.05, q <0.2), implicating reactivity to these epitopes in age-related defects in response to H1N1 influenza. We also employed multivariate linear regression with cross-validation to build models based on age and pre-vaccine peptide reactivity that predicted vaccine-induced neutralization of seasonal H1N1 and H3N2 influenza strains with a high level of accuracy (84.7% and 74.0%, respectively).
Our methods provide powerful tools for rapid and accurate measurement of broad antibody-based immune responses to influenza, and may be useful in measuring response to other vaccines and infectious agents.
Intracellular delivery of functional macromolecules, such as DNA and RNA, across the cell membrane and into the cytosol, is a critical process in both biology and medicine. Herein, we develop and use ...microfluidic chips containing post arrays to induce microfluidic vortex shedding, or μVS, for cell membrane poration that permits delivery of mRNA into primary human T lymphocytes. We demonstrate transfection with μVS by delivery of a 996-nucleotide mRNA construct encoding enhanced green fluorescent protein (EGFP) and assessed transfection efficiencies by quantifying levels of EGFP protein expression. We achieved high transfection efficiency (63.6 ± 3.44% EGFP + viable cells) with high cell viability (77.3 ± 0.58%) and recovery (88.7 ± 3.21%) in CD3 + T cells 19 hrs after μVS processing. Importantly, we show that processing cells via μVS does not negatively affect cell growth rates or alter cell states. We also demonstrate processing speeds of greater than 2.0 × 10
cells s
at volumes ranging from 0.1 to 1.5 milliliters. Altogether, these results highlight the use of μVS as a rapid and gentle delivery method with promising potential to engineer primary human cells for research and clinical applications.
High-throughput screening for interactions of peptides with a variety of antibody targets could greatly facilitate proteomic analysis for epitope mapping, enzyme profiling, drug discovery and ...biomarker identification. Peptide microarrays are suited for such undertaking because of their high-throughput capability. However, existing peptide microarrays lack the sensitivity needed for detecting low abundance proteins or low affinity peptide-protein interactions. This work presents a new peptide microarray platform constructed on nanostructured plasmonic gold substrates capable of metal enhanced NIR fluorescence enhancement (NIR-FE) by hundreds of folds for screening peptide-antibody interactions with ultrahigh sensitivity. Further, an integrated histone peptide and whole antigen array is developed on the same plasmonic gold chip for profiling human antibodies in the sera of systemic lupus erythematosus (SLE) patients, revealing that collectively a panel of biomarkers against unmodified and post-translationally modified histone peptides and several whole antigens allow more accurate differentiation of SLE patients from healthy individuals than profiling biomarkers against peptides or whole antigens alone.
Abstract
Microfluidic vortex shedding (
µVS
) can rapidly deliver mRNA to T cells with high yield and minimal perturbation of the cell state. The mechanistic underpinning of
µVS
intracellular ...delivery remains undefined and
µVS
-Cas9 genome editing requires further studies. Herein, we evaluated a series of
µVS
devices containing splitter plates to attenuate vortex shedding and understand the contribution of computed force and frequency on efficiency and viability. We then selected a
µVS
design to knockout the expression of the endogenous T cell receptor in primary human T cells via delivery of Cas9 ribonucleoprotein (RNP) with and without brief exposure to an electric field (
eµVS
).
µVS
alone resulted in an equivalent yield of genome-edited T cells relative to electroporation with improved cell quality. A 1.8-fold increase in editing efficiency was demonstrated with
eµVS
with negligible impact on cell viability. Herein, we demonstrate efficient processing of 5 × 10
6
cells suspend in 100 µl of cGMP OptiMEM in under 5 s, with the capacity of a single device to process between 10
6
to 10
8
in 1 to 30 s. Cumulatively, these results demonstrate the rapid and robust utility of
µVS
and
eµVS
for genome editing human primary T cells with Cas9 RNPs.
Protein microarrays based on fluorescence detection have been widely utilized for high-throughput functional proteomic analysis. However, a drawback of such assays has been low sensitivity and narrow ...dynamic range, limiting their capabilities, especially for detecting low abundance biological molecules such as cytokines in human samples. Here, we present fluorescence-enhancing microarrays on plasmonic gold films for multiplexed cytokine detection with up to three orders of magnitude higher sensitivity than on conventional nitrocellulose and glass substrates. Cytokine detection on the gold plasmonic substrate is about one to two orders of magnitude more sensitive than enzyme-linked immunosorbent assay （ELISA） and can be multiplexed. A panel of six cytokines （Vascular endothelial growth factor （VEGF）, Interleukin 1β （IL-1β）, Interleukin 4 （IL-4）, Interleukin 6 （IL-6）, Interferon γ （IFN-γ）, and Tumor necrosis factor （TNF）） were detected in the culture media of cancer cells. This work establishes a new method of high throughput multiplexed cytokine detection with higher sensitivity and dynamic range than ELISA.
Imatinib has been investigated for the treatment of systemic sclerosis (SSc) because of its ability to inhibit the platelet-derived growth factor receptor and transforming growth factor-β signaling ...pathways, which have been implicated in SSc pathogenesis. In a 12-month open-label clinical trial assessing the safety and efficacy of imatinib in the treatment of diffuse cutaneous SSc (dcSSc), significant improvements in skin thickening were observed. Here, we report our analysis of sera collected during the clinical trial.
We measured the levels of 46 cytokines, chemokines, and growth factors in the sera of individuals with dcSSc using Luminex and ELISA. Autoantigen microarrays were used to measure immunoglobulin G reactivity to 28 autoantigens. Elastic net regularization was used to identify a signature that was predictive of clinical improvement (reduction in the modified Rodnan skin score ≥ 5) during treatment with imatinib. The signature was also tested using sera from a clinical trial of nilotinib, a tyrosine kinase inhibitor that is structurally related to imatinib, in dcSSc.
The elastic net algorithm identified a signature, based on levels of CD40 ligand, chemokine (C-X-C motif) ligand 4 (CXCL4), and anti-PM/Scl-100, that was significantly higher in individuals who experienced clinical improvement than in those who did not (p = 0.0011). The signature was validated using samples from a clinical trial of nilotinib.
Identification of patients with SSc with the greatest probability of benefit from treatment with imatinib has the potential to guide individualized treatment. Validation of the signature will require testing in randomized, placebo-controlled studies. Clinicaltrials.gov NCT00555581 and NCT01166139.
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