Definition of Naturally Processed Peptides Reveals Convergent Presentation of Autoantigenic Topoisomerase I Epitopes in Scleroderma.

ObjectiveAutoimmune responses to DNA topoisomerase I (topo I) are found in a subset of scleroderma patients who are at high risk for interstitial lung disease (ILD) and mortality. Anti-topo I antibodies (ATAs) are associated with specific HLA-DRB1 alleles, and the frequency of HLA-DR-restricted topo I-specific CD4+ T cells is associated with the presence, severity, and progression of ILD. Although this strongly implicates the presentation of topo I peptides by HLA-DR in scleroderma pathogenesis, the processing and presentation of topo I has not been studied.MethodsWe developed a natural antigen processing assay (NAPA) to identify putative CD4+ T cell epitopes of topo I presented by monocyte-derived dendritic cells (mo-DCs) from 6 ATA-positive patients with scleroderma. Mo-DCs were pulsed with topo I protein, HLA-DR-peptide complexes were isolated, and eluted peptides were analyzed by mass spectrometry. We then examined the ability of these naturally presented peptides to induce CD4+ T cell activation in 11 ATA-positive and 11 ATA-negative scleroderma patients.ResultsWe found that a common set of 10 topo I epitopes was presented by Mo-DCs from scleroderma patients with diverse HLA-DR variants. Sequence analysis revealed shared peptide-binding motifs within the HLA-DRβ chains of ATA-positive patients and a subset of topo I epitopes with distinct sets of anchor residues capable of binding to multiple different HLA-DR variants. The NAPA-derived epitopes elicited robust CD4+ T cell responses in 73% of ATA-positive patients (8 of 11), and the number of epitopes recognized correlated with ILD severity (P = 0.025).ConclusionThese findings mechanistically implicate the presentation of a convergent set of topo I epitopes in the development of scleroderma

Association of Cross-Reactive Antibodies Targeting Peptidyl-Arginine Deiminase 3 and 4 with Rheumatoid Arthritis-Associated Interstitial Lung Disease

Background: A subset of rheumatoid arthritis (RA) patients have detectable antibodies directed against the peptidyl-arginine deiminase (PAD) enzyme isoforms 3 and 4. Anti-PAD3/4 cross-reactive antibodies (anti-PAD3/4XR) have been shown to lower the calcium threshold required for PAD4 activation, an effect potentially relevant to the pathogenesis of RA-associated interstitial lung disease (ILD). Methods: RA patients underwent multi-detector computed tomography (MDCT) of the chest with interpretation by a pulmonary radiologist for ILD features. A semi-quantitative ILD Score (range 0–32) was calculated. Concurrent serum samples were assessed for antibodies against PAD by immunoprecipitation with radiolabeled PAD3 and PAD4. Results: Among the 176 RA patients studied, any ILD was observed in 58 (33%) and anti-PAD3/4XR was detected in 19 (11%). The frequency of any ILD among those with anti-PAD3/4XR was 68% vs. 29% among those with no anti-PAD (crude OR = 5.39; p = 0.002) and vs. 27% among those with anti-PAD4 that was not cross-reactive with PAD3 (crude OR = 5.74; p = 0.001). Both associations were stronger after adjustment for relevant confounders (adjusted ORs = 7.22 and 6.61, respectively; both p-values<0.01). Among ever smokers with anti-PAD3/4XR, the adjusted frequency of any ILD was 93% vs. 17% for never smokers without the antibody (adjusted OR = 61.4; p = 0.001, p-value for the interaction of smoking with anti-PAD3/4XR<0.05). Conclusions: The prevalence and extent of ILD was markedly higher among RA patients with anti-PAD3/4 cross-reactive antibodies, even after accounting for relevant confounders, particularly among ever smokers. These findings may suggest etiopathologic mechanisms of RA-ILD, and their clinical utility for predicting ILD warrants additional study

Smoking is not linked to the development of anti-peptidylarginine deiminase 4 autoantibodies in rheumatoid arthritis

Background: Defining environmental factors responsible for development of autoimmunity in rheumatoid arthritis (RA) is critical for understanding mechanisms of disease initiation and propagation. Notably, a history of cigarette smoking has been implicated in the genesis of RA and is associated with worse disease outcomes. Antibodies to peptidylarginine deiminase 4 (PAD4) are also associated with more severe RA. A subset of patients who have PAD4 autoantibodies that cross-react with PAD3 (anti-PAD3/4) are at the highest risk for interstitial lung disease, and this risk is augmented by a history of cigarette smoking. It is unclear, however, if smoking is etiologically linked to the development of anti-PAD4 antibodies. Methods: Patients were included in this study if they had physician-diagnosed RA as well as DNA, serum, and a date-matched clinical assessment (n = 274). Anti-PAD4 and anti-CCP antibodies were measured by immunoprecipitation and ELISA, respectively; shared epitope (SE) status was determined by HLA-DRβ1 genotyping. Logistic regression analysis was used to evaluate associations of smoking with PAD4 antibodies, with adjustment for relevant demographic and clinical features. Stratified analyses by disease duration and shared epitope status were also performed. Results: Anti-PAD4 antibodies were present in 25% of RA patients, with 50% of these individuals having anti-PAD3/4 cross-reactive antibodies. Anti-PAD4 antibodies were significantly associated with a longer disease duration, SE alleles, and anti-CCP antibodies. Importantly, there were no significant differences in smoking history between anti-PAD4 positive and negative groups in univariate analyses, stratified analyses, or multivariable models. However, an inverse relationship between smoking and anti-PAD4 antibodies was suggested by a lower prevalence of current smokers among patients with anti-PAD3/4 antibodies compared to antibody negative individuals (p = 0.04). Further, the lowest levels of anti-PAD4 antibodies were observed in current smokers (p = 0.14), and a significant association of SE and anti-PAD4 antibodies was only present among never smokers (p = 0.01). Conclusions: Smoking history was not associated with anti-PAD4 antibodies in patients with RA. The finding that anti-PAD4 antibodies were not associated with smoking suggests that other environmental factors may contribute to the development of autoimmunity to PAD4 in these patients

Autoantibodies to Peptidylarginine Deiminase 2 Are Associated With Less Severe Disease in Rheumatoid Arthritis

Objective: Peptidylarginine deiminases (PAD) 2 and 4 are key enzymes in rheumatoid arthritis (RA) pathogenesis due to their ability to generate the protein targets of anti-citrullinated protein antibodies (ACPA). Anti-PAD4 antibodies that cross-react with PAD3 (anti-PAD3/4) have been identified and are associated with severe joint and lung disease. Here, we examined whether anti-PAD2 antibodies were present in patients with RA and defined their clinical significance.Patients and Methods: A PAD2 ELISA was established to screen for anti-PAD2 IgG in sera from RA patients from a prospective observational cohort study (n = 184) and healthy controls (n = 100). RA patient characteristics were compared according to anti-PAD2 antibody status. Multivariable models were constructed to explore the independent associations of anti-PAD2 antibodies with clinical variables.Results: Anti-PAD2 antibodies were found in 18.5% of RA patients and 3% of healthy controls (p &lt; 0.001). Among RA patients, anti-PAD2 antibodies were not associated with traditional genetic or serologic RA risk factors, including HLA-DRβ1 shared epitope alleles, ACPA, rheumatoid factor (RF), or anti-PAD3/4 antibodies. In addition, antibodies to PAD2 were associated with fewer swollen joints, a lower prevalence of interstitial lung disease, and less progression of joint damage. In subset analyses in which patients were stratified by the baseline presence of ACPA/RF or anti-PAD3/4 antibodies, anti-PAD2 antibodies provided additional value in identifying patients with the least progressive joint disease.Conclusions: Anti-PAD2 antibodies represent a novel serologic marker in RA that identifies a genetically and clinically unique subset of patients with less severe joint and lung disease

Microneedle Array Design Determines the Induction of Protective Memory CD8+ T Cell Responses Induced by a Recombinant Live Malaria Vaccine in Mice

BACKGROUND: Vaccine delivery into the skin has received renewed interest due to ease of access to the immune system and microvasculature, however the stratum corneum (SC), must be breached for successful vaccination. This has been achieved by removing the SC by abrasion or scarification or by delivering the vaccine intradermally (ID) with traditional needle-and-syringes or with long microneedle devices. Microneedle patch-based transdermal vaccine studies have predominantly focused on antibody induction by inactivated or subunit vaccines. Here, our principal aim is to determine if the design of a microneedle patch affects the CD8(+) T cell responses to a malaria antigen induced by a live vaccine. METHODOLOGY AND FINDINGS: Recombinant modified vaccinia virus Ankara (MVA) expressing a malaria antigen was percutaneously administered to mice using a range of silicon microneedle patches, termed ImmuPatch, that differed in microneedle height, density, patch area and total pore volume. We demonstrate that microneedle arrays that have small total pore volumes induce a significantly greater proportion of central memory T cells that vigorously expand to secondary immunization. Microneedle-mediated vaccine priming induced significantly greater T cell immunity post-boost and equivalent protection against malaria challenge compared to ID vaccination. Notably, unlike ID administration, ImmuPatch-mediated vaccination did not induce inflammatory responses at the site of immunization or in draining lymph nodes. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that the design of microneedle patches significantly influences the magnitude and memory of vaccine-induced CD8(+) T cell responses and can be optimised for the induction of desired immune responses. Furthermore, ImmuPatch-mediated delivery may be of benefit to reducing unwanted vaccine reactogenicity. In addition to the advantages of low cost and lack of pain, the development of optimised microneedle array designs for the induction of T cell responses by live vaccines aids the development of solutions to current obstacles of immunization programmes

Soluble Markers of Antibody Secreting Cell Function as Predictors of Infection Risk in Rheumatoid Arthritis

Background. Rheumatoid arthritis (RA) is a systemic autoimmune disease associated with immune dysregulation and increased risk of infections. The presence of autoantibodies and immunoglobulin abnormalities indicates B-cell and antibody-secreting cell (ASC) dysfunction. We hypothesize that soluble factors associated with B-cell and ASC activity are decreased in RA patients and that this is linked to higher susceptibility to infections. Methods. Using the Johns Hopkins Arthritis Cohort and Biorepository, we contrasted serum protein levels of soluble factors involved in B-cell activation (CD40, CD40L) and B-cell/ASC homing (CXCL10, CXCL11, and CXCL13) or survival (BAFF, APRIL, TACI, and BCMA) in 10 healthy subjects and 23 adult RA patients (aged 24-65 years). We subdivided RA patients into those with (n=17) and those without infections (n=6) within a 2-year period. In order to reduce the effect of RA treatment, we only included patients receiving methotrexate monotherapy or no RA treatments at baseline. Soluble serum protein levels of B-cell/ASC factors were quantified by multiplex immunoassays. Results. We identified that (1) serum levels of soluble BCMA, APRIL, CD40, and CD40L were significantly decreased in RA patients relative to healthy individuals; (2) serum soluble BCMA, predominantly released by ASC, correlated with serum concentrations of class-switched immunoglobulins, IgG and IgA; and (3) RA patients with a history of infections had significantly lower soluble BCMA levels compared with healthy donors and with RA patients without infections. Conclusions. Our study using soluble factors linked to B-cell/ASC activation and survival suggests that there is a paucity of ASC in a subset of RA patients and that this may be linked to altered antibody production and increased risk of infections. Further delineating the link between ASC and infection susceptibility in RA may optimize disease management and provide novel insights into disease pathogenesis that are susceptible to intervention