Ancestry-based differences in the immune phenotype are associated with lupus activity.

Systemic lupus erythematosus (SLE) affects 1 in 537 Black women, which is >2-fold more than White women. Black patients develop the disease at a younger age, have more severe symptoms, and have a greater chance of early mortality. We used a multiomics approach to uncover ancestry-associated immune alterations in patients with SLE and healthy controls that may contribute biologically to disease disparities. Cell composition, signaling, epigenetics, and proteomics were evaluated by mass cytometry; droplet-based single-cell transcriptomics and proteomics; and bead-based multiplex soluble mediator levels in plasma. We observed altered whole blood frequencies and enhanced activity in CD8+ T cells, B cells, monocytes, and DCs in Black patients with more active disease. Epigenetic modifications in CD8+ T cells (H3K27ac) could distinguish disease activity level in Black patients and differentiate Black from White patient samples. TLR3/4/7/8/9-related gene expression was elevated in immune cells from Black patients with SLE, and TLR7/8/9 and IFN-α phospho-signaling and cytokine responses were heightened even in immune cells from healthy Black control patients compared with White individuals. TLR stimulation of healthy immune cells recapitulated the ancestry-associated SLE immunophenotypes. This multiomic resource defines ancestry-associated immune phenotypes that differ between Black and White patients with SLE, which may influence the course and severity of SLE and other diseases.

Unique Serum Immune Phenotypes and Stratification of Oklahoma Native American Rheumatic Disease Patients.

OBJECTIVE: Native American (NA) populations have higher rates of rheumatic disease and present with overlapping disease symptoms and nontraditional serologic features, thus presenting an urgent need for better biomarkers in NA diagnostics. This study used a machine learning approach to identify immune signatures that more effectively stratify NA patients with rheumatic disease. METHODS: Adult NA patients with autoantibody-positive (AAB+) rheumatoid arthritis (RA; n = 28), autoantibody negative (AAB-) RA (n = 18), systemic autoimmune rheumatic disease (n = 28), arthralgia/osteoarthritis (n = 28), or polyarthritis/undifferentiated connective tissue disease (n = 28), and control patients (n = 28) provided serum samples for cytokine, chemokine, and AAB assessment. Random forest clustering and soluble mediator groups were used to identify patients and control patients with similar biologic signatures. The American College of Rheumatology criteria specific for systemic disease and RA identified differences in disease manifestations across clusters. RESULTS: Serum soluble mediators were not homogenous between different NA rheumatic disease diagnostic groups, reflecting the heterogeneity of autoimmune diseases. Clustering by serum biomarkers created 5 analogous immune phenotypes. Soluble mediators and pathways associated with chronic inflammation and involvement of the innate, B cell, T follicular helper cell, and interferon-associated pathways, along with regulatory signatures, distinguished the 5 immune signatures among patients. Select clinical features were associated with individual immune profiles. Patients with low inflammatory and higher regulatory signatures were more likely to have few clinical manifestations, whereas those with T cell pathway involvement had more arthritis. CONCLUSION: Serum protein signatures distinguished NA patients with rheumatic disease into distinct immune subsets. Following these immune profiles over time may assist with earlier diagnoses and help guide more personalized treatment approaches.

Antibody Responses to Epstein-Barr Virus in the Preclinical Period of Rheumatoid Arthritis Suggest the Presence of Increased Viral Reactivation Cycles.

OBJECTIVE: Patients with established rheumatoid arthritis (RA) demonstrate altered immune responses to Epstein-Barr virus (EBV), but the presence and roles of EBV have not been fully explored during the pre-clinical disease period. This study was undertaken to determine if EBV infection, as evidenced by an altered anti-EBV antibody response, either plays an important role in driving the development of RA or is a result of expanded RA-related autoimmunity. METHODS: A total of 83 subjects with RA according to the 1987 American College of Rheumatology (ACR) criteria and 83 age-, sex-, and race-matched control subjects without RA were included in our study. We collected sera from RA subjects and matched controls during the pre-RA and post-RA diagnosis periods and tested the sera for the presence of 5 anti-EBV antibodies (anti-EBV nuclear antigen 1 IgG isotype, anti-viral capsid antigen [anti-VCA] isotypes IgG and IgA, and anti-early antigen [EA] isotypes IgG and IgA), 7 RA-related autoantibodies (rheumatoid factor measured by nephelometry [RF-Neph] as well as isotype-specific IgA-RF, IgM-RF, and IgG-RF, and anti-cyclic citrullinated peptide [anti-CCP] antibodies, including anti-CCP2, anti-CCP3, and anti-CCP3.1), 22 cytokines/chemokines, 36 individual anti-citrullinated protein antibodies, and IgG-cytomegalovirus (CMV) antibodies. Random forest classification, mixed modeling, and joint mixed modeling were used to determine differences in anti-EBV antibody levels between RA subjects and controls. RESULTS: Random forest analysis identified the presence of preclinical EBV antibodies in the serum that differentiated RA subjects from controls without RA. Specifically, IgG-EA antibody levels were higher in RA subjects (mean ± SD 0.82 ± 0.72 international standardized ratio [ISR]) compared to controls (mean ± SD 0.49 ± 0.28 ISR). In subjects with RA, elevated serum IgG-EA levels in the preclinical period before seroconversion were significantly correlated with increased serum IgM-RF levels (P = 0.007), whereas this correlation was not seen in control subjects without RA (P = 0.15). IgG-CMV antibody levels did not differ between groups. CONCLUSION: Subjects whose serum IgG-EA antibody levels are elevated in the preclinical period will eventually develop RA, which suggests that EBV reactivation cycles are increased during the preclinical period of RA. A combination of RF and EBV reactivation may play an important role in the development of RA.

Serologic markers of Epstein-Barr virus reactivation are associated with increased disease activity, inflammation, and interferon pathway activation in patients with systemic lupus erythematosus.

SLE is a clinically heterogeneous disease characterized by an unpredictable relapsing-remitting disease course. Although the etiology and mechanisms of SLE flares remain elusive, Epstein-Barr virus (EBV) reactivation is implicated in SLE pathogenesis. This study examined the relationships between serological measures of EBV reactivation, disease activity, and interferon (IFN)-associated immune pathways in SLE patients. Sera from adult SLE patients (n = 175) and matched unaffected controls (n = 47) were collected and tested for antibodies against EBV-viral capsid antigen (EBV-VCA; IgG and IgA), EBV-early antigen (EBV-EA; IgG), cytomegalovirus (CMV; IgG), and herpes simplex virus (HSV-1; IgG). Serological evidence of EBV reactivation was more common in SLE patients compared to controls as demonstrated by seropositivity to EBV-EA IgG (39% vs 13%; p = 0.0011) and EBV-VCA IgA (37% vs 17%; p = 0.018). EBV-VCA, CMV1, and HSV-1 IgG seropositivity rates did not differ between SLE patients and controls. Furthermore, concentrations of EBV-VCA (IgG and IgA) and EBV-EA (IgG) were higher in SLE patients. SLE patients with high disease activity had increased concentrations of EBV-VCA IgA (mean ISR 1.34 vs. 0.97; p = 0.041) and EBV-EA IgG levels (mean ISR 1.38 vs. 0.90; p = 0.007) compared with those with lower disease activity. EBV reactivation was associated with enhanced levels of the IFN-associated molecule IP-10 (p < 0.001) and the soluble mediators BLyS (p < 0.001) and IL-10 (p = 0.0011). In addition, EBV-EA IgG responses were enriched in two previously defined patient clusters with robust expression of IFN and inflammatory or lymphoid and monocyte responses. Patients in these clusters were also more likely to have major organ involvement, such as renal disease. This study supports a possible role for EBV reactivation in SLE disease activity.

Expanded Autoantibody Profiles for Subsetting of Native American, African American, and European American Patients With Systemic Lupus Erythematosus.

OBJECTIVE: Many Native American (NA) patients with systemic lupus erythematosus (SLE) do not exhibit the classical SLE autoantibody profiles of European American (EA) and African American (AA) patients with SLE. The poorer SLE disease outcomes noted in NA patients highlights a need for more equitable diagnostic and prognostic tools for NA patients with SLE. The objective was to identify informative autoantibody profiles for NA, AA, and EA patients with SLE using an expanded set of autoantigens. METHODS: Sera from 49 NA, 49 AA, and 49 EA age-, sex-, and antinuclear autoantibody titer-matched patients with SLE who met the American College of Rheumatology classification criteria and 10 ethnicity-, sex-, and age-matched controls were tested for autoantibody reactivity by autoantigen microarrays. Autoantibodies that were significantly elevated in patients with SLE compared with ethnicity-specific controls were selected for hierarchical clustering. Differences in clinical criteria between patient clusters were determined by Fisher's exact test and corrected for multiple comparisons. RESULTS: NA, AA, and EA patients with SLE each had a cluster distinguished by higher levels of anti-Ro52 and another cluster distinguished by nucleic acid-specific autoantibodies. Additional clusters were distinguished in NA patients by elevated extracellular matrix autoantibodies and were distinguished in AA patients by elevated Sm/RNP autoantibody and elevated nucleolin/histone autoantibody. Two EA patient clusters with similar nucleic acid- and Ro52-specific autoantibodies were distinguished by either high or low histone 2A reactivity. Renal manifestations trended higher in the NA Ro52 cluster and were significantly enriched in the AA nucleolin/histone cluster. The AA nucleolin/histone cluster and EA H2A cluster had higher disease activity. CONCLUSION: Expanded autoantibody profiles can identify informative subsets of patients with SLE.

Unique clinical characteristics, autoantibodies and medication use in Native American patients with systemic lupus erythematosus.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with varied morbidity and mortality. We assessed clinical presentations, autoantibody specificities and therapeutic interventions in Native American (NA) patients with SLE. METHODS: Patients with SLE meeting 1997 American College of Rheumatology classification criteria (n=3148) were enrolled between 1992 and 2010 in the multiethnic, cross-sectional Lupus Family Registry and Repository. Clinical, demographic and therapeutic information were extracted from medical records using a standardised form and formalised training. Autoantibodies were assessed by indirect immunofluorescence (antinuclear antibodies (ANA) and antidouble-stranded DNA), precipitin (ENA) and ELISA (IgG and IgM anticardiolipins). RESULTS: NA patients met SLE classification at a younger age (29.89±12.3 years) than European Americans (EA; 32.02±12.87, P=0.0157) and a similar age to African-Americans (AAs) and Hispanics (HIS). More NA patients had concurrent rheumatic diseases or symptoms, such as Raynaud's phenomenon, interstitial lung disease, SjÓ§gren's syndrome and systemic sclerosis. Compared with EAs, NAs were more likely to have high-titre ANA (≥1:3240; P<0.0001) and had more SLE-associated autoantibodies. Autoantibodies with unknown specificities were more common in NAs (41%) compared with other racial/ethnic groups in this collection (AA: 24%, P=0.0006; EA: 17%, P<0.0001; HIS: 23%, P=0.0050). Fewer NA patients used hydroxychloroquine (68%) compared with others (AA: 74%, P=0.0308; EA: 79%, P=0.0001, HIS: 77%, P=0.0173); this was influenced by lower hydroxychloroquine use in NA patients from Latin America (32%). NA patients had higher rates of methotrexate use (28%) compared with AA (18%, P=0.0006) and HIS patients (14%, P=0.0003), higher azathioprine use (38%) compared with EA patients (30%, P=0.0105) and higher mycophenolate mofetil use (26%) compared with EA (17%, P=0.0012) and HIS patients (11%, P<0.0001). CONCLUSIONS: NA patients are diagnosed with SLE earlier in life and present worse concurrent rheumatic disease symptoms than EA patients. NA patients also are more likely to have expanded autoantibody profiles and precipitins of unknown specificities.

Human monoclonal antibodies generated following vaccination with AVA provide neutralization by blocking furin cleavage but not by preventing oligomerization.

In order to identify the combination of antibody-mediated mechanisms of neutralization that result from vaccination with anthrax vaccine adsorbed (AVA), we isolated antibody secreting cells from a single donor seven days after booster vaccination with AVA and generated nine fully human monoclonal antibodies (hmAb) with high specificity for protective antigen (PA). Two of the antibodies were able to neutralize lethal toxin in vitro at low concentrations (IC(50): p6C01, 0.12 µg/ml and p6F01, 0.45 µg/ml). Passive transfer of either of these hmAbs to A/J mice prior to challenge with lethal toxin conferred 80-90% protection. We demonstrate that hmAb p6C01 is neutralizing by preventing furin cleavage of PA in a dose-dependent manner, but the mechanism of p6F01 is unclear. Three additional antibodies were found to bind to domain 3 of PA and prevent oligomerization, although they did not confer significant protection in vivo and showed a significant prozone-like effect in vitro. These fully human antibodies provide insight into the neutralizing response to AVA for future subunit vaccine and passive immunotherapeutic cocktail design.