POS0001 PHENOTYPE AND FUNCTIONAL CHARACTERISTICS OF ANTIGEN-SPECIFIC, AUTO-REACTIVE B CELL RESPONSES REVEAL DIFFERENTIAL IMMUNOLOGICAL ACTIVITY IN PATIENTS WITH SYSTEMIC SCLEROSIS

Background:Systemic Sclerosis (SSc) is a systemic autoimmune disease that carries the highest mortality burden among the rheumatic diseases. Disease risk and course are difficult to predict in individual patients, and anti-inflammatory and B-cell depleting therapies show varying results. >95% of SSc patients harbor autoantibodies. Among those, anti-topoisomerase antibodies (ATA) and anti-centromere antibodies (ACA) are most prevalent, mutually exclusive in individual patients and associate with distinct disease phenotypes. Despite these associations, the clinical value of both ATA and ACA for patient stratification within these phenotypes is limited. Here, we hypothesized that phenotypic and functional characteristics of the underlying autoreactive B cell responses could allow insights in differential ‘immunological disease activity’ in individual patients, thereby providing indications as to potential drivers of these responses as well as granularity as to which patients may benefit from targeted interventions.Objectives:To assess phenotypic and functional characteristics of anti-topoisomerase and anticentromere specific B cell responses in individual patients with SSc.Methods:Peripheral blood mononuclear cells (PBMC) from ATA- and ACA-positive SSc patients were cultured without stimulation or in the presence of CD40L-expressing fibroblasts, IL-21 and BAFF. Following culture, ATA- and ACA-IgG and -IgA were measured in culture supernatants by ELISA. In addition, PBMC were depleted of circulating plasmablasts by fluorescence activated cell sorting (FACS), and isolated plasmablasts were cultured separately. Furthermore, the presence of antigen-specific plasmablasts was confirmed by ELISPOT. Finally, the degree of spontaneous ATA secretion was correlated to the presence or absence of interstitial lung disease (ILD; based on high-resolution computed tomography). Healthy donors and patients with rheumatoid arthritis served as controls.Results:We observed that individual ATA- and ACA-positive SSc patients harbored circulating B cells that secrete either ATA-IgG or ACA-IgG upon stimulation, depending on their serotype. In addition, we noted spontaneous secretion of ATA-IgG and, more remarkably, extensive secretion of ATA-IgA in ATA-positive patients. This degree of spontaneous, antigen-specific IgA secretion was specific for the ATA response, while spontaneous ACA-IgA secretion was undetectable in patients harboring ACA. FACS experiments and ELISPOT showed that the spontaneous ATA-IgA and -IgG secretion was attributable to circulating plasmablasts. Of note, the degree of spontaneous ATA-IgG secretion was remarkably higher in patients with ILD than in those without.Conclusion:Our findings demonstrate that individual ATA-positive SSc patients harbor activated ATA-IgG and ATA-IgA B cell responses, as indicated by the spontaneous secretion of both ATA isotypes by circulating plasmablasts. Importantly, by taking the presence of plasmablasts as a proxy for recent B cell activation, our data suggest a link between the activity of the antigen-specific B cell response and the presence of ILD. In contrast, the ACA B cell response was far less active and lacked the active IgA component, which suggests a difference in the triggers driving these autoreactive B cell responses in patients. In fact, the remarkable ATA-IgA secretion points towards a potential mucosal trigger of the ATA response, which may be continuously active in individual patients.Disclosure of Interests:None declared.

Temporal kinetics of bovine mammary IgG secretion into colostrum and transition milk

Neonatal calf survival and health is predominantly dependent on sufficient consumption of immunoglobulin G (IgG) and the resulting transfer of passive immunity (TPI). In this study we investigate the potential for continued IgG secretion and temporal kinetics of mammary IgG output in sequential milkings performed at 0, 4, 16, 28, 40 and 52 hours post-calving in Holstein dairy cows. For colostrum (0 hour), we also scrutinize the relationships between IgG concentration, volume, refractometer readings (˚Bx values, Brix ®) and concentration of sugars (lactose and glucose). Mammary transcripts postpartum (0 hour) indicated that active IgG secretion continues beyond the first milking (colostrum; n=4-5). IgG measurements at the different timepoints indicated that colostrum represents only 25.1% of the total IgG produced across the six sequential milking timepoints, with a substantial 48.9% being secreted into transition milk over the next three timepoints (4-, 6- and 28-hour) combined. The differences on the basis of IgG concentrations across 0-, 4- and 16-hour milking timepoints were not statistically significant (p=0.1522; n=9). For colostrum, volume remained highly variable, even with induced let-down prior to milking (n=27). Nonetheless, colostrum IgG secretion was significantly co-regulated with volume (R 2=0.915; p<0.001; n=18), an association that was stronger than that measured for lactose (R 2=0.803; p<0.001; n=18) and glucose (R 2=0.467; p=0.002; n=17). Comparing colostrum ˚Bx values to absolute IgG concentrations showed no correlation (R 2=0.127; p=0.07; n=27); biochemical separation of colostrum components indicated that both proteins and non-protein solutes could affect ˚Bx values (p<0.0001 for both; n=5). This suggests that ˚Bx values do not reasonably indicate IgG concentration to serve as a measure of “colostrum quality.” Additionally, our finding that early transition milk (4-, 6- and 28-hour) can contribute substantially more IgG than colostrum forces a rethink of existing feeding paradigms and means to maximize TPI in calves. Collectively, our results reveal the remarkable value of early transition milk and caveats to colostrum assessments that could advance application in enhancing neonatal calf health.

Association of Tuberculosis and Infections of Herpes Simplex, Varicella Zoster Viruses and Cytomegalovirus

To determine frequency of Herpes simplex (HSV), Varicella zoster viral (VZV) and Сytomegaloviral (CMV) coinfection with tuberculosis (TB) we examined 45 patients with pulmonary TB and 62 healthy donors, 25 of them were healthcare workers in tuberculosis hospital and the rest 37 had no previous contact with TB. None of the participants had vesicular rash of skin or mucosa. For diagnosis of herpes viral infection enzyme-linked immunosorbent assay for detection of HSV, VZV, CMV IgG in PBMC supernatants was performed. A significant increase in CMV (53.3%) and HSV (40%) infection in TB patients was observed comparing to healthy donors (p < 0.05; 19.4 and 16.1% respectively). Frequency of VZV infection in TB patients (17.8%) and healthy donors (8.1%) differed statistically unsignificantly. Levels of specific IgG secretion in PBMC culture in both groups in case of VZV and CMV infections didn’t differ, but in case of HSV coinfection in TB patients mean level of HSV IgG secretion (1.106 ± 0.297 OD) significantly exceeded mean level of HSV IgG in PBMC supernatants of healthy donors with asymptomatic HSV infection (0.285 ± 0.048 OD, р < 0,05). The obtained data give evidence of an association of pulmonary TB and herpes viral infections. Influence of these coinfections on course and morbidity of TB requires further research.