Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has become pandemic. Cytokine release syndrome occurring in a minority of SARS-CoV-2 infections is associated with severe disease and high mortality. We profiled the composition, activation, and proliferation of T cells in 20 patients with severe or critical COVID-19 and 40 matched healthy controls by flow cytometry. Unsupervised hierarchical cluster analysis based on 18 T cell subsets resulted in separation of healthy controls and COVID-19 patients. Compared to healthy controls, patients suffering from severe and critical COVID-19 had increased frequencies of activated and proliferating CD38+Ki67+ CD4+ and CD8+ T cells, suggesting active antiviral T cell defense. Frequencies of CD38+Ki67+ Th1 and CD4+ cells correlated negatively with plasma IL-6. Thus, our data suggest that patients suffering from COVID-19 have a distinct T cell composition that is potentially modulated by IL-6.

Immunocompromised patients are considered high-risk and prioritized for vaccination against COVID-19. We aimed to analyze B-cell subsets in these patients to identify potential predictors of humoral vaccination response. Patients (n=120) suffering from hematologic malignancies or other causes of immunodeficiency and healthy controls (n=79) received a full vaccination series with an mRNA vaccine. B-cell subsets were analyzed prior to vaccination. Two independent anti-SARS-CoV-2 immunoassays targeting the receptor-binding domain (RBD) or trimeric S protein (TSP) were performed three to four weeks after the second vaccination. Seroconversion occurred in 100% of healthy controls, in contrast to 67% (RBD) and 82% (TSP) of immunocompromised patients, while only 32% (RBD) and 22% (TSP) achieved antibody levels comparable to those of healthy controls. The number of circulating CD19+IgD+CD27- naïve B cells was strongly associated with antibody levels (ρ=0.761, P<0.001) and the only independent predictor for achieving antibody levels comparable to healthy controls (OR 1.07 per 10-µL increase, 95%CI 1.02–1.12, P=0.009). Receiver operating characteristic analysis identified a cut-off at ≥61 naïve B cells per µl to discriminate between patients with and without an optimal antibody response. Consequently, measuring of naïve B cells in immunocompromised hematologic patients could be useful in predicting their humoral vaccination response.

Objectives Immunocompromised patients are at risk of severe coronavirus disease 2019 and are considered a high priority for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination. Humoral vaccination response is impaired in these patients when circulating B cells are lacking. We aimed to analyze B-cell subsets at the time of vaccination to identify potential predictors of humoral vaccination response. Methods Patients (n=120) receiving B-cell-depleting therapy (n=41), those suffering from inborn errors of immunity (n=25) and hematologic malignancies (n=56), and healthy controls (n=79) were vaccinated twice with BNT162b2 or mRNA 1273. B-cell subsets were analyzed prior to vaccination. Two independent anti-SARS-CoV-2 S immunoassays targeting the receptor-binding domain (RBD) or trimeric S protein (TSP) were performed three to four weeks after the second vaccination. Results Seroconversion occurred in 100% of the healthy controls, in contrast to 67% (RBD) and 82% (TSP) of the patients, while only 32% (RBD) and 22% (TSP) achieved antibody levels comparable to those of healthy controls. The number of circulating naive B cell was strongly associated with antibody levels (r=0.761, P<0.001) across all immunosuppressive treatments or conditions. In multivariable analysis, the number of naive B cells was an independent predictor for achieving antibody levels comparable to healthy controls, and receiver operating characteristic analysis predicted that at least six naive B cells per microL were required. Conclusions Assessing the abundance of naive B cells in immunocompromised patients could be useful in predicting the optimal vaccination response.

BackgroundNumerous cytokines that influence disease activity in psoriatic arthritis (PsA) are modulators of the Janus Kinases/Signal Transducers and Activators of Transcription (JAK/STAT) pathway. The JAK1/STAT1/STAT3/STAT5 network can drive the expansion of Th17 and regulatory T cells via proinflammatory cytokines in PsA joints,[1], [2] while hyperphosphorylation of STAT3 in immune cells has previously been shown to promote PsA pathogenesis through the Interleukin (IL)-23/IL-17/IL-22 axis.[3] Therefore, the phosphorylation status of STAT molecules in leucocytes of PsA patients may indicate active disease and could potentially guide treatment with JAK inhibitors.ObjectivesTo analyse phosphorylated STAT (pSTAT) levels of circulating leucocyte subsets in PsA patients with active and inactive diseaseMethodsWhole blood was drawn on consecutive PsA patients fulfilling the CASPAR criteria[4] to perform flow cytometry analysis using the BD FACSLyric platform. Disease activity was assessed using the Disease activity for psoriasis arthritis (DAPSA) score.[5] All steps from storage of drawn blood to cell fixation were performed at 4°C to prevent auto-activation of leucocytes. The geometric mean fluorescence intensities (gMFI) of pSTATs in granulocytes, monocytes, B cells and CD4+/- naïve/memory T cells were compared between patients with moderate to high (MoDA/HDA) and remission to low disease activity (REM/LDA). Correlation analysis between gMFIs and DAPSA scores were performed.ResultsForty-two patients (female ratio: 0.48) with established PsA (median ± standard deviation, age: 56 ± 12.54 years, disease duration: 8.50 ± 7.10 years) were included in this study. Twenty-one percent of patients were in MoDA/HDA, while the remaining 79% were in REM/LDA. Patients in MoDA/HDA showed significantly higher pSTAT3 levels in CD4+ naïve (gMFI median ± standard deviation: 284.5 ± 79.9 vs 238 ± 92.9, p = 0.011), CD4- naïve (297 ± 107.5 vs 238 ± 98.4, p = 0.04), CD4+ memory (227 ± 62.9 vs 190.5 ± 72.2, p = 0.009) and CD4- memory T cells (209 ± 66.8 vs 167.0 ± 64.9, p = 0.036). On the other hand, PsA patients in remission or low disease activity displayed higher pSTAT1 levels in granulocytes (2509 ± 1887 vs 1330.5 ± 784.1, p = 0.040) and monocytes (255 ± 230 vs 144 ± 62.5, p = 0.049). Positive correlations were found between DAPSA scores and pSTAT3 in CD4+ naïve and memory T cells (Spearman’s correlation coefficient rho (ρ) = 0.5, p = 0.0012 and ρ = 0.47, p = 0.0025 resp.) whereas pSTAT1 in granulocytes and monocytes were negatively correlated with the DAPSA scores (ρ = -0.45, p = 0.0074 and ρ = -0.34, p = 0.05).ConclusionDifferential phosphorylation of STAT3 and STAT1 molecules in circulating leucocyte subsets indicates PsA disease activity. Further studies to examine the value of STAT phosphorylation patterns guiding JAK inhibitor therapy are underway.References[1]U. Fiocco et al., “Ex vivo signaling protein mapping in T lymphocytes in the psoriatic arthritis joints,” J. Rheumatol., vol. 93, pp. 48–52, 2015, doi: 10.3899/jrheum.150636.[2]S. K. Raychaudhuri, C. Abria, and S. P. Raychaudhuri, “Regulatory role of the JAK STAT kinase signalling system on the IL-23/IL-17 cytokine axis in psoriatic arthritis,” Ann. Rheum. Dis., vol. 76, no. 10, pp. e36–e36, 2017.[3]E. Calautti, L. Avalle, and V. Poli, “Psoriasis: A STAT3-centric view,” International Journal of Molecular Sciences, vol. 19, no. 1. MDPI AG, Jan. 06, 2018, doi: 10.3390/ijms19010171.[4]W. Taylor, D. Gladman, P. Helliwell, A. Marchesoni, P. Mease, and H. Mielants, “Classification criteria for psoriatic arthritis: Development of new criteria from a large international study,” Arthritis Rheum., vol. 54, no. 8, pp. 2665–2673, 2006, doi: 10.1002/art.21972.[5]M. M. Schoels, D. Aletaha, F. Alasti, and J. S. Smolen, “Disease activity in psoriatic arthritis (PsA): Defining remission and treatment success using the DAPSA score,” Ann. Rheum. Dis., vol. 75, no. 5, pp. 811–818, 2016, doi: 10.1136/annrheumdis-2015-207507.Disclosure of InterestsBarbara Dreo: None declared, Daniel Ruben Pietsch: None declared, Rusmir Husic Speakers bureau: MSD, Lilly und Abbvie, Angelika Lackner: None declared, Johannes Fessler: None declared, Janine Rupp: None declared, Anirudh Subramanian Muralikrishnan: None declared, Jens Thiel Speakers bureau: GSK, BMS, AbbVie, Novartis, Consultant of: GSK, Novartis, Grant/research support from: BMS, Martin Stradner Speakers bureau: Eli Lilly, Pfizer, MSD, BMS, AbbVie, Janssen, Consultant of: Eli Lilly, AbbVie, Janssen, Philipp Bosch Grant/research support from: Pfizer

Objective To evaluate tender joints (TJ) and swollen joints (SJ) for the assessment of ultrasound (US) defined inflammation in PsA. Methods Eighty-three PsA patients underwent clinical and US examinations at two scheduled study visits 12 months apart. Tenderness and swelling were assessed at 68 and 66 joints respectively and US examinations were conducted at all 68 joints. At patient level, associations with clinical composites and US scores were performed using correlations and by analysing patients with predominantly tender (pTender) or swollen joints (pSwollen). At joint level, a PD value ≥ 1 was defined as active synovitis. A generalized linear mixed model was created to assess the predictive value of TJ and SJ for active synovitis after 12 months. Results SJC showed better correlations with GS/PD scores (r = 0.37/0.47) than with TJC (PD: r = 0.33), while TJC correlated better with patient reported outcomes (PROMs) like patient global assessment (TJC: r = 0.57; SJC r = 0.39). Patients with pTender showed poorer results for PROMs and disease activity scores than patients with pSwollen, but not for laboratory or US markers of inflammation. Swollen joints showed active synovitis in 35% of cases, while only 16% of tender joints were active according to US. Swelling at baseline better predicted active synovitis at the same joint after 12 months (OR 6.33, p&lt; 0.001) as compared with tenderness (OR 3.58, p&lt; 0.001). Conclusions SJ are more closely linked with US signs of inflammation as compared with TJ in PsA. Joint swelling is a better predictor for signs of US inflammation than tenderness after one year of follow-up.

Background:B-cells play a major role in the pathogenesis and perpetuation of the immune response in systemic lupus erythematosus (SLE). So far, B-cell subtypes have been studied well, but the precise mechanisms of the B-cell alterations during disease activity and during remission, depending on different medication, are still unclear.Objectives:The aim of our study was to investigate the drug dependent alterations in the B-cell repertoire of SLE patients with low disease activity (SLEDAI – 2K ≤4).Methods:Peripheral blood samples from 39 patients suffering from SLE (mean±SD; age 43±13 years, 87.2% females, disease duration 11.1±7 years) were drawn over 2 years. All SLE patients were in remission or low disease activity (median±SE, SLEDAI of 2.0±1.5). B-cells were characterized using CD19, CD20, CD5, CD27 antibodies and were grouped in naïve (IgD+27-), non-switched memory (IgD+, CD27+), memory (IgD-,CD27+), B1 (CD5+27-) and MBL-like (CD5++) B-cells. A quantitative flow cytometric bead-based assay (QuantiBRITE PE kit from Becton Dickinson) was used for the estimation of CD19 antibodies bound per cell. Further, CD38 and CD86 antibodies were used to characterize the B-cell subsets. All cytometric measurements were performed using a standardized BD LSR Fortessa platform. After 3 years of follow-up, patients’ data about disease activity and current medication were obtained.Results:22 SLE patients were treated with hydroxychloroquine (85.8%) and 19 patients received mycophenolate mofetil (MMF; n=14; 54.6%) or azathioprine (AZA; n= 5; 19.5 %). 5 patients were treated with other DMARDs. Independently of hydroxychloroquine and/or MMF, no significant differences were seen in naïve, non-switched memory, post-switched memory, plasma blasts, B1- or MBL-like B-cells. Patients treated with AZA had significantly lower naïve B-cells (mean±SD, 39.3±6.7vs. 73.1±19.3 %; p = 0.028), but had significantly higher IgD-post switched B-cells (31.2±9.1 vs.12.5 ±9.2 %; p = 0.028, respectively) compared with no AZA-treatment. Interestingly, activated B-cells (5.5±1.5 vs. 1.8±1.1%; p = 0.009) were significantly higher in AZA-treated. After 3 years of follow-up, almost all patients were in remission (median±SE, SLEDAI of 2.0±2.0), except of 3 patients with a SLEDAI of ≥ 6. Interestingly, those patients had at baseline, statistically higher naïve B-cells (p = 0.041) and lower B1-like B-cells (p =0.020) compared with patients with low disease activity.Conclusion:Our results suggest that independently of hydroxychloroquine and/or MMF treatment, all patients with low disease activity had similar normal B-cell subsets. Interestingly, in the small group of patients who were treated with AZA, a reduced regeneration of B-cells was shown. Patients with higher disease and high naïve B-cells showed an increased disease activity after three years.Acknowledgments:The research was performed in “CBmed” and funded by the Austrian Federal Government within the COMET K1 Centre Program, Land Steiermark and Land Wien.Disclosure of Interests:None declared

Background:Under physiological conditions, T regulatory cells (Tregs) are responsible for the downregulation of the immune response. In autoimmune diseases, such as rheumatoid arthritis (RA), auto-inflammation is driven by an imbalance of activation and downregulation of immunological pathways. Thus, treatment plans for autoimmune diseases often involve the enhancement of immunoregulatory pathways by administering inhibitors of costimulation, i.e. CTLA-4-Ig (abatacept, ABA). ABA binds specifically to CD80 and CD86 on antigen presenting cells (APC). Consequently, T cell activation via the CD28 receptor is blocked. Previous studies have demonstrated surprising effects of abatacept on Tregs, specifically decreased frequency of these cells but enhancement in their function1. Whether these alterations can only be found in patients with ABA treatment, or whether they are also present in patients receiving other anti-inflammatory drugs is currently unknown.Objectives:The aim of our research was to delineate the impact of ABA on the different subsets of effector and regulatory T cells in RA and compare these findings with patients receiving tocilizumab (TCZ) or rituximab (RTX).Methods:Peripheral blood samples from 56 RA patients (median ± SE; age: 60.5 ± 1.3 years, female ratio: 0.7, disease duration: 17.9 ± 2.1 years; respectively) were drawn over a sampling period of 2 years. Freshly isolated PBMCs of RA patients were stained with fluorochrome-labelled antibodies and T cell subsets were identified by flow cytometric means. CD3+CD4+T cells were further classified using different T cell markers (CD25, CD127, CD39, CD95). All cytometric measurements were performed using a standardized BD LSR-Fortessa platform. RA patients were compared according to their treatment with ABA, TCZ or RTX.Results:Eighteen out of 56 RA patients (32%) received ABA, 25 patients (45%) received TCZ and 13 patients (23%) were under CD20+ cell depletion therapy with RTX. RA patients receiving ABA displayed a significant decrease in CD3+CD4+CD25+CD127dimTregs (3.7% ± 0.4) compared to patients with TCZ (5.4% ± 0.4, p = 0.041) and patients under RTX treatment (7.52% ± 0.93, p = 0.026). CD39+Tregs were significantly higher in RA patients treated with TCZ (49.5% + 3.2, p = 0.000) or RTX (50.5% ± 5.3, p = 0.026) compared to patients receiving ABA (24.5% ± 3.1). In addition, the frequency of CD95+Tregs was significantly reduced in ABA patients compared to RTX patients (59.6% ± 3.1 vs.76.7% ± 3.6, p = 0.014; respectively). Interestingly, T cells displaying an effector T cell phenotype (CD3+CD4+CD25+/-CD127+) were increased in ABA treated patients compared to RTX treated patients (59.6% ± 3.1 and 76.7% ± 3.6, p = 0.002). Since none of our patients were a non-responder or had high disease activity, we could not analyse whether these changes are associated with treatment outcome.Conclusion:Our data demonstrate that blockage of T cell stimulation via ABA leads to characteristic alterations in different regulatory and effector T cells not seen in patients treated with TCZ or RTX. Further studies must clarify whether the analysis of regulatory and effector T cell subpopulations before treatment initiation can be used as biomarker for treatment response.References:[1]Álvarez-Quiroga C, Abud-Mendoza C, Doníz-Padilla L, et al. CTLA-4-Ig therapy diminishes the frequency but enhances the function of treg cells in patients with rheumatoid arthritis.J Clin Immunol. 2011;31(4):588-595.doi:10.1007/s10875-011-9527-5Acknowledgments:Work done in “CBmed” was funded by the Austrian Federal Government within the COMET K1 Centre Program, Land Steiermark and Land Wien.Disclosure of Interests:None declared

Schnitzler syndrome (SchS) is a rare autoinflammatory disease, characterized by urticarial rash, recurrent fever, osteo-articular pain/arthritis with bone condensation, and monoclonal gammopathy. Diagnosis may be difficult due to overlapping signs with other diseases. Here, we describe the case of a 62-year-old man with SchS, who was initially misdiagnosed with multicentric Castleman disease (MCD). As excessive release of IL-6 is characteristic of MCD, in contrast to IL-1 in SchS, we measured the phosphorylation of intracellular signaling proteins of the respective pathways by flow cytometry. We found a distinct increase of phosphorylated IRAK-4 in our patient’s B cells and monocytes while phosphorylation of STAT-3 was low, suggesting predominant IL-1 signaling. In accordance with these results and the classification criteria, we established the diagnosis of SchS instead of MCD and commenced therapy with the IL-1 receptor antagonist anakinra. We observed a rapid remission of signs accompanied by a reduction of phosphorylated IRAK-4 to normal levels. In conclusion, we propose phosphorylated IRAK-4 in B cells and monocytes as a potential marker for diagnosis of SchS and for treatment response to IL-1 blockade.