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Background and Aims

Parathormone (PTH) has been clinically associated with autoimmune diseases but its pathophysiological impact has not been established. Our aim was to evaluate the expression of the PTH receptor (PTH1R) on B cells in patients with primary Sjögren’s Syndrome (pSS) and Systemic Lupus Erythematosus (SLE), and PTH effects on B cells in vitro.

Methods

We evaluated PTH1R expression on B cells and the receptor-expressing B cell subsets’ distribution (Bm1-Bm5) by flow cytometry in peripheral blood of SLE, pSS and healthy controls (HC). Analysis in vitro was performed in a B-cell line (Ramos) with PTH stimulus; effects on proliferation, apoptosis, and subpopulations were evaluated. We correlated PTH1R levels with ESSDAI and SLEDAI. Statistical analysis was performed using Kruskal Wallis and Spearman’s tests.

Results

We included 12 patients with SLE, 19 with pSS, and 20 HC. PTH1R expression on B-cells from SLE was 13% compared to pSS (4.4%), p=0,019; and HC (5%), p=0,013. Distribution of B cell subsets expressing PTH1R was significantly lower in Bm1 (23%), and higher in Bm2 (31.1%), and Bm2´ (6.4%) in pSS compared to HC (39%, 18%, 1.7%, respectively), p=0.010, 0.05, 0.028. Correlation between PTH1R levels and ESSDAI was r=0.511, p=0.026; SLEDAI was not correlated. Higher PTH concentrations tended to maintain B cells in Bm3-4 subset compared to unstimulated B cells that differentiated to eBm5 subset.

Conclusions

PTHR1 expression could be a useful biomarker in SLE and activity marker in pSS. PTH effects on B-cell subsets modulation suggest a potential role of the hormone in the pathophysiology of autoimmune diseases.

Background and Aims

B cells may be involved in inflammatory arthritis induced by checkpoint inhibitors (CPI-IA). Our aim was to investigate the phenotype of circulating B cells in patients receiving CPI treatment and the relationship with CPI-IA.

Methods

Peripheral blood mononuclear cells were analyzed with flow-cytometry in CPI-IA patients and in patients who had not developed immune-related adverse events upon CPI (non-irAE). B cells were identified as CD19⁺ and divided into naïve (CD27^-IgD⁺), memory (CD27⁺IgD^+/-) and transitionals (CD10⁺CD24⁺CD38^+/hi). Levels of the activation marker CD21 were also analyzed. Plasma levels of IL-6, IL-10 and BAFF were measured by ELISA. Non-parametric tests were used for comparisons.

Results

Six CPI-IA and 7 non-irAE patients matched for age, gender and CPI treatment were included. CPI-IA patients displayed increased levels of circulating B cells (p=0.002) and of transitional B cells vs. non-irAE (median %, range: 7.8 (4.5-11.4) vs. 3.2 (1.6-4.3), p=0.007), while no remarkable changes were seen across other subsets. Transitional B cells significantly decreased when CPI-IA resolved (p=0.008). Levels of CD21 on transitional B cells were increased in CPI-IA vs. non-irAE (p=0.01). There were no significant differences in cytokine levels between patient groups. IL-10 tended to increase while IL-6 tended to decrease at CPI-IA resolution, yet not reaching significance. BAFF levels significantly increased from active to quiescent CPI-IA (p=0.02).

Conclusions

Transitional B cells are expanded in CPI-IA patients. BAFF increase may parallel their decrease at CPI-IA resolution. Monitoring of transitional B cells could facilitate early diagnosis and treatment of CPI-IA.

Background and Aims

Myositis are chronic autoimmune diseases characterized by muscular weakness, inflammation, and extra-muscular involvement. Extracellular vesicles (EVs) are heterogeneous lipid bilayer nanoparticles, ranging from 30 nm to 1000 nm, naturally released from cells. Convoying their cargo, EVs allow intercellular communication. Their role in the pathogenesis of autoimmune diseases is recognized. The study aims to investigate EVs as potential myositis biomarkers.

Methods

EVs were isolated from platelet-free plasma of myositis patients and healthy donors (HDs) through size exclusion chromatography and enriched by ultrafiltration. They were observed by transmission electron microscopy (TEM) and quantify by tunable resistive pulse sensing (TRPS).

Results

By TEM, EVs were seen more concentrated in patients (n=4) than in HDs (n=5) samples. EVs appeared intact, roundish, and heterogeneous in size, with a prevalence of smaller EVs in both groups. TRPS measurements showed an EVs concentration of 1.76e+14 [EVs/mL] and a mode diameter of 80 nm, in the patient sample (n=1). The mean EVs concentration of HDs samples (n=2) was 1.12e+14 [EVs/mL] with a mean mode diameter of 77 nm (Figure 1).

Figure 1. TRPS measurement of EVs report (qNano, Izon instruments). Quantification of a myositis patient (on the left) and HD (on the right) samples. Particles distribution graphs show particles’ concentration [EVs/mL] related to their size.

Conclusions

Preliminary data based on TEM observation and TRPS measurements showed that EVs are predominantly small in both patient and HDs samples. Moreover, the EVs concentration appeared slightly higher in the patient rather than in HDs. Further analyzed employing classical and imaging flow cytometry are ongoing.