Silvia Piantoni

Abatacept Reduces Levels of Switched Memory B Cells, Autoantibodies, and Immunoglobulins in Patients with Rheumatoid Arthritis

Objective. Abatacept (ABA) is a chimeric molecule, able to block the CD28-mediated costimulatory pathway. To evaluate the hypothesis that, through this mechanism of action, ABA may down-modulate the immune responses of B lymphocytes in rheumatoid arthritis (RA), we investigated the serum levels of immunoglobulins (Ig), free light chains (FLC), anticitrullinated protein antibodies (ACPA), and rheumatoid factor (RF), as well as the number of B lymphocytes differentiated into post-switch memory cells in patients treated with ABA. Methods. The serum levels of Ig, FLC, different ACPA, RF isotypes, and the B cell phenotype were longitudinally evaluated in 30 patients with RA treated with ABA. Results. At baseline, the proportion of total and post-switch memory B cells was lower in RA than in healthy individuals. After 6 months of ABA treatment we observed significant reductions of serum levels of IgG, IgA, and IgM, as well as FLC, with a normalization in many patients who had initially abnormal values. A significant reduction of the titers of IgG- and IgA-ACPA, as well as of IgM-, IgA-, and IgG-RF was also observed. A decrease of autoantibodies below the upper limits of normal values was found in 2 of 26 patients (8%) initially seropositive for IgG-ACPA, 1 of 14 (7%) for IgA-ACPA, 5 of 22 (23%) for IgM-RF, 7 of 22 (30%) for IgA-RF, and 5 of 16 (31%) for IgG-RF. After treatment, the proportion of circulating post-switch memory B cells was also further significantly decreased. Conclusion. ABA treatment in patients with RA can reduce signs of polyclonal B cell activation, inducing a trend toward normalization of serum levels of different classes of Ig and of FLC, decreasing titers of ACPA and RF, and percentages of post-switch memory B cells.

A 24-month prospective study on the efficacy and safety of two different monthly regimens of vitamin D supplementation in pre-menopausal women with systemic lupus erythematosus

Background Low vitamin D (vit.D) serum levels are common in patients with systemic lupus erythematosus (SLE) and seem to correlate with higher disease activity. We investigated the effects of different regimens of vit.D supplementation in SLE patients with inactive disease. Methods This 24-month prospective study included 34 SLE women who were randomized to receive, together with their ongoing treatment, a standard regimen (SR) of cholecalcipherol (25,000 UI monthly) or an intensive regimen (IR) (300,000 UI initial bolus followed by 50,000 UI monthly) for one year and then were switched to the other regimen in the second year. Patients were seen quarterly for assessment of 25-OH vit.D levels, disease activity, SLE serology and bone metabolism markers. Results By intra-patient comparison, only the IR was found able to significantly raise vit.D serum levels. After 12 months, values above 30 ng/ml were found in 75% of patients in IR while in only 28% in SR. No significant differences in disease activity and SLE serology were found at any time point between SR and IR. No changes in the mineral metabolism were observed. Conclusions The IR was safe and effective in obtaining sufficient levels of vit.D in most SLE patients. However, both regimens of supplementation did not differently affect disease activity nor SLE serology.

Clinical Significance of IgA Anti-Cardiolipin and IgA Anti-β2Glycoprotein I Antibodies

IgA antiphospholipid antibodies (aPL) are not currently recognized as formal laboratory criteria for the Antiphospholipid Syndrome (APS). This is mainly due to methodological issues (different study designs, use of various non-standardized IgA assays). However, there are experimental data showing the pathogenic role of IgA anti-cardiolipin antibodies (aCL) and IgA anti-β2glycoprotein I antibodies (anti-β2GPI). Isolated IgA aCL are not very common, therefore their testing could be useful in the case of strong suspicion of APS but negative results for other aPL tests. IgA anti-β2GPI seem to be the most prevalent isotype in patients with Systemic Lupus Erythematosus (SLE), with a significant association with thrombotic events. Such a clinical relevance has been recently recognized by the inclusion of these autoantibodies among the aPL tests in the novel SLICC classification criteria for SLE. Emerging interest has been raised by IgA anti-β2GPI against domain 4/5 as a novel subgroup of clinically relevant aPL.

Phenotype modifications of T-cells and their shift toward a Th2 response in patients with systemic lupus erythematosus supplemented with different monthly regimens of vitamin D

Background Vitamin D receptor is constitutively expressed on the lymphocyte surface. Recent studies highlight that vitamin D may exert actions on T-cells, inhibiting Th1 and Th17 response and enhancing Th2 and T-regulatory (T-reg) function. Methods Thirty-four patients with systemic lupus erythematosus (SLE) were randomly enrolled in a two-year prospective study. In the first year, 16 patients were supplemented with an intensive regimen of cholecalciferol (IR) (300.000 UI of cholecalciferol at baseline and 50.000 UI/monthly as maintenance, 850.000 UI annually), whereas 18 with a standard regimen (SR) (25.000 UI of cholecalciferol monthly, 300.000 UI annually). During the second year, patients were switched to the other arm of treatment. Phenotypic analysis of peripheral T lymphocyte and the quantification of cytokine production from peripheral blood mononuclear cells (PBMCs) were evaluated by flow cytometry. Results At baseline, no significant difference between the two groups emerged among main T-cell subtypes. Over two years of treatment, we saw an increase in the number of T-reg cells, in the total amount of CD4+CD45RA+CCR7− T-cells, whereas a significant reduction of CD8+CD28− T-cells was observed. In addition, the analysis of PBMCs from eight patients following the IR showed the reduction of the IFN-γ/IL-4 ratio (p = 0.01) among CD8+ T-cells after 12 months. Conclusions After a long-term of monthly treatment with vitamin D in SLE patients, an enhancement of T-reg cells and the production of Th2 cytokines should be expected.

Circulating CD4+ T-cell number decreases in rheumatoid patients with clinical response to rituximab

patients were identified (female: 85 %; age: 51 (41–66); disease duration: 10 (1–20) years; RF+: 85 %; ACPA+: 82 %; previous therapy with anti-TNFα agents: 70 %, with other biologic therapies: 21 %; concomitant therapy with methotrexate: 79 % (dosage: 15 (10–15) mg/week) and with prednisone: 94 % (dosage: 6.7 (3.6–10) mg/day)). Patients received a 1.000-mg infusion of rituximab preceded by a 100-mg intravenous pulse of methylprednisolone, at baseline (T = 0) and week 2. Tand B-cell counts were determined by four-color flow cytometry (Cytomics FC-500, Beckman Coulter Inc., USA) at T = 0 and 6 months after infusion (T = 6). Absolute cell count was determined by single-platform analysis using Flow-Count beads. At T = 6, according the EULAR Criteria of Response to the treatment, twenty-six patients obtained a moderate or good response, whereas seven patients did not respond. Patients of two groups had similar clinical disease activity at baseline (CRP-DAS28 = 5.32 (4.1–6.5) vs. 5.68 (5.1– 7.3), p = 0.12), as well as positivity for RF (85 vs. 86 %; p = 1) or ACPA (88 vs. 57 %; p = 0.09). Dividing patients by the positivity for autoantibodies or not, there was no significant difference with respect to the T-cell subpopulations at baseline and after 6 months. Comparing responders and non-responders, there was no difference in the percentage or absolute number of CD4+ T cells between the two groups at T0 (p = 0.88 and p = 0.5, respectively), but it should be acknowledged that the small number of non-responders may limit the possibility to detect differences. At T = 6, B-cell number was below 5 μl in 30/33 patients. The variations of CD3+CD4+ cells tended to be different in responders as compared with non-responders [median: −126 μl (−609.3 to +33) vs. +181 μl (+112 to +530.4); p:0.06]. The reduction in responders was Rituximab is a chimeric anti-CD20 monoclonal antibody that induces the depletion of mature B and pre-B cells and has been proven to be effective in the treatment of rheumatoid arthritis (RA) [1]. In the pathophysiology of RA, B cells play a central role, which is not limited to the production of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (ACPA), but includes secretion of cytokines and the capability to activate T cells [2, 3]. The roles of B and T cells should therefore be considered tightly linked. However, studies of lymphocyte subpopulation counts during rituximab treatment in patients with RA have been focused mainly on the B-cell compartment only. In a recent report, Melet et al. [4] described that rituximab induced a decrease in circulating T-cell number, mainly of CD4+ cells, in RA. The depletion of CD4+ T cells was substantial in some patient, with a decrease below 200 μl, a threshold carrying a risk for opportunistic infections in HIV+ individuals; [5] in 5.8 % of the cases. Of note, the decrease in CD4+ T cells was associated with clinical response. We have retrospectively evaluated our experience in RA patients who received their first course of rituximab at our institution between 2006 and 2013. Data are presented as percentage or median (10th–90th percentile). Nonparametric tests were used for the comparisons. Thirty-three

Effector T-cells are expanded in systemic lupus erythematosus patients with high disease activity and damage indexes

Background and objectives T-cell activation may be one of the pathogenic mechanisms of systemic lupus erythematosus (SLE). After repeated antigenic stimulation, T-cells undergo different modifications, leading to the differentiation into effector memory T-cells (CCR7−CD45RA−) and terminally differentiated effector memory (TDEM) T-cells (CCR7−CD45RA+). Similarly, down-modulation of CD28 may lead to the expansion of the CD28− T-cells, a subpopulation with peculiar effector activities. The aim of this study was the characterization of T-cell phenotype in a cohort of patients with SLE according to disease activity and damage index. Materials and methods Phenotypic analysis of peripheral blood T lymphocytes of 51 SLE patients and 21 healthy controls was done by flow-cytometry. SLE disease activity was evaluated by SLE Disease Activity Index-2000 (SLEDAI-2K) and damage by the Systemic Lupus International Collaborating Clinics/American College of Rheumatology damage index (SDI). The variations between different groups were evaluated by Mann–Whitney test. Bonferroni correction was applied to adjust for multiple comparisons (padj). Spearman rank test was used to evaluate the correlations between quantitative variables. Results CD4+ lymphopenia was found among SLE patients. Patients showed a trend for a higher percentage of TDEM among the CD4+ T-cell subpopulation in comparison with healthy controls (p = .04). SLE patients were divided into two groups according to disease activity: patients with SLEDAI-2K ≥ 6 (n = 13) had a higher percentage of circulating CD4+ T-cells with CD28− phenotype (padj = .005) as well as those with an effector memory (padj = .004) and TDEM (padj = .002) phenotype and a trend of decrease of regulatory T-cells (TREGs) (p = .02), in comparison with patients with low disease activity (n = 38). Patients with damage (SDI ≥ 1) tended to show an expansion of TDEM among CD4+ T-cells as compared with patients with no damage (p = .01). In SLE patients an inverse correlation was found between the percentages of TREGs and those of TDEM (p < .01) or CD4 + CD28− (p < .01) T-cells. Conclusions CD4+ T-cell subpopulations displaying phenotype characteristics of effector lymphocytes are proportionally expanded in patients with active SLE and a higher damage index. These findings may suggest a role of effector T-cells in the pathogenesis of the disease and in the mechanisms of damage in SLE.

The limited cutaneous form of systemic sclerosis is associated with urinary incontinence: an international multicentre study

Objectives The aim of this study was to explore the association between urinary incontinence (UI) and the main clinical and serological subsets of SSc, to assess risk factors for UI and its impact on quality of life (QoL). Methods UI and QoL were assessed through self-administered questionnaires in 334 patients with SSc from five European tertiary centres. Logistic regressions were performed to test the association between clinical forms, serological status and UI and to adjust for confounders. Further independent predefined SSc risk factors for UI were tested through a multivariable logistic model. Results The prevalence of UI was 63% (95% CI: 60, 68%). lcSSc and ACAs were both significantly associated with UI even after adjusting for age, sex, disability, diabetes, BMI, caffeine consumption, dyspnoea, faecal incontinence, abnormal bowel movement, presence of overlapping rheumatological disease and pulmonary hypertension [adjusted odds ratio (OR) = 2.4; 95% CI: 1.2, 4.7]. ACA and lcSSc doubled the risk of frequent and heavy urinary leaks. Factors independently associated with UI were as follows: lcSSc (OR = 2.2; 95% CI: 1.1, 3.2), ACA (OR = 2.8; 95% CI: 1.4, 5.8), female sex (OR = 10.8; 95% CI: 2.8, 41.3), worsening of dyspnoea (OR = 6.8; 95% CI: 1.2, 36.7), higher HAQ-DI (OR = 3.2; 95% CI: 1.5, 6.7), BMI (OR = 1.1; 95% CI: 1.0, 1.1) and active finger ulceration (OR = 0.3; 95% CI: 0.1, 0.7). Patients suffering from UI had decreased QoL. Conclusion Self-reported UI is frequent in SSc and disproportionally affects the limited cutaneous form of the disease and patients positive for ACA. Trial registration ClinicalTrials.gov, http://clinicaltrials.gov, NCT01971294.

A8.25 The effect of abatacept therapy on granzyme B serum levels in patients with rheumatoid arthritis

Background and objectives Rheumatoid arthritis (RA) is characterised by increased numbers of circulating CD28neg T-cells, a population displaying functional characteristic of cytotoxic memory cells, including expression of granzymes, a family of serine proteases playing key roles in the induction of target-cell death. Soluble granzymes (as granzyme B (GrB)) are elevated in sera from patients with RA, in which are associated with development of radiographic erosions. Abatacept (CTLA4-Ig; ABA) competing with the engagement of CD28 on T-cells, influences the subsequent T-cell activation. In patients treated with ABA, the number of circulating CD28neg T-cells decreases, suggesting that the drug can prevent the generation of CD28neg T-cell populations. Since CD28neg T-cells may be an important source of GrB, we have evaluated the effect of ABA therapy on GrB serum levels in patients with RA. Materials and methods Fifty-three RA patients, treated for at least 3 months with ABA were evaluated. Disease activity and response to the treatment were measured with DAS28 (based on CRP) and EULAR Criteria. T-cell counts were determined by flow cytometry. Serum GrB samples were collected before the first administration of ABA (T0) and then after 6 months (T6), and measured by an indirect solid-phase enzyme immunoassay with a sensitivity limit of 20 pg/ml. Results The percentage and the absolute number of circulating CD4+CD28-neg T-cells decreased after ABA therapy (T0 vs. T6: p = 0.018; p = 0.018, respectively), as well as those of CD8+CD28-neg T cells (T0 vs. T6: p = 0.005; p = 0.008, respectively). At T0, GrB serum levels were detectable in all RA patients, and were correlated with disease activity (p = 0.0022), and percentages of circulating CD4+CD28- (p = 0.007) and CD8+CD28- T-cells (p = 0.031). In 25 patients serum level of GrB were evaluated at T6: in 18 patients with a moderate or good clinical response to ABA the levels of GrB significantly decreased from T0 (median: 62.8 pg/mL[10th-90th percentile:45.8–116]) to T6 (53.8 [46.4–96.6]; p = 0.023), whereas no variation was observed in 7 non responders. The variation of GrB levels was directly correlated with that of DAS28-PCR (p = 0.040), but not with those of circulating CD28-neg T-cell subsets. Conclusions Costimulation blockade by ABA can decrease the serum levels of GrB in RA patients responding to the treatment, suggesting that this might be one of the mechanism by which ABA can prevent radiographic erosions. However, the lack of correlation of such decrease with the numbers of circulating CD28-neg T-cells suggests that these cells are not the main source of serum GrB.

Prevalence and disease-specific risk factors for Lower Urinary Tract symptoms in Systemic Sclerosis: an international multi-centric study

To determine the prevalence of lower urinary tract symptoms (LUTS) in systemic sclerosis (SSc), to find specific risk factors, and to assess their impact on quality of life (QoL).

A2.21 The increase of circulating CD4+ T-cells with effector phenotype in patients with systemic lupus erythematosus may be reverted after belimumab therapy

Background and objectives T-cell activation may be one of the pathogenic mechanisms of systemic lupus erythematosus (SLE). After repeated antigenic stimulation, T-cells undergo different modifications, leading to the differentiation into effector memory T-cells (CCR7-CD45RA-) and highly experienced memory T-cells (CCR7-CD45RA+). Similarly, down-modulation of CD28 may lead to the expansion of the CD28-T-cells, a subpopulation with peculiar effector activities. Recent studies showed that memory CD4+ T-cells are increased in the peripheral blood of SLE patients, whereas contradictory data are reported on CD28-T-cells. Belimumab is an anti-Blys therapy approved for SLE. The aims of this study were the characterisation of T-cell phenotype in a cohort of patients with SLE, according with disease activity, and the analysis of T-cell phenotype modifications after 6 months of therapy with belimumab. Materials and methods Phenotypic analysis of peripheral blood T lymphocytes was made by flow-cytometry. First, a cross-sectional study on 41 consecutive SLE patients was performed. Second, 7 patients treated with belimumab were longitudinally followed. Disease activity was evaluated by SLEDAI-2K score. Results SLE patients were divided in two groups according disease activity: patients with SLEDAI-2K ≥6 (n.6) had a higher percentage of circulating CD4+T-cells with CD28- phenotype (11 vs 2.5%, p = 0.01), as well as of those with an effector memory (34 vs 18%, p = 0.03), or highly experienced memory (8 vs 1%, p = 0.01) phenotype, in comparison with patients with low disease activity (n.35). After 6 months of treatment with belimumab, a trend toward a reduction of the CD4+CD28- T-cells was observed (from 10.5% to 4.6%; p:0.12). In particular, a reduction of CD4+CD28- T-cells showing an effector memory phenotype (from 31.6 to 26% of CD4+CD28- cells, p = 0.01) was found. Conclusions CD4+ T-cells subpopulations displaying phenotype characteristics of effector lymphocytes are proportionally expanded in patients with active SLE, but some of these abnormalities seems to be reverted by anti-BlyS therapy. Since the presence of BlyS receptor 3 on T cells and of a BLyS-dependent T-cell activation pathway have been well demonstrated, further studies are warranted to understand the possible effects of anti-BlyS therapy on T cells from SLE patients.