Sustained Remission Without Corticosteroids Among Patients With Pemphigus Who Had Rituximab as First-Line Therapy: Follow-Up of the Ritux 3 Trial.

Importance: The Ritux 3 trial demonstrated the short-term efficacy and safety of first-line treatment with rituximab compared with a standard corticosteroid regimen in pemphigus. No data on the long-term follow-up of patients who received rituximab as first line are available. Objective: To assess the long-term efficacy and safety of the Ritux 3 treatment regimen. Design, Setting, and Participants: This 7-year follow-up study of the Ritux 3 trial included patients with pemphigus from 25 dermatology departments in France from January 1, 2010, to December 31, 2015. Exposure: Patients were initially randomized in the rituximab plus prednisone group or prednisone-alone group. Main outcomes and measures: The primary outcome was the 5- and 7-year disease-free survival (DFS) without corticosteroids, assessed by Kaplan-Meier curves. Secondary outcomes were occurrence of relapse, occurrence of severe adverse events (SAEs), and evolution of antidesmoglein (Dsg) antibody enzyme-linked immunosorbent assay values to predict long-term relapse. Results: Of the 90 patients in the Ritux 3 trial, 83 were evaluated at the end of follow-up study visit (44 in the rituximab plus prednisone group; 39 in the prednisone-alone group) with a median (IQR) follow-up of 87.3 (79.1-97.5) months. Forty-three patients (93%) from the rituximab plus prednisone and 17 patients (39%) from the prednisone-alone group had achieved complete remission without corticosteroids at any time during the follow-up. Patients from the rituximab group had much longer 5- and 7-year DFS without corticosteroids than patients from the prednisone-alone group (76.7% and 72.1% vs 35.3% and 35.3%, respectively; P < .001), and had about half the relapses (42.2% vs 83.7%; P < .001). Patients who received rituximab as second-line treatment had shorter DFS than patients treated as first line (P = .007). Fewer SAEs were reported in the rituximab plus prednisone group compared with the prednisone-alone group, 31 vs 58 respectively, corresponding to 0.67 and 1.32 SAEs per patient, respectively (P = .003). The combination of anti-Dsg1 values of 20 or more IU/mL and/or anti-Dsg3 values of 48 or more IU/mL yielded 0.83 positive predictive value and 0.94 negative predictive value to predict long-term relapse. Conclusions and Relevance: In this secondary analysis of the Ritux 3 trail, first-line treatment of patients with pemphigus with the Ritux 3 regimen was associated with long-term sustained complete remission without corticosteroid therapy without any additional maintenance infusion of rituximab.

Modifications of the BAFF/BAFF-Receptor Axis in Patients With Pemphigus Treated With Rituximab Versus Standard Corticosteroid Regimen.

The efficacy of the B-cell-depleting agent rituximab has been reported in immune diseases but relapses are frequent, suggesting the need for repeated infusions. The B-cell activating factor (BAFF) is an important factor for B cell survival, class switch recombination and selection of autoreactive B cells, as well as maintaining long-lived plasma cells. It has been hypothesized that relapses after rituximab might be due to the increase of serum BAFF levels. From the Ritux3 trial, we showed that baseline serum BAFF levels were higher in pemphigus patients than in healthy donors (308 ± 13 pg/mL versus 252 ± 28 pg/mL, p=0.037) and in patients with early relapse compared who didn't (368 ± 92 vs 297 ± 118 pg/mL, p=0.036). Rituximab and high doses of CS alone have different effects on the BAFF/BAFF-R axis. Rituximab led to an increase of BAFF levels associated to a decreased mRNA (Day 0: 12.3 ± 7.6 AU vs Month 36: 3.3 ± 4.3 AU, p=0.01) and mean fluorescence intensity of BAFF-R in non-autoreactive (Day 0: 3232 vs Month 36: 1527, mean difference: 1705, 95%CI: 624 to 2786; p=0.002) as well as on reappearing autoreactive DSG-specific B cells (Day 0: 3873 vs Month 36: 2688, mean difference: 1185, 95%CI: -380 to 2750; p=0.20). Starting high doses of corticosteroids allowed a transitory decrease of serum BAFF levels that re-increased after doses tapering whereas it did not modify BAFF-R expression in autoreactive and non-autoreactive B cells. Our results suggest that the activation of autoreactive B cells at the onset of pemphigus is likely to be related to the presence of high BAFF serum levels and that the decreased BAFF-R expression after rituximab might be responsible for the delayed generation of memory B cells, resulting in a rather long period of mild pemphigus activity after rituximab therapy. Conversely, the incomplete B cell depletion and persistent BAFF-R expression associated with high BAFF serum levels might explain the high number of relapses in patients treated with CS alone.

Rituximab and Corticosteroid Effect on Desmoglein-Specific B Cells and Desmoglein-Specific T Follicular Helper Cells in Pemphigus.

Pemphigus is an autoimmune blistering disease mediated by autoantibodies directed against desmogleins (DSGs). We recently showed that first-line treatment with rituximab (RTX) enables more patients to achieve long-lasting remission off therapy than corticosteroids alone. To understand the immunological mechanisms that mediate long-lasting clinical remission after RTX treatment, we analyzed the phenotype of DSG-specific memory B cells and DSG-specific T follicular helper cells by flow cytometry and measured antibody-secreting cells by enzyme-linked immune absorbent spot in patients treated with corticosteroids alone or RTX. This post hoc analysis of the RITUX3 trial showed that RTX induced a significant decrease of IgG-switched DSG-specific memory B cells. Accordingly, anti-DSG antibody-secreting cells were no longer detected in patients in complete remission after RTX. In contrast, corticosteroids did not modify the frequency or the phenotype of DSG-specific memory B cells, and anti-DSG antibody-secreting cells were still detected after treatment, even in patients in remission. Using peptide-HLADRB1∗0402 tetramer staining, we identified DSG-3-specific T follicular helper cells, which dramatically decreased after RTX, while remaining stable after corticosteroid treatment. Our findings suggest that long-lasting response to RTX in pemphigus relies on the decrease of DSG-specific circulating T follicular helper cells, which correlates with a sustained depletion of IgG-switched memory autoreactive B cells, leading to the disappearance of anti-DSG antibody-secreting cells.

Methods to Study the Transcriptome of Regulatory B Cells.

Regulatory B cells do not constitute a distinct cell lineage because no unique marker or set of markers can exclusively identify neither murine nor human regulatory B cells, and efficient IL-10 production is their only known distinguishing feature. After purification of IL-10-secreting B cells, one may want to characterize them by analyzing their gene expression profile. This goal can be achieved by using different technologies: RT-qPCR, microarrays, Nanostring's nCounter technology, Biomark HD are techniques that will allow you to analyze their gene expression, whether in a targeted (RT-qPCR), extended but targeted (Nanostring's nCounter technology, Biomark HD) or exhaustive (Microarray) way. Aim of this chapter is the description of these techniques in the view of their application to the study and characterization of regulatory B cells.

B-cell depletion induces a shift in self antigen specific B-cell repertoire and cytokine pattern in patients with bullous pemphigoid.

Bullous Pemphigoid is the most common auto-immune bullous skin disease. It is characterized by the production of auto-antibodies directed against 2 proteins of the hemi-desmosome (BP180 and BP230). We assessed the efficacy and mechanisms of action of rituximab, an anti-CD20 monoclonal antibody, in 17 patients with severe and relapsing type of bullous pemphigoid. The phenotype, cytokine gene expression, and rearrangement of BP180-specific B-cell receptor genes were performed over 2 years following treatment. At the end of the study, 5 patients had died, 3 had withdrawn from the study, and 9 patients were in complete remission. The one- and two-year relapse rates were 44.1% (95% Confidence Interval (CI): 21.0-76.0%) and 66.5%, (95% CI: 38.4-91.4%), respectively. Phenotypic analyses confirmed dramatic B-cell depletion, which lasted for 9 to 12 months. The ELISA values of serum anti-BP180 antibodies and the frequency of BP180-specific circulating B cells decreased dramatically following treatment, which paralleled the improvement of skin lesions. During B-cell reconstitution, a polyclonal IgM repertoire appeared and a shift in the rearrangement of the B-cell receptor genes of BP180-specific circulating B cells was observed. Concurrently, we observed a decrease of IL-15, IL-6 and TNFα expressing BP180-specific B cells, and the emergence of IL-10 and IL-1RA-expressing BP180-specific IgM+ B cells in patients in complete remission off therapy, suggesting the functional plasticity of BP180-specific auto-immune B cells after rituximab treatment.

First-line rituximab combined with short-term prednisone versus prednisone alone for the treatment of pemphigus (Ritux 3): a prospective, multicentre, parallel-group, open-label randomised trial.

BACKGROUND: High doses of corticosteroids are considered the standard treatment for pemphigus. Because long-term corticosteroid treatment can cause severe and even life-threatening side-effects in patients with this disease, we assessed whether first-line use of rituximab as adjuvant therapy could improve the proportion of patients achieving complete remission off-therapy, compared with corticosteroid treatment alone, while decreasing treatment side-effects of corticosteroids. METHODS: We did a prospective, multicentre, parallel-group, open-label, randomised trial in 25 dermatology hospital departments in France (Ritux 3). Eligible participants were patients with newly diagnosed pemphigus aged 18-80 years being treated for the first time (not at the time of a relapse). We randomly assigned participants (1:1) to receive either oral prednisone alone, 1·0 or 1·5 mg/kg per day tapered over 12 or 18 months (prednisone alone group), or 1000 mg of intravenous rituximab on days 0 and 14, and 500 mg at months 12 and 18, combined with a short-term prednisone regimen, 0·5 or 1·0 mg/kg per day tapered over 3 or 6 months (rituximab plus short-term prednisone group). Follow-up was for 3 years (study visits were scheduled weekly during the first month of the study, then monthly until month 24, then an additional visit at month 36). Treatment was assigned through central computer-generated randomisation, with stratification according to disease-severity (severe or moderate, based on Harman's criteria). The primary endpoint was the proportion of patients who achieved complete remission off-therapy at month 24 (intention-to-treat analysis). This study is registered with ClinicalTrials.gov, number NCT00784589. FINDINGS: Between May 10, 2010, and Dec 7, 2012, we enrolled 91 patients and randomly assigned 90 to treatment (90 were analysed; 1 patient withdrew consent before the random assignment). At month 24, 41 (89%) of 46 patients assigned to rituximab plus short-term prednisone were in complete remission off-therapy versus 15 (34%) of 44 assigned to prednisone alone (absolute difference 55 percentage points, 95% CI 38·4-71·7; p<0·0001. This difference corresponded to a relative risk of success of 2·61 (95% CI 1·71-3·99, p<0·0001), corresponding to 1·82 patients (95% CI 1·39-2·60) who would need to be treated with rituximab plus prednisone (rather than prednisone alone) for one additional success. No patient died during the study. More severe adverse events of grade 3-4 were reported in the prednisone-alone group (53 events in 29 patients; mean 1·20 [SD 1·25]) than in the rituximab plus prednisone group (27 events in 16 patients; mean 0·59 [1·15]; p=0·0021). The most common of these events in both groups were diabetes and endocrine disorder (11 [21%] with prednisone alone vs six [22%] with rituximab plus prednisone), myopathy (ten [19%] vs three [11%]), and bone disorders (five [9%] vs five [19%]). INTERPRETATION: Data from our trial suggest that first-line use of rituximab plus short-term prednisone for patients with pemphigus is more effective than using prednisone alone, with fewer adverse events. FUNDING: French Ministry of Health, French Society of Dermatology, Roche.

Human regulatory B cells combine phenotypic and genetic hallmarks with a distinct differentiation fate.

Regulatory B cells (B-reg) produce IL-10 and suppress inflammation in both mice and humans, but limited data on the phenotype and function of these cells have precluded detailed assessment of their contribution to host immunity. In this article, we report that human B-reg cannot be defined based on a phenotype composed of conventional B cell markers, and that IL-10 production can be elicited in both the CD27(+) memory population and naive B cell subset after only a brief stimulation in vitro. We therefore sought to obtain a better definition of IL-10-producing human B-regs using a multiparameter analysis of B cell phenotype, function, and gene expression profile. Exposure to CpG and anti-Ig are the most potent stimuli for IL-10 secretion in human B cells, but microarray analysis revealed that human B cells cotreated with these reagents resulted in only ∼0.7% of genes being differentially expressed between IL-10(+) and IL-10(-) cells. Instead, connectivity map analysis revealed that IL-10-secreting B cells are those undergoing specific differentiation toward a germinal center fate, and we identified a CD11c(+) B cell subset that was not capable of producing IL-10 even under optimal conditions. Our findings will assist in the identification of a broader range of human pro-B-reg populations that may represent novel targets for immunotherapy.

Drug Reaction with Eosinophilia and Systemic Symptoms: an update on pathogenesis.

The syndrome termed 'Drug Reaction with Eosinophilia and Systemic Symptoms' (DRESS) is an unpredictable, life-threatening condition associated with adverse reactions to therapy. Although the etiology of DRESS is poorly understood, genetic susceptibility markers have been identified within the HLA complex and there are several prevailing models of pathogenesis. Modification of host antigens by haptens (drugs or their metabolites), or non-covalent drug binding to endogenous proteins (the p-i concept), may drive pro-inflammatory immune responses in patients. Alternatively, a viral trigger for DRESS has been proposed based on the concomitant detection of herpesviruses and the recent demonstration of Epstein-Barr virus-specific immune responses in DRESS patients. In the present review, we discuss the latest findings concerning the pathogenesis of drug reactions and known risk factors for DRESS.

Critical role of TLR2 and TLR4 in autoantibody production and glomerulonephritis in lpr mutation-induced mouse lupus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathogenic autoantibodies directed against nuclear Ags and immune complex deposits in damaged organs. Environmental factors have been thought to play a role in the onset of the disease. The recognition of these factors is mediated by TLRs, in particular TLR2 and TLR4 which bind pathogen-associated molecular patterns of Gram(+) and Gram(-) bacteria, respectively. We attempted to determine the role of these TLRs in SLE by creating TLR2- or TLR4-deficient C57BL/6(lpr/lpr) mice. These mice developed a less severe disease and fewer immunological alterations. Indeed, in C57BL/6(lpr/lpr)-TLR2 or -TLR4-deficient mice, glomerular IgG deposits and mesangial cell proliferation were dramatically decreased and antinuclear, anti-dsDNA, and anti-cardiolipin autoantibody titers were significantly reduced. However, the response against nucleosome remained unaffected, indicating a role of TLR2 and TLR4 in the production of Abs directed against only certain categories of SLE-related autoantigens. Analysis of B cell phenotype showed a significant reduction of marginal zone B cells, particularly in C57BL/6(lpr/lpr)-TLR4-deficient mice, suggesting an important role of TLR4 in the sustained activation of these cells likely involved in autoantibody production. Interestingly, the lack of TLR4 also affected the production of cytokines involved in the development of lupus disease.

Functional tolerance of CD8+ T cells induced by muscle-specific antigen expression.

Skeletal muscles account for more than 30% of the human body, yet mechanisms of immunological tolerance to this tissue remain mainly unexplored. To investigate the mechanisms of tolerance to muscle-specific proteins, we generated transgenic mice expressing the neo-autoantigen OVA exclusively in skeletal muscle (SM-OVA mice). SM-OVA mice were bred with OT-I or OT-II mice that possess a transgenic TCR specific for OVA peptides presented by MHC class I or class II, respectively. Tolerance to OVA did not involve clonal deletion, anergy or an increased regulatory T cell compartment. Rather, CD4+ T cell tolerance resulted from a mechanism of ignorance revealed by their response following OVA immunization. In marked contrast, CD8+ T cells exhibited a loss of OVA-specific cytotoxic activity associated with up-regulation of the immunoregulatory programmed death-1 molecule. Adoptive transfer experiments further showed that OVA expression in skeletal muscle was required to maintain this functional tolerance. These results establish a novel asymmetric model of immunological tolerance to muscle autoantigens involving Ag ignorance for CD4+ T cells, whereas muscle autoantigens recognized by CD8+ T cells results in blockade of their cytotoxic function. These observations may be helpful for understanding the breakage of tolerance in autoimmune muscle diseases.

Active Ca2+/calmodulin-dependent protein kinase II gamma B impairs positive selection of T cells by modulating TCR signaling.

T cell development is regulated at two critical checkpoints that involve signaling events through the TCR. These signals are propagated by kinases of the Src and Syk families, which activate several adaptor molecules to trigger Ca(2+) release and, in turn, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation. In this study, we show that a constitutively active form of CaMKII antagonizes TCR signaling and impairs positive selection of thymocytes in mice. Following TCR engagement, active CaMKII decreases TCR-mediated CD3zeta chain phosphorylation and ZAP70 recruitment, preventing further downstream events. Therefore, we propose that CaMKII belongs to a negative-feedback loop that modulates the strength of the TCR signal through the tyrosine phosphatase Src homology 2 domain-containing phosphatase 2 (SHP-2).