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IntroductionHepatitis C virus (HCV) causes mixed cryoglobulinemia (MC) by driving clonal expansion of B cells expressing B cell receptors (BCRs), often encoded by the VH1-69 variable gene, endowed with both rheumatoid factor (RF) and anti-HCV specificity. These cells display an atypical CD21low phenotype and functional exhaustion evidenced by unresponsiveness to BCR and Toll-like receptor 9 (TLR9) stimuli. Although antiviral therapy is effective on MC vasculitis, pathogenic B cell clones persist long thereafter and can cause virus-independent disease relapses.MethodsClonal B cells from patients with HCV-associated type 2 MC or healthy donors were stimulated with CpG or heath-aggregated IgG (as surrogate immune complexes) alone or in combination; proliferation and differentiation were then evaluated by flow cytometry. Phosphorylation of AKT and of the p65 NF-kB subunit were measured by flow cytometry. TLR9 was quantified by qPCR and by intracellular flow cytometry, and MyD88 isoforms were analyzed using RT-PCR.DiscussionWe found that dual triggering with autoantigen and CpG restored the capacity of exhausted VH1-69pos B cells to proliferate. The signaling mechanism for this BCR/TLR9 crosstalk remains elusive, since TLR9 mRNA and protein as well as MyD88 mRNA were normally expressed and CpG-induced phosphorylation of p65 NF-kB was intact in MC clonal B cells, whereas BCR-induced p65 NF-kB phosphorylation was impaired and PI3K/Akt signaling was intact. Our findings indicate that autoantigen and CpG of microbial or cellular origin may unite to foster persistence of pathogenic RF B cells in HCV-cured MC patients. BCR/TLR9 crosstalk might represent a more general mechanism enhancing systemic autoimmunity by the rescue of exhausted autoreactive CD21low B cells.

Summary The objective of this study was to evaluate the predictive role of CD21low B cells as markers of new digital ulcers in systemic sclerosis patients. Peripheral blood B cell subpopulations and clinical assessments have been evaluated in 74 systemic sclerosis patients at baseline and after a 12‐month follow‐up. After a 12‐month follow‐up, 23 (31.1%) systemic sclerosis patients developed new digital ulcers. The median percentage of CD21low B cells was significantly higher in patients with than without new digital ulcers [10.1 (4.3–13.6) versus 4.8 (3.5–7.4); p, CD21low B cells are increased in SSc patients. CD21low B cells are higher in SSc patients with major vascular complications. CD21low B cells may be a possible predictive marker of new DUs.

Objectives The biomarkers of an immunological dysregulation due to a chronic HBV infection are indeed understudied. If untreated, this condition may evolve into liver impairment co-occurring with extrahepatic involvements. Here, we aim to identify a new panel of biomarkers [including immunoglobulin G (IgG) subclasses, RF, and Free Light Chains (FLCs)] that may be useful and reliable for clinical evaluation of HBV-related cryoglobulinemia. Methods We retrospectively analysed clinical data from 44 HBV-positive patients. The patients were stratified (according to the presence/absence of mixed cryoglobulinemia) into two groups: 22 with cryoglobulins (CGs) and 22 without CGs. Samples from 20 healthy blood donors (HDs) were used as negative controls. Serum samples were tested for IgG subclasses, RF (-IgM, -IgG, and -IgA type), and FLCs. Results We detected a strikingly different distribution of serum IgG subclasses between HDs and HBV-positive patients, together with different RF isotypes; in addition, FLCs were significantly increased in HBV-positive patients compared with HDs, while no significant difference was shown between HBV-positive patients with/without mixed cryoglobulinemia. Conclusion The immune-inflammatory response triggered by HBV may be monitored by a peculiar profile of biomarkers. Our results open a new perspective in the precision medicine era; in these challenging times, they could also be employed to monitor the clinical course of those COVID-19 patients who are at high risk of HBV reactivation due to liver impairment and/or immunosuppressive therapies.

CD38 is a multifunctional protein expressed on the surface of B cells in healthy individuals but also in B cell malignancies. Previous studies have suggested a connection between CD38 and components of the IgM class B cell antigen receptor (IgM-BCR) and its coreceptor complex. Here, we provide evidence that CD38 is closely associated with CD19 in resting B cells and with the IgM-BCR upon engagement. We show that targeting CD38 with an antibody, or removing this molecule with CRISPR/Cas9, inhibits the association of CD19 with the IgM-BCR, impairing BCR signaling in normal and malignant B cells. Together, our data suggest that CD38 is a new member of the BCR coreceptor complex, where it exerts a modulatory effect on B cell activation upon antigen recognition by regulating CD19. Our study also reveals a new mechanism where α-CD38 antibodies could be a valuable option in therapeutic approaches to B cell malignancies driven by aberrant BCR signaling.

Hepatitis C virus (HCV) causes B-cell lymphoproliferative disorders (LPDs) expressing stereotyped B-cell receptors (BCRs) endowed with rheumatoid factor (RF) activity and putatively recognizing the HCV E2 protein. To further untangle the shaping and function of these BCRs, we analyzed immunoglobulin gene rearrangements of monoclonal B cells from 13 patients with HCV-associated LPDs and correlated their features with the clinical outcomes of antiviral therapy. While only two patients shared a stereotyped heavy-chain complementarity determining region 3 (CDR3) sequence, two kappa chain CDR3 stereotyped sequences accounted for 77% of BCRs. Light chains were enriched in sequences homologous to anti-HCV E2 antibodies compared with heavy chains (7/13 vs. 0/13; p = 0.005). Anti-HCV E2 homology was uniquely associated (7/7 vs. 0/6; p = 0.0006) with a stereotyped CDR3 sequence encoded by IGKV3-20/3D-20 gene(s) accounting for 54% of BCRs. An IGKV3-15/IGKJ1-encoded stereotyped sequence homologous to WA RF accounted for 23% of BCRs. LPDs expressing KCDR3s homologous to anti-HCV E2 antibodies responded more frequently to the eradication of HCV by antiviral therapy (6/6 vs. 1/6; p = 0.015). These findings, although limited by the small sample size, suggest that a stereotyped KCDR3 may predominantly shape anti-HCV specificity of BCRs, possibly providing a signature that may help identifying bona fide HCV-dependent LPDs.

Patients with autoimmune systemic diseases (ASDs) represent a frail population during the ongoing COVID-19 pandemic. The vaccination is the major preventive measure; however, a significant number of ASD patients show an impaired production of anti-COVID-19 neutralizing antibodies (NAb), possibly counterbalanced by adequate T-cell response. The present study aimed at evaluating both humoral and cellular response to COVID-19 vaccine booster dose in this particular setting.Serum NAb titer and T-cell response (measuring interferon gamma -IFN-γ- release) were evaluated 3 weeks after the COVID-19 vaccine booster dose, in 17 patients (12 F, mean age 68.8 ± 15.3 SD yrs) with different ASDs, compared to 17 healthy controls (HCs).The analysis excluded one patient reporting symptoms of COVID-19 only after the immunogenicity tests had been performed.The NAb levels were significantly lower in ASD compared to HCs (p 0.0001); moreover, patients showed a higher percentage of negative/sub-optimal humoral response (31% vs 0% of HCs; p = 0.0184).The study of cellular response showed lower levels of IFN-γ for both Ag1 (The impaired immunogenicity to COVID-19 vaccine booster and even more the concomitant lack of both humoral and cellular response might represent a high risk for severe COVID-19, particularly in ASD patients undergoing DMT.These frail subjects should be tightly monitored for their immune protection and prioritized for the fourth dose of COVID-19 vaccine. Moreover, in the occurrence of SARS-CoV2 infection, treatments with specific monoclonal antibodies and/or antivirals may be highly recommendable.

Autoimmune systemic diseases (ASD) may show impaired immunogenicity to COVID-19 vaccines. Our prospective observational multicenter study aimed to evaluate the seroconversion after the vaccination cycle and at 6-12-month follow-up, as well the safety and efficacy of vaccines in preventing COVID-19. The study included 478 unselected ASD patients (mean age 59 ± 15 years), namely 101 rheumatoid arthritis (RA), 38 systemic lupus erythematosus (SLE), 265 systemic sclerosis (SSc), 61 cryoglobulinemic vasculitis (CV), and a miscellanea of 13 systemic vasculitis. The control group included 502 individuals from the general population (mean age 59 ± 14SD years). The immunogenicity of mRNA COVID-19 vaccines (BNT162b2 and mRNA-1273) was evaluated by measuring serum IgG-neutralizing antibody (NAb) (SARS-CoV-2 IgG II Quant antibody test kit; Abbott Laboratories, Chicago, IL) on samples obtained within 3 weeks after vaccination cycle. The short-term results of our prospective study revealed significantly lower NAb levels in ASD series compared to controls [286 (53–1203) vs 825 (451–1542) BAU/mL, p < 0.0001], as well as between single ASD subgroups and controls. More interestingly, higher percentage of non-responders to vaccine was recorded in ASD patients compared to controls [13.2% (63/478), vs 2.8% (14/502); p < 0.0001]. Increased prevalence of non-response to vaccine was also observed in different ASD subgroups, in patients with ASD-related interstitial lung disease (p = 0.009), and in those treated with glucocorticoids (p = 0.002), mycophenolate-mofetil (p < 0.0001), or rituximab (p < 0.0001). Comparable percentages of vaccine-related adverse effects were recorded among responder and non-responder ASD patients. Patients with weak/absent seroconversion, believed to be immune to SARS-CoV-2 infection, are at high risk to develop COVID-19. Early determination of serum NAb after vaccination cycle may allow to identify three main groups of ASD patients: responders, subjects with suboptimal response, non-responders. Patients with suboptimal response should be prioritized for a booster-dose of vaccine, while a different type of vaccine could be administered to non-responder individuals.

Studies on the safety and immunogenicity of SARS-CoV-2 vaccination in patients with inflammatory rheumatic diseases have so far not included mixed cryoglobulinaemia (MC) vasculitis.1–3 We report a prospective observational multicentre study on this disorder. Participants were followed at four tertiary referral centres and were instructed to promptly inform the attending physicians about unusual events felt as possibly related to vaccination. Seventy-one patients were recruited: they had infection-cured hepatitis C virus (HCV)-related MC, either uncomplicated (HCV-MC, n=50) or complicated by low-grade non-Hodgkin’s lymphoma (MC-NHL, n=8), or essential MC (EMC, n=13). The characteristics of the patients, exclusion criteria and definition of bona fide vaccination-related flare are described in online supplemental methods.### Supplementary data [annrheumdis-2021-221248supp001.pdf] Overall, 9 of 71 (12.7%) patients had postvaccination MC vasculitis flare. However, 8 of 71 patients had experienced within 12 months before vaccination spontaneous flares, where 7 cases required rituximab and 3 of them (37.5%) had postvaccination flare (see online supplemental information). Thus, to exclude the confounding effects of high proneness to spontaneous flare as the facilitator and of rituximab as the preventor, we further restricted the evaluation of postvaccination flare rate to 63 patients off-therapy and without spontaneous flares for 20–48 months before …

In the past months, mass vaccination represented the turning point of the global battle against the COVID-19 pandemic, an unprecedented challenge for physicians, healthcare professionals, health systems and pharmaceutical companies. More than 6 billion doses of vaccine have been administered to date, covering nearly 50% of the world’s population. Although the vaccination campaign is still thwarted by spread of fake news disseminated by a ubiquitous antivaxxer movement, accumulating real-life data1 confirm the favourable safety profile already demonstrated in phase III clinical trials.2 Despite the lack of a steady literature evidence,3 the potential role of vaccines in promoting autoimmunity continues to intrigue many researchers. The theoretical basis of this association relies on the possible molecular mimicry between macromolecular components of the vaccine and specific human proteins and the exuberant immune response elicited by adjuvants contained in vaccines.4 Adverse events (AEs) associated with COVID-19 vaccines are usually mild and mainly restricted to injection site reactions. Interestingly, among systemic AEs, arthralgia is one of the most common.2 To the best of our knowledge, only isolated cases5 of arthritis developed after COVID-19 …

Mixed cryoglobulinaemic vasculitis (MCV) is an immune-complex-mediated systemic vasculitis characterised by heterogeneous clinical manifestations mainly involving lymphatic system, skin, kidney and peripheral nervous system. Although MCV patients have been included in priority programs for vaccination against SARS-CoV-2 in Italy, limited information is available for these patients. Aims of this multicentre Italian study were to investigate SARS-CoV-2 vaccination rate in MCV patients and its safety profile.All MCV patients referring to participating centres were assessed with an interview-based survey about vaccination, reasons for not getting vaccinated, adverse events (AE), and disease flares within a month after vaccination.A total of 416 patients were included in the study. Among participants, 7.7% did not get vaccinated, mainly for fear related to vaccine side-effects (50%) or medical decision (18.8%). They were more frequently treated with chronic glucocorticoids or rituximab (p=0.049 and p=0.043, respectively). Mild and self-limiting AE were recorded in 31.7% of cases, while post-vaccination vasculitis flares were observed in 5.3% of subjects. Disease relapses were mainly observed in patients with peripheral neuropathy or skin vasculitis (40% and 25%, respectively).Vaccination against SARS-CoV-2 has been performed in a high percentage of MCV patients with encouraging safety profile. Vasculitis flares rate was in line with that observed for other autoimmune diseases, despite patients with purpura or peripheral neuropathy seem to be at risk for symptoms' exacerbation. Patients' hesitancy, rituximab and glucocorticoids treatment were the main reasons for delaying vaccination.

Autoimmune systemic diseases (ASD) show impaired immunogenicity to COVID-19 vaccines. Our prospective observational multicenter study aimed at evaluating the seroconversion elicited by COVID-19 vaccine over the entire vaccination cycle including the booster dose. Among 478 unselected ASD patients originally evaluated at the end of the first vaccination cycle (time 1), 344 individuals were re-evaluated after a 6-month period (time 2), and 244 after the booster vaccine dose (time 3). The immunogenicity of mRNA COVID-19 vaccines (BNT162b2 and mRNA-1273) was assessed by measuring serum IgG-neutralizing antibody (NAb) on samples obtained at the three time points in both patients and 502 age-matched controls. In the 244 ASD group that received booster vaccine and monitored over the entire follow-up, the mean serum NAb levels (time 1, 2, and 3: 696.8±52.68, 370.8±41.92, and 1527±74.16SD BAU/mL, respectively; p&nbsp

Summary Several reports have highlighted the abnormal increments of serum immunoglobulin free light chains (FLCs) in the course of systemic autoimmune rheumatic diseases (SARD), but a comparative analysis among different conditions is still lacking. A strong association between elevated FLC and hepatitis C virus (HCV)-related mixed cryoglobulinaemia (HCVMC) has been well established. Here, we aimed to analyse serum FLC levels in patients with four different SARD in comparison with HCVMC. Using a turbidimetric assay, free κ and λ chains were quantified in sera from 198 SARD patients (37 rheumatoid arthritis, RA; 47 systemic lupus erythematosus, SLE; 52 anti-phospholipid syndrome, APS; 62 primary Sjogren's syndrome, pSS), 62 HCVMC and 50 healthy blood donors (HD). All patient groups showed increased κ levels when compared to HD: 33·5 ± 2·6 mg/l in HCVMC, 26·7 ± 2·3 mg/l in RA, 29·7 ± 1·9 mg/l in SLE, 23·8 ± 1·1 mg/l in APS, 24·2 ± 1·1 mg/l in pSS; 10·1 ± 0·6 mg/l in HD. Free λ levels displayed a significant increase only for HCVMC (20·4 ± 1·4 mg/l) and SLE (18·4 ± 1·0 mg/l) compared to HD (13·6 ± 0·9 mg/l). The increase of κ compared to λ takes into account a κ /λ ratio of 1·6 for all groups. Our results substantially analyse and strengthen the association between FLC and SARD focusing the questions regarding their role in the pathogenesis and diagnosis of human diseases. Unfortunately, the biochemical differences distinguishing normal from pathological FLC have not been identified. Production of different isotypes is probably connected to still-unknown pathways.

Summary Circulating free light chains (FLCs), considered biomarkers of B cell activity, are frequently elevated in patients affected by systemic inflammatory autoimmune diseases. As the systemic sclerosis (SSc) clinical course can be variable, this study is aimed at evaluating FLCs levels in affected individuals as biomarkers of disease activity. We assessed FLC levels in serum and urine of 72 SSc patients and 30 healthy controls (HC). Results were analyzed in comparison with overall clinical and laboratory findings, disease activity index (DAI) and disease severity scale (DSS). SSc patients displayed increased levels of κ and λ FLC in serum significantly higher than HC (p = 0.0001) alongside the mean values of free κ/λ ratio and κ + λ sum (p = 0.0001). SSc patients showed increased free κ in urine with a κ/λ higher than HC (p = 0.0001). SSc patients with increased κ + λ in serum showed that erythro-sedimentation rate (p = 0.034), C-reactive protein (p = 0.003), DAI (p = 0.024) and DSS (p = 0.015) were higher if compared to SSc patients with normal levels of FLC. A positive linear correlation was found between serum levels of free κ and DAI (r = 0.29, p = 0.014). In addition, SSc patients with increased free κ in urine had higher DAI (p = 0.048) than SSc patients with normal κ levels. Our results strengthen the role of serum FLC as useful biomarker in clinical practice to early diagnosis and monitor disease activity, showing for the first time that also urine FLC levels correlated with disease activity in SSc patients.

Background: Glucocorticoids (GCs) plus rituximab (RTX) represent the first-line treatment of nonviral mixed cryoglobulinemia vasculitis (CryoVas). However, data on therapeutic management and outcome of patients refractory to RTX are lacking. Methods: We conducted a European collaborative retrospective multicenter study of patients with nonviral mixed CryoVas refractory to RTX and performed a literature review. Results: Twenty-six original cases and 7 additional patients from the literature were included. All patients but one had type 2 cryoglobulinemia, and causes were autoimmune disease (51%), malignant hemopathy (12%) or essential CryoVas (42%). CryoVas was primary refractory to RTX in 42%, while 58% had an initial response to RTX before immune escape. After RTX failure, patients received a median of 1 (IQR, 1–3) line of treatment, representing 65 treatment periods during follow-up. Main treatments used were GCs in 92%, alkylating agents in 43%, RTX in combination with other treatments in 46%, and belimumab in 17%. Combination of anti-CD20 plus belimumab, alkylating agents alone and anti-CD20 plus alkylating agents provided the highest rates of clinical response in 100% 82% and 73%, respectively, but showed poor immunological response, in 50%, 30% and 38%, respectively. Rates of severe infection were 57%, 9% and 0% in patients receiving anti-CD20 plus belimumab, alkylating agents alone and anti-CD20 plus alkylating agents, respectively. Conclusion: In patients with nonviral mixed CryoVas refractory to RTX, anti-CD20 plus belimumab, and alkylating agents associated or not with anti-CD20, provide the highest rates of clinical response. However, anti-CD20 plus belimumab was frequently associated with severe infections.

Objectives To evaluate expansion of CD21low B cells and their role in B cell homeostasis, apoptosis, clinical manifestations and serum vascular endothelial growth factor (VEGF) in systemic sclerosis (SSc). Materials and methods B-cells subpopulations and apoptosis have been assessed in 74 SSc patients and 20 healthy donors. Renal Doppler ultrasound, echocardiography, pulmonary function test and VEGF were performed. Results SSc patients with expanded CD21low B cells (SSc-CD21low) show a distinct B cell profile with increased memory B cells compared to patients without CD21low B cells (SSc-CD21+). Renal resistive index, systolic pulmonary arterial pressure and FVC/DLCO ratio were significantly higher in SSc-CD21low group than SSc-CD21+, DLCO was lower in SSc-CD21low group than SSc-CD21+. We found a positive linear correlation between CD21low and sPAP, RI and FVC/DLCO ratio whereas a negative correlation was observed between CD21low and DLCO and VEGF levels. Conclusions CD21low B cells are increased in SSc patients with visceral vascular manifestations.

Hepatitis C virus (HCV) represents the major risk factor for mixed cryoglobulinemia (MC), a small-vessel vasculitis that may evolve into an overt B-cell non-Hodgkin's lymphoma. Here, we aimed to identify a biomarker signature for the early diagnosis of minimal residual disease (MRD). We assessed free light chains (FLCs), IgM k,and IgM λ heavy/light chain (HLC) pairs, and vascular endothelial growth factor (VEGF) in sera from 34 patients with MC vasculitis (32 HCV- and 2 HBV-related), treated with low-dose rituximab (RTX). FLCs and IgM HLCs were measured by turbidimetric assay; VEGF by an enzyme-linked immunosorbent assay. After RTX, the positive (complete + partial) clinical and laboratory responses were of 85.29% and 50%, respectively; in contrast, the mean levels of FLCs, IgM HLCs, and VEGF were substantially unaffected in most patients and still above the normal range. In those achieving a reduction of FLCs and IgM k and λ chains values within the range of normality, we found that post-treatment free λ chains and IgM k values correlated with clinical and laboratory response. Our results suggest that high levels of FLCs, IgM HLCs, and VEGF could represent the signature of "dormant" B cell clones' activity that could be very useful to identify MRD indicative of possible relapse or worsening outcome.

Hepatitis C virus (HCV) infection affects about 70 million people worldwide. HCV is responsible for both hepatitis and extra-hepatic manifestations. Chronic infection has been shown to develop in about 70% of cases and can progress to cirrhosis or hepatocellular carcinoma. Ten percent of HCV patients may develop extra-hepatic manifestations, including mixed cryoglobulinemia (MC) and non-Hodgkin lymphomas. Many studies have demonstrated that, after antiviral therapy, MC can disappear along with HCV eradication. After the introduction of the new direct antiviral agents (DAAs), the combination of pegylated interferon and ribavirin has been abandoned. Several studies on new DAAs have reported remarkable 90% to 100% eradication rates, regardless of HCV genotype. Treatment with DAAs has comparable efficacy on viral eradication in patients with MC, but definite clinical improvements of vasculitis can be observed only in half the patients. On the contrary, the regression of renal disease and lympho-proliferative disorders, induced by HCV, appears to have a lower remission rate after viral eradication with DAAs and most cases need immunosuppressive treatments. In HCV related CV, the main clinical goal must be early eradication of HCV, to avoid organ complication and manifestation of lympho-proliferative diseases. This review focuses on the role of DAAs in treatment of HCV-related cryoglobulinemic vasculitis.

Cryoglobulinemic vasculitis (CV) can develop in 1.2-4% of hepatitis B virus (HBV)-infected patients. HBV infection affects about 350 million people worldwide. It can progress from acute or fulminant hepatitis to chronic hepatitis, cirrhosis or hepatocellular carcinoma. Twenty per cent of HBV patients may develop extra-hepatic manifestations, such as polyarteritis nodosa, glomerulonephritis, dermatitis, polyarthralgias and arthritis, lung disease, aplastic anaemia. Our review focuses on the role of antiviral agent nucleot(s)ide analogues (NAs) in treatment of HBV-related CV. The studies in literature have demonstrated that NAs therapy in HBV-related CV yields high virological and satisfying clinical responses in most patients with mild-and-moderate CV, but a low response in severe CV. Overall, NAs represent a promising therapeutic option for HBV-related CV. Obtaining early suppression of HBV viral load should be the main virological and clinical goal in order to prevent organ complications and lymphoproliferative disorders.

Hepatitis C virus (HCV)-associated mixed cryoglobulinemia (MC) vasculitis commonly regresses upon virus eradication, but conventional therapy with pegylated interferon and ribavirin yields approximately 40% sustained virologic responses (SVR). We prospectively evaluated the efficacy and safety of sofosbuvir-based direct-acting antiviral therapy, individually tailored according to the latest guidelines, in a cohort of 44 consecutive patients with HCV-associated MC. In two patients MC had evolved into an indolent lymphoma with monoclonal B-cell lymphocytosis. All patients had negative HCV viremia at week 12 (SVR12) and at week 24 (SVR24) posttreatment, at which time all had a clinical response of vasculitis. The mean (±standard deviation) Birmingham Vasculitis Activity Score decreased from 5.41 (±3.53) at baseline to 2.35 (±2.25) (P < 0.001) at week 4 on treatment to 1.39 (±1.48) (P < 0.001) at SVR12 and to 1.27 (±1.68) (P < 0.001) at SVR24. The mean cryocrit value fell from 7.2 (±15.4)% at baseline to 2.9 (±7.4)% (P < 0.01) at SVR12 and to 1.8 (±5.1)% (P < 0.001) at SVR24. Intriguingly, in the 2 patients with MC and lymphoma there was a partial clinical response of vasculitis and ∼50% decrease of cryocrit, although none experienced a significant decrease of monoclonal B-cell lymphocytosis. Adverse events occurred in 59% of patients and were generally mild, with the exception of 1 patient with ribavirin-related anemia requiring blood transfusion. Conclusion: Interferon-free, guideline-tailored therapy with direct-acting antivirals is highly effective and safe for HCV-associated MC patients; the overall 100% rate of clinical response of vasculitis, on an intention-to-treat basis, opens the perspective for curing the large majority of these so far difficult-to-treat patients. (Hepatology 2016;64:1473-1482)

Depletion of B cells is beneficial in rheumatoid arthritis (RA) patients with autoantibodies to citrullinated proteins (ACPA) and/or the Fc portion of immunoglobulins (rheumatoid factor [RF]), suggesting a role for B cells in disease pathogenesis. To date, however, the identity of specifically pathogenic B cell subsets has not been discovered. One candidate population is identified by the low expression or absence of complement receptor 2 (CD21−/low B cells). In this study, we sought to determine whether there was any correlation between CD21−/low B cells and clinical outcome in patients with established RA, either ACPA+/RF+ (n = 27) or ACPA−/RF− (n = 10). Healthy donors (n = 17) were included as controls. The proportion of the CD21−/low CD27−IgD− memory B cell subset in peripheral blood (PB) was significantly increased in ACPA+/RF+ RA patients compared with healthy donors, and the frequency of this subset correlated with joint destruction (r = 0.57, P < 0.04). The levels of the chemokines CXCL-9 and CXCL-10 were higher in synovial fluid than in plasma, and PB CD21−/low cells expressed the receptor, CXCR3. In synovial fluid, most of the B cells were CD21−/low, approximately 40% of that population was CD27−IgD−, and a third of those expressed the pro-osteoclastogenic factor receptor activator of the nuclear factor κB ligand (RANKL). This subset also secreted RANKL, in addition to other factors such as IL-6, even in the absence of stimulation. We interpret these data as reason to propose the hypothesis that the CD27−IgD− subset of CD21−/low B cells may mediate joint destruction in patients with ACPA+/RF+ RA. (Less)

BACKGROUND & AIMS Hepatitis C virus (HCV)-related mixed cryoglobulinaemia vasculitis (MCV) is characterized by the expansion of rheumatoid factor-producing B-cell clones. The aim of this study was to assess whether B-cell clones may persist in these patients after the clearance of the virus with antiviral therapy, and whether their persistence influences clinical outcomes. METHODS Forty-five HCV-cured MCV patients were followed up for a median of 18.5 (range 9-38) months after the clearance of HCV. Circulating B-cell clones were detected using flow cytometry either by the skewing of kappa/lambda ratio or by the expression of a VH 1-69-encoded idiotype. RESULTS The clinical response of vasculitis was 78% complete, 18% partial and 4% null. However, cryoglobulins remained detectable in 42% of patients for more than 12 months. Circulating B-cell clones were detected in 18 of 45 patients, and in 17 of them persisted through the follow-up; nine of the latter patients cleared cryoglobulins and had complete response of vasculitis. Several months later, two of these patients had relapse of MCV. CONCLUSIONS B-cell clones persist in MCV patients long after HCV infection has been cleared but halt the production of pathogenic antibody. These 'dormant' cells may be reactivated by events that perturb B-cell homeostasis and can give rise to the relapse of cryoglobulinaemic vasculitis.

In this issue of Blood, Ren et al report the results of a broad genomic and transcriptomic analysis of hepatitis B virus (HBV)–associated diffuse large B-cell lymphomas (DLBCLs) in Chinese patients, providing for the first time a distinctive molecular profile of these tumors.1

Purpose The optimal immune globulin replacement dosages required over time to minimize infection risks in patients with Primary Antibody Deficiencies are not definitely established. As with many interventions, there may be specific subgroups of patients who are more likely to benefit from treatment with higher or lower dosages. The aim of the study was to verify the efficacy of a rationale for individualized immune globulin utilization and to elucidate the effects of care on patient outcome. Methods Single centre interventional study on 108 patients with Primary Antibody Deficiencies. The objective was to determine for each patient the best interval between immune globulins administration in order to: • Keep IgG trough levels >500 mg/dL, • Minimize of major infections (pneumonias and infections requiring hospitalization), • Minimize of adverse events (AE). Results Ninthly eight per cent of patients achieved the objective of the study. Patients who had low switched memory B cells and low IgA serum levels and/or are affected by bronchiectasis and/or enteropathy and/or continued to experience adverse events despite pre-medications, achieved the study objective by shortening the administration intervals to 2-weeks or to 1-week without the need to increase the monthly cumulative immunoglobulin dosage and its relative cost. The adverse events were reduced by administrating low Ig dosages in a single setting. Patients without risk factors achieved the study objective with immune globulin replacement administered with the widely used interval of 3 or 4 weeks. Conclusions The exact timing and optimal immunoglobulin prophylaxis regimen might be tailored according to clinical and immunological markers.

Hepatitis C virus (HCV) causes mixed cryoglobulinemia (MC) by driving clonal expansion of IgM(+)CD27(+) B cells. These cells display both the features of anergy induced by continual engagement of the B-cell receptor (BCR), such as high expression of phosphorylated extracellular signal-regulated kinase (pERK) and reduced lifespan, and of virus-specific exhaustion, such as CD21(low) phenotype and a defective response to ligation of BCR and Toll-like receptor 9 (TLR9). MC usually regresses after eradication of HCV with interferon, whose immunomodulatory activity might contribute to this effect. We investigated the phenotypic and functional changes in clonal B cells of MC patients with sustained virologic responses to direct-acting antivirals (DAAs), which lack immunomodulatory properties. We found that high pERK expression and accelerated apoptosis revert within 4 weeks after beginning therapy, whereas clonal B cells unresponsive to TLR9 stimulation persist for at least 24 weeks, although they may partially rescue normal CD21 expression. Thus, similar to mouse models, features of anergy in MC B cells rapidly revert after disengagement from HCV, whereas virus-specific exhaustion imparts a durable inhibitory imprint on cell function. Treatment of HCV(+) MC with DAAs provides a valuable tool for untangling the molecular mechanisms of anergy and exhaustion in human B cells.

Hepatitis C virus (HCV) causes monoclonal B cell lymphoproliferative disorders ranging from benign, such as in mixed cryoglobulinemia (MC), to indolent or aggressive lymphomas. MC and indolent lymphomas commonly regress when HCV is eradicated with interferon (IFN) therapy; however, sustained virologic response (SVR) to IFN is achieved only in ~50% of patients. The new all oral direct-acting antivirals (DAA), yielding nearly 100% SVR, promise a breakthrough in the treatment of HCV-associated lymphoproliferative disorders, but experience is still scanty. Areas covered: A literature search was performed to summarize current pathogenetic hypotheses in HCV-associated indolent lymphoproliferative disorders and to identify clinical trials focused on the use of antiviral therapy. Hematological outcomes of IFN-based and IFN-free DAA-based regimens were compared. Expert commentary: While MC appears to regress in most patients after DAA therapy, the still very limited experience with indolent lymphomas suggests that hematologic responses might be less than those observed with IFN. Furthermore, anecdotal observations of early progression to aggressive lymphoma after DAA are disquieting. Large studies are needed to determine the values and limits of DAA for treating HCV-associated indolent lymphomas and to identify subgroups at risk of non-response.

This study aims to evaluate the efficacy and safety of repeated treatments with low-dose rituximab for relapsing mixed cryoglobulinemia vasculitis. Thirty-seven patients with mixed cryoglobulinemia vasculitis refractory to standard of care treatment, 34 of which were HCV-positive, were treated with rituximab at the reduced dosage of 250 mg/m2 given twice 1 week apart per cycle. Thirty patients (81%) achieved a clinical response; 5 of them remain in remission, 3 were lost to follow-up or died, and 22 relapsed after a mean of 15.7 months. Eleven relapsers were retreated with one (6 patients), 2 (3 patients), or 3 (2 patients) additional rituximab cycles given at each relapse. Clinical and laboratory efficacy and side effects of long-term treatment were evaluated. Clinical response to retreatment was 91% (10/11) at the first relapse, 80% (4/5) at the second relapse, and 100% (2/2) at the third relapse. The mean (±SD) time to relapse was 17.1 ± 14.1 months in 30 patients who were treated with only one cycle (from first cycle to the first relapse) and 45.7 ± 30.6 months (from first cycle to the last observed relapse) in 11 patients treated with 2 or more cycles (p = 0.0037). Severe adverse reactions occurred in 3 patients, in 2 of whom at the first cycle. Our results suggest that repeated treatment of relapsing mixed cryoglobulinemia with a low-dose rituximab regimen is efficacious, safe, and cost-effective for the long-term management of this disorder.

Monoclonal marginal zone (MZ) B cells expressing a V(H)1-69-encoded idiotype accumulate in HCV-associated mixed cryoglobulinemia (MC). These cells recognize the E2 protein of HCV and their massive clonal expansion reflects the propensity of MZ B cells to proliferate robustly upon antigenic stimulation by microorganisms, a property that makes them prone to neoplastic transformation. V(H)1-69(+) B cells of MC patients are phenotypically heterogeneous and resemble either mature MZ B cells (IgM(+)CD27(+)CD21(high)) or the unusual CD21(low) B cells that accumulate in other immunological disorders such as common variable immunodeficiency (CVID) or HIV infection. The CD21(low) V(H)1-69(+) B cells of MC patients, like those of CVID and HIV patients, are anergic to BCR and TLR9 stimulation and display deregulation of several anergy-related genes; proliferative anergy is also observed in CD21(high) MZ-like V(H)1-69(+) B cells, that over-express the antiproliferative transcriptional repressor Stra13. Upon evolution to splenic marginal zone lymphoma, MZ-like V(H)1-69(+) B cells down-regulate Stra13 and partially recover their capacity to proliferate in response to TLR9 ligation. Like yin and yang, robust clonal expansion and early proliferative anergy may be viewed as the opposite forces balancing the responses of human MZ B cells to chronic microbial stimuli. Disruption of this balance facilitates autoimmunity and lymphoproliferation.

International audience; Regression of hepatitis C virus (HCV)-associated lymphoma with interferon (IFN)-based antiviral treatment supports an etiological link between lymphoma and HCV infection. In addition, a favorable impact of antiviral treatment on overall survival of patients with HCV-related lymphoma has been reported. Data on IFN-free regimens combining direct-acting antivirals (DAAs) in HCV-associated lymphoproliferative disorders are scanty. We analyzed the virological and lymphoproliferative disease response (LDR) of 46 patients with indolent B-cell non-Hodgkin lymphomas (NHLs) or chronic lymphocytic leukemia (CLL) and chronic HCV infection treated with DAAs. The histological distribution was 37 marginal zone lymphomas (MZLs), 2 lymphoplasmacytic lymphomas, 2 follicular lymphomas, 4 CLL/small lymphocytic lymphomas (CLL/SLLs), and 1 low-grade NHL not otherwise specified. Thirty-nine patients received a sofosbuvir-based regimen and 7 patients received other DAAs. The median duration of DAA therapy was 12 weeks (range, 6-24 weeks). A sustained virological response at week 12 after finishing DAAs was obtained in 45 patients (98%); the overall LDR rate was 67%, including 12 patients (26%) who achieved a complete response. The LDR rate was 73% among patients with MZL, whereas no response was observed in CLL/SLL patients. Seven patients cleared cryoglobulins out of 15 who were initially positive. After a median follow-up of 8 months, 1-year progression-free and overall survival rates were 75% (95% confidence interval [CI], 51-88] and 98% [95% CI, 86-100], respectively. DAA therapy induces a high LDR rate in HCV-associated indolent lymphomas. These data provide a strong rationale for prospective trials with DAAs in this setting.

A clonal population of B cells expressing a VH1-69-encoded idiotype accumulates in hepatitis C virus (HCV) associated mixed cryoglobulinemia (MC). These cells are phenotypically heterogeneous, resembling either typical marginal zone (MZ) B cells (IgM+IgD+CD27+CD21+) or the exhausted CD21low B cells that accumulate in HIV infection or in common variable immunodeficiency. We show that both the MZ-like and the CD21low VH1-69+ B cells of MC patients are functionally exhausted, since they fail to respond to TLR and BCR ligands. The proliferative defect of VH1-69+ B cells can be overcome by co-stimulation of TLR9 and BCR in the presence of interleukin(IL)-2 and IL-10. The MZ-like VH1-69+ B cells do not express the inhibitory receptors distinctive of CD21low B cells, but display constitutive activation of extracellular signal regulated kinase (ERK) and attenuated BCR/ERK signaling. These cells also express abundant transcripts of Stra13 (DEC1, Bhlhb2, Sharp2, Clast5), a basic helix-loop-helix transcription factor that acts as a powerful negative regulator of B-cell proliferation and homeostasis. Our findings suggest that MZ B cells activated by HCV undergo functional exhaustion associated with BCR signaling defects and overexpression of a key antiproliferative gene, and may subsequently become terminally spent CD21low B cells. Premature exhaustion may serve to prevent the outgrowth of chronically stimulated MZ B cells.

Functionally exhausted and mostly autoreactive B-cells with a peculiar CD21(low)CD11c(+) phenotype accumulate in several human immunological disorders including common variable immunodeficiency, HIV infection and rheumatoid arthritis. In HCV-associated mixed cryoglobulinemia (MC) there is accumulation of exhausted clonal B cells expressing a V(H)1-69-encoded cross-reactive idiotype; these cells are phenotypically heterogeneous, displaying either a CD21(low)CD11c(+) or a marginal zone (MZ)-like (IgM(+)CD27(+)CD21(+)CD11c(-)) phenotype. Irrespective of their phenotype, V(H)1-69(+) B-cells are unresponsive to the stimulation of Toll-like receptor 9 (TLR9). We investigated the fate of these cells after the eradication of HCV.Fourteen MC patients were studied before and after antiviral therapy. V(H)1-69(+) B-cells were identified using the G6 monoclonal antibody and their phenotype and responsiveness to the stimulation of TLR9 were investigated.In seven virological non-responders, cryoglobulin levels and the number and phenotype of V(H)1-69(+) B cells remained substantially unchanged. By contrast, in sustained viral responders cryoglobulinemia subsided and the number of V(H)1-69(+) B cells declined. However, high proportions of MZ-like V(H)1-69(+) B cells retaining unresponsiveness to TLR9 stimulation persisted for several months in these patients.Clonal expansion of CD21(low) V(H)1-69(+) B cells may depend on continual stimulation by HCV, whereas their MZ-like counterparts may persist for years after the eradication of infection. Prolonged survival of exhausted MZ-like B cells after withdrawal of the initial inciting stimulus may contribute to the accumulation of autoreactive B cells in immunological disorders.

The homeostasis of circulating B cell subsets in the peripheral blood of healthy adults is well regulated, but in disease it can be severely disturbed. Thus, a subgroup of patients with common variable immunodeficiency (CVID) presents with an extraordinary expansion of an unusual B cell population characterized by the low expression of CD21. CD21 low B cells are polyclonal, unmutated IgM + IgD + B cells but carry a highly distinct gene expression profile which differs from conventional naïve B cells. Interestingly, while clearly not representing a memory population, they do share several features with the recently defined memory-like tissue, Fc receptor-like 4 positive B cell population in the tonsils of healthy donors. CD21 low B cells show signs of previous activation and proliferation in vivo, while exhibiting defective calcium signaling and poor proliferation in response to B cell receptor stimulation. CD21 low B cells express decreased amounts of homeostatic but increased levels of inflammatory chemokine receptors. This might explain their preferential homing to peripheral tissues like the bronchoalveolar space of CVID or the synovium of rheumatoid arthritis patients. Therefore, as a result of the close resemblance to the gene expression profile, phenotype, function and preferential tissue homing of murine B1 B cells, we suggest that CD21 low B cells represent a human innate-like B cell population.

Objective. To investigate the contribution of inherited and acquired thrombophilic defects to the clinical manifestations of mixed cryoglobulinaemia vasculitis. Methods. The following thrombophilic defects were investigated in 64 consecutive patients with HCV-associated mixed cryoglobulinaemia: aPLs, lupus anti-coagulant, homocysteinaemia, protein C and protein S concentrations, activated protein C resistance, plasminogen activator inhibitor-1 4G4G and 5G5G genotypes, and the presence of mutations of factor V (Leiden and H1299R), of prothrombin (G20210A) and of methyl tetrahydrofolate reductase (C677T and A1298C). Additional variables were demographic data, duration of the disease, cryocrit level and vascular risk factors (diabetes, hypertension, hypercholesterolaemia and smoking habit). The following clinical manifestations of mixed cryoglobulinaemia were analysed as dependent covariates: severity of purpura, presence of necrotic skin ulcers, presence of peripheral neuropathy and presence of kidney disease. Results. Logistic regression analysis identified hyperhomocysteinaemia as a risk factor for severe purpura (P < 0.0001) and for the presence of skin ulcers (P < 0.0001), whereas none of the other thrombophilic defects influenced the clinical presentation of mixed cryoglobulinaemia. Purpura improved in two patients after lowering homocysteine with vitamin supplementation. Conclusions. Hyperhomocysteinaemia may be a risk factor for severe cutaneous manifestations in mixed cryoglobulinaemia.

INTRODUCTION: The multicenter prospective study provides information on adverse reactions to intravenous and subcutaneous immunoglobulin treatment in a cohort of 262 patients with common variable immunodeficiency. Severe adverse reactions are a rare but unpredictable event that might occur also in patients who tolerate substitutive intravenous or subcutaneous immunoglobulin therapy for months or years. RESULTS: Subcutaneous therapy has been proved to be a safe option in the 13 patients who had to stop intravenous treatment and who remained out of immunoglobulin replacement for long periods of time. However, severe reactions to subcutaneous therapy occurred at the first or after several subcutaneous immunoglobulin administrations in 2 out of 13 patients. CONCLUSION: Therefore, patients with previous severe reactions to intravenous immunoglobulin should be considered at particularly high risk for reaction to subcutaneous administration. In these cases, switching from in-hospital administration to home self-administration should be done with extreme care.

Since its widespread introduction in the early 1980s, immunoglobulin therapy (IG) has been extensively investigated to understand its mechanism of action and clinical benefits. Research has also continued to improve its production. The survival benefits of IG for patients with primary immunodeficiency (PID) are accepted (1). The basic principle of replacing the missing protein has led to proposals for progressive increases in dosage, delivered intravenously (2), or subcutaneously (3). It is suggested that continuously increasing IG trough levels decreases pulmonary infections and damage. In contrast, other studies on large patient cohorts found no correlation between IG trough level and the incidence of pneumonia and serious infections when trough levels were raised above 400 mg/dl (4, 5). Dosage and other aspects of the therapeutic regimen remain open questions even in the mainstream indication of substitution therapy in PID. These issues have influenced the development of product modifications such as highly concentrated solutions and fast infusion rates. They have also contributed to the increased usage of 16–20% IG infused subcutaneously. Simple replacement of the antibody defect in PID is now known to be an incomplete explanation of the mechanism of IG. A range of immunomodulatory and anti-inflammatory mechanisms are involved (6). These mechanisms are important in the role of IG in autoimmune disorders, particularly neuropathies including chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome (GBS), and multifocal motor neuropathy (MMN). These indications represent the largest area of IG use in the established economies. They contribute greatly to the steady increase in demand for immunoglobulins experienced in the past 20 years, despite uncertainty in mechanisms of action. The increase in adverse events, such as thrombogenicity (7) and haemolysis (8), experienced in recent years makes a better understanding of mechanism and dosage even more important. The substantial increase in the usage of expensive IG products has also influenced developments in formulation and infusion practices. Faster infusion of more concentrated solutions will decrease hospital stay and costs. The subcutaneous route is supposedly easier and more convenient to deliver in home therapy settings (9), also potentially decreasing hospital stay. Although approved by regulatory agencies, these developments have yet to be validated through the long period of clinical practice experienced with the previous range of IG products. The increased demand for IG has also seen the rapid development of new manufacturing methods replacing the traditional Cohn fractionation system (10). This system has demonstrated decades of safety and efficacy and caution is warranted as new methods are introduced into production and clinical use. This Research Topic of Frontiers in Immunology has been assembled by an editorial team which have experienced sufficient “dark forebodings” (11) regarding the uncertainties outlined above. They have called upon a group of international experts to assess some of these issues from their perspective. The mechanism of IG on the immune system is explored by Nagelkerke and Kuijpers (12) who describe the different Fcγ receptors variants on immune cells and the direct IG effects at the level of the activating Fcγ receptors, including the more recently described FcgRIIc. Mitrevski et al. (13) assess analogous mechanisms in the action of IG in PID, showing that IG at replacement dosages could prime B cells to an anergic, apoptotic state through the generation of an increase in CD21low B cells. Matucci et al. (14) discuss the role of benefits additional to the direct substitution of deficient IG, such as the immunomodulatory and anti-inflammatory effects of IG preparations, while Paquin-Proulx and Sandberg (15) discuss the role of immune activation in the pathology of commonest PID – common variable immunodeficiency (CVID) – and its alleviation by IG therapies. Taken together, this body of work mitigates our “dark forebodings” regarding the lack of clarity on the mechanism of action of IG. More work is also needed to optimize therapeutic practice. Kerr et al. (16) note the desirability of progressing beyond simple, mandated, weight-based dosages in PID, and the need to approach more individualized therapeutic regimens for different PID patients. Their approach is augmented by the review of Wolf et al. (17) demonstrating how the identification of impaired IG formation may differentiate patients requiring IG from those who do not. Patient data collected through long term monitoring led Lucas et al. (18) to conclude that “The goal of replacement therapy should be to improve clinical outcome and not to reach a particular IgG trough level” supporting previous work tailoring optimal IG prophylaxis regimens to clinical and immunological markers (19). Studies such as those cited should influence treatment protocols and a negate a “one size fits all” therapeutic approach as reflected in many current guidelines. Kerr et al. (16) also note how little evidence underpins the dosage regimens used in autoimmune indications. It is to be wondered that empirically driven doses developed for the treatment of idiopathic thrombocytopenic purpura have been extended into the treatment of the various autoimmune neuropathies. The importance of individualizing treatment is also discussed by Compagno et al. (20) in their review of the use of IG in the various acquired hypogammaglobulinemic diseases. We are aware of large discrepancies between and within countries in this area, leading to the need for better evidence based on individualized treatment. Overall, these particular contributions support the need to improve outcomes in PID as shown by the study of Tabolli et al. (21) who conclude that Health-Related Quality of Life in PID patients is not related to IG therapy but to more sophisticated personal and clinical preferences. The final component of this Research Topic of Frontiers in Immunology examines the manufacture of IG therapies and its effect on the efficacy and safety of the products. Goldacker et al. (22) show that, despite the mandatory large plasma donation pool size for IG products, the content of specific, therapeutically important antibodies is still subject to geographical influence. This is very important in the context of continuing to ensure that IG therapies are relevant in the protection of PID patients in different countries. We suggest that this requires the attention of regulatory authorities. Finally, Farrugia and Quinti (23) and Spath et al. (24) discuss the history of the manufacture of IG therapies. They suggest ways whereby changes introduced by companies to optimize yield and minimize hospital related costs might alter the repertoire of specificities and the efficacy of IG. These changes may also have played a role in recently observed surges in adverse events. These observations suggest that a more cautionary approach in the usage IG therapies is warranted. Overall, the therapeutics of diseases caused by a deficiency or an inappropriate type of antibodies continues to be an exciting and fruitful area of clinical research, and well suited to contribute to the ongoing evolution in medicine toward more individualized and patient centric paradigms. We hope that this Research Topic of Frontiers in Immunology contributes to this debate. Such a wide debate between experts is necessary to enable the new challenges in immunoglobulin usage.

Intravenous immunoglobulin (IVIG) is used as replacement therapy in patients with antibody deficiencies and at higher dosages in immune-mediated disorders. Although different mechanisms have been described in vitro, the in vivo immunomodulatory effects of IVIG are poorly understood. Different studies have suggested that IVIG modulates B-cell functions as activation, proliferation, and apoptosis. Recently, it was shown that IVIG induces in vitro B-cell unresponsiveness similar to anergy. In accord with this, we recently reported that IVIG therapy in patients affected by common variable immunodeficiency (CVID) interferes in vivo with the B-cell receptor (BCR) signaling by increasing constitutive ERK activation and by reducing the phosphorylated ERK increment induced by BCR cross-linking. Moreover, we observed that IVIG induces in CVID patients an increase of circulating CD21(low) B-cells, an unusual population of anergic-like B-cells prone to apoptosis. Therefore, IVIG at replacement dose in vivo could prime B-cells to an anergic, apoptotic program. Here, we discuss these recent findings, which may improve our understanding of the immunomodulatory effects of IVIG, individualizing single involved molecules for more specific treatments.

Objective To evaluate whether rituximab at a low dose of 250 mg/m 2 × 2 may be as effective as at higher dosages, most commonly 375 mg/m 2 × 4, used in previous studies on the treatment of patients with refractory mixed cryoglobulinemia (MC) vasculitis associated with hepatitis C virus (HCV) infection. Methods We conducted a phase 2, single-arm two-stage trial (EUDRACT n. 2008-000086-38) of low-dose rituximab in 52 patients with HCV-associated MC who were ineligible/intolerant or non-responder to antiviral therapy. The primary outcomes were response of vasculitis evaluated by the Birmingham Vasculitis Activity Score (BVAS) at months 3, 6 and 12, rate of relapses and time to relapse, and rate of adverse events. Our data were compared with those reported in 19 published studies selected among 291 reviewed in a literature search. Results The cumulative response rate (complete and partial) at month 3 was 81% in our patients, and 86% in 208 patients from studies using high-dose rituximab. The relapse rate and median time to relapse were, respectively, 41% and 6 months in our study, and 32% and 7 months in high-dose studies. Treatment-related adverse events were 11.5% in our study and 19.9% in high-dose studies. None of these differences was statistically significant. Conclusion Rituximab at a low dosage of 250 mg/m 2 × 2 is as effective as at higher dosages for treating MC vasculitis. This low-dose regimen may improve the cost/benefit profile of rituximab therapy for MC.

The host genetic basis of mixed cryoglobulin vasculitis is not well understood and has not been studied in large cohorts. A genome-wide association study was conducted among 356 hepatitis C virus (HCV) RNA-positive individuals with cryoglobulin-related vasculitis and 447 ethnically matched, HCV RNA-positive controls. All cases had both serum cryoglobulins and a vasculitis syndrome. A total of 899 641 markers from the Illumina HumanOmni1-Quad chip were analyzed using logistic regression adjusted for sex, as well as genetically determined ancestry. Replication of select single-nucleotide polymorphisms (SNPs) was conducted using 91 cases and 180 controls, adjusting for sex and country of origin. The most significant associations were identified on chromosome 6 near the NOTCH4 and MHC class II genes. A genome-wide significant association was detected on chromosome 6 at SNP rs9461776 (odds ratio=2.16, P=1.16E-07) between HLA-DRB1 and DQA1: this association was further replicated in additional independent samples (meta-analysis P=7.1 × 10(-9)). A genome-wide significant association with cryoglobulin-related vasculitis was identified with SNPs near NOTCH4 and MHC Class II genes. The two regions are correlated and it is difficult to disentangle which gene is responsible for the association with mixed cryoglobulinemia vasculitis in this extended major histocompatibility complex region.

Intravenous immunoglobulin (IVIG), besides its use as replacement therapy in patients with antibody deficiencies, is broadly used as an immunomodulatory agent for the treatment of autoimmune and inflammatory disorders. The mechanisms of action of IVIG include Fc receptor blockade, inhibition of cytokines and growth factors, modulation of macrophages and dendritic cells, enhancement of regulatory T cells, and modulation of B cells through the FcγRIIB receptor and CD22. Recent studies suggest that in vitro exposure of human B cells to IVIG determines functional changes reminiscent of anergy and that IVIG treatment of patients with common variable immunodeficiency (CVID) induces in B cells ERK activation, a feature of anergy. Here, we show that IVIG therapy drives the B cells of patients with CVID to down-regulate CD21 expression and to assume the peculiar phenotype of the anergic-like, apoptosis-prone CD21(low) B cells that are spontaneously expanded in a subset of CVID and in some other immunological disorders. The CD21(low) B cells newly generated after IVIG infusion undergo spontaneous apoptosis upon in vitro culture. Furthermore, IVIG infusion is rapidly followed by a significant, although discrete, decrease in the number of circulating B cells, but not of T cells or of natural killer cells. These findings suggest that IVIG therapy may constrain antibody responses by inducing B cell depletion through differentiation into CD21(low) B cells that undergo accelerated apoptosis.

Hepatitis C virus (HCV) is associated with the B-cell lymphoproliferative disorders mixed cryoglobulinemia (MC) and non-Hodgkin lymphoma. We have previously reported that HCV+MC+ patients have clonal expansions of hypermutated, rheumatoid factor–bearing marginal zone-like IgM+CD27+ peripheral B cells using the VH1-69 gene. Here we coupled transcriptional profiling with immunophenotypic and functional studies to ascertain these cells' role in MC pathogenesis. Despite their fundamental role in MC disease, these B cells have overall transcriptional features of anergy and apoptosis instead of neoplastic transformation. Highly up-regulated genes include SOX5, CD11C, galectin-1, and FGR, similar to a previously described FCRL4+ memory B-cell subset and to an “exhausted,” anergic CD21low memory B-cell subset in HIV+ patients. Moreover, HCV+MC+ patients' clonal peripheral B cells are enriched with CD21low, CD11c+, FCRL4high, IL-4Rlow memory B cells. In contrast to the functional, rheumatoid factor–secreting CD27+CD21high subset, the CD27+CD21low subpopulation exhibits decreased calcium mobilization and does not efficiently differentiate into rheumatoid factor–secreting plasmablasts, suggesting that a large proportion of HCV+MC+ patients' clonally expanded peripheral B cells is prone to anergy and/or apoptosis. Down-regulation of multiple activation pathways may represent a homeostatic mechanism attenuating otherwise uncontrolled stimulation of circulating HCV-containing immune complexes. This study was registered at www.clinicaltrials.gov as #NCT00435201.

Eradication of hepatitis C virus (HCV) by antiviral therapy is the treatment of choice for mixed cryoglobulinemia secondary to this infection, but many patients fail to achieve sustained viral responses and need second-line treatments. Several studies have demonstrated that the infusion of the anti-CD20 monoclonal antibody rituximab is highly effective for refractory mixed cryoglobulinemia, with a clinical response in approximately 80% of patients, although the relapse rate is high. Virtually all published studies employed a rituximab dosage of 375mg/m(2) given four times, a schedule used for treating non-Hodgkin's lymphomas. Based on a prior pilot study, we designed a phase II single-arm two-stage study (EUDRACT n. 2008-000086-38) to evaluate the efficacy of a lower dosage of rituximab, 250mg/m(2) given twice, for refractory mixed cryoglobulinemia. We present here the preliminary results in the first 27 patients enrolled. The overall response rate in 24 evaluable patients was 79%, and the mean time to relapse was 6.5months, similar to the 6.7months reported in studies with high-dose rituximab. Side effects were comparable to those seen in patients treated with high-dose. Increase of HCV viral load, reported in some high-dose studies, was not observed in our patients. Low-dose rituximab may provide a more cost/effective and possibly safer alternative for treating refractory HCV-associated mixed cryoglobulinemia.

A subset of patients with common variable immunodeficiency (CVID), group 1a of the Freiburg classification, is characterized by increased B cells expressing low levels of CD21 (CD21(low) ), lymphoproliferation and autoimmunity. The CD21(low) B cells have been shown to be profoundly anergic, and defects of BCR-mediated calcium signaling and of T cells have been described in CVID 1a. We found that also the classical naïve B cells from CVID 1a patients, but not from CVID non-1a patients, proliferated poorly. The B cells of CVID 1a patients had a reduced capacity to divide reminiscent of the proliferative arrest associated with replicative senescence. Thus, we investigated whether lymphocyte dysfunction in CVID 1a was related to telomere-dependent replicative senescence, and found that both the B and the T cells from CVID 1a patients had significantly shorter telomeres compared with B and T cells from CVID non-1a patients. Telomere lengths in B and T cells were significantly correlated, indicating that the rate of telomere attrition in lymphocytes is an individual characteristic of CVID patients. Our findings suggest that telomere-dependent replicative senescence contributes to the immune dysfunction of CVID 1a patients, and may provide an important clue for a better understanding of the pathogenesis of CVID.

Toll-like receptor 9 (TLR-9) and TLR-7 may have a role in the production of anti-DNA and anti-RNA autoantibodies, respectively, but murine models do not clearly demonstrate their contribution to the development of systemic lupus erythematosus (SLE). Herein we describe a patient with SLE who had long-lasting remission of her autoimmune disease after development of an antibody deficiency resembling common variable immunodeficiency (CVID). After CVID had developed, anti–double-stranded DNA antibodies disappeared, although antinuclear antibodies remained positive for >10 years. In vitro studies revealed that the patient's B cells proliferated poorly and failed to differentiate into plasmablasts after stimulation of either TLR-9 or TLR-7, providing evidence for an acquired defect of the signaling pathway downstream of these TLRs. These observations suggest, although indirectly, that signaling through TLR-9 and TLR-7 is important in the pathogenesis of human SLE, and indicate that investigation of potential treatment strategies with TLR antagonists is warranted.

Chronic infection with hepatitis C virus (HCV) is related to monoclonal B-cell lymphoproliferative disorders including a benign monoclonal lymphoproliferation such as type II mixed cryoglobulinemia, and B-cell non-Hodgkin's lymphomas (NHL).