High prevalence of past hepatitis B virus infection in diffuse large B cell lymphoma: a retrospective study from Italy.

Studies from high endemic areas, mostly China, indicate that surface antigen positive (HBsAgpos) chronic hepatitis B virus (HBV) infection is associated with an increased risk of developing diffuse large B-cell lymphoma (DLBCL), whereas studies in low endemic areas have provided conflicting results. Past infection, serologically defined by negative HBsAg and positive anti-core antibody (HBsAgnegHBcAbpos), has also been suggested to increase the risk of B-cell non-Hodgkin's lymphoma (NHL) in high endemic areas. We retrospectively reviewed unselected clinical records of 253 patients with DLBCL (54% male, aged 60.3 ± 14.6 years at diagnosis) and 694 patients with different types of indolent B-cell NHL (46% male, aged 61.7 ± 12.8 years). Patients were seen at a single center in Italy between 2001 and 2022 and HBV serological status (HBsAg, HBsAb, HBcAb, HBeAg, HBeAb, and HBV DNA) was analyzed through enzyme-linked immunosorbent assays and molecular assays; patients infected with hepatitis C virus or human immunodeficiency virus were excluded. We used an unconditional multiple logistic regression model including as matching variables gender, age at diagnosis, immigrant status, and HBV serological status. Patients with DLBCL had, compared to indolent NHL, a higher prevalence of HBsAgpos active infection (odds ratio (OR) 2.8, 95% confidence interval (95% CI) 1.2-6.3, p = 0.014). Strikingly, patients with DLBCL had also a significantly higher prevalence of past infection (OR 2.4, 95% CI 1.5-4.0, p = 0.0006). Male gender was associated with increased risk of DLBCL independently of the HBV serological status. These findings suggest that both past and active HBV infection may increase the risk of DLBCL in a low endemic area. Our study needs confirmation by studies in areas or populations with different rates of chronic or past HBV infection.

Opposite Effects of mRNA-Based and Adenovirus-Vectored SARS-CoV-2 Vaccines on Regulatory T Cells: A Pilot Study.

New-generation mRNA and adenovirus vectored vaccines against SARS-CoV-2 spike protein are endowed with immunogenic, inflammatory and immunomodulatory properties. Recently, BioNTech developed a noninflammatory tolerogenic mRNA vaccine (MOGm1Ψ) that induces in mice robust expansion of antigen-specific regulatory T (Treg) cells. The Pfizer/BioNTech BNT162b2 mRNA vaccine against SARS-CoV-2 is identical to MOGm1Ψ except for the lipid carrier, which differs for containing lipid nanoparticles rather than lipoplex. Here we report that vaccination with BNT162b2 led to an increase in the frequency and absolute count of CD4posCD25highCD127low putative Treg cells; in sharp contrast, vaccination with the adenovirus-vectored ChAdOx1 nCoV-19 vaccine led to a significant decrease of CD4posCD25high cells. This pilot study is very preliminary, suffers from important limitations and, frustratingly, very hardly can be refined in Italy because of the >90% vaccination coverage. Thus, the provocative perspective that BNT162b2 and MOGm1Ψ may share the capacity to promote expansion of Treg cells deserves confirmatory studies in other settings.

Dual stimulation by autoantigen and CpG fosters the proliferation of exhausted rheumatoid factor-specific CD21low B cells in hepatitis C virus-cured mixed cryoglobulinemia.

Introduction: Hepatitis 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. Methods: Clonal 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. Discussion: We 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.

Management of mixed cryoglobulinemia with rituximab: evidence and consensus-based recommendations from the Italian Study Group of Cryoglobulinemia (GISC).

Cryoglobulinemic vasculitis (CV) or mixed cryoglobulinemic syndrome (MCS) is a systemic small-vessel vasculitis characterized by the proliferation of B-cell clones producing pathogenic immune complexes, called cryoglobulins. It is often secondary to hepatitis C virus (HCV), autoimmune diseases, and hematological malignancies. CV usually has a mild benign clinical course, but severe organ damage and life-threatening manifestations can occur. Recently, evidence in favor of rituximab (RTX), an anti-CD 20 monoclonal antibody, is emerging in CV: nevertheless, questions upon the safety of this therapeutic approach, especially in HCV patients, are still being issued and universally accepted recommendations that can help physicians in MCS treatment are lacking. A Consensus Committee provided a prioritized list of research questions to perform a systematic literature review (SLR). A search was made in Medline, Embase, and Cochrane library, updated to August 2021. Of 1227 article abstracts evaluated, 27 studies were included in the SLR, of which one SLR, 4 RCTs, and 22 observational studies. Seventeen recommendations for the management of mixed cryoglobulinemia with rituximab from the Italian Study Group of Cryoglobulinemia (GISC) were developed to give a valuable tool to the physician approaching RTX treatment in CV.

Clinico-immunological outcomes of HCV-cured cryoglobulinemia: Lower relapse rate with interferon-based than interferon-free therapy.

Sustained virological response (SVR) obtained with interferon (IFN) or with direct-acting antivirals (DAAs) is commonly followed by response of hepatitis C virus (HCV)-associated mixed cryoglobulinemia vasculitis (MCV), but relapse of MCV despite SVR has been reported in several patients after DAAs and rarely after IFN. Since relapses could have been overlooked in studies with IFN, we retrospectively compared the outcomes of MCV in SVR patients treated with DAAs (n = 70) or IFN (n = 39) followed-up, respectively, for 30.5 (range 11-51) or 48 months. Groups were comparable for demographics and clinics and response rates of MCV were similar (92% and 86%); however, DAA-treated patients less efficiently reduced cryoglobulins (P = .006) and circulating B-cell clones (P = .004), and had more frequently relapses of MCV (18% vs 3%, P = .028) and need for rituximab therapy (P = .01). Although largely inferior on an intention-to-treat basis, IFN may be superior to DAAs on clinico-immunological outcomes possibly owing to its antiproliferative activity.

A stereotyped light chain may shape virus-specific B-cell receptors in HCV-dependent lymphoproliferative disorders.

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.

Rheumatoid factor-producing CD21low anergic clonal B-cells in essential mixed cryoglobulinaemia: a model for autoantigen-driven pathogenesis of infectious and non-infectious cryoglobulinaemias.

OBJECTIVES: Essential mixed cryoglobulinaemia (EMC) is a disorder of B-cells producing rheumatoid factor (RF), and is clinically and immunologically similar to mixed cryoglobulinaemia (MC) related to hepatitis C virus (HCV-MC). We report here the first comprehensive analysis of B-cell clonality, phenotype and function in EMC. METHODS: The study population included 16 patients with EMC and 24 patients with HCV-MC. Molecular analysis was done for the detection of circulating clonal B cells and for B cell receptor sequencing. B-cell phenotype, proliferative response, apoptosis and ERK signaling were analysed by flow cytometry. RESULTS: Molecular analysis of immunoglobulin genes rearrangements revealed circulating B-cell clones in about half of patients, on average of smaller size than those found in HCV-MC patients. Sequence analysis showed usage of the same stereotyped RF-encoding B-cell receptors frequently expressed in HCV-MC and in primary Sjögren's syndrome. B-cells with low expression of CD21 (CD21low) and unusual homing and inhibitory receptors were increased in EMC and in HCV-MC, but at a significantly lower extent in the former. The CD21low B-cells of EMC and HCV-MC patients shared functional features of exhaustion and anergy, namely reduced proliferation upon ligation of Toll-like receptor 9, high constitutive expression of phosphorylated ERK, and proneness to spontaneous apoptosis. CONCLUSIONS: Our findings suggest a common pathogenetic mechanism in EMC, HCV-MC and primary Sjögren's syndrome, consisting of autoantigen-driven clonal expansion and exhaustion of selected RF-producing B-cells. The more massive clonal expansion in HCV-MC may be due to co-stimulatory signals provided by the virus.

Long-Term Outcome of WHIM Syndrome in 18 Patients: High Risk of Lung Disease and HPV-Related Malignancies.

BACKGROUND: In the warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, variable phenotypic expression may delay diagnosis. Panleukopenia, malignancy, and chronic lung disease all affect morbidity and mortality risks. Routinely used treatments include immunoglobulins, granulocyte-colony stimulating factor (G-CSF), and antibiotics; recent trials with a target C-X-C chemokine receptor type 4 (CXCR4) antagonist show promising results. OBJECTIVE: We sought to characterize the largest cohort of patients with WHIM and evaluate their diagnostic and therapeutic management. METHODS: Data were collected from an international cohort of 18 patients with CXCR4 mutations. RESULTS: The clinical features manifested at 2.2 ± 2.6 years of age, whereas the disease diagnosis was delayed until 12.5 ± 10.4 years of age. Patients with WHIM commonly presented with a severe bacterial infection (78%). Pneumonia recurrence was observed in 61% of patients and was complicated with bronchiectasis in 27%. Skin warts were observed in 61% of patients at a mean age of 11 years, whereas human papilloma virus (HPV)-related malignancies manifested in 16% of patients. All the patients had severe neutropenia (195 ± 102 cells/mm3 at onset), whereas lymphopenia and hypogammaglobulinemia were detected in 88% and 58% of patients, respectively. Approximately 50% of patients received antibiotic prophylaxis, whereas G-CSF and immunoglobulin treatments were used in 72% and 55% of patients, respectively. CONCLUSIONS: The WHIM syndrome onsets early in life and should be suspected in patients with chronic neutropenia. Patients with WHIM need careful monitoring and timely intervention for complications, mainly lung disease and HPV-related malignancies. We suggest that immunoglobulin therapy should be promptly considered to control the frequency of bacterial infections and prevent chronic lung damage.

Long-lasting persistence of large B-cell clones in hepatitis C virus-cured patients with complete response of mixed cryoglobulinaemia vasculitis.

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.

DEC1/STRA13 is a key negative regulator of activation-induced proliferation of human B cells highly expressed in anergic cells.

The transcription factor DEC1/STRA13 (also known as BHLHE40 and SHARP2) is involved in a number of processes including inhibition of cell proliferation and delay of cell cycle, and is a negative regulator of B cell activation and development in mice. We show here that, unlike in mice, DEC1/STRA13 expression is induced in human naïve and memory resting B cells by activation through the B-cell receptor (BCR) or Toll-like receptor 9 (TLR9). siRNA silencing of DEC1/STRA13 increases the capacity of activated B cells to perform a high number of divisions after TLR9 ligation. This identifies DEC1/STRA13 as a critical negative regulator of clonal expansion of activated human B cells. We also show that DEC1/STRA13 is upregulated in human anergic CD21low B cells clonally expanded in patients with HCV-associated mixed cryoglobulinemia, which fail to proliferate in response to BCR or TLR9 ligation. siRNA knockdown of DEC1/STRA13, however, fails to restore responsiveness to stimuli in these cells, although it might improve the proliferative capacity in a subset of anergic cells with less pronounced proliferative defect.

From the pathogenesis to the cure of indolent B-cell lymphoproliferative disorders associated with hepatitis C virus infection: which role for direct-acting antivirals?

INTRODUCTION: 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.

Reversion of anergy signatures in clonal CD21low B cells of mixed cryoglobulinemia after clearance of HCV viremia.

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 CD21low 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.

Efficacy and safety of long-term treatment with low-dose rituximab for relapsing mixed cryoglobulinemia vasculitis.

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.

Prospective study of guideline-tailored therapy with direct-acting antivirals for hepatitis C virus-associated mixed cryoglobulinemia.

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).

Hepatitis B virus causes mixed cryoglobulinaemia by driving clonal expansion of innate B-cells producing a VH1-69-encoded antibody.

OBJECTIVES: To investigate the expression of a VH1-69-encoded idiotype, and the phenotypic and functional features of monoclonal B-cells from patients with type II mixed cryoglobulinaemia (MC) secondary to chronic hepatitis B virus (HBV) infection. METHODS: B-cell immunophenotype and expression of a VH1-69-encoded idiotype were investigated by flow cytometry. B-cell proliferative responses to stimuli were investigated by the CFSE dilution assay. RESULTS: Two out of five patients with chronic HBV studied had massive monoclonal expansion of VH1-69-expressing B-cells. These cells had the peculiar CD21(low) phenotype and low responsiveness to stimuli typical of the VH1-69-expressing B-cells commonly expanded in MC secondary to hepatitis C virus (HCV) infection. In both patients, anti-HBV therapy led to the regression of MC and of VH1-69+ B-cell expansion. CONCLUSIONS: VH1-69-encoded antibodies are known to preferentially recognise a variety of viral proteins including HCV E2, influenza A virus haemagglutinin and HIV gp41/gp120, and may serve as innate first line antiviral defense. Thus, like HCV, HBV may cause MC by protracted antigenic stimulation of VH1-69-expressing B-cells.

Efficacy of low-dose rituximab for the treatment of mixed cryoglobulinemia vasculitis: Phase II clinical trial and systematic review.

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.