Marcella Visentini

Circulating CD21low B cells in common variable immunodeficiency resemble tissue homing, innate-like B cells.

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. CD21low 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. CD21low 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. CD21low 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 CD21low B cells represent a human innate-like B cell population.

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)

A phase II, single-arm multicenter study of low-dose rituximab for refractory mixed cryoglobulinemia secondary to hepatitis C virus infection.

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-Hodgkins 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.

Genome-wide association study of hepatitis C virus- and cryoglobulin-related vasculitis

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.

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.

Prospective Study on CVID Patients with Adverse Reactions to Intravenous or Subcutaneous IgG Administration

IntroductionThe 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.ResultsSubcutaneous 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.ConclusionTherefore, 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.

Clonal expansion and functional exhaustion of monoclonal marginal zone B cells in mixed cryoglobulinemia: the yin and yang of HCV-driven lymphoproliferation and autoimmunity.

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.

Clonal B cells of HCV-associated mixed cryoglobulinemia patients contain exhausted marginal zone-like and CD21 low cells overexpressing Stra13

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.

Telomere-dependent replicative senescence of B and T cells from patients with type 1a common variable immunodeficiency

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 (CD21low), lymphoproliferation and autoimmunity. The CD21low 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.

Regression of systemic lupus erythematosus after development of an acquired Toll‐like receptor signaling defect and antibody deficiency

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 patients 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.