Massimo Fiorilli

Hepatitis C Virus Drives the Unconstrained Monoclonal Expansion of VH1–69-Expressing Memory B Cells in Type II Cryoglobulinemia: A Model of Infection-Driven Lymphomagenesis

Chronic hepatitis C virus infection causes B cell lymphoproliferative disorders that include type II mixed cryoglobulinemia and lymphoma. This virus drives the monoclonal expansion and, occasionally, the malignant transformation of B cells producing a polyreactive natural Ab commonly encoded by the VH1–69 variable gene. Owing to their property of producing natural Ab, these cells are reminiscent of murine B-1 and marginal zone B cells. We used anti-Id Abs to track the stages of differentiation and clonal expansion of VH1–69+ cells in patients with type II mixed cryoglobulinemia. By immunophenotyping and cell size analysis, we could define three discrete stages of differentiation of VH1–69+ B cells: naive (small, IgMhighIgDhighCD38+CD27−CD21highCD95−CD5−), “early memory” (medium-sized, IgMhighIgDlowCD38−CD27+CD21lowCD95+CD5+), and “late memory” (large-sized, IgMlowIgDlow-negCD38−CD27lowCD21low-negCD5−CD95−). The B cells expanded in cryoglobulinemia patients have a “memory” phenotype; this fact, together with the evidence for intraclonal variation, suggests that antigenic stimulation by hepatitis C virus causes the unconstrained expansion of activated VH1–69+ B cells. In some cases, these cells replace the entire pool of circulating B cells, although the absolute B cell number remains within normal limits. Absolute monoclonal VH1–69+ B lymphocytosis was seen in three patients with cryoglobulinemia and splenic lymphoma; in two of these patients, expanded cells carried trisomy 3q. The data presented here indicate that the hepatitis C virus-driven clonal expansion of memory B cells producing a VH1–69+ natural Ab escapes control mechanisms and subverts B cell homeostasis. Genetic alterations may provide a further growth advantage leading to an overt lymphoproliferative disorder.

Relative Increase of T Cells Expressing the Gamma/Delta Rather Than the Alpha/Beta Receptor in Ataxia-Telangiectasia

In ataxia-telangiectasia, B-cell and T-cell deficiencies are thought to be due to a defect of rearrangements of immunoglobulin and T-cell receptor genes. T cells recognize antigens through two types of CD3-associated receptors: alpha/beta chains on mature cells and gamma/delta chains mostly on immature cells. We studied 10 patients with ataxia-telangiectasia and found that most had a relative increase of circulating T cells bearing gamma/delta receptors rather than alpha/beta receptors, as compared with normal subjects (P less than 0.001). Patients with other immune deficits, including eight with common variable immunodeficiency, one with Wiskott-Aldrich syndrome, two with hyperimmunoglobulinemia E syndrome, and one with severe combined immunodeficiency, had normal ratios of gamma/delta-bearing to alpha/beta-bearing cells. A marked predominance of gamma/delta-bearing T cells was found in a patient with a primary T-cell defect. The relative increase in gamma/delta-bearing T cells in the patients with ataxia-telangiectasia was largely accounted for by cells that reacted with the monoclonal antibody BB3, an apparently distinct subset of T cells that selectively express the C gamma 1 gene product of the T-cell receptor. Although they had normal ratios of gamma/delta-bearing to alpha/beta-bearing T cells, the patients with common variable immunodeficiency had a significant increase (P = 0.01) in the number of T cells expressing C gamma 2 that reacted with the monoclonal antibody delta-TCS-1. We conclude that the increased ratio of gamma/delta-bearing to alpha/beta-bearing T cells in ataxia-telangiectasia may reflect both a recombinational defect that interferes with T-cell and B-cell gene rearrangements and an inability to repair damage to the DNA.

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.

Variant of ataxia-telangiectasia with low-level radiosensitivity

SummaryIn the present study we examined cells from several patients clinically diagnosed as having ataxia-telangiectasia (AT), for the capacity of their cells to inhibit DNA synthesis following exposure to gamma irradiation, and for the rate of spontaneous or blcomycin-induced chromosomal aberrations. Cells from two patients showed normal inhibition of DNA synthesis and levels of induced chromosomal aberrations intermediate between normal and AT cells. These two patients had only minimal immunologic impairment. These findings appear to define one distinct subset of AT.

Heterogeneity of immunological abnormalities in ataxia-telangiectasia

Peripheral blood mononuclear cells from five patients with ataxia-telangiectasia were evaluated for their reactivity with a panel of monoclonal antibodies directed against T-cell subsets and for theirin vitro functions in a pokeweed mitogen-induced immunoglobulin biosynthesis assay. All the patients had significantly reduced proportions of cells identified by monoclonal antibodies to subpopulations of T lymphocytes with helper activity (OKT4 and 5/9) and produced low amounts or no IgA and IgGin vitro. Immunoglobulin biosynthesis was increased by the addition of normal x-irradiated peripheral blood mononuclear cells in one of three patients, suggesting a helper T-cell deficiency in this patient and intrinsic B-cell defects in the other two. Two patients had increased proportions of cells identified by a monoclonal antibody to a subpopulation of T lymphocytes which includes suppressor T cells (OKT8), and their cells were able to suppress immunoglobulin biosynthesis by peripheral blood mononuclear cells from normal donors. These findings indicate heterogeneous disturbances of immunoregulatory mechanisms in ataxia-telangiectasia.

Selective deficiency of CD4+/CD45RA+ lymphocytes in patients with ataxia-telangiectasia

Several immunological abnormalities have been observed in ataxia-telangiectasia (AT), the most consistent being defects of immunoglobulin isotypes, decreased T-cell numbers, and reduced proliferative responses to mitogens. We examined the distribution of T lymphocytes expressing distinctive surface Ag characteristic of “naive” (CD45RA+) and “memory” (CD29+, CD45RO+) T cells, in both CD4+ and CD8+ (bright and dim) lymphocytes from 13 AT patients, compared with healthy agematched controls. We found that, irrespective of age, patients with AT had a severe deficiency of CD4+/CD45RA+ lymphocytes. This decrease accounted for the reduction of total CD4+ cells, since the absolute numbers ofmemory CD4+ cells were not significantly different in AT and in controls. Functional tests revealed poor proliferative responses to phytohemagglutinin and normal responses to soluble Ag (tetanus toxoid) in AT patients. These data fit with the distribution ofnaive andmemory cells, which are known to respond predominantly to mitogens or to recall Ag, respectively. CD45RA molecules were normally expressed on CD8+ lymphocytes. This rules out a generalized defect of regulation or differential splicing as the cause of defective expression of CD45RA on CD4+ cells. The selective deficiency of CD4+CD45RA+ may provide a cellular basis for some functional T-cell abnormalities of AT patients. Furthermore, it might practically serve for an early, or even prenatal, diagnosis of this disease.

Mixed cryoglobulinemia secondary to visceral Leishmaniasis.

We describe a case of type II mixed cryoglobulinemia, with monoclonal IgMkappa rheumatoid factor, associated with visceral leishmaniasis caused by Leishmania infantum. Involvement of Leishmania antigen(s) in the formation of cryoprecipitable immune complexes was suggested by the fact that cryoglobulinemic vasculitis subsided after antiparasite therapy and that anti-Leishmania antibodies, as well as rheumatoid factor, were enriched in the cryoprecipitate. We observed 2 additional patients with visceral leishmaniasis and cryoglobulinemic vasculitis. All 3 patients had seemingly contracted leishmaniasis in Italy, were hepatitis C virus negative, and were initially diagnosed as having autoimmune disorders. These findings indicate that Leishmania can be an etiologic agent of type II mixed cryoglobulinemia. This parasitosis should be taken into consideration in the differential diagnosis of vasculitides in endemic areas.

THYMOPOIETIN PENTAPEPTIDE TREATMENT OF PRIMARY IMMUNODEFICIENCIES

26 patients with primary immunodeficiencies (3 infants with severe combined immunodeficiency [SCID] 3 with DiGeorge syndrome, 6 with T-cell defect or SCID with B cells, 4 with common variable hypogammaglobulinaemia and associated T-cell defect, 5 with ataxia-telangiectasia, and 5 with hyper-IgE syndrome) were treated with thymopoietin pentapeptide (TP-5) at a dose of 0 . 5 mg/kg daily for 2 weeks and then 3 times a week at 0 . 5 mg/kg for 10 weeks, 3 patients with DiGeorge syndrome and 3 with primary T-cell defect demonstrated pronounced clinical and immunological improvement during treatment. None of the patients with SCID and 3 of 6 patients with SCID with B cells or primary T-cell defect showed any clinical or immunological changes during therapy. In 5 patients with ataxia-telangiectasia clinical manifestations and immunological tests were unchanged by TP-5. Abnormality of T cells in cases of hyper-IgE syndrome was not corrected by TP-5 treatment.

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.