Maurizio Carbonari

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.

T-lymphocyte reactivity to the recombinant mycobacterial 65- and 70-kDa heat shock proteins in multiple sclerosis

Owing to their conservation and immunogenicity, heat shock proteins (hsps) represent a class of potential autoantigens. Moreover, they could be targets for gamma delta T lymphocytes, which are prominent in various immune disorders. We studied the T cell proliferative primary responses to recombinant M. bovis 65 kDa hsp (hsp65) and M. tuberculosis 70 kDa hsp (hsp70) in 31 patients with multiple sclerosis (MS), 19 patients with other neurological diseases (OND) and 19 healthy individuals. Positive responses to hsp70, but not to hsp65 were significantly more frequent in patients with MS than in patients with OND or in healthy individuals. In order to verify and refine these results and to characterize the hsp reactive T lymphocytes, we screened 147 PPD-specific long-term T cell lines (76 from 10 patients with MS and 71 from 12 healthy donors) for their proliferative response to hsp65 and hsp70. hsp70-reactive T lines were significantly more common in patients with MS than in healthy controls. The number of T lines responding to hsp65 increased in the MS group only slightly. In 19 T lymphocyte lines from patients with MS and healthy donors, a cytofluorometric analysis was performed with special attention paid to distinct T cell receptor gamma delta determinants. With one exception, in each line the population of gamma delta T cells remained a minority. We conclude that an increased T cell response to mycobacterial hsp70 may be present in patients with multiple sclerosis.

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.

Deficiency of natural killer activity, but not of natural killer binding, in patients with lymphoadenopathy syndrome positive for antibodies to HTLV-III

Blood lymphocytes (BL) of eleven patients with lymphoadenopathy syndrome (LAS) were studied for natural killer (NK) activity against the K562 cell line (using both the standard 51Cr release assay and the single-cell cytotoxicity assay on poly-L-lysine-coated coverslips) and for surface phenotype (employing OKT4, OKT8 and Leu7 monoclonal antibodies). A significant reduction in NK activity and in NK active cells was detected, while the percentage of target binding cells was not affected. Furthermore, the OKT4/OKT8 ratio was found to be inverted, and the Leu7+ subpopulation expanded. The patients had high titers of anti-HTLV-III antibodies. This study indicates that defective NK activity in LAS is secondary to an abnormality in the lytic event itself and not in target binding.

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.

MYCN Sensitizes Human Neuroblastoma to Apoptosis by HIPK2 Activation through a DNA Damage Response

MYCN amplification occurs in approximately 20% of human neuroblastomas and is associated with early tumor progression and poor outcome, despite intensive multimodal treatment. However, MYCN overexpression also sensitizes neuroblastoma cells to apoptosis. Thus, uncovering the molecular mechanisms linking MYCN to apoptosis might contribute to designing more efficient therapies for MYCN-amplified tumors. Here we show that MYCN-dependent sensitization to apoptosis requires activation of p53 and its phosphorylation at serine 46. The p53S46 kinase HIPK2 accumulates on MYCN expression, and its depletion by RNA interference impairs p53S46 phosphorylation and apoptosis. Remarkably, MYCN induces a DNA damage response that accounts for the inhibition of HIPK2 degradation through an ATM- and NBS1-dependent pathway. Prompted by the rare occurrence of p53 mutations and by the broad expression of HIPK2 in our human neuroblastoma series, we evaluated the effects of the p53-reactivating compound Nutlin-3 on this pathway. At variance from other tumor histotypes, in MYCN-amplified neuroblastoma, Nutlin-3 further induced HIPK2 accumulation, p53S46 phosphorylation, and apoptosis, and in combination with clastogenic agents purged virtually the entire cell population. Altogether, our data uncover a novel mechanism linking MYCN to apoptosis that can be triggered by the p53-reactivating compound Nutlin-3, supporting its use in the most difficult-to-treat subset of neuroblastoma. Mol Cancer Res; 9(1); 67–77 ©2010 AACR.

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.

A unified procedure for conservative (morphology) and integral (DNA and immunophenotype) cell staining for flow cytometry

BACKGROUND Current methods for multiparameter DNA flow cytometry suffer from several limitations. These include significant modifications of cell morphological parameters, the impossibility to counterstain cells with certain fluorochromes, and laborious tuning of the instrument that, for some procedures, must be equipped with an ultraviolet (UV) laser. To overcome these problems, we developed a novel method for the simultaneous analysis of morphological parameters, four-color immunophenotyping, and stoichiometric DNA labeling using a bench-top flow cytometer. METHODS The method consists of a mild permeabilization/fixation treatment at room temperature, followed by labeling with fluorochrome-conjugated monoclonal antibodies (mAbs) and with the DNA dye 7-aminoactinomycin D (7-AAD) at 56 degrees C. RESULTS Using this method, we analyzed resting peripheral blood mononucleated cells (PBMC), proliferating T cells cultured in the presence of interleukin-2 (IL-2), and lymphoblastoid B cells. Lymphocytes, monocytes, and lymphoblasts treated by this procedure retained differential light scattering (DLS) characteristics virtually identical to those of untreated cells. This allowed regions to be drawn on forward scatter (FSC) and side scatter (SSC) cytograms resolving different cell populations. DLS were preserved well enough to distinguish large lymphoblasts in the S or G2/M phases from small G0/G1 cells. Also, stainability with fluorescein-isothiocyanate (FITC), R-phycoerythrin (PE), allophycocyanin (APC)-conjugated mAbs was generally preserved. DNA labeling with 7-AAD was of quality good enough to permit accurate cell cycle analysis. CONCLUSIONS The method described here, which we called integral hot staining (IHS), represents a very simple, reproducible, and conservative assay for multiparameter DNA analysis using a bench-top flow cytometer. Last but not least, the cytometer tuning for multiparameter acquisition is straightforward.

Phenotypically immature IgG-bearing B cells in patients with hypogammaglobulinemia

We investigated the prevalence of phenotypically immature IgG B cells (i.e., coexpressing surface IgG and IgM) in the peripheral blood of 12 patients with hypogammaglobulinemia and in normal individuals. Patients had ataxia-telangiectasia (N=1), hyper-IgM combined immunodeficiency (N=1), or common variable immunodeficiency (CVI). IgG/IgM-positive B cells were evaluated by two-color immunofluorescence using fluoresceinor rhodamine-conjugated goat antiserum; to minimize artifacts due toin vivo cytophilic binding of autologous IgG, cell-bound cytophilic Ig were eluted at pH 4 and Fc receptors were blocked by heat-aggregated rabbit IgG before fluorescent staining. All patients, except two with late-onset CVI, had markedly increased proportions of double-stained IgG B cells (56 to 100% of IgG-bearing B cells) in comparison with normal individuals (11 to 33%).