c-myc overexpression activates alternative pathways for intracellular proteolysis in lymphoma cells.

Burkitt's lymphoma (BL) is a highly malignant B-cell tumour characterized by chromosomal translocations that constitutively activate the c-myc oncogene. Here we show that BL cells are resistant to apoptosis and do not accumulate ubiquitin conjugates in response to otherwise toxic doses of inhibitors of the proteasome. Deubiquitinating enzymes and the cytosolic subtilisin-like protease tripeptidylpeptidase II are upregulated in BLs, and could be rapidly induced by the overexpression of c-myc in normal B cells carrying oestrogen-driven recombinant Epstein-Barr virus. Apoptosis was induced by inhibiting tripeptidylpeptidase II, suggesting that the activity of this protease may be required for the survival of BL cells. We thus show that there is a regulatory link between c-myc activation and changes in proteolysis that may affect malignant transformation.

Cytolethal distending toxins.

The cytolethal distending toxins (CDTs) constitute the most recently discovered family of bacterial protein toxins. CDTs are unique among bacterial toxins as they have the ability to induce DNA double strand breaks (DSBs) in both proliferating and nonproliferating cells, thereby causing irreversible cell cycle arrest or death of the target cells. CDTs are encoded by three linked genes ( cdtA, cdtB and cdtC) which have been identified among a variety of Gram-negative pathogenic bacteria. All three of these gene products are required to constitute the fully active holotoxin, and this is in agreement with the recently determined crystal structure of CDT. The CdtB component has functional homology with mammalian deoxyribonuclease I (DNase I). Mutation of the conserved sites necessary for this catalytic activity prevents the induction of DSBs as well as all subsequent intoxication responses of target cells. CDT is endocytosed via clathrin-coated pits and requires an intact Golgi complex to exert the cytotoxic activity. Several issues remain to be elucidated regarding CDT biology, such as the detailed function(s) of the CdtA and CdtC subunits, the identity of the cell surface receptor(s) for CDT, the final steps in the cellular internalization pathway, and a molecular understanding of how CDT interacts with DNA. Moreover, the role of CDTs in the pathogenesis of diseases still remains unclear.

Identification and characterization of an Epstein-Barr virus-specific T-cell response in the pathologic tissue of a patient with Hodgkin's disease.

Several lines of evidence indicate that an impairment of EBV-specific immune responses may contribute to the pathogenesis of Hodgkin's disease (HD). At present, however, it is not clear whether a defective immunity to EBV is a characteristic restricted to EBV-associated HD cases or a more generalized phenomenon, part of the inherent immune deficiency of HD patients. In this study, we have addressed this issue by analyzing EBV-specific responses in infiltrating T lymphocytes (TILs) from one HD biopsy, where the virus was confined to a small proportion of apparently normal lymphocytes. TIL cultures were established using low amounts of recombinant interleukin 2 and in the absence of specific stimulation, conditions that preferentially induce the proliferation of in vivo activated T cells. An EBV-specific cytotoxic component was revealed by the capacity of these TILs to lyse autologous EBV-positive lymphoblastoid cell lines (LCLs) obtained by spontaneous transformation from the lesion but not HLA-mismatched LCLs and autologous phytohemagglutinin blasts. This cytotoxic activity closely resembled that of EBV-specific memory T cells, which may be reactivated from the blood lymphocytes of healthy donors by in vitro stimulation with autologous LCLs. The use of a panel of appropriately HLA-matched B95.8-transformed LCLs as targets in standard 51Cr release assays revealed EBV-specific cytotoxic responses to be restricted mainly through the A11 and B44 HLA alleles with a minor HLA-A26-restricted component. Using autologous fibroblasts infected with recombinant vaccinia viruses expressing the EBV latent antigens, the TIL culture was shown to recognize latent membrane protein 2 and, to a lesser extent, EBV-encoded nuclear antigen 6. In addition, a strong proliferative response was induced by coculture of TILs with autologous but not with allogeneic LCLs or autologous phytohemagglutinin blasts. Six CD4-positive, EBV-specific T-cell clones were isolated by limiting dilution. The study of cytokine mRNA expression, carried out by reverse transcriptase-assisted PCR, revealed that three of these T-cell clones expressed a Th0 phenotype, whereas 1 had a Th2 phenotype. These findings are consistent with the presence in this HD lesion of an ongoing immune response against EBV-carrying cells and suggest that the complex immune deficiency that characterizes HD patients probably does not include a generalized, constitutional defect of EBV-specific T-cell responses.

Bacterial genotoxin functions as immune-modulator and promotes host survival.

Bacterial genotoxins are effectors that cause DNA damage in target cells. Many aspects of the biology of these toxins have been characterised in vitro, such as structure, cellular internalisation pathways and effects on the target cells. However, little is known about their function in vivo. Salmonella enterica serovar Typhi (S. Typhi) is a Gram-negative, intracellular bacterium that causes typhoid fever, a debilitating disease infecting more than 20 million people every year. S. Typhiproduce a genotoxin named typhoid toxin (TT), but its role in the contest of host infection is poorly characterized. The major obstacle in addressing this issue is that S. Typhi is exclusively a human pathogen. To overcome this limitation, we have used as model bacterium S. Typhimurium, and engineered it to produce endogenous levels of an active and inactive typhoid toxin, hereby named as TT (or genotoxic) and cdtB (or control), respectively. To our surprise, infection with the genotoxin strain strongly suppressed intestinal inflammation, leading to a better survival of the host during the acute phase of infection, suggesting typhoid toxin may exert a protective role. The presence of a functional genotoxin was also associated with an increased frequency of asymptomatic carriers.

Type 2 Epstein-Barr virus genome and latent membrane protein-1 expression in a T-cell-rich lymphoma of probable B-cell lineage.

In a 79-year-old white woman, a lymphoproliferative disorder that was associated with type 2 Epstein-Barr virus (EBV) infection or reactivation, documented by three subsequent lymph node biopsies, was studied. After an initial phase with features of reactive lymphadenopathy with exhaustion of the follicular germinal centers and depletion of the B-cell lymphoid population, the disease evolved to a T-cell-rich lymphoma in which a clonal cell population of probable B-cell origin was identified. Such clonal cell population harbored the viral genome and expressed EBV latent membrane protein-1 but not EBV nuclear antigen-2. The implications of immunologic interactions between the clonal EBV-infected cells and the reactive T-cell component in the pathogenetic process are discussed.

The cytolethal distending toxins induce DNA damage and cell cycle arrest.

The cytolethal distending toxins (CDTs) are a newly discovered family of bacterial protein toxins with the unique ability to interfere with the cell cycle, causing irreversible cell cycle arrest and consequently death of the target cells. CDTs are encoded by three linked genes (cdtA, cdtB and cdtC) and are produced by a variety of Gram negative bacteria. The mechanism of action of this toxin family only now begins to be elucidated. CDTs are internalized by endocytosis and require an intact Golgi complex to exert their cytotoxic activity. The CdtB component was shown to have functional homology with the mammalian deoxyribonuclease I (DNase I) and the induction of cell cycle arrest in mammalian cells mimicked that induced by DNA damaging agents, suggesting that DNA is the cellular target. Still there are many issues that need to be clarified, such as identification of the function(s) of CdtA and CdtC, characterization of the receptor(s), understanding of the final steps of the internalization pathway and localization of the active component. This review focuses mainly on the effect of CDTs on mammalian cells, highlighting the questions that remain to be answered regarding their molecular mode of action.

Variations in proteasome subunit composition and enzymatic activity in B-lymphoma lines and normal B cells.

We investigated the expression of interferon gamma (IFN-gamma)-regulated subunits and the enzymatic activity of proteasomes purified from tumor-derived and normal B lymphocytes representing different stages of B-cell activation/differentiation. The catalytic beta subunits (Lmp2 and Lmp7) and the regulatory subunits (PA28alpha and PA28beta) were expressed at equally high levels in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs), freshly isolated B-chronic lymphocytic leukemia (B-CLL) cells and normal CD23(-) B lymphocytes. Lmp2 and Lmp7 were selectively down-regulated in germinal center cell-derived Burkitt's lymphoma (BL) and Hodgkin's lymphoma (HD) cell lines. There was a direct correlation between the expression of Lmp2/7 and the chymotrypsin and trypsin-like activities in proteasomes purified from LCLs, BLs and CLL cells, whereas 5 HD cell lines expressing B or T-cell markers exhibited a variable pattern of subunit expression and enzymatic activity. Poor hydrolysis of the fluorogenic substrates by proteasomes from BL cells correlated with a distinct pattern of cleavage of a reference 50mer peptide, production of different sets of degradation products and significantly reduced recovery of a known cytotoxic T-lymphocyte (CTL) target epitope. The enzymatic activity of proteasomes from normal CD23(-) "resting" B lymphocytes resembled that of BL cells in spite of high Lmp2/7 expression. This pattern was not reversed by treatment with the B-cell mitogen, lipopolysaccharide (LPS). The results suggest that different stages of B-cell activation/differentiation are associated with distinct profiles of IFN-gamma-regulated subunit composition and enzymatic activity of the proteasome. This may have important implications for the analysis and manipulation of tumor-specific immune responses.

Phenotype-dependent differences in proteasome subunit composition and cleavage specificity in B cell lines.

We have compared the subunit composition and enzymatic activity of purified 26S proteasomes from Burkitt's lymphoma (BL) cells and in vitro EBV-transformed lymphoblastoid cell lines (LCLs) of normal B cell origin. Low expression of the IFN-gamma-regulated beta low molecular mass polypeptide (Lmp)2, Lmp7, and MECL-1 was demonstrated in a panel of seven BL lines that express the germinal center cell phenotype of the original tumor. Coexpression of Lmp2 and Lmp7 with the constitutively expressed subunits delta and MB1 was demonstrated in the BL lines by immunoprecipitation and two-dimensional gel fractionation of the 20S proteasomes. Coexpression of these subunits correlated with reduced levels of chymotrypsin- and trypsin-like activities detected by the cleavage of fluorogenic substrates. Down-regulation of Lmp2 and Lmp7 and decreased chymotrypsin- and trypsin-like activities were also observed in purified proteasomes from a c-myc-transfected subline of the ER/EB2-5 LCL that has adopted a BL-like phenotype. A synthetic peptide analogue of the immunodominant epitope from the EBV nuclear Ag 4 (E4416-424Y) was cleaved by proteasomes from BLs and A1, while proteasomes from LCLs were inactive. Cleavage of the E4416-424Y peptide was not affected by treatment of the BL cells with IFN-gamma despite both significant up-regulation of Lmp2 and Lmp7 and reconstitution of chymotrypsin and trypsin-like activities against fluorogenic substrates to LCL-like levels. The results demonstrate that B cell lines representing different stages of B cell activation and differentiation express proteasomes with different subunit compositions and enzymatic activity. This may result in the generation of a distinct set of endogenous peptides and influence the immunogenicity of these cells.

The Haemophilus ducreyi cytolethal distending toxin induces cell cycle arrest and apoptosis via the DNA damage checkpoint pathways.

The cytolethal distending toxins (CDTs) induce cell cycle arrest by a mechanism still not well characterized. We demonstrate that the effect of the Haemophilus ducreyi CDT (HdCDT) is cell type-specific: B cell lines underwent apoptosis, epithelial cells and keratinocytes arrested exclusively in G(2), whereas normal fibroblasts arrested both in G(1) and G(2). We studied normal keratinocytes and fibroblasts, which are relevant for understanding the pathogenicity of H. ducreyi. The response to HdCDT resembles the checkpoint response activated by ionizing radiation. Both responses were characterized by an early induction of the p53 gene and the cyclin-dependent kinase inhibitor p21 in fibroblasts, and activation of the chk2 kinase in epithelial cells. In the Ataxia Telangiectasia-mutated gene (ATM)-deficient lymphoblastoid cell lines, intoxication was significantly delayed compared with ATM wild type cells, and was associated with a slower kinetic of p53 stabilization, suggesting that the early response to HdCDT is ATM-dependent. Activation of ATM-dependent pathways was further confirmed by the ability of caffeine to partially override the HdCDT-mediated cell cycle arrest. Our data shed new light on the mechanism of action of this novel family of bacterial toxins, limiting the target candidates to DNA or molecules directly involved in activation of checkpoint responses.

In vivo phenotypic characteristics of AKR T-cell lymphomas with different leukemic potential: possible role of alpha 4 beta 7 integrin in the progression towards the leukemic phenotype.

The present study was undertaken in order to determine whether the expression of specific surface molecules which mediate immune recognition as well as cell-cell and cell-matrix interactions is associated with the leukemic evolution of T-cell lymphomas. To this end, we have investigated the in vivo phenotypic characteristics and the in vitro natural-killer(NK)-cell susceptibility of a group of MCF-247-induced AKR/J T-cell lymphomas with different leukemic potential. We found that in the AKR/J model, the biological aggressiveness of leukemic cells is not dependent upon an escape from host immune surveillance as a consequence of an in vivo down-regulation of H2-Kk determinants or a resistance to NK lysis. Moreover, NK susceptibility of AKR/J lymphomas does not seem to correlate with the level of H2-antigen expression. No obvious correlation was found between the leukemic phenotype and the amount of MEL-14, LFA-1, ICAM-1, Pgp-1/CD44 and THAM/CD26 antigen expression. An in vivo coordinated up-regulation of alpha 4 and beta 7 integrin chains, with the highest levels of expression detected in secondary sites of leukemic infiltration, was observed in the highly leukemic lymphoma NQ22 and, albeit to a lesser extent, in lymphomas with moderate leukemic potential. Conversely, non-leukemic lymphomas were repeatedly alpha 4- and beta 7-negative. These findings suggest that in the AKR/J system the expression of alpha 4 beta 7 integrin may contribute to leukemic spreading of T-cell lymphomas.

The clonal composition of a peptide-specific oligoclonal CTL repertoire selected in response to persistent EBV infection is stable over time.

The TCR repertoire of a peptide-specific HLA A11-restricted CTL response to persistent infection with EBV was followed for a period of 57 mo. Sequencing of TCR V alpha and V beta chains and alanine scanning mutagenesis analysis of 83 CTL clones isolated in five reactivation experiments demonstrated that this repertoire is composed of at least four distinct CTL clonotypes that are constantly reactivated from donor's blood and express structurally heterogeneous TCRs. Target cell recognition and CD8 blocking experiments indicate that the four clonotypes possess different avidity and TCR affinity for the specific Ag. This demonstrates that at least in some individuals a heterogeneous peptide-specific memory CTL repertoire selected by a persistent Ag can be remarkably stable in time and accommodate a range of TCR affinities and T cell avidities. Our results suggest that competition for the specific Ag may be not the major force driving the maintenance of memory CTLs and that the nature of the first antigenic challenge may largely determine the clonal composition of memory.

CD40 cross-linking enhances the immunogenicity of Burkitt's-lymphoma cell lines.

Epstein-Barr-virus (EBV)-positive Burkitt's-lymphoma (BL) cell lines are not recognized by EBV-specific T cells, due to their non-immunogenic phenotype and restricted expression of latent EBV genes. We tested whether triggering of CD40 can alter the phenotype of the tumor cells with regard to: (i) expression of surface markers, (ii) expression of viral antigens, (iii) presentation of endogenous antigens to MHC-class-1 restricted cytotoxic T lymphocytes (CTLs), (iv) stimulatory capacity in allogeneic mixed-lymphocyte cultures (MLCs), (v) sensitivity to natural-killer (NK)-cell-mediated lysis. Co-culture of EBV-positive BL cells with CD40-ligand-transfected L cells induced up-regulation of CD54 and CD80 but did not affect the expression of viral genes. In spite of significant up-regulation of TAP1 and TAP2, and increased expression of MHC class 1, the BL cells remained unable to present endogenously expressed viral antigens to EBV-specific CTL. However, the up-regulation of adhesion and co-stimulatory molecules was associated with increased stimulatory capacity in MLC and enhanced sensitivity to NK cells. These findings indicate that, while inducing only a modest phenotype shift, cross-linking of CD40 under physiologic conditions may selectively enhance the sensitivity of BL cells to anti-tumor immune responses.

Defective presentation of MHC class I-restricted cytotoxic T-cell epitopes in Burkitt's lymphoma cells.

Defects of antigen processing/presentation have been suggested to play a role in the escape of Burkitt's lymphoma (BL) from cytotoxic T lymphocyte (CTL)-mediated rejection. Impaired presentation of an immunodominant HLA A11-restricted epitope from the resident or recombinant vaccinia virus-expressed Epstein-Barr virus nuclear antigen (EBNA)4 was demonstrated in the EBV-positive E95B-BL28 and its EBV-negative parental BL28 cell lines. We have investigated whether this was due to (i) impaired production of the antigenic peptide, (ii) poor peptide translocation into the ER lumen or (iii) inefficient maturation and transport of the MHC-peptide complexes at the cell surface. The defect was not overcome by cytosolic expression of a pre-formed epitope, suggesting that presentation of EBNA4 is not limited by inefficient production of the antigenic peptide. BL28 expressed 5- to 10-fold lower levels of the transporter associated with antigen presentation (TAP) 1 and TAP2 proteins and behaved poorly in a streptolysin-O-mediated peptide translocation assay, whereas E95B-BL28 showed higher TAP expression and virtually normal transporter function. Up-regulation of HLA A11 and reconstitution of TAP activity by treatment with IFN-gamma did not restore presentation of the resident EBNA4 in E95SB-BL28 and did not enhance presentation of the vaccinia virus-expressed intact protein or preformed epitope. Efficient maturation of class I molecules to Endo H-resistant species was demonstrated in pulse-chase experiments. Taken together, our findings identify a previously uncharacterized defect of antigen presentation which appears to affect events occurring after proteasomal degradation but before TAP-dependent peptide transport and MHC class I assembly and maturation.

Local suppression of Epstein-Barr virus (EBV)-specific cytotoxicity in biopsies of EBV-positive Hodgkin's disease.

Epstein-Barr virus (EBV)-positive Hodgkin's and Reed-Sternberg (HRS) cells express the virus-encoded latent membrane proteins LMP1 and LMP2 that could serve as rejection targets in Hodgkin's disease (HD). To examine whether EBV-triggered reactivities can be detected in the tumor, we have compared cytokine mRNA expression, cell phenotype, and cytotoxic activity in biopsies from 8 EBV-carrying and 6 EBV-HD patients. Neither the pattern of lymphokine production nor the cell phenotype of the in vivo-activated interleukin-2-responding populations provided a clear discrimination between EBV+ and EBV- cases. HLA class I-restricted EBV-specific cytotoxicity was shown in interleukin-2-dependent cultures from 3 of 3 EBV- tumors, whereas cultures from 6 of 6 EBV+ tumors were either noncytotoxic or exerted LAK-type cytotoxicity. EBV-specific cytotoxic T lymphocyte precursors were present in the blood of 1 patient carrying an EBV+ tumor. The results suggest that a tumor-associated suppression of EBV-specific T-cell responses may play an important role in the pathogenesis of EBV+ HD.

Epstein-Barr virus (EBV) load in bone marrow transplant recipients at risk to develop posttransplant lymphoproliferative disease: prophylactic infusion of EBV-specific cytotoxic T cells.

A semiquantitative polymerase chain reaction assay was used to monitor the blood levels of Epstein-Barr virus (EBV)-DNA in 9 patients receiving allogeneic bone marrow transplants (BMT). Four of 5 recipients of HLA-mismatched T-cell-depleted grafts showed a 4- to 5-log increase of EBV-DNA within 1 to 3 months after BMT. Administration of 2 to 4 infusions of 10(7) EBV-specific cytotoxic T-lymphocytes (CTLs)/m(2) starting from the time of maximal virus load resulted in a 2- to 3-log decrease of virus titers in 3 patients. One patient, who received a T-cell culture lacking a major EBV-specific component, progressed to fatal EBV-positive lymphoma. Administration of EBV-CTLs before the onset of the EBV-DNA peak resulted in stabilization of the virus titers within 2 to 3 logs above the normal levels in the fifth patient. A moderate increase of virus titers was also detected in 3 of 4 patients receiving unmanipulated HLA-matched grafts, whereas 1 patient with Wiskott-Aldrich syndrome reached a 5-log increase of EBV-DNA load within 70 days after BMT. Our results suggest that a rapid increase of circulating EBV-DNA occurs in the absence of EBV-specific T-cell precursors or in the presence of congenital immune defects that prevent the reestablishment of virus-specific immunity. Prophylactic administration of EBV-CTLs early after BMT appears to provide the most effective protection against the development of EBV-associated lymphoproliferative disease.