Martin Rowe

Antigen Processing Defects in Cervical Carcinomas Limit the Presentation of a CTL Epitope from Human Papillomavirus 16 E6

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with the development of cervical cancer. The E6 and E7 proteins of HPV are constitutively expressed in cervical carcinoma cells making them attractive targets for CTL-based immunotherapy. However, few studies have addressed whether cervical carcinomas can process and present HPV E6/E7-derived Ags for recognition by CTL. We generated HLA-A*0201-restricted CTL clones against HPV16 E629–38 that recognized HPV16 E6 Ags transfected into B lymphoblastoid cells. These CTL were unable to recognize HLA-A*0201+ HPV16 E6+ cervical carcinoma cell lines even when the level of endogenous HPV16 E6 in these cells was increased by transfection. This defect in presentation of HPV16 E629–38 correlated with low level expression of HLA class I, proteasome subunits low molecular mass protein 2 and 7, and the transporter proteins TAP1 and TAP2 in the cervical carcinoma cell lines. The expression of all of these proteins could be up-regulated by IFN-γ, but this was insufficient for CTL recognition unless the level of HPV16 E6 Ag was also increased by transfection. CTL recognition of the HPV16 E629–38 epitope in 721.174 B cells was dependent on TAP expression but independent of immunoproteasome expression. Collectively, these findings suggest that presentation of the HPV16 E629–38 epitope in cervical carcinoma cell lines is limited both by the level of TAP expression and by the low level or availability of the source HPV E6 oncoprotein. These observations place constraints on the use of this, and potentially other, HPV-derived CTL epitopes for the immunotherapy of cervical cancer.

Epstein-Barr virus LMP1 blocks p16INK4a-RB pathway by promoting nuclear export of E2F4/5.

The p16INK4a–RB pathway plays a critical role in preventing inappropriate cell proliferation and is often targeted by viral oncoproteins during immortalization. Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is often present in EBV-associated proliferative diseases and is critical for the immortalizing and transforming activity of EBV. Unlike other DNA tumor virus oncoproteins, which possess immortalizing activity, LMP1 does not bind to retinoblastoma tumor suppressor protein, but instead blocks the expression of p16INK4a tumor suppressor gene. However, it has been unclear how LMP1 represses the p16INK4a gene expression. Here, we report that LMP1 promotes the CRM1-dependent nuclear export of Ets2, which is an important transcription factor for p16INK4a gene expression, thereby reducing the level of p16INK4a expression. We further demonstrate that LMP1 also blocks the function of E2F4 and E2F5 (E2F4/5) transcription factors through promoting their nuclear export in a CRM1-dependent manner. As E2F4/5 are essential downstream mediators for a p16INK4a-induced cell cycle arrest, these results indicate that the action of LMP1 on nuclear export has two effects on the p16INK4a–RB pathway: (1) repression of p16INK4a expression and (2) blocking the downstream mediator of the p16INK4a–RB pathway. These results reveal a novel activity of LMP1 and increase an understanding of how viral oncoproteins perturb the p16INK4a–RB pathway.

Isolation and analysis of two strongly transforming isoforms of the Epstein‐Barr‐virus(EBV)‐encoded latent membrane protein‐1 (LMP1) from a single Hodgkin's lymphoma

Two genes encoding the latent membrane protein 1 (LMP1) of the Epstein‐Barr virus (EBV) were isolated from a single case of Hodgkins disease (HD) and were tested for their biological activities. The LMP1 gene from the Reed‐Sternberg cells contained point mutations relative to the prototype LMP1 gene, leading to amino‐acid exchanges. The LMP1 gene from passenger lymphocytes showed identical point mutations, but also had an in‐frame insertion of 132 base pairs within the 33‐bp repeat region. This insert encoding 44 amino acids contained the sequence PSQQS, corresponding to the potential TRAF‐binding motif PXQXT/S. When compared to the B95.8 gene, both HD‐derived LMP1 genes showed an increase in the transformation of Rat‐1 rodent fibroblasts. The transforming ability of the LMP1 gene with the insertion was greater than that of the other HD‐derived LMP1, and was comparable with the highly transforming LMP1‐Cao gene derived from a nasopharyngeal carcinoma. The HD‐derived genes stimulated expression of the cell‐surface markers, CD40 and CD54, similarly to the LMP1‐B95.8 gene, while the LMP1‐Cao gene had a significantly reduced ability to induce these proteins. In contrast, the LMP1‐Cao transactivated an NF‐κB‐response element more efficiently than did the HD‐derived genes. Transfer of the 132‐bp insert alone into the B95.8 gene did not increase its transforming activity to the LMP1‐Cao level, indicating that additional mutations in the LMP1 gene are necessary for modulating this function. Int. J. Cancer 76:194–200, 1998.© 1998 Wiley‐Liss, Inc.

Epstein-Barr Virus Latent Membrane Protein-1 Mediates Upregulation of Tumor Necrosis Factor-α in EBV-Infected T Cells: Implications for the Pathogenesis of Hemophagocytic Syndrome

The infection of human T cells by Epstein-Barr virus (EBV) may result in a fatal hemophagocytic syndrome (HS). We have previously shown that EBV can selectively upregulate the tumor necrosis factor-alpha (TNFalpha) gene and lead to activation of macrophages in a manner similar to the pathobiology of HS in EBV-infected T lymphoproliferative disorders (LPDs). This study was designed to further clarify the specific EBV gene product(s) responsible for TNFalpha upregulation. RT-PCR analysis of EBV gene expression was performed on 2 CR2-transfected EBV-infected T lymphoma lines and 2 EBV-infected B cell lines. To identify the EBV gene responsible for upregulation of TNFalpha, 2 reporter recombinant plasmids, pTNF-CAT and pTNFalpha-Luc, were then constructed and cotransfected with the expression plasmids of the EBV latent and lytic genes (EBNA-1, EBNA-2, LMP-1, LMP-2A, and BZLF-1) in both T and B cell lines. Analyses using ELISA and Western blotting were further performed to detect the secreted TNFalpha. The results revealed that EBNA-1 and LMP-1 were consistently expressed in EBV-infected T cell lines (type II latency), while a type III latency with expression of EBNA-1, EBNA-2, LMP-1, and lytic BZLF transcripts was detected in EBV-infected B cell lines. LMP-1 was demonstrated to be the only EBV gene product to transactivate the TNFalpha gene, and this phenomenon was observed only in T, not in B, cells. Enhanced secretion of TNF-alpha protein was also detected in LMP1-transfected T cell lines. We concluded that LMP1 is the candidate protein in the upregulation of the TNFalpha gene in T cells and is probably responsible for the pathogenesis of HS in EBV-infected T lymphoproliferative disorders.

NF-κB is required for cell death induction by latent membrane protein 1 of Epstein-Barr virus

Abstract NF-κB is a transcription factor known to promote or antagonize cell death depending on cell types and stimuli. Here, we demonstrate that expression of latent membrane protein 1 (LMP1), an Epstein–Barr virus (EBV)-encoded membrane protein, triggers programmed cell death in an NF-κB-dependent manner. Co-expression of NF-κB inhibitors completely prevented activation of NF-κB and LMP1-induced cell death. Addition therein of RelA, an active subunit of NF-κB, restored the NF-κB activation and cell death induction by LMP1, but RelA alone did not induce cell death. These results indicate that the activation of NF-κB is required for cell death induced by LMP1. Moreover, LMP1 induced activation of caspase-3 via the activation of NF-κB. Studies with z-VAD-fmk, a caspase inhibitor, indicated that NF-κB mediated both caspase-dependent and -independent death pathways. In conclusion, the cell death induced by LMP1 uncovered caspase-dependent and -independent death pathways both of which require NF-κB.

Analysis of human tumour necrosis factor receptor 1 dominant-negative mutants reveals a major region controlling cell surface expression

Tumour necrosis factor receptor 1 (TNFR1) plays a critical role in host defence and inflammation. We have identified a membrane proximal region (aa 218–324) of TNFR1 that restricts surface expression. This was prompted by comparing the dominant‐negative properties of a C‐terminal truncation of TNFR1 with a point mutant that prevents signalling. C‐terminal truncation (aa 218–426) generates a better dominant‐negative TNFR1 mutant than inactivation of the death domain by point mutation. The increased dominant‐negative activity correlates with increased cell surface expression. The membrane proximal region is the most important region of the receptor for restricting expression.

Characterization of a CD40-Dominant Inhibitory Receptor Mutant

CD40 is an important mediator of immune and inflammatory responses. It is a costimulatory molecule for B cell proliferation and survival. Blockade of CD40 has been shown to induce tolerance and its role in other pathogenic conditions has led to the proposal that CD40 inhibition could be valuable therapeutically. As a first step to this end, we have characterized a CD40-dominant negative receptor. This inhibitory mutant lacks the identified CD40 signaling domains. It inhibits both cotransfected and endogenous CD40 activation of NF-κB. This mutant is specific, as it does not affect TNF or latent membrane protein 1 signaling. Its potential usefulness is illustrated by its ability to inhibit the CD40 ligand-stimulated increases of HLA and CD54 expression, molecules involved in Ag recognition and lymphocyte recruitment leading to organ rejection. The inhibitory mutant has no TNFR-associated factor 2-binding capabilities and inhibits the recruitment of TNFR-associated factor 2 to the CD40 signaling complex after stimulation. These studies show that the CD40 inhibitory receptor molecule is effective, specific, and useful both for research and potentially as a clinical tool. And furthermore, it is likely that similar dominant inhibitory receptors can be generated for all of the members of the TNFR superfamily.