The neoplastically transformed (CD30hi) Marek’s disease lymphoma cell phenotype most closely resembles T-regulatory cells

INTRODUCTION: Marek's disease (MD), a herpesvirus-induced lymphoma of chickens is a unique natural model of CD30-overexpressing (CD30hi) lymphoma. We have previously proposed that the CD30hi neoplastically transformed CD4+ T cells in MD lymphomas have a phenotype antagonistic to cell mediated immunity. Here were test the hypothesis that the CD30hi neoplastically transformed MD lymphoma cells have a phenotype more closely resembling T-helper (Th)-2 or regulatory T (T-reg) cells. MATERIALS AND METHODS: We separated ex vivo-derived CD30hi, from the CD30lo/- (non-transformed), MD lymphoma cells and then quantified the relative amounts of mRNA and proteins for cytokines and other genes that define CD4+ Th-1, Th-2 or T-reg phenotypes. RESULTS AND DISCUSSION: Gene Ontology-based modeling of our data shows that the CD30hi MD lymphoma cells having a phenotype more similar to T-reg. Sequences that could be bound by the MD virus putative oncoprotein Meq in each of these genes' promoters suggests that the MD herpesvirus may play a direct role in maintaining this T-reg-like phenotype.     

Expression of the Marek''s disease virus homolog of herpes simplex virus glycoprotein B in Escherichia coli and its identification as B antigen.

Marek's disease (MD) is an oncogenic disease of chickens caused by MD virus (MDV). Among the major glycoproteins found in MDV-infected cells are gp100, gp60, and gp49, detected by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis with antisera previously shown to be reactive with B antigen in immunodiffusion analysis. Following treatment with tunicamycin (TM), an inhibitor of N-linked glycosylation, the same sera were reported to detect two molecules called pr88 and pr44. However, the gene encoding B antigen was not unequivocally identified. Recently, an MDV homolog of the gene encoding herpes simplex virus glycoprotein B (gB) was identified and sequenced (L. J. N. Ross, M. Sanderson, S. D. Scott, M. M. Binns, T. Doel, and B. Milne, J. Gen. Virol. 70:1789-1804, 1989). To determine whether the MDV gB homolog gene might encode the B antigen, antisera against trpE fusion proteins of the MDV gB homolog (trpE-MDV-gB) were prepared. These antisera immunoprecipitated gp100, gp60, gp49, and a 92-kDa precursor polypeptide (pr88, now designated 92-kDa pr88, in the presence of TM) from MDV-infected cell lysates. On the basis of size comparison, trpE-MDV-gB competition and blocking assays, and the fact that gp100, gp60, gp49, and 92-kDa pr88 could be detected in MDV-infected cells with antisera specific to both MDV B antigen and the gB homolog, it was concluded that (i) the MDV gB homolog gene encodes MDV B antigen and (ii) 92-kDa pr88 is the primary precursor polypeptide. The antisera against trpE-MDV-gB also contained antibody reactive with the herpesvirus of turkey gB homolog, consistent with the known antigenic relatedness between the MDV and herpesvirus of turkey B antigens. TM inhibition data and results from pulse-chase analysis with MDV-infected cells show that MDV gB homolog processing involves cotranslational glycosylation of 92-kDa pr88 to form gp100, which is then cleaved to form gp60 and gp49, the N- and C-terminal halves, respectively, of gp100. This processing pathway is consistent with those of other gB homologs, further supporting the gene identification described above. The conclusions of this study will facilitate future research on the immunobiology of MD, especially studies on the mechanism of immunoprotection.     

Latent Marek's disease virus can be activated from its chromosomally integrated state in herpesvirus-transformed lymphoma cells.

Marek's disease virus (MDV), a lymphotropic herpesvirus, induces T-cell lymphomas in chicken, its natural host. The lymphoma cells are latently infected with MDV but the viral contribution to the transformed phenotype is not understood. To investigate the virus-cell interaction, we focused on the status of MDV in the transformed cells. By the use of highly sensitive fluorescent in situ hybridization with metaphase chromosomes, we found (i) MDV DNA to be randomly integrated at multiple sites in the chromosomes of primary lymphoma cells from chicken tissues; (ii) extrachromosomal, circular MDV genomes were absent and linear virion DNA was usually not detectable in the latently infected lymphoma cells; (iii) the pattern of integration sites revealed the clonal origin of the tumour cells; which (iv) was retained in in vitro established cell lines derived from primary lymphomas; (v) activation of the lytic phase of MDV's life cycle occurred in vitro suggesting that MDV can escape from its integrated status by an unknown mechanism.     

Identification of a unique Marek's disease virus gene which encodes a 38-kilodalton phosphoprotein and is expressed in both lytically infected cells and latently infected lymphoblastoid tumor cells.

The identification of unique Marek's disease (MD) virus (MDV) antigens expressed not only in lytically infected cells but also in latently infected MD lymphoblastoid tumor cell lines is important in understanding the molecular mechanisms of latency and transformation by MDV, an oncogenic lymphotropic herpesvirus of chickens. Through cDNA and nucleotide sequence analysis, an open reading frame (designated the pp38 ORF) which encodes a predicted polypeptide of 290 amino acids was identified in BamHI-H. Demonstration that the pp38 ORF spans the junction of the MDV long unique and long internal repeat regions (MDV has an alphaherpesvirus genome structure) precludes the presence of the gene encoding the B-antigen complex (gp100, gp60, and gp49) in the same region of BamHI-H, where it was originally thought to exist. Duplication of the complete pp38 ORF was not observed in BamHI-D, but part of it (encoding 45 amino acids) was found in the long terminal repeat region of the fragment. By use of trpE-pp38 fusion proteins, antisera against pp38 were prepared. By immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a predominant virus-specific 38,000-dalton polypeptide (designated pp38) and a minor 24,000-dalton polypeptide (designated p24) were found. No precursor-product relationship was found between pp38 and p24 by pulse-chase analysis, and only pp38 was detected by Western blot (immunoblot) analysis with antiserum to pp38. pp38 was found to be phosphorylated and present in oncogenic serotype 1-but not nononcogenic serotype 3-infected cells. Expression of the gene encoding pp38 was relatively insensitive to phosphonoacetic acid inhibition, suggesting that pp38 may belong to one of the early classes of herpesvirus proteins. pp38 was also detected in the latently infected MSB-1 lymphoblastoid tumor cell line. The detection of antibody against pp38 in immune chicken sera indicates that pp38 is an immunogen in birds with MD. Most of the properties described here for a protein detected by methods based on finding the ORF first are identical to those of a 38-kDa phosphoprotein reported by others, suggesting that they are the same. Collectively, the data reported here provide (i) more definitive information on the complete ORF of another MDV gene and the protein that it encodes, (ii) clarification of the gene content within a specific region of the MDV genome, and (iii) the molecular means to conduct further studies to determine whether pp38 plays a role in MDV latency and transformation.     

Expression of the Marek''s disease virus (MDV) homolog of glycoprotein B of herpes simplex virus by a recombinant baculovirus and its identification as the B antigen (gp100, gp60, gp49) of MDV.

A gene encoding a homolog of glycoprotein B of herpes simplex virus (gB homolog) has been identified on the Marek's disease virus (MDV) genome (L. J. N. Ross, M. Sanderson, S. D. Scott, M. M. Binns, T. Doel, and B. Milne, J. Gen. Virol. 70:1789-1804, 1989); however, the molecular and immunological characteristics of the gene product(s) are still not clear. In the present study, the gB homolog of MDV was expressed in insect cells by a recombinant baculovirus, and it was characterized to determine its molecular and antigenic properties. The expressed recombinant protein had three molecular sizes (88 to 110, 58, and 49 kDa) and was recognized by antisera from chickens inoculated with each of the three serotypes of MDV. By immunofluorescence analysis, it was shown that the protein was expressed in the cytoplasm and on the surface of the recombinant baculovirus-infected cells. The gB homolog of MDV was processed similarly to pseudorabies virus and varicella-zoster virus with respect to cleavage and the intramolecular disulfide bond between the cleaved products. Interestingly, the expressed protein reacted with monoclonal antibody M51, specific to the B antigen (gp100, gp60, gp49) of MDV, although the locations of the gene encoding the B antigen and of the gene encoding the gB homolog were reported to be different. Moreover, competitive experiments revealed that anti-gB homolog serum and monoclonal antibody M51 recognized the same molecules. From these results, the gB homolog and the B antigen of MDV seem to be the same glycoprotein.     

Experimental infection of NOD/SCID mice reconstituted with human CD34+ cells with Epstein-Barr virus

Epstein-Barr virus (EBV)-induced lymphoproliferative disease is an important complication in the context of immune deficiency. Impaired T-cell immunity allows the outgrowth of transformed cells with the subsequent production of predominantly B-cell lymphomas. Currently there is no in vivo model that can adequately recapitulate EBV infection and its association with B-cell lymphomas. NOD/SCID mice engrafted with human CD34(+) cells and reconstituted mainly with human B lymphocytes may serve as a useful xenograft model to study EBV infection and pathogenesis. We therefore infected reconstituted mice with EBV. High levels of viral DNA were detected in the peripheral blood of all infected mice. All infected mice lost weight and showed decreased activity levels. Infected mice presented large visible tumors in multiple organs, most prominently in the spleen. These tumors stained positive for human CD79a, CD20, CD30, and EBV-encoded RNAs and were light chain restricted. Their characterization is consistent with that of large cell immunoblastic lymphoma. In addition, tumor cells expressed EBNA1, LMP1, and LMP2a mRNAs, which is consistent with a type II latency program. EBV(+) lymphoblastoid cell lines expressing human CD45, CD19, CD21, CD23, CD5, and CD30 were readily established from the bone marrow and spleens of infected animals. Finally, we also demonstrate that infection with an enhanced green fluorescent protein (EGFP)-tagged virus can be monitored by the detection of infected EGFP(+) cells and EGFP(+) tumors. These data demonstrate that NOD/SCID mice that are reconstituted with human CD34(+) cells are susceptible to infection by EBV and accurately recapitulate important aspects of EBV pathogenesis.     

The RNA subunit of telomerase is encoded by Marek's disease virus

Marek's disease virus (MDV) is a herpesvirus of chickens that induces T lymphomas and tumors within 4 to 5 weeks of infection. Although the ability of MDV to induce tumors was demonstrated many years ago and although a number of viral oncogenic proteins have been identified, the mechanism by which the MDV is implicated in tumorigenesis is still unknown. We report the identification of a virus-encoded RNA telomerase subunit (vTR) within the genome of MDV. This gene is found in the genomic DNA of the oncogenic MDV strains, whereas it is not carried by the nononcogenic MDV strains. The vTR sequence exhibits 88% sequence identity with the chicken gene (cTR). Our functional analysis suggests that this telomerase RNA can reconstitute telomerase activity in a heterologous system (the knockout murine TR(-/-) cell line) by interacting with the telomerase protein component encoded by the host cell. We have also demonstrated that the vTR promoter region is efficient whatever the species of cell line considered and that vTR is expressed in vivo in peripheral blood leukocytes from chickens infected with the oncogenic MDV-RB1B and the vaccine MDV-Rispens strains. The functionality of the vTR gene and the potential implication of vTR in the oncogenesis induced by MDV is discussed.     

Recombinant Marek’s Disease Virus (MDV)-Derived Lymphoblastoid Cell Lines: Regulation of a Marker Gene within the Context of the MDV Genome

Marek’s disease is a herpesvirus (Marek’s disease virus [MDV])-induced pathology of chickens characterized by paralysis and the rapid appearance of T-cell lymphomas. Lymphoblastoid cell lines (LBCLs) derived from MDV-induced tumors have served as models of MDV latency and transformation. We have recently reported the construction of mutant MDVs having a deletion (M. S. Parcells et al., J. Virol. 69:7888–7898, 1995) and an insertion (A. S. Anderson et al., J. Virol. 72:2548–2553, 1998) within the unique short region of the virus genome. These mutant MDVs retained oncogenicity, and LBCLs have been established from the mutant-induced tumors. We report the characterization of these cell lines with respect to (i) virus structure within and reactivated from the cell lines, (ii) surface antigen expression, (iii) kinetics of MDV and marker gene induction, (iv) localization and colocalization of induced MDV antigens and β-galactosidase (β-Gal), and (v) methylation status of the region of lacZ insertion in recombinant- and non-recombinant-derived cell lines. Our results indicate that (i) recombinant-derived cell lines contain no parental virus, (ii) the established cell lines are predominantly CD4⁺ CD8⁻, (iii) the percentage of Lac-expressing cells is low (1 to 3%) but increases dramatically upon 5′-iododeoxyuridine (IUdR) treatment, (iv) lacZ expression is induced with the same kinetics as several MDV lytic-phase genes (pp38, US1, gB, gI, and US10), and (v) the regulation of lacZ expression is not mediated by methylation. Furthermore, the MDV-encoded oncoprotein, Meq, could be detected in cells expressing β-Gal and various lytic antigens but did not appear to be induced by IUdR treatment. Our results indicate that regulation of the lacZ marker gene can serve as sensitive measure of virus lytic-phase induction and the reactivation from latency.     

The early expression of glycoprotein B from herpes simplex virus can be detected by antigen-specific CD8+ T cells

The immune response to cutaneous herpes simplex virus type 1 (HSV-1) infection begins with remarkable rapidity. Activation of specific cytotoxic T lymphocytes (CTL) begins within hours of infection, even though the response within the draining lymph nodes peaks nearly 5 days later. HSV gene products are classified into three main groups, alpha, beta, and gamma, based on their kinetics and requirements for expression. In C57BL/6 mice, the immunodominant epitope from HSV is derived from glycoprotein B (gB(498-505)). While gB is considered a gamma or "late" gene product, previous reports have indicated that some level of gene expression may occur soon after infection. Using brefeldin A as a specific inhibitor of viral antigen presentation to major histocompatibility complex class I-restricted CTL, we have formally addressed the timing of gB peptide expression in an immunologically relevant manner following infection. Presentation of gB peptide detected by T-cell activation was first observed within 2 h of infection. Comparison with another viral epitope expressed early during infection, HSV-1 ribonucleotide reductase, demonstrated that gB is presented with the same kinetics as this classical early-gene product. Moreover, this rapidity of gB expression was further illustrated via rapid priming of na?ve transgenic CD8(+) T cells in vivo after HSV-1 infection of mice. These results establish that gB is expressed rapidly following HSV-1 infection, at levels capable of effectively stimulating CD8(+) T cells.     

Ex vivo profiling of CD8+-T-cell responses to human cytomegalovirus reveals broad and multispecific reactivities in healthy virus carriers

Human cytomegalovirus (HCMV) can establish both nonproductive (latent) and productive (lytic) infections. Many of the proteins expressed during these phases of infection could be expected to be targets of the immune response; however, much of our understanding of the CD8(+)-T-cell response to HCMV is mainly based on the pp65 antigen. Very little is known about T-cell control over other antigens expressed during the different stages of virus infection; this imbalance in our understanding undermines the importance of these antigens in several aspects of HCMV disease pathogenesis. In the present study, an efficient and rapid strategy based on predictive bioinformatics and ex vivo functional T-cell assays was adopted to profile CD8(+)-T-cell responses to a large panel of HCMV antigens expressed during different phases of replication. These studies revealed that CD8(+)-T-cell responses to HCMV often contained multiple antigen-specific reactivities, which were not just constrained to the previously identified pp65 or IE-1 antigens. Unexpectedly, a number of viral proteins including structural, early/late antigens and HCMV-encoded immunomodulators (pp28, pp50, gH, gB, US2, US3, US6, and UL18) were also identified as potential targets for HCMV-specific CD8(+)-T-cell immunity. Based on this extensive analysis, numerous novel HCMV peptide epitopes and their HLA-restricting determinants recognized by these T cells have been defined. These observations contrast with previous findings that viral interference with the antigen-processing pathway during lytic infection would render immediate-early and early/late proteins less immunogenic. This work strongly suggests that successful HCMV-specific immune control in healthy virus carriers is dependent on a strong T-cell response towards a broad repertoire of antigens.     

Transformation of T-lymphocyte subsets by Marek''s disease herpesvirus.

Marek's disease herpesvirus (MDV)-transformed lymphoblastoid tumor cell lines were characterized for the presence of the surface markers. Monoclonal antibodies were used for CD3 (T-cell receptor [TCR] complex), TCR1, TCR2, and TCR3, CD4, CD8, and Ia antigen by indirect fluorescence staining followed by microscopic examination or flow cytometry. The lymphoblastoid cell lines were obtained from tumors from chickens infected with MDV (n = 44) or from local lesions induced by inoculation of allogeneic, MDV-infected chick kidney cells (n = 56). Lymphocytes were harvested from these lesions between 4 and 16 days postinoculation and cultured in vitro to establish cell lines. All cell lines expressed Ia antigen and CD3 and/or TCR and thus are activated T cells. Most of the cell lines developed from tumors were CD4+ CD8-; only one cell line was negative for both markers. Sixteen percent of the cell lines were TCR3+, while the remainder were TCR2+. The cell lines developed from local lesions were much more heterogeneous: 45% were CD4- CD8+, 34% were CD4- CD8-, and only 21% were CD4+ CD8-. The number of TCR3+ cell lines was larger than expected for the CD4- CD8+ and CD4- CD8- cell lines, as judged from the presence of these cells in the blood. These results indicate that several subsets of T lymphocytes can be transformed by MDV, depending on the pathogenesis of infection. Activation of T cells as a consequence of the normal pathogenesis or by allogeneic stimulation seem to be a first important step in the process of transformation.     

Protection against Marek''s disease by a fowlpox virus recombinant expressing the glycoprotein B of Marek''s disease virus.

Fowlpox virus (FPV) recombinants expressing the glycoprotein B and the phosphorylated protein (pp38) of the GA strain of Marek's disease virus (MDV) were assayed for their ability to protect chickens against challenge with virulent MDV. The recombinant FPV expressing the glycoprotein B gene elicited neutralizing antibodies against MDV, significantly reduced the level of cell-associated viremia, and, similar to the conventional herpesvirus of turkeys, protected chickens against challenge with the GA strain and the highly virulent RB1B and Md5 strains of MDV. The recombinant FPV expressing the pp38 gene failed to either elicit neutralizing antibodies against MDV or protect the vaccinated chickens against challenge with MDV.     

Defining the herpes simplex virus-specific CD8+ T cell repertoire in C57BL/6 mice

HSV type 1 (HSV-1) expresses its genes sequentially as immediate early (α), early (β), leaky late (γ1), and true late (γ2), where viral DNA synthesis is an absolute prerequisite only for γ2 gene expression. The γ1 protein glycoprotein B (gB) contains a strongly immunodominant CD8(+) T cell epitope (gB(498-505)) that is recognized by 50% of both the CD8(+) effector T cells in acutely infected trigeminal ganglia (TG) and the CD8(+) memory T cells in latently infected TG. Of 376 predicted HSV-1 CD8(+) T cell epitopes in C57BL/6 mice, 19 (gB(498-505) and 18 subdominant epitopes) stimulated CD8(+) T cells in the spleens and TG of HSV-1 acutely infected mice. These 19 epitopes identified virtually all CD8(+) T cells in the infected TG that represent all or the vast majority of the HSV-specific CD8(+) TCR repertoire. Only 11 of ～84 HSV-1 proteins are recognized by CD8(+) T cells, and most (～80%) are expressed before viral DNA synthesis. Neither the immunodominance of gB(498-505) nor the dominance hierarchy of the subdominant epitopes is due solely to MHC or TCR affinity. We conclude that the vast majority of CD8(+) T cells in HSV-1 acutely infected TG are HSV specific, that HSV-1 β and γ1 proteins that are expressed before viral DNA synthesis are favored targets of CD8(+) T cells, and that dominance within the TCR repertoire is likely due to the frequency or expansion and survival characteristics of CD8(+) T cell precursors.     

Identification of chicken lymphocyte antigen 6 complex, locus E (LY6E, alias SCA2) as a putative Marek's disease resistance gene via a virus-host protein interaction screen

Marek's disease virus (MDV) is a naturally occurring oncogenic avian herpesvirus that causes neurological disorders and T cell lymphoma disease in domestic chickens. Identification and functional characterization of the individual factors involved in Marek's disease (MD) resistance or pathogenesis will enhance our understanding of MDV pathogenesis and further genetic improvement of chickens. To study the genetic basis for resistance to MD, a strategy that combined protein-protein interaction screens followed by linkage analysis was performed. The MDV protein US10 was used as the bait in an E. COLI two-hybrid screening of a cDNA library derived from activated splenic T cells. The chicken LY6E, also known as SCA2 and TSA1, was found to specifically interact with US10. This interaction was confirmed by an in vitro protein-binding assay. Furthermore, LY6E was found to be significantly associated with MD traits in an MD resource population comprised of commercial chickens. Previously, LY6E was implicated in two independent DNA microarray experiments evaluating differential gene expression following MDV infection. Given that LY6E is involved in T cell differentiation and activation, we suggest that LY6E is a candidate gene for MD resistance and deserves further investigation on its role in MDV pathogenesis, especially with respect to the binding of US10.     

A new subset of CD103+CD8alpha+ dendritic cells in the small intestine expresses TLR3, TLR7, and TLR9 and induces Th1 response and CTL activity

CD103(+) dendritic cells (DCs) are the major conventional DC population in the intestinal lamina propria (LP). Our previous report showed that a small number of cells in the LP could be classified into four subsets based on the difference in CD11c/CD11b expression patterns: CD11c(hi)CD11b(lo) DCs, CD11c(hi)CD11b(hi) DCs, CD11c(int)CD11b(int) macrophages, and CD11c(int)CD11b(hi) eosinophils. The CD11c(hi)CD11b(hi) DCs, which are CD103(+), specifically express TLR5 and induce the differentiation of naive B cells into IgA(+) plasma cells. These DCs also mediate the differentiation of Ag-specific Th17 and Th1 cells in response to flagellin. We found that small intestine CD103(+) DCs of the LP (LPDCs) could be divided into a small subset of CD8α(+) cells and a larger subset of CD8α(-) cells. Flow cytometry analysis revealed that CD103(+)CD8α(+) and CD103(+)CD8α(-) LPDCs were equivalent to CD11c(hi)CD11b(lo) and CD11c(hi)CD11b(hi) subsets, respectively. We analyzed a novel subset of CD8α(+) LPDCs to elucidate their immunological function. CD103(+)CD8α(+) LPDCs expressed TLR3, TLR7, and TLR9 and produced IL-6 and IL-12p40, but not TNF-α, IL-10, or IL-23, following TLR ligand stimulation. CD103(+)CD8α(+) LPDCs did not express the gene encoding retinoic acid-converting enzyme Raldh2 and were not involved in T cell-independent IgA synthesis or Foxp3(+) regulatory T cell induction. Furthermore, CD103(+)CD8α(+) LPDCs induced Ag-specific IgG in serum, a Th1 response, and CTL activity in vivo. Accordingly, CD103(+)CD8α(+) LPDCs exhibit a different function from CD103(+)CD8α(-) LPDCs in active immunity. This is the first analysis, to our knowledge, of CD8α(+) DCs in the LP of the small intestine.     

Persistence of Marek''s disease virus in a subpopulation of B cells that is transformed by avian leukosis virus, but not in normal bursal B cells.

Previous studies have described an augmentation of avian leukosis virus (ALV)-induced lymphoid leukosis in chickens that were coinfected with a serotype 2 Marek's disease virus (MDV) strain, SB-1. As a first step toward understanding the mechanism of this augmentation, we have analyzed the tropism of the MDV for the ALV-transformed B cell. After hatching, chickens were coinfected with ALV and a nonpathogenic strain of MDV, SB-1. Seventy primary and metastatic ALV-induced lymphomas that developed in chickens between 14 and 20 weeks of age were found, with only one exception, to carry SB-1 DNA. The MDV genome was maintained in cell lines derived from the tumors. However, MDV DNA could not be detected in nontransformed bursal B cells from chickens carrying ALV lymphomas. Moreover, during and after the lytic phase of MDV infection, SB-1 DNA was near or below the level of detection in bursal cells, suggesting that MDV most likely infects only a small subpopulation of bursal cells. By contrast, ALV-transformed B cells from MDV-free chickens could be persistently infected with MDV in vitro. These findings indicate that ALV lymphoma cells, unlike nontransformed bursal B cells, are susceptible to persistent MDV infection and can serve as a reservoir of MDV that can potentially influence the physiology of the transformed cell.