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Biomedical subjects

M Rowe

Publications and source records attributed to M Rowe.

At least 91 records · Page 5Linked to original sources
PubMed1994

Lymphotoxin acts as an autocrine growth factor for Epstein-Barr virus-transformed B cells and differentiated Burkitt lymphoma cell lines.

A critical event in B cell immortalization by Epstein-Barr virus (EBV) is the establishment of an autocrine loop where cells produce a growth factor which supports their own proliferation. We investigated the potential of lymphoblastoid cell lines (LCL) and Burkitt lymphoma (BL) cell lines to produce and respond to the cytotoxins, tumor necrosis factor-alpha (TNF-alpha) and lymphotoxin (LT). Transformation in vitro of peripheral blood B cells by EBV from seven different donors resulted in spontaneous production of both LT (11,542 pg/ml +/- 7546, mean +/- SD) and, to a lesser extent, TNF-alpha (197 pg/ml +/- 174). Similarly BL cell lines derived from in vivo transformation which developed a 'LCL-like' phenotype in vitro (group III) produced more LT (1990 pg/ml +/- 1740) than the 'group I' BL cell lines (< 40 pg/ml LT) which had maintained the original BL biopsy cell phenotype in vitro. Transformation of peripheral blood B cells to generate LCL also resulted in an increase in surface p75 (p < 0.02) and to a lesser extent p55 (not significant, ns) TNF receptor (TNF-R) expression. Similar increases in surface TNF-R (p75 p < 0.02, p55 ns) were observed on the 'group III' BL cell lines compared with the 'group I' BL cell lines. Proliferation of an LCL and a 'group III' BL cell line in vitro was via an autocrine loop since inhibition of LT reduced proliferation. This proliferation could also be blocked in the presence of the antagonistic anti-p55 TNF-R antibody, H398, but not the antagonistic antibody anti-p75 TNF-R antibody UTR-1. Furthermore, proliferation could be induced with the p55 agonistic antibody, HTR-9. In contrast to these observations with p55 TNF-R antibodies, two out of six of the 'group III' BL lines (Jijoye and Oba) only expressed the p75 TNF-R and proliferation of these cells could only be blocked by the antagonistic anti-p75 TNF-R antibody UTR-1. These data suggest that LT is an autocrine growth factor for lymphoblastoid cells, and BL cell lines which display an LCL phenotype. Furthermore, although both TNF-R are increased on the surface of these cells, this autocrine growth signal is mediated principally through binding to the p55 TNF-R.

B-Lymphocytes↗
PubMed1994

HIV-1 induces down-regulation of bcl-2 expression and death by apoptosis of EBV-immortalized B cells: a model for a persistent "self-limiting" HIV-1 infection.

Interactions between HIV-1 and EBV were studied in HIV-1-infected EBV-positive lymphoblastoid B cells. Following in vitro exposure of B cells to HIV-1, the number of infected cells reached a plateau (25-35%) in approximately 20 days and remained fairly stable thereafter, despite the presence of infectious virus in culture supernatants. HIV-1-positive (gp120+) were separated from HIV-1-negative (gp120-) cells, and the two fractions were further characterized for EBV antigens, bcl-2 expression, and growth capacity in vitro. Compared to gp120- cells, EBNA 1, EBNA 2, and LMP 1 were down-regulated, and the episomal form of EBV-DNA was dramatically decreased in the gp120+ cells. When plated in culture gp120+, but not gp120-, cells died; BZLF1 antigen was not expressed, thus ruling out a reactivation of the EBV lytic cycle. Cytofluorometric, morphological, and molecular analyses disclosed that gp120+ cell death was due instead to apoptosis; evidence of bcl-2 down-regulation in these cells was consistent with this finding. gp120+ cell apoptosis contributed to keeping the level of HIV-1-infected cells at a steady state in the unfractionated culture, where persistent infection was maintained by HIV-1 transmission to B cells newly arising from the proliferation of HIV-1-uninfected cells.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Reduced signal transduction through glucocorticoid receptor in Burkitt's lymphoma cell lines.

Signal transduction through the glucocorticoid receptor (GR) was shown to be significantly reduced in Epstein-Barr virus (EBV)-positive group I Burkitt's lymphoma (BL) cell lines compared to human B-lymphocytes immortalized by EBV (LCLs). On the basis of hormone binding assays, nuclear DNA binding activity, and transactivation assays the response was reduced 5- to 10-fold. Direct sequence analysis of the expressed glucocorticoid receptor mRNA in two BL cell lines indicated that the phenotype did not result from mutation of the GR gene. By preparing a high-titer polyclonal antiserum against the t-1 region of the human GR, we further showed that the deficient GR response in BLs is largely reflected in reduced GR steady-state protein levels in BL cells compared to LCLs. However, the level of GR mRNA varies less between the BL cell lines and the LCLs. The Cp promoter of EBV which normally drives expression of the EBNA gene family in EBV-immortalized LCLs contains a functional glucocorticoid response element. Transfection of GR expression constructs to group I BL cells converted the GR response to approximately LCL levels both with respect to hormone binding and glucocorticoid-dependent transcription of a glucocorticoid-dependent promoter. A modest activation of EBNA-2 expression was seen in some such cell lines, suggesting that the lower GR response contributes to the down-regulation of EBNA expression observed in BL.

Antigens, Viral↗
PubMed1994

Inhaled nitric oxide in congenital diaphragmatic hernia.

Pulmonary hypertension is a major complication of congenital diaphragmatic hernia (CDH). Inhaled nitric oxide (NO) is a selective pulmonary vasodilator because it produces vasodilatation of the pulmonary vasculature without systemic hypotension. In experimental and clinical studies, inhaled NO ameliorates pulmonary hypertension and improves gas exchange. The goal of the present study was to determine the extent to which infants with CDH respond to inhaled NO. Four newborn infants with CDH complicated by severe respiratory insufficiency and right-to-left shunting received inhaled NO. In three patients, postductal oxygenation improved in response to small concentrations of NO (5 to 10 ppm); two received NO after operative repair, and the third both before and after repair. However, tachyphylaxis developed in all cases within 1 to 6 days. A fourth patient received inhaled NO in an attempt at weaning from ECMO. He did not respond, remaining hypoxic despite 80 ppm NO, and continued to require ECMO. In the three patients who responded to inhaled NO, plasma nitrites and nitrates (stable oxidative end products of NO) accumulated over time, but not in the patient who did not respond. The accumulation of nitrite and nitrate in plasma may reflect alveolar-capillary NO absorption, and may identify patients who will respond to continued inhaled NO. Methemoglobin remained below 1.9% in all four babies. Selected infants with CDH may respond to NO, but the benefit may be temporary.

Administration, Inhalation↗
PubMed1994

Precipitation of the Epstein-Barr virus protein EBNA 2 by an EBNA 3c-specific monoclonal antibody.

Two monoclonal antibodies, E3cD8 and E3cA10, were generated to the EBNA 3c nuclear protein from the B95.8 isolate of Epstein-Barr virus (EBV). Both antibodies efficiently precipitate EBNA 3c from B95.8-transformed lymphoblastoid cell lines, and E3cA10 also detects EBNA 3c on Western blots. Whereas E3cD8 reacts with all 11 Type-1 isolates of EBV tested, and E3cA10 reacts with 14 of 17 Type-1 isolates, neither antibody detects the EBNA 3c protein encoded by Type-2 isolates. E3cD8 recognizes a peptide sequence (PA/PPQAPYQGY) in a repeat region of the B95.8 EBNA 3c coding sequence which is not present in the prototype Type-2 AG876 sequence. The E3cA10 antibody epitope has been mapped to the minimal five amino acid B95.8 peptide, WAPSV, which has an alanine to valine substitution in the AG876 virus isolate. This substitution was also found in three Type-1 EBV isolates that expressed EBNA 3c proteins not detected by E3cA10. In immunoprecipitation studies E3cA10 additionally coprecipitated the EBNA 2 protein from Type-1 isolates of EBV. The possibility of a direct interaction between EBNA 2 and EBNA 3c was ruled out by the demonstration that the antibody precipitated EBNA 2 from the Raji cell line which carries a virus with a deleted EBNA 3c gene. Since the WAPSV epitope identified in EBNA 3c is not present in EBNA 2, and no EBNA 2 linear peptide reactivity was detected in ELISA, it seems likely that E3cA10 recognizes a conformational epitope on EBNA 2. However, from the present data we cannot exclude the possibility that the antibody reacts with a cellular protein that physically associates with EBNA 2.

Amino Acid Sequence↗
PubMed1994

Upregulation of bcl-2 by the Epstein-Barr virus latent membrane protein LMP1: a B-cell-specific response that is delayed relative to NF-kappa B activation and to induction of cell surface markers.

An ability of the Epstein-Barr virus latent membrane protein LMP1 to enhance the survival of infected B cells through upregulation of the bcl-2 oncogene was first suggested by experiments involving gene transfection and the selection of stable LMP1+ clones (S. Henderson, M. Rowe, C. Gregory, F. Wang, E. Kieff, and A. Rickinson, Cell 65:1107-1115, 1991). However, it was not possible to ascertain whether Bcl-2 upregulation was a specific consequence of LMP1 expression or an artifact of the selection procedure whereby rare Bcl-2+ cells already present in the starting population might best be able to tolerate the potentially toxic effects of LMP1. We therefore reexamined this issue by using two different experimental approaches that allowed LMP1-induced effects to be monitored immediately following expression of the viral protein and in the absence of selective pressures; activation of the NF-kappa B transcription factor and upregulation of the cell adhesion molecule ICAM-1 were used as early indices of LMP1 function. In the first approach, stable clones of two B-cell lines carrying an LMP1 gene under the control of an inducible metallothionein promoter were induced to express LMP1 in all cells. Activation of NK-kappa B and upregulation of ICAM-1 occurred within 24 h and were followed at 48 to 72 h by upregulation of Bcl-2. In the second approach, we tested the generality of this phenomenon by transiently expressing LMP1 from a strong constitutively active promoter in a range of different cell types. All six B-cell lines tested showed NF-kappa B activation in response to LMP1 expression, and this was followed in five of six lines by expression of ICAM-1 and Bcl-2. In the same experiments, all three non-B-cell lines showed NF-kappa B activation and ICAM-1 upregulation but never any effect upon Bcl-2. We therefore conclude that Bcl-2 upregulation is part of the panoply of cellular changes induced by LMP1 but that the effect is cell type specific. Our data also suggest that whilst NF-kappa B may be an essential component of LMP1 signal transduction, other cell-specific factors may be required to effect some functions of the viral protein.

Antigens, CD↗
PubMed1993

Epstein-Barr virus-coded BHRF1 protein, a viral homologue of Bcl-2, protects human B cells from programmed cell death.

Epstein-Barr virus, a human herpesvirus that persists within the B-lymphoid system, can enhance the survival potential of latently infected B cells in vitro through up-regulation of the cellular survival protein Bcl-2. The possibility that an analogous effect is operative in lytically infected cells was suggested by the observation of distant sequence homology between an Epstein-Barr virus-coded early lytic cycle protein, BHRF1, and Bcl-2. Here we show by gene transfer that BHRF1 resembles Bcl-2 both in its subcellular localization and in its capacity to enhance B-cell survival. Thus confocal microscopic analysis of cells acutely cotransfected with BHRF1 and Bcl-2 expression vectors revealed substantial colocalization of the two proteins in the cytoplasm. In subsequent experiments, stable BHRF1 gene transfectants of Burkitt lymphoma cells paralleled Bcl-2 transfectants in their enhanced survival under conditions that induce cell death by apoptosis. Despite their limited sequence conservation, therefore, the two proteins appear to be functionally homologous. We suggest that BHRF1 provides an alternative, Bcl-2-independent, means of enhancing B-cell survival that may operate during the virus lytic cycle.

Animals↗
PubMed1993

EBV peptide epitope sensitization restores human cytotoxic T cell recognition of Burkitt's lymphoma cells. Evidence for a critical role for ICAM-2.

The pathogenesis of EBV+ Burkitt's lymphoma (BL) suggests evasion of the CTL response against EBV. Two important features of this tumor have been previously suggested to explain this immune evasion, (a) absence/low expression of cellular adhesion molecules and (b) restricted expression of EBV latent Ag. To determine the relative importance of these features in relation to evasion of EBV-specific CTL, a group of BL cell lines with variable expression of the aforementioned phenotypic characteristics were assayed for specific CTL lysis after exogenous addition of EBV peptide epitopes. In spite of down-regulated expression of the adhesion molecules LFA-1, LFA-3, and/or ICAM-1, peptide-sensitized BL cells were recognized and lysed by EBV-specific CTL. Moreover, there was no significant difference between the CTL lysis of the BL cells and that of adhesion molecule-positive control cells over a wide range of peptide epitope concentrations. Blocking experiments with mAb to individual adhesion molecules suggested that virus-specific CTL recognition of lymphoblastoid cell lines was dependent on an intact LFA-3/CD2 pathway. In contrast, the CTL recognition of peptide-sensitized BL cells was critically dependent on the LFA-1/ICAM pathway, with an insignificant contribution by CD2/LFA-3. The consistently high expression of ICAM-2 on all BL cell lines suggests that the accessory function in CTL recognition of these cells is mediated by the LFA-1/ICAM-2 pathway. Thus, down-regulation of LFA-1, LFA-3, and/or ICAM-1 expression on BL cells does not provide an absolute barrier to tumor cell recognition by virus-specific CTL. The ability of virus-specific CTL to recognize peptide epitope-sensitized BL cells as efficiently as normal cells has demonstrated the importance of latent Ag expression in the CTL control of EBV+ tumors.

Amino Acid Sequence↗
PubMed1993

HLA-A11 epitope loss isolates of Epstein-Barr virus from a highly A11+ population.

Cytotoxic T lymphocytes (CTLs) control viral infections by recognizing viral peptides presented by major histocompatibility complex (MHC) class I molecules. Human leukocyte antigen (HLA)-A11-restricted CTLs that recognize peptide residues 416 to 424 of the Epstein-Barr virus (EBV) nuclear antigen-4 frequently dominate EBV-induced responses in A11+ Caucasian donors. This epitope is conserved in type A EBV strains from Caucasians and central African populations, where A11 is relatively infrequent. However, strains from highly A11+ populations in New Guinea carry a lysine-to-threonine mutation at residue 424 that abrogates CTL recognition and binding of the peptide to nascent A11 molecules. The results suggest that evolution of a widespread and genetically stable virus such as EBV is influenced by pressure from MHC-restricted CTL responses.

Africa↗
PubMed1993

Persistence of antibody at 18 months following vaccination of young Gambian infants with PRP-OMPC Haemophilus influenzae type b conjugate vaccine.

The rate of decline in anti-PRP antibody levels was measured in two groups of Gambian children who had been given PRP-OMPC at 1 and 3 months or 2 and 4 months of age. In the younger group (n = 70), the geometric mean titre fell from 1.32 micrograms/ml at 4 months to 0.44 micrograms/ml at 18 months. In the older group (n = 54), the geometric mean titre fell from 1.18 micrograms/ml at 5 months to 0.46 micrograms/ml at 18 months. The proportion of vaccinated children with antibody levels over 1.0 microgram/ml fell from 54% 1 month after the second dose of vaccine to 27% at the age of 18 months, while the proportion with levels over 0.15 micrograms/ml fell from 82% to 60%, with no significant differences observed between the vaccination groups. For those children who did not show evidence of environmental boosting, the half-life of anti-PRP antibody was about 100 days. This did not differ between the groups. These findings suggest that to provide lasting immunity PRP-OMPC should be given with a late booster dose at 12-15 months, as is the current practice in the USA. The need for a late booster dose may limit the value of this vaccine in developing countries where vaccination of children is difficult after the 1st year of life.

Antibodies, Bacterial↗
PubMed1993

MHC class II-restricted presentation of endogenously synthesized antigen: Epstein-Barr virus transformed B cell lines can present the viral glycoprotein gp340 by two distinct pathways.

Epstein-Barr virus (EBV) transformed B lymphoblastoid cell lines (LCL) efficiently process exogenous antigens for MHC class II-restricted presentation via the chloroquine-sensitive endosomal pathway. Using MHC class II-restricted T cell clones specific for EBV structural proteins, however, we frequently observed significant responses to autologous LCL cells without the addition of exogenous virus. Such responses were reduced by pre-treating the LCL with acyclovir (ACV), a drug blocking productive EBV infection. This suggested T cell recognition of antigen synthesized by LCL cells spontaneously entering virus productive cycle, and led us to question by what route(s) MHC class II-restricted presentation of endogenously synthesized virion proteins was occurring. Cell sorting experiments, using the viral envelope glycoprotein gp340 as a surface marker of productively-infected cells, confirmed that stimulatory activity lay within the gp340-positive fraction. However, closer analysis revealed that most of these cells were not productively-infected but were EBV receptor-positive and had bound released virus. We infer that receptor-mediated delivery of released virus into the endosomal pathway is one route whereby an LCL can present endogenously synthesized EBV proteins on MHC class II molecules. To ask whether another, more direct, route of processing was possible, we used a recombinant vaccinia viral vector to express gp340 de novo in ACV-treated LCLs. Significantly, these cells presented the endogenously synthesized antigen to autologous gp340-specific T cell clones via a chloroquine-resistant pathway. In the same experiments, vaccinia-mediated expression of a signal peptide-deleted form of gp340 did not lead to T cell stimulation, suggesting that this second route of processing required entry of endogenously synthesized antigen into the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1993

HLA A2.1-restricted cytotoxic T cells recognizing a range of Epstein-Barr virus isolates through a defined epitope in latent membrane protein LMP2.

Cytotoxic T-lymphocyte (CTL) responses induced by persistent Epstein-Barr virus (EBV) infection in normal B-lymphoid tissues could potentially be directed against EBV-positive malignancies if expression of the relevant viral target proteins is maintained in tumor cells. For malignancies such as nasopharyngeal carcinoma and Hodgkin's disease, this will require CTL targeting against the nuclear antigen EBNA1 or the latent membrane proteins LMP1 and LMP2. Here we analyze in detail a B95.8 EBV-reactivated CTL response which is specific for LMP2 and restricted through a common HLA allele, A2.1. We found that in vitro-reactivated CTL preparations from several A2.1-positive virus-immune donors contained detectable reactivity against A2.1-bearing target cells expressing either LMP2A or the smaller LMP2B protein from recombinant vaccinia virus vectors. Peptide sensitization experiments then mapped the A2.1-restricted response to a single epitope, the nonamer CLGGLLTMV (LMP2A residues 426 to 434), whose sequence accords well with the proposed peptide binding motif for A2.1. Most Caucasian and African virus isolates (whether of type 1 or type 2) were identical in sequence to B95.8 across this LMP2 epitope region, although 2 of 12 such isolates encoded a Leu-->Ile change at epitope position 6. In contrast, most Southeast Asian and New Guinean isolates (whether of type 1 or type 2) constituted a different virus group with a Cys-->Ser mutation at epitope position 1. CTLs raised against the B95.8-encoded epitope were nevertheless able to recognize these variant epitope sequences in the context of A2.1 whether they were provided exogenously as synthetic peptides or generated endogenously in B cells transformed with the variant viruses. A CTL response of this kind could have therapeutic potential in that it is directed against a protein expressed in many EBV-positive malignancies, is reactive across a range of virus isolates, and is restricted through a relatively common HLA allele.

Amino Acid Sequence↗
PubMed1993

Cytogenetic rearrangement of C-MYC oncogene occurs prior to infection with Epstein-Barr virus in the monoclonal malignant B cells from an AIDS patient.

Two cell lines were originated from the peripheral blood (PB-LAM) and bone-marrow (BM-LAM) of a patient with Burkitt-type acute lymphoblastic leukemia and AIDS. 26 and 7 clones were isolated from PB-LAM and BM-LAM respectively by limiting dilution. All of these had surface IgM lambda and the CD10 marker with low to absent CD23, CD30, CD39 and surface adhesion molecules. Furthermore, they shared the same chromosomal abnormalities (trisomy 7 and t(8;14) translocation) and the same rearrangements of immunoglobulin L and H chain and of c-myc gene loci. These features are those most frequently found in Burkitt's lymphoma (BL) cells and were different from those of the parental cell lines, which, besides cells identical to those of the malignant clones, also contained normal lymphoblastoid cells. Therefore, the cloning procedure used selected for the growth of cells with malignant features. EBV latent antigens were detected in all clones by Western blotting and their pattern of expression resembled that usually observed in BL cells. All the clones were positive for the EBV genome by Southern blotting and had monomorphic EBV-fused termini as determined by using cDNA probes specific for sequences at either end of the viral genome. However, the clones derived from PB-LAM had EBV fused termini of a different size from that of the clones derived from BM-LAM. The presence of different EBV-fused termini in otherwise monoclonal malignant cells indicate that EBV infection was possibly a late event in lymphomagenesis following rearrangement of the c-myc and the Ig gene loci.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Epstein-Barr virus and the lacrimal gland pathology of Sjögren's syndrome.

The lacrimal gland (LG) immunopathology of Sjögren's syndrome (SS) consists of a proliferation of B and CD4 lymphocytes surrounding epithelial structures (Pepose JS, et al: Ophthalmology 1990, 97:1599-1605). Based on the detection of EBV genomes in a greater percentage of SS than normal LG biopsies, we previously postulated that Epstein-Barr virus (EBV) is a risk factor for LG lymphoproliferation in SS (Pflugfelder SC, et al: Ophthalmology 1990, 97:976-984). The purpose of this study was to determine the cellular site(s) of infection, virus type, and antigen expression of EBV infecting normal and SS LGs. EBV DNA was detected by in situ hybridization in intraductal epithelia in 13-33% of lobules in 21% of normal LGs and in cells in areas of B lymphoproliferation as well as the majority of epithelia in 86% of SS LGs. EBV genomic sequences were amplified from 36% of normal and 88% of SS LG biopsies by polymerase chain reaction. Only type 1 EBV sequences were amplified in SS LGs; in contrast EBV nuclear antigen 2-deleted but not type 1 sequences were amplified in normal LGs. Immunohistochemistry with EBV-specific monoclonal antibodies was performed on normal and SS LGs. No EBV antigens were detected in normal LGs. In contrast, latent antigens (latent membrane protein, EBV nuclear antigen 2) were detected in lymphocytes in areas of B lymphoproliferation, and early and late lytic cycle antigens were observed in epithelia in SS LGs. These studies suggest that EBV may play a role in the LG B lymphoproliferation and epithelial pathologic changes observed in SS.

Adult↗
PubMed1992

Identification of target antigens for the human cytotoxic T cell response to Epstein-Barr virus (EBV): implications for the immune control of EBV-positive malignancies.

Epstein-Barr virus (EBV), a human herpes virus with oncogenic potential, persists in B lymphoid tissues and is controlled by virus-specific cytotoxic T lymphocyte (CTL) surveillance. On reactivation in vitro, these CTLs recognize EBV-transformed lymphoblastoid cell lines (LCLs) in an HLA class I antigen-restricted fashion, but the viral antigens providing target epitopes for such recognition remain largely undefined. Here we have tested EBV-induced polyclonal CTL preparations from 16 virus-immune donors on appropriate fibroblast targets in which the eight EBV latent proteins normally found in LCLs (Epstein-Barr nuclear antigen [EBNA] 1, 2, 3A, 3B, 3C, leader protein [LP], and latent membrane protein [LMP] 1 and 2) have been expressed individually from recombinant vaccinia virus vectors. Most donors gave multicomponent responses with two or more separate reactivities against different viral antigens. Although precise target antigen choice was clearly influenced by the donor's HLA class I type, a subset of latent proteins, namely EBNA 3A, 3B, and 3C, provided the dominant targets on a range of HLA backgrounds; thus, 15 of 16 donors gave CTL responses that contained reactivities to one or more proteins of this subset. Examples of responses to other latent proteins, namely LMP 2 and EBNA 2, were detected through specific HLA determinants, but we did not observe reactivities to EBNA 1, EBNA LP, or LMP 1. The bulk polyclonal CTL response in one donor, and components of that response in others, did not map to any of the known latent proteins, suggesting that other viral target antigens remain to be identified. This work has important implications for CTL control over EBV-positive malignancies where virus gene expression is often limited to specific subsets of latent proteins.

Antigens, Viral↗
PubMed1992

Epstein-Barr viral DNA in acute large granular lymphocyte (natural killer) leukemic cells.

Serologic studies in a male Caucasian presenting with an acute hepatitis-like illness, associated with an increase in peripheral blood large granular lymphocytes (LGLs), suggested a chronic or reactived Epstein-Barr virus (EBV) infection. The LGL were shown to have a natural killer (NK) cell, CD3- CD16- CD56+ CD57- phenotype and mediated strong nonspecific major histocompatability complex-unrestricted (NK) cytotoxic activity. A progressive increase in the peripheral blood LGL count was associated with a rapid deterioration, hepatic necrosis, and death. Widespread organ infiltration with LGLs suggested a malignant lymphoproliferative condition, but no lymphoid (T-cell receptor or IgH) gene rearrangement or cytogenetic marker was detected. However, molecular analysis identified EBV genomic DNA present in a single episomal form within the LGL, establishing the clonal nature of the LGL proliferation. Confirmation that the EBV had infected the leukemic LGL was obtained by in situ hybridization studies that showed EBV RNA within the LGLs. Immunoblotting of LGL protein extracts established that, of the EBV gene products, EBV nuclear antigen-1 (EBNA-1) was expressed but EBNA-2 and the latent membrane protein (LMP-1) were not detectable in the leukemic cells. These results suggest that EBV may be involved directly in LGL cell transformation, in a manner similar to EBV-associated B-cell lymphomas, although other molecular changes probably contribute to the evolution of a fully malignant leukemic clone.

Antigens, CD↗
PubMed1992

Restoration of the LFA-3 adhesion pathway in Burkitt's lymphoma cells using an LFA-3 recombinant vaccinia virus: consequences for T cell recognition.

Conjugate formation between cytotoxic T lymphocytes (CTL) and target B cells, as observed in vitro, is mediated by interactions between adhesion molecules on the two cell surfaces rather than involving immune recognition through the T cell receptor. It is still not clear to what extent such adhesive contacts facilitate the process of immune recognition and target cell lysis. However, work on the Epstein-Barr virus (EBV)-associated malignancy Burkitt's lymphoma (BL) has suggested that down-regulation of one particular adhesion molecule, the lymphocyte function-associated antigen LFA-3, on the tumor cell surface is a key factor in allowing these target cells to escape EBV-specific T cell surveillance. To examine this directly, we used a cDNA for the full-length transmembrane form of LFA-3 to construct a recombinant vaccinia virus (Vacc-LFA 3), which is capable of restoring surface LFA-3 in adhesion molecule-negative BL cell lines to levels as high as seen in EBV-transformed lymphoblastoid cell lines (LCL); biochemical studies confirmed expression of the authentic N-glycosylated protein. The recombinant vaccinia-encoded LFA-3 was functional as an adhesion molecule since BL cells acutely infected with Vacc-LFA-3 then acquired the ability to form conjugates with activated T cells in vitro. However, there was no clear dependence upon LFA-3 when such BL cell lines were tested as targets for cytotoxic T lymphocytes (CTL). Firstly, LFA-3- BL cells could be killed by allospecific CTL recognizing HLA class I alloantigens, in some cases as efficiently as the corresponding LCL. In other cases where lysis was slightly below that of the LCL, Vacc-LFA-3 infection of the BL cells increased lysis up to, but never beyond, LCL values. Secondly, we studied the sensitivity of BL to EBV-specific HLA class I-restricted CTL using a BL target line which was LFA-3- but which expressed the same spectrum of EBV target proteins as an LCL. This line was not recognized by appropriately HLA-matched effectors, even after restoration of LFA-3 expression. We conclude that the LFA-3 status of BL cells influences their conjugate forming ability in in vitro assays but not necessarily their sensitivity to immune T cell-mediated cytolysis.

Antigens, Surface↗