Identification of Epstein-Barr virus-infected cells in tonsils of acute infectious mononucleosis by in situ hybridization

In situ hybridization with 35S-labeled Epstein-Barr virus (EBV) probes was applied to paraffin sections of tonsils from seven patients with clinical, serologic, and morphologic evidence of acute infectious mononucleosis. EBV genomes were demonstrated in activated lymphoid B blasts in the interfollicular and perifollicular zones in all these cases. However, in no case could EBV be identified in epithelial cells. These results are at variance with the current concept which attributes a central role to the tonsillar epithelium in primary EBV infection.     

Terminal deoxynucleotidyl transferase-positive cells in human tonsils.

To study the possible cellular origin of recently recognized indolent terminal deoxynucleotidyl transferase (TdT)-positive T-lymphoblastic proliferations of the tonsils and oropharynx, we studied normal human tonsils for the presence of TdT-positive cells. TdT-positive cells were readily demonstrated in the tonsils from 15 children and adults by immunohistochemical staining. TdT-positive cells were distributed in discrete foci at the periphery of lobules of lymphoid tissue and adjacent to fibrous septa and had the morphologic features of small to medium-sized lymphocytes. Double-antibody staining indicated the TdT-positive cells had the phenotype of uncommitted early lymphoid precursors (CD3-, CD79a-, CD10-). Foci of TdT-positive cells were not identified in 6 reactive lymph nodes studied as controls. These studies indicate that tonsils, like bone marrow and thymus, are sites of lymphopoiesis. The presence of TdT-positive precursor cells in human tonsils may be a factor in the pathogenesis of recently described indolent T-lymphoblastic proliferations involving the tonsils and oropharynx. The presence of TdT-positive cells in human tonsils should not be misinterpreted as evidence of lymphoblastic lymphoma or leukemia.     

Epstein-Barr nuclear antigen (EBNA) carrying lymphocytes in human palatine tonsils.

The presence of Epstein-Barr virus (EBV) antigens in human palatine tonsilderived lymphocytes (TDL) was investigated using the indirect fluorescent antibody (FA) technique. The TDL were screened for the presence of EBV early antigen (EA), virus capsid antigen (VCA), and EBV nuclear antigen (EBNA). In 76% of the patients diagnosed as recurrent exudative tonsillitis, and in 33% diagnosed as recurrent tonsillitis and/or serous otitis media, EBNA was demonstrated in the purified TDLs. No EA- or VCA-producing cells were found in either the glass adsorbed or TDL cell preparations from all of the patients. These data suggest that in our patient sample, the tonsils may serve as a reservoir for EBV carrying lymphocytes and a basis for recurrent disease.     

Interleukin-18 expression induced by Epstein-Barr virus-infected cells

Human Epstein-Barr virus (EBV)-negative Burkitt lymphomas cells usually grow as malignant subcutaneous tumors in athymic mice, but these tumors regress when the Burkitt cells are injected in conjunction with EBV-positive lymphoblastoid cells or when the Burkitt cells are transfected with the EBV latent membrane protein-1 (LMP-1) gene. Tumor regression is mediated, in part, by murine interferon gamma (IFN-gamma) and the IFN-gamma-induced murine chemokine IFN-gamma-inducible protein-10 (IP-10). The mechanisms by which EBV-LMP-1 promotes the expression of IFN-gamma has remained unclear. Here we show that murine interleukin (IL)-18 was consistently expressed in regressing Burkitt tumors but was either expressed at low levels or absent from progressively growing Burkitt tumors. By immunohistochemical methods, IL-18 protein was visualized in regressing but not in progressively growing Burkitt tumors. In contrast, IL-12 p35 and IL-12 p40 were only rarely expressed in regressing Burkitt tumors. In splenocyte cultures, EBV-infected lymphoblastoid cells and LMP-1-transfected Burkitt cells promoted the expression of IL-18 but not the expression of IL-12 p35 and IL-12 p40. A neutralizing antibody directed at murine IL-18 reduced murine IP-10 expression induced by EBV-immortalized cells in splenocyte cultures. These results provide evidence for IL-18 expression in response to a viral latency protein and suggest that IL-18 may play an important role as an endogenous inducer of IFN-gamma expression, thereby contributing to tumor regression.     

Lymphoid progenitor cells in human tonsils

To investigate the occurrence of lymphoid progenitor cells in human tonsils, we studied tonsils from children and adults by immunohistochemistry by using a panel of antibodies to antigens associated with lymphoid progenitor cells, including terminal deoxynucleotidyl transferase (TdT), CD10 (CALLA), CD34, CD99 (p30/32mic2), and CD117 (c-kit), and compared them to reactive lymph nodes. Lymphoid progenitor cells, positive for TdT, CD10, and CD99, but not CD34 or CD117, were readily identified in tonsils from children and adults (TdT, 14 of 15; CD10, 15 of 15; CD99, 11 of 15), but were rarely present in lymph nodes (TdT, 1 of 8; CD10, 1 of 8; CD99, 0 of 8). Lymphoid progenitor cells in tonsils were localized to discrete foci at the periphery of lymphoid lobules adjacent to fibrous septae. Lymphoid progenitor cells are present in human tonsils, and the tonsils are a potential site of postnatal lymphopoiesis. The presence of lymphoid progenitor cells in human tonsils should not be confused with lymphoblastic lymphoma or leukemia.     

Immunoglobulin-containing cells in human tonsils as demonstrated by immunohistochemistry.

Intracellular immunoglobulin has been demonstrated in human palatine tonsils by the unlabelled antibody peroxidase-antiperoxidase complex (PAP) method in which rabbit antiserum to a range of human immunoglobulins (Igs) was linked to the PAP complex by an intermediate stage of swine antiserum to rabbit Ig. The effects of different methods of fixation and processing have been compared, formol-saline fixation giving the best results. The PAP technique proved greatly superior to the fluorescein isothiocyanate (FITC)-based technique, not only in sensitivity but in permitting study of the finer histological and cytological features. The lymphoid follicles are shown to have three distinct zones, two forming the follicle centre (zones (a) and (b)), and the third (zone (c)) the lymphocyte cap. Ig synthesis appeared to begin in the cells in zone (b). IgG, IgA, IgM, IgE and IgD were present in all tonsils, with IgG predominating, confirming that the tonsil resembles lymph nodes more closely than it does alimentary lymphoid tissue. Some follicles contained more than one type of Ig. The tonsil appears to have a well-developed T-dependent area, the lymphoid follicles forming a B-cell area. The structure of the tonsil would seem to facilitate contact between its lymphoid tissue and antigens in the crypts, and it is postulated that some T cells within the crypt epithelium, after contact with antigen, may leave the tonsil by the efferent lymphatics and enter the peripheral circulation by the thoracic duct, whilst other primed T cells interact with B cells in the follicle centres. Some B cells may then start to synthesize immunoglobulin, whilst others become memory cells in the lymphocyte 'cap' of the follicle.     

Induction of a deoxyuridine triphosphate nucleotidohydrolase activity in Epstein-Barr virus-infected cells

Superinfection of Raji cells with Epstein-Barr virus (EBV) or chemical induction of HR-1 cells with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) results in the induction of a deoxyuridine triphosphate nucleotidohydrolase (dUTPase) which is not observed in mock-treated cells or TPA-treated EBV genome-negative BJAB cells. The EBV-induced dUTPase could be distinguished from the host dUTPase based upon differences in their migration in polyacrylamide gels and sensitivity to the 5-mercurithioguanosine derivitive of dUTP. The expression of the EBV-specified dUTPase is prevented by phosphonoacetic acid indicating that its expression is dependent upon EBV-DNA replication.     

Human T-cell hybrids with HLA-restricted proliferative response to Epstein-Barr virus-infected B cells.

Human T-cell hybrids were constructed from an HGPRT-negative mutant of the acute lymphoblastoid leukaemia cell-line CEM and an uncloned population of T cells from donor SW (SW-T; partner cell) known to have a strong specificity for the autologous Epstein-Barr virus (EBV)-transformed B cell, SWEBV. The resulting hybrids, 1A9, 1D12 and 2C8, were shown not to be cytotoxic to SWEBV, nor did they have natural killer-like (NK) activity. However, when presented with the target SWEBV in a mixed lymphocyte reaction (MLR), all of the hybrids rapidly increased their rate of proliferation by up to a factor of seven. Hybrid 1D12 also produced interleukin-2-like material (IL2) under these conditions. The hybrids did not react with the autologous PHA-blasts (SWPHA), nor with various unrelated targets. When tested against a bank of EBV-transformed B-cell targets, it was observed that the human T-cell hybrids 1A9 and 2C8 responded only to those targets bearing the antigen HLA Bw35. This response could be blocked by treating the target with the monoclonal antibody W6/32, specific for a shared determinant of the HLA-A, -B and -C antigens. Similarly, the human T-cell hybrid 1D12 reacted only against those targets bearing the antigen HLA DrW2, and this activity could be blocked by the monoclonal antibody DA6.231, specific for a common region of the HLA-DR and SB antigens. Thus, human T-cell hybrids can be produced which exhibit HLA-restricted responses to antigenic stimulation.     

Detection of Epstein-Barr virus-infected mucosal lymphocytes in nasal polyps.

Primary nasal lymphomas of T or NK cell origin are known to be associated with Epstein-Barr virus (EBV). However, it is not known whether EBV is normally present in nasal mucosa as distinct to nasopharyngeal tissue. This study investigates the prevalence of EBV infection in 13 cases of nasal polyps. EBV DNA was detected in 2 of 13 (15%) by Southern blot hybridization and in 9 of 13 (69%) by polymerase chain reaction. In situ hybridization for EBV-encoded small nuclear RNAs (EBER) was positive in 11 of 13 (85%) cases; the virus was present in stromal lymphocytes only and not in the epithelial cells. Immunohistochemistry for EBV proteins in 7 cases revealed EBV nuclear antigen (EBNA)-2, latent membrane protein (LMP)-1, and ZEBRA (the switch protein encoded by gene BZLF1) expression in rare isolated stromal lymphocytes in 3 cases. Double immunostaining in 1 case showed that the LMP-1+ cells were B or T cells. Immunohistochemistry for EBV lytic proteins showed very rare viral capsid antigen (VCA)+ and membrane antigen (MA)+ cells in 1 case and very rare diffuse early antigen (EA-D)+ and VCA+ cells in 1 other case. The expression of ZEBRA, EA-D, VCA, and MA suggested a disruption of latency in very rare stromal lymphocytes leading to a productive cycle. Although the incidence of EBV positivity in nasal polyps in our population is high (85%), very low numbers of EBV+ cells are found in each case. Nevertheless, they indicate that nasal mucosa could be one of the sites of EBV persistence through a low level of infection of the resident lymphocytes and thereby provide a possible setting for the emergence of virally associated tumors in this site.     

Killing of measles virus-infected cells by human cytotoxic T cells.

Lymphocytes from normal individuals were tested for the capacity to generate measles virus-specific cytotoxic T-cell responses after in vitro stimulation with measles virus. Approximately 12% (5 of 40) of the normal adults tested produced significant cytotoxic responses. The cytotoxic response was measles virus specific both at the level of stimulation and at the effector level. Studies of the specificity of cytotoxic effectors from five normal donors by direct lysis or cold target inhibition or both indicated that most, if not all, of the virus-specific activity was self-specific. A detailed analysis of one donor (W6) indicated that measles-specific cytotoxicity was largely HLA-A and -B restricted; unexplained cross-reactive lysis was observed with some targets, but this lysis appeared to be HLA related, since all of the targets expressed HLA-B12. An analysis of the cellular requirements for the production of measles-immune cytotoxic T lymphocytes demonstrated that T cells and macrophages (depleted of natural killer and K cells) were sufficient for the generation of killer cells. Most of the cytotoxic effector activity was mediated by OKT3+ OKT4- OKT8+ cells.     

Cytotoxic T cell recognition of Epstein-Barr virus-infected B cells. I. Specificity and HLA restriction of effector cells reactivated in vitro

The experiments show that the phenomenon of regression, seen exclusively in Epstein-Barr (EB) virus-infected cultures of mononuclear cells from EB virus antibody-positive donors, is mediated by cytotoxic T cells reactivated in vitro and specifically recognizing an EB virus-induced lymphocyte-detected membrane antigen LYDMA. Thus, effector T cells from regressing cultures kill autologous EB virus-transformed cells but not autologous pokeweed mitogen-stimulates lymphoblasts nor any of a range of EB virus genome-negative human hemopoietic cell lines (K562, HSB2, BJAB, EB4) particularly sensitive to nonspecific natural killer-like activities. Moreover, these reactivated effector cells exhibit classical HLA restriction of target cell recognition; in a survey of 14 effector cell donors, preferential lysis of the autologous virus-transformed line was a consistent feature, while the relative degree of lysis of allogeneic lines was in general directly related to the number of HLA-A and B antigens shared between effector and target cells. The pattern of reactivity shown by effector T cell preparations from any one donor was strikingly reproducible, and the results from a number of donors revealed differences between particular HLA-A and B antigens with respect to the level of EB virus-specific killing which was associated with sharing through these determinants.     

Membrane antigen expression in Epstein-Barr virus-infected Raji cells in the presence of phosphonoacetic acid

The expression of the Epstein-Barr virus (EBV)-induced membrane antigen (MA) in Raji cells experimentally infected with EBV concentrates was inhibited by phosphonoacetic acid (PAA) as determined by membrane immunofluorescence and inhibition of antibody-dependent lymphocyte cytotoxicity. PAA was only effective if present during the first 24 hr following virus adsorption, indicating that the synthesis of MA was primarily a late viral gene function requiring viral DNA synthesis.     

Correlation between allergy and persistent Epstein-Barr virus infections in chronic-active Epstein-Barr virus-infected patients

Forty-six anti-Epstein Barr nuclear antigen-positive allergic patients, 11 of whom having clinical and laboratory evidence of chronic-active Epstein-Barr virus (CA-EBV) infections, were characterized by EBV serology, percentages of T cells, B cells, and IgE+ cells, serum levels of IgE, and allergen-induced responsiveness of lymphocytes. Results demonstrated patients with CA-EBV have significantly increased responsiveness toward specific allergens, responses toward greater numbers of allergens, numbers of IgE+ T and B cells, and levels of background DNA activity in nonstimulated lymphocytes than do subjects who suffer from allergies in the absence of the CA-EBV syndrome. Further comparison between subjects with laboratory-determined mild and moderate allergy and those with CA-EBV demonstrated a progressive increase in the serum levels of IgE as the degree of allergy increased, no difference in concentrations of T and B cells, and titers of anti-viral capsid antigen and anti-early antigen to be significantly greater in patients with CA-EBV. Statistical analysis demonstrated that patients with CA-EBV could be separated from subjects with allergies by metabolic and immunologic variables. The data suggested that allergen-induced responses may contribute to the CA-EBV syndrome.     

Isolation of follicular dendritic cells from human tonsils and adenoids. II. Immunocytochemical characterization

Follicular dendritic cells (FDC) are specialized cells found only within lymphoid follicles. They bind immune complexes and play a role in the presentation of antigen to follicular B cells and in the generation of B cell memory. In the present report the isolation of FDC from human tonsils and adenoids is described. These isolated cells have an unusual spherical arrangement and enclose lymphocytes within extensions of their membranes. Their ultrastructural features are similar to those observed in situ. The reactivity of isolated FDC with a number of monoclonal antibodies was analyzed by immunofluorescence and by immunostaining (at the electron microscopic level) with colloidal gold. In keeping with the results of previous investigations on tissue sections IgM, IgG and IgA (but not IgD) can be detected on the surface of isolated FDC, as can C3b receptors and the FDC-associated antigen detected by monoclonal antibody R4/23. The immunoglobulins associated with FDC are mostly embedded in an electron-dense material. The majority of the lymphoid cells enclosed within the membrane extensions of FDC are of B cell type. These results suggest that isolated FDC may be suitable for further in vitro investigation of their role in the humoral immune response.