B cells infected by Epstein-Barr virus (EBV), a transforming virus endemic in humans, are rapidly cleared by the immune system, but some cells harboring the virus persist for life. Under conditions of immunosuppression, EBV can spread from these cells and cause life-threatening pathologies. We have generated mice expressing the transforming EBV latent membrane protein 1 (LMP1), mimicking a constitutively active CD40 coreceptor, specifically in B cells. Like human EBV-infected cells, LMP1+ B cells were efficiently eliminated by T cells, and breaking immune surveillance resulted in rapid, fatal lymphoproliferation and lymphomagenesis. The lymphoma cells expressed ligands for a natural killer (NK) cell receptor, NKG2D, and could be targeted by an NKG2D-Fc fusion protein. These experiments indicate a central role for LMP1 in the surveillance and transformation of EBV-infected B cells in vivo, establish a preclinical model for B cell lymphomagenesis in immunosuppressed patients, and validate a new therapeutic approach.

A human hematopoietic cell line (U-937) with exceptional characteristics was derived from a patient with generalized histiocytic lymphoma. The morphology of the cell line was identical to that of the tumor cells in the pleural effusion from which the line was derived. Since Epstein-Barr virus (EBV) carrying diploid lymphoblastoid cell lines unrelated to the tumor population often become established in vitro from non-Burkitt lymphoma explants, several parameters were studied to discriminate the U-937 from such lines: morphology in vitro, growth characteristics, cytochemistry, surface receptor pattern, Ig production, lysozyme production, beta2-microglobulin production, presence of EBV genome and karyotype. In all these respects U-937 differed from prototype lymphoblastoid cell lines. The histiocytic origin of the cell line was shown by its capacity for lysozyme production and the strong esterase activity (naphtol AS-D acetate esterase inhibited by NaF) of the cells. It is therefore concluded that the U-937 is a neoplastic, histiocytic cell line.

Antigen-presenting cells contain a specialized late endocytic compartment, MIIC (major histocompatibility complex [MHC] class II-enriched compartment), that harbors newly synthesized MHC class II molecules in transit to the plasma membrane. MIICs have a limiting membrane enclosing characteristic internal membrane vesicles. Both the limiting membrane and the internal vesicles contain MHC class II. In this study on B lymphoblastoid cells, we demonstrate by immunoelectron microscopy that the limiting membrane of MIICs can fuse directly with the plasma membrane, resulting in release from the cells of internal MHC class II-containing vesicles. These secreted vesicles, named exosomes, were isolated from the cell culture media by differential centrifugation followed by flotation on sucrose density gradients. The overall surface protein composition of exosomes differed significantly from that of the plasma membrane. Exosome-bound MHC class II was in a compact, peptide-bound conformation. Metabolically labeled MHC class II was released into the extracellular medium with relatively slow kinetics, 10 +/- 4% in 24 h, indicating that direct fusion of MIICs with the plasma membrane is not the major pathway by which MHC class II reaches the plasma membrane. Exosomes derived from both human and murine B lymphocytes induced antigen-specific MHC class II-restricted T cell responses. These data suggest a role for exosomes in antigen presentation in vivo.

BACKGROUND. Lymphoma associated with Epstein-Barr virus (EBV) is a complication of bone marrow transplantation that responds poorly to standard forms of therapy. The lymphoma is usually of donor origin. We hypothesized that treatment with infusions of donor leukocytes, which contain cytotoxic T cells presensitized to EBV, might be an effective treatment. METHODS. We studied five patients in whom EBV-associated lymphoproliferative disorders developed after they received a T-cell-depleted allogeneic bone marrow transplant. Biopsy specimens were immunophenotyped, subjected to the polymerase chain reaction to determine the origin of the lymphoma (donor or host) and to detect the presence of EBV, and analyzed by Southern blotting for the presence of the clonal EBV genome and immunoglobulin-gene rearrangement. Patients were treated with infusions of unirradiated donor leukocytes at doses calculated to provide approximately 1.0 x 10(6) CD3+ T cells per kilogram of body weight. RESULTS. Histopathological examination of biopsy specimens from all five patients demonstrated monomorphic, malignant lymphomas of B-cell origin. Each of the four specimens that could be evaluated was of donor-cell origin. Evidence of clonality was found in two of the three samples adequate for study. EBV DNA was detected by the polymerase chain reaction in all five samples. In all five patients there were complete pathological or clinical responses. The responses were first documented histologically within 8 to 21 days after infusion. Clinical remissions were achieved within 14 to 30 days after the infusions and were sustained without further therapy in the three surviving patients for 10, 16, and 16 months. CONCLUSIONS. In a small number of patients, infusions of unirradiated donor leukocytes were an effective treatment for EBV-associated lymphoproliferative disease that arose after allogeneic bone marrow transplantation.

Traffic of MHC molecules dictates the source of peptides that are presented to T cells. The intracellular distribution of MHC class I and class II molecules reflects the dichotomy in presentation of antigen from endogenous and exogenous origin, respectively. In human B lymphoblastoid cells, class I molecules are present in compartments constituting the biosynthetic pathway, whereas class II molecules enter structures related to lysosomes during their biosynthesis.

It is generally accepted that tumours arise through the accumulation of several changes affecting the control of cell growth. Recent advances in molecular biology have made it possible to define some of these changes in molecular terms and to trace the steps by which certain tumours evolve.

A method for producing random subclones using sonication to fragment the DNA is presented. The sonication is combined with enzymatic repair of the fragment ends and a rigorous size fractionation step to prepare subclones of relatively homogeneous and specific size. Under some conditions sonication is shown to shear A + T-rich sequences preferentially, although under most conditions it will create a random subclone library. The use of these subclone libraries for an improved "shotgun" DNA sequencing strategy is tested on a 17.2-kb (kilobase) fragment of Epstein-Barr virus.

Posttransplant lymphoproliferative disorders (PTLDs) were diagnosed in 43 patients from the Pittsburgh-Denver series between June 1980 and March 1987. This constitutes a detection rate of 1.7%. Major categories of clinical presentation included a mononucleosislike syndrome, gastrointestinal/abdominal disease, and solid organ disease. The median time of onset in patients initially immunosuppressed with cyclosporine-A (CsA)-containing regimens was 4.4 months after transplant, regardless of tumor clonality. A strong association of PTLD with Epstein-Barr virus (EBV) was observed. A histologic spectrum of lesions from polymorphic to monomorphic was observed. Whereas polymorphic lesions could be either clonal or nonclonal, monomorphic lesions appeared to be clonal in composition. The presence of large atypical cells (atypical immunoblasts) or necrosis did not appreciably worsen the prognosis. Twelve patients had clonal, 13 had nonclonal, and five had both clonal and nonclonal tumors. Clonality was indeterminate in 13 cases. Most patients were treated with a regimen based on reduced immunosuppression and supportive surgery. Almost all nonclonal and about half of the clonal lesions respond to this conservative therapy, indicating that it is an appropriate first line of treatment. This behavior suggests that a spectrum of lesions ranging from infectious mononucleosis to malignant lymphoma constitutes the entity known as PTLD. Some monoclonal tumors can undergo regression, however, apparently in response to host immune control mechanisms. Because of its short latency and strong association with EBV, PTLD is an important model for the study of virus-associated tumor progression in humans.

The expression of angiotensin-I converting enzyme (ACE; EC 3.4.15.1) in human circulating mononuclear cells was studied. T-lymphocytes contained the highest level of enzyme, approx. 28 times more per cell than monocytes. No activity was detected in B-lymphocytes. ACE was present mainly in the microsomal fraction, where it was found to be the major membrane-bound bradykinin-inactivating enzyme. An mRNA for ACE was detected and characterized after reverse transcription and amplification by PCR in T-lymphocytes and several T-cell leukaemia cell lines. We have previously observed that the interindividual variability in the levels of ACE in plasma is, in part, genetically determined and influenced by an insertion/deletion polymorphism of the ACE gene. To investigate the mechanisms involved in the regulation of ACE biosynthesis, the ACE levels of T-lymphocytes from 35 healthy subjects having different ACE genotypes were studied. These levels varied widely between individuals but were highly reproducible and influenced by the polymorphism of the ACE gene. T-lymphocyte levels of ACE were significantly higher in subjects who were homozygote for the deletion than in the other subjects. These results show that ACE is expressed in T-lymphocytes and indicate that the level of ACE expression in cells synthesizing the enzyme is genetically determined.