EBV-transformed B-cell Lines Research Articles

Defective actin polymerization in EBV-transformed B-cell lines from patients with the Wiskott-Aldrich syndrome.

The Wiskott-Aldrich syndrome (WAS) is a rare X-linked recessive disorder characterized by eczema, thrombocytopenia, and immunodeficiency. An allelic variant of the disease is characterized by isolated thrombocytopenia (XLT). The gene responsible for WAS/XLT (WASP) encodes for a 502 amino acid protein (WASP) that is possibly involved in actin binding and cytoskeleton organization. The expression of WASP and the distribution of F-actin and alpha-actinin (which binds to and stabilizes actin filaments) have been analysed in lymphoblastoid cell lines from six patients with WAS and one with XLT. Western blot and immunocytochemistry did not reveal WASP expression in four WAS patients, whereas two WAS patients (with a moderate clinical course) expressed trace amounts of mutant WASP. In contrast, the XLT patient expressed normal amounts of WASP. Furthermore, cell lines from WAS and XLT patients also markedly differed in F-actin polymerization and alpha-actinin distribution. In particular, severe defects of cytoplasmic F-actin expression and of F-actin-positive microvillus formation, and impaired capping of alpha-actinin, were observed in all patients who lacked WASP. As a whole, the degree of impairment of WASP protein expression in WAS/XLT seems to correlate with anomalies of cytoskeletal organization, strongly supporting a role for WASP in the regulation of F-actin polymerization.

EFFECT OF CYCLOSPORINE AND TACROLIMUS ON THE GROWTH OF EPSTEIN-BARR VIRUS-TRANSFORMED B-CELL LINES1,2

Transplant patients receiving immunosuppressive drugs are at increased risk for Epstein-Barr virus (EBV)-associated disorders including posttransplant lymphoproliferative disorder. The function of T lymphocytes, which are critical to preventing the expansion of EBV-infected B cells, is inhibited by immunosuppressive drugs. The purpose of this study was to determine whether immunosuppressive drugs have direct effects on EBV-infected B cells. The growth and proliferation of EBV-infected spontaneous lymphoblastoid cell lines (SLCLs), cultured in the presence or absence of cyclosporine (CsA) and tacrolimus (TAC), were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and [3H]thymidine incorporation assays. The effect of CsA and TAC on the viability of SLCLs was determined by cell counts with trypan blue. Apoptosis of SLCLs was induced with an anti-Fas agonist monoclonal antibody in the presence or absence of CsA and TAC and measured by flow cytometry after terminal deoxynucleotidyl transferase end-labeling and propidium iodide staining. CsA and TAC, but not sirolimus, increased the growth of SLCLs. The increased growth in the presence of CsA and TAC was attributable to enhanced cell viability and not increased cell division of SLCLs. In addition, CsA and TAC inhibited Fas-mediated apoptosis of SLCLs. CsA and TAC enhance the survival of EBV-transformed B-cell lines. CsA and TAC promote or augment SLCL growth through protection from cell death but do not affect cell division. The inhibition of cell death by CsA and TAC may contribute to the expansion of EBV-infected cells in immunosuppressed individuals.

Expression and signal transduction of T-cell antigen receptor (TCR)/CD3 complexes on fresh or in vitro expanded T lymphocytes from patients with Hodgkin's and non-Hodgkin's lymphomas.

T-cell responses against soluble antigens, alloantigens and mitogens are frequently diminished in patients with certain types of cancer. In the present study, the authors investigated possible mechanisms for the partial T-cell immunodeficiency in patients with Hodgkin's or non-Hodgkin's lymphomas. It was found that T-cells from lymphoma patients had significantly reduced proliferative responses to EBV-transformed B-cell lines and to anti-TCR/CD3 MoAb; a 30-50% reduction of cells expressing membrane T-cell receptor (TCR) complexes; and a significantly reduced signal transduction function. Long-term in vitro culture conditions were developed to expand T cells in TCR/CD3-dependent or TCR/CD3-independent manners. With such methods, it was found that the decreased T-cell responses in patients with Hodgkin's and non-Hodgkin's lymphomas appeared to be an intrinsic T-cell defect (not at the antigen presenting cell level), and the T-cell responses could be recovered after only a few days in culture. Thus, it is suggested that the T-cell response-defect in Hodgkin or non-Hodgkin lymphoma patients is a reversible phenomenon, dependent on the patient's tumour-bearing environment.

Engagement of the signaling lymphocytic activation molecule (SLAM) on activated T cells results in IL-2-independent, cyclosporin A-sensitive T cell proliferation and IFN-gamma production.

The expression and function of the novel signaling lymphocytic activation molecule (SLAM) are described. SLAM is present on immature thymocytes, CD45R0(high) memory T cells, T cell clones, CD56+ T cells, EBV-transformed B-cell lines and on a proportion of B cells. SLAM is rapidly induced on naive T cells, TCR gammadelta+ T cells, and B cells following activation. Engagement of SLAM by the agonistic anti-SLAM mAb A12 resulted in proliferation of T cell clones and Ag- or PHA-activated peripheral blood T cells. mAb A12-induced T cell proliferation is independent of IL-2 and sensitive to inhibition by cyclosporin A. The extent of the anti-SLAM-induced proliferation was related to the activation state of the T cells, since fully rested T cell clones failed to proliferate in response to mAb A12 stimulation, despite unchanged SLAM expression on their surface. Ligation of SLAM on activated peripheral T cells or T cell clones by mAb A12 induced IFN-gamma production in the absence of other exogenous stimuli, even in allergen-specific Th2 clones. These data indicate that stimulation via SLAM reverses the cytokine production profile of Th2 clones to a Th0 phenotype, whereas it further polarizes cytokine production by Th1 clones. Thus, SLAM is a novel receptor that mediates IL-2-independent expansion of activated T cells during immune responses, while concomitantly directing these proliferating cells to a Th0/Th1 pathway.

Characteristics of human EBV-specific cytotoxic T lymphocytes utilized for adoptive immunotherapy of EBV-induced lymphoproliferations in xenografted SCID mice

Human Epstein-Barr virus-specific cytotoxic T lymphocytes (EBV-CTLs) prolong the survival of mice with severe combined immune deficiency bearing the autologous, but not HLA-mismatched, human EBV-induced lymphoproliferative disorders (EBV-LPDs). In the present study, we demonstrate that the HLA-restricted activity displayed by EBV-CTLs both in vitro and in vivo correlates with their in vivo homing pattern, and further characterize these effectors. EBV-CTLs were CD3+, CD16/56-, TCR alpha/beta+, predominantly CD8+ and CD4-, and had a high expression of T-cell activation antigens. EBV-CTLs were positive for CD11a/CD18, CD54, CD58, CD44, CD49d, CD28, and CD45RO, and negative for CD45RA, CD11b, CD11c. After 26 days in culture, EBV-CTLs displayed strong cytotoxicity against the autologous EBV-transformed B-cell line (EBV-LCL), which was inhibited by the addition of anti-CD3 MoAb and mostly HLA class I-restricted. Unirradiated and irradiated EBV-CTLs in the absence of IL-2 failed to proliferate after more than 2 days in culture with the autologous EBV-LCLs, while unirradiated EBV-CTLs with IL-2 formed large colonies and had a high thymidine incorporation both on days 5 and 8. The cytotoxicity of irradiated EBV-CTLs against the autologous EBV-LCLs was conserved. It remains to be determined whether irradiated EBV-CTLs are capable of homing to EBV-LPDs in vivo and to mediate a therapeutic response comparable to that observed with unirradiated EBV-CTLs.

BCR-ABL-positive lymphoblastoid cells display limited proliferative capacity under in vitro culture conditions.

We studied the kinetics of EBV-transformed B-cell lines from patients with chronic myeloid leukaemia (CML) using RT-PCR for BCR-ABL transcripts and immunoglobulin heavy chain (IgH) gene rearrangements. Peripheral blood mononuclear cells, obtained from four patients with CML in chronic phase and from one in accelerated phase, were incubated with supernatant from the B95-8 EBV producing cell line. In 11/25 (44%) B-cell cultures established we demonstrated the presence of BCR-ABL transcripts at intervals ranging from 32 to 125 d post EBV transformation. In all but two cases, evidence of BCR-ABL transcripts disappeared with time. Cultures were initially polyclonal with respect to IgH rearrangements but became progressively oligoclonal, suggesting the longer-term survival of fewer clones, all of which were BCR-ABL negative. We conclude that BCR-ABL-positive lymphoid cultures can be established in the short term from the majority of patients with CML but they have limited capacity to survive in the longer term. Therefore, in lymphoid cells the presence of the BCR-ABL chimaeric gene appears to confer no survival advantage.

Detection of panel-reactive anti-HLA class I antibodies by enzyme-linked immunosorbent assay or lymphocytotoxicity: Results of a blinded, controlled multicenter study

A soluble HLA ELISA for the detection of anti-HLA class I IgG antibodies was developed and compared to complement-dependent microlymphocytotoxicity. ELISA plates were coated with a panel of sHLA class I antigens isolated from the culture supernatants of 46 different EBV-transformed phenotyped B-cell lines. After the incubation of the coated plates with test serum, bound antibodies were detected using a peroxidase-conjugated anti-human IgG antibody. Absorbance was read using an ELISA plate reader and assay results were analyzed by computer. Antibody specificities were determined by Fisher's exact test tail analysis. The reproducibility of ELISA assay results was evaluated in a blinded, controlled multicenter study. A total of 102 serum specimens from patients on waiting lists to receive kidney transplants were tested five times by ELISA in five different laboratories. The correlation coefficients ( r) of %PRA values determined by ELISA ranged from 0.89 to 0.96, and the average agreement on qualitative assay results (antibody positive vs antibody negative) was 98%. Endpoint deration of several serum specimens demonstrated equivalent sensitivity of ELISA and microlymphocytotoxicity (using the anti-globulin antibody protocol). Most of the antibody specificities determined by ELISA were in agreement with specificities determined by microlymphocytotoxicity. To evaluate the correlation of ELISA and microlymphocytotoxicity (CDC) assay results the same 102 specimens were tested six times by CDC in five different laboratories. The interlaboracory correlation coefficient ( r) of %PRA values determined by microlymphocytotoxicity ranged from 0.57 to 0.94, and the average agreement on qualitative assay results was 85%. A comparison of ELISA with microlymphocytotoxicity was performed using consensus microlymphocytotoxicity results. This showed a high correlation (r = 0.81) of %PRA values determined by ELISA and microlymphocytotoxicity. This demonstrates that the detection of anti-HLA class I antibodies by soluble HLA ELISA is a reliable alternative to microlymphocytotoxicity testing.

Isolation and characterization of allergen-binding cells from normal and allergic donors.

Flow cytometry of the immune system so far has been limited to the analysis of subpopulations according to lineage markers. The cells involved in a particular immune response could not be assayed due to their low frequency. Here we show the potential of antigen-specific high gradient magnetic cell sorting to enrich cells for visualisation in multiparameter cytometry, functional studies and immortalization. The aim of this study was the development of an efficient technology for staining and isolation of antigen-binding cells from human peripheral blood. In particular, allergen-specific cells from normal and allergic donors should be analysed and compared to develop a cellular diagnosis of allergy. The rare antigen-specific cells were sorted by high-gradient magnetic cell sorting with MACS. Haptenized phospholipase A2 (PLA2), the major allergen of bee venom, or haptenized ParoI, the major allergenic component of Parietaria officinalis, were used as antigens. The cells from normal and allergic donors, binding to the allergen were characterized phenotypically by immuno-fluorescence. Allergen-specific B-cells were immortalized by EBV transformation. Allergen-specific cells can be enriched from blood of both allergic and normal donors to purities of up to 75%, by high gradient magnetic cell sorting. The specificity of labelling with allergen was confirmed by establishing allergen-specific EBV-transformed B-cell lines from the sorted cells. Clear differences exist in the cellular composition of allergen-binding cells from normal compared to allergic donors. In normal donors the allergen-binding cells are B-cells expressing CD19 and CD21. In allergic donors, in addition to allergen-binding B-cells, occurring in about equal absolute numbers as in normal donors, basophilic granulocytes are labeled by allergen. These cells express CD38, CD9 and CD25 on their surface, and stain for IgE.

Autotumour reactive T-cell clones among tumour-infiltrating T lymphocytes in B-cell non-Hodgkin's lymphomas.

Seventy-three T-cell clones (TCC) were established from tumour-infiltrating lymphocytes-T (TIL-T) derived from lymph nodes involved by B-cell non-Hodgkin's lymphomas (B-NHL) in nine patients with different histological subtypes and clinical stages. 40 TCC (55%) expressed the CD25 Ag and were also able to proliferate in the presence of irradiated autologous B-NHL cells. Among them, 23 autotumour (AuTu) proliferative TCC were found not to proliferate to autologous EBV-transformed B-cell lines, indicating that the proliferative reactivity of these TCC was preferentially directed at autologous B-NHL cells. Tested against autologous B-NHL cells, only three AuTu proliferative TCC (CD8+) showed a significant level of cytotoxicity (specific lysis > 15%). In blocking experiments, the AuTu proliferative reactivity of three TCC from one patient was strongly inhibited by anti-DR and anti-DQ mAbs, whereas that of three TCC from another patient was not affected by either anti-MHC class I or class II (DR, DP, DQ) mAbs. These findings suggest that the recognition of autologous B-NHL cells by AuTu proliferative TCC may occur through MHC-restricted as well as MHC-unrestricted mechanisms.

Identification of thyroid stimulating hormone receptor-specific T cells in Graves' disease thyroid using autoantigen-transfected Epstein-Barr virus-transformed B cell lines.

The importance of thyrotropin receptor (TSHR) agonist antibodies in the manifestations of Graves' disease (GD) is recognized. There are, however, no convincing reports of TSHR-specific T cells. We have previously cloned T cells specific for thyroglobulin and thyroid peroxidase (TPO) from GD lymphoid infiltrates and used autologous EBV-transformed B cell lines (EBVL) transfected with an expression vector encoding TPO to efficiently detect TPO-specific T cells. Here we used EBVL transfected with TSHR to seek TSHR-specific T cells in the GD infiltrates, after cloning the in vivo activated T cells without antigen. 3 out of 30 clones responded vigorously and reproducibly to EBVL-TSHR, with a mean stimulation index > 7. Their release of IL-2, IL-4, and IL-10 after stimulation with soluble anti-CD3 and phorbol ester was indistinguishable from the other clones from this thyroid. However, they produced relatively little IFN gamma (median IL-4/IFN gamma ratio of 0.80) compared with the other clones (median IL-4/IFN gamma ratio 0.06). Thus, this new potent method of antigen presentation, using autoantigen-transfected EBVL, has permitted the first unequivocal identification of TSHR T cells in GD thyroid, with distinct Th0/Th2 characteristics, unlike previously cloned TPO-responsive cells which have Th1 characteristics.

B lymphocytes with latent EBV infection appearing in long-term bone marrow cultures (HLTBMCs) from haematological patients induce lysis of stromal microenvironment.

Human long-term bone marrow cultures (HLTBMCs) are a valuable in vitro model for studying the role of the haemopoietic microenvironment. Here we report the spontaneous appearance of EBV-positive B cells in 6/40 HLTBMCs from patients with various haematological diseases after 3-5 months of culture. After subcultivation of these cells, a novel type of cell line could be characterized, which displayed surface markers and morphological features typical for EBV transformed B-cell lines. As the deproteinized and ultrafiltrated culture supernatants of these cell lines were found to contain an agent with stroma toxic properties, they were termed SSB lines (stroma-toxic-agent-secreting B-cell lines). This agent also exhibited a colony-inhibitory activity on in vitro myelopoiesis and erythropoiesis. These properties are typical for the two polyamines spermine and spermidine which were detected at elevated levels in the culture supernatants of SSB lines. The hypothesis that latent presence of EBV in bone marrow may induce an increased synthesis of spermine and spermidine, which are known to be associated with malignant haematological diseases and bone marrow aplasia, is discussed.

Functional role of CD26 on human B lymphocytes

CD26 is a well-known activation marker on T cells and natural killer (NK) cells [1]. It is identical with the ectopeptidase dipeptidyl peptidase IV (DP IV). The expression of CD26 on B cells has been discussed controversially [2,3]. We have studied the expression of this enzyme on B cells from the peripheral blood of healthy donors and of CVID patients, on cells of the Daudi Burkitt line and the EBV-transformed B-cell lines Jojo and Laz509. DP IV was detected by using anti-CD26 monoclonal antibodies and with help of specific enzyme substrates. Further the influence of specific synthetic DP IV inhibitors on mitogenic activation of purified B cells and DNA synthesis of cell lines was studied. We could show that in both groups 0–5% of freshly isolated CD20-positive B cells do express the CD26 antigen. After stimulation with pokeweed mitogen or St. aureus protein, the fraction of CD26-positive cells was enhanced up to 51% and 36%, respectively. Interestingly, induction of CD26 expression on B cells from CVID patients occurs in a manner similar to the B cells from healthy donors. Treatment of peripheral blood B cells and B-cell lines with highly specific competitive DP IV inhibitors leads to a significant inhibition of DNA synthesis in a dose-dependent manner. These data show that CD26 can be considered to be an activation marker not only of T- and NK cells but also of a main population of B cells, suggesting an involvement of CD26 in B-cell activation.

CD23 molecule acts as a galactose-binding lectin in the cell aggregation of EBV-transformed human B-cell lines.

Epstein-Barr virus (EBV)-transformed human B-cell lines, L-KT9 and DH3 cells express CD23 antigen, and grow in a mixture of single and aggregated cells. The CD23 molecule has high amino acid sequence homology with C-type lectin and recently we have shown that the solubilized CD23 molecule can really interact with galactose residues on glycoproteins. In this study, therefore, we tested whether CD23 antigen on the cell surface really acts as a galactose-binding lectin in the aggregation of these cells. The EBV-transformed cells (L-KT9) were separated into an aggregated-cell-rich fraction and a single-cell-rich fraction. Aggregated cells disaggregated after removal of galactose by beta-galactosidase treatment, whereas single cells made large aggregation on sialidase treatment, and this aggregation was inhibited in the presence of asialo-fetuin. On the other hand, naturally aggregated cells become single cells with anti-CD23 monoclonal antibody (mAB) as well as the soluble form of CD23, but not with anti-CD21 mAB. In addition, L-KT9 and DH3 cells bound to asialo-fetuin-coupled Sepharose (ASF-Sepharose) and this binding was significantly inhibited by pre-treatment of cells with anti-CD23, but not with anti-CD21 or other anti-adhesion molecules. From these results, we conclude that the naturally aggregated state of EBV-transformed cells occurs mainly through the interaction of CD23 as a lectin molecule and galactose residues as its ligand.

Epstein-Barr virus infection induces expression in B lymphocytes of a novel gene encoding an evolutionarily conserved 55-kilodalton actin-bundling protein.

A novel human mRNA whose expression is induced over 200-fold in B lymphocytes by latent Epstein-Barr virus (EBV) infection was reverse transcribed, cloned, and sequenced. The mRNA is predicted to encode a protein containing four peptides which precisely match amino acid sequences from a previously identified 55-kDa actin-bundling protein, p55. In vitro translation of the cDNA results in a 55-kDa protein which binds to actin filaments in the presence of purified p55 from HeLa cells. The p55 mRNA is undetectable in non-EBV-infected B- and T-cell lines or in a myelomonocytic cell line (U937). Newly infected primary human B lymphocytes, EBV-transformed B-cell lines, latently infected Burkitt tumor cells expressing EBNA2 and LMP1, a chronic myelogenous leukemia cell line (K562), and an osteosarcoma cell line (TK143) contain high levels of p55 mRNA or protein. In EBV-transformed B cells, p55 localizes to perinuclear cytoplasm and to cell surface processes that resemble filopodia. The p55 mRNA is detected at high levels in spleen and brain tissues, at moderate levels in lung and placenta tissues, and at low levels in skeletal muscle, liver, and tonsil tissues and is undetectable in heart, kidney, pancreas, and bone marrow tissues. Immunohistochemical staining of human brain tissue demonstrates p55 localization to the perinuclear cytoplasm and dendritic processes of many, but not all, types of cortical or cerebellar neurons, to glial cells, and to capillary endothelial cells. In cultured primary rat neurons, p55 is distributed throughout the perinuclear cytoplasm and in subcortical filamentous structures of dendrites and growth cones. p55 is highly evolutionarily conserved since it shows 40% amino acid sequence identity to the Drosophila singed gene product and 37% identity to fascin, an echinoderm actin-bundling protein. The evolutionary conservation of p55 and its lack of extensive homology to other actin-binding proteins suggest that p55 has specific microfilament-associated functions in cells in which it is differentially expressed, including neural cells and EBV-transformed B lymphocytes.

A 16mer peptide of the human autoantigen calreticulin is a most prominent HLA-DR4Dw4-associated self-peptide

The human Ca 2+-binding (storage) protein calreticulin, located in the lumen of the endoplasmic reticulum, is proposed to play a role as autoantigen: anticalreticulin autoantibodies occur in the sera of patients with SLE and patients with onchocerciasis (calreticulin shows a high sequence homology to the Onchocerca volvulus antigen RAL-1). Here we present sequencing data of a HLA-DR4Dw4-associated calreticulin peptide fragment, Cal(295–310), purified from a DR4Dw4 self-peptide pool. Cal(295–310) proved to be one of three commonest self-peptides associated with DR4Dw4 molecules that were isolated from the EBV-transformed B-cell line BSM (DR4Dw4, DRw53). We tested the binding of Cal(295–309) and the analogous RAL-1 peptide to HLA-DR molecules: Cal(295–309) exhibited specific binding characteristics for DR4Dw4. Binding assays using self-peptide analogues with replaced amino acids led us to a DR4Dw4-binding motif with anchor residues at relative positions 1 and 6. The sequencing data suggest that calreticulin is a frequently processed intracellular protein. The abundance of calreticulin makes the presentation of different calreticulin peptides associated with HLA-D molecules likely to occur, supporting the immunologic relevance of this molecule.

Progressive B-cell chronic lymphocytic leukaemia frequently exhibits aberrant p53 expression.

We have analysed the p53 status in non-progressive and progressive chronic B-cell leukemia (B-CLL) by ELISA, immunoprecipitation, FACS and cDNA sequencing in relation to in vitro proliferation in response to Staphylococcus aureus strain Cowan I (SAC) and IL-2. In FACS, cells from progressive leukaemia were found to over-express p53 with a conformation recognized by PAB240. In a PAb240-based ELISA, 60% of progressive B-CLL were positive. DNA sequencing of p53 exons 5 to 9 revealed a codon 179 His to Gln change in one of the ELISA-positive, progressive B-CLL but failed to reveal any mutations in 4 other ELISA-positive, progressive B-CLL. Among progressive B-CLL populations, 10/14 responded by proliferation in vitro to SAC/IL-2. In non-progressive cells, low levels of p53 were found by FACS, none was positive in the PAb240 ELISA and only one case showed a weak proliferative response to SAC/IL-2. Low p53 expression was also seen in different types of normal B cells, resting and activated, and in EBV-transformed B-cell lines, in contrast to the high expression observed in Burkitt lymphoma cell lines with verified p53 mutations. We conclude that progressive B-CLL is characterized by aberrant p53 expression which may be a significant prognostic factor.

Isolation and characterization of tumor-infiltrating lymphocytes from cervical carcinoma.

The evidence that virus-induced tumors generally elicit T-cell responses prompts the notion that HPV-related cervical carcinoma would be amenable to treatment by T-cell-mediated adoptive therapy. Therefore, we cultured and cloned tumor-infiltrating lymphocytes (TIL) from a patient with cervical carcinoma and studied the in vitro characteristics of these TIL by using the established autologous tumor-cell line. After stimulation of bulk TIL cultures with 1,000 Units/ml recombinant interleukin 2 (rIL-2), followed by limiting dilution, T-cell clones were generated in the presence of 20 U/ml rIL-2 and irradiated autologous tumor cells, PBLs and EBV-transformed B-cell lines. Phenotypically, all clones were CD3/CD8-positive with a heterogeneous CD56 expression. All expressed preferential cytolytic activity against autologous tumor cells, did not lyse autologous lymphoblasts, and were cytotoxic against the NK-sensitive cell line K562. A minor lytic capacity was detectable on allogeneic cervical tumor-cell lines or tumor-cell lines of other histologic types. Cytotoxicity against the autologous tumor could be inhibited by anti-CD3, anti-CD8 and anti-ICAM1 but not by anti-HLA class-1 (W6/32, B9.12.1), anti-allele-specific HLA determinants and anti-LFA-3 antibodies. We demonstrate a highly specific autologous lytic activity of cervical carcinoma TIL, in which a CD3-associated surface antigen recognition is involved. These results may prove useful in further studies on adoptive immunotherapy of cervical cancer patients.

Transfection of thyroid autoantigens into EBV-transformed B cell lines. Recognition by Graves' disease thyroid T cells.

Autologous EBV-transformed B cell lines (EBVL) from a patient with Graves' disease (GD) were transfected with the expression vector pREP4 encoding thyroid peroxidase (TPO), the thyroid-stimulating hormone receptor (TSHR), or chloramphenicol acetyl transferase. The relevant proteins were expressed. The capacity of EBVL to present transfected Ag was assessed by using EBVL transfected with TPO (EBVL-TPO) to stimulate TPO-specific T cells cloned from autologous thyroid tissue; stimulation indices greater than 100 were consistently obtained, even at low APC:T cell ratios. EBVL-TPO and EBVL transfected with TSHR (EBVL-TSHR) were used to characterize the Ag specificity of five thyroid epithelial cell-specific T cell clones. Despite previous unresponsiveness to recombinant TPO or purified thyroglobulin, four of these clones responded vigorously to EBVL-TPO but none responded to EBVL-TSHR. Experiments were then performed to determine whether transfected EBVL were presenting endogenously synthesized TPO, or were taking up and reprocessing exogenous Ag shed from the surface of adjacent cells. The stimulatory capacity of EBVL-TPO was not transferable to untransfected EBVL by cell-free culture supernatant and to only a minor degree by EBVL-TPO-derived cell membrane fragments, indicating that the majority of the TPO presented by the transfected EBVL is endogenously synthesized. The clear and reproducible proliferative responses obtained illustrate the usefulness and potency of this method of Ag presentation to T cells. Because this system obviates the need for large numbers of autologous APC or purified recombinant Ag, it will be useful for obtaining autoantigen-reactive T cells and to help define the autoantigenic epitopes recognized.

TELOMERIC ASSOCIATION OF CHROMOSOMES IS AN EARLY MANIFESTATION OF PROGRAMMED CELL-DEATH

Programmed cell death is an inherent characteristic of all somatic cells, including some immortalized cell lines. Cell death by apoptosis is attributed to nuclear disintegration, chromatin condensation, DNA fragmentation and cytoplasmic boiling. The purpose of this communication is to provide another cytogenetic marker of extensive telomeric associations between chromosomes of cells that are undergoing apoptosis. This can occur in normal fibroblasts, EBV-transformed lymphoblastoid B-cell lines, and even in certain tumor cell lines (including cell lines derived from pancreatic and head and neck tumors). From our preliminary data, we conclude that acquisition of ability to form dicentric chromosomes is associated with programmed cell death that can occur not only in normal fibroblasts but even in some tumor cell lines.

Epitopes recognized by HIV-SF2 nef-specific CD4+ T-cell clones generated from HIV-1-uninfected donors.

Human T-cell clones with specificity to the HIV-1 nef protein were generated by the in vitro stimulation of peripheral blood mononuclear cells (PBMCs) from HIV-1-seronegative donors with purified nef from the HIV-SF2 isolate produced in genetically engineered yeast. Here the characterization is described of a total of seven discrete clones derived from five different donors. Each clone was CD3+ CD4+ CD8- as determined by FACS analysis. The epitopes recognized by these clones were identified using synthetic overlapping peptides spanning the entire length of nef. Six discrete helper T-cell epitopes located in five distinct regions of nef were identified by this approach. Three of these epitopes are more than 80% conserved among all HIV-1 nef proteins for which sequence data are available. The remaining epitopes are in regions of nef that vary among isolates. Many of the epitopes recognized by our clones overlap T-cell epitopes identified by others examining T-cell responses to nef in HIV-1-infected patients and immunized animals. Using partially class II-matched EBV-transformed B-cell lines, we were able to identify five different HLA class II alleles which encode restricting elements for the in vitro nef-specific proliferative response of these clones (DR1, DRw15(2), DRw6, DQw7, DP5).