Epstein-Barr Virus-transformed B-lymphoblastoid Cell Lines Research Articles

Human B cells and dendritic cells are susceptible and permissive to enterovirus D68 infection.

Enterovirus D68 (EV-D68) is predominantly associated with mild respiratory infections, but can also cause severe respiratory disease and extra-respiratory complications, including acute flaccid myelitis. Systemic dissemination of EV-D68 is crucial for the development of extra-respiratory diseases, but it is currently unclear how EV-D68 spreads systemically (viremia). We hypothesize that immune cells contribute to the systemic dissemination of EV-D68, as this is a mechanism commonly used by other enteroviruses. Therefore, we investigated the susceptibility and permissiveness of human primary immune cells for different EV-D68 isolates. In human peripheral blood mononuclear cells inoculated with EV-D68, only B cells were susceptible but virus replication was limited. However, in B cell-rich cultures, such as Epstein-Barr virus-transformed B-lymphoblastoid cell line (BLCL) and primary lentivirus-transduced B cells, which better represent lymphoid B cells, were productively infected. Subsequently, we showed that dendritic cells (DCs), particularly immature DCs, are susceptible and permissive for EV-D68 infection and that they can spread EV-D68 to autologous BLCL. Altogether, our findings suggest that immune cells, especially B cells and DCs, could play an important role in the pathogenesis of EV-D68 infection. Infection of these cells may contribute to systemic dissemination of EV-D68, which is an essential step toward the development of extra-respiratory complications.IMPORTANCEEnterovirus D68 (EV-D68) is an emerging respiratory virus that has caused outbreaks worldwide since 2014. EV-D68 infects primarily respiratory epithelial cells resulting in mild respiratory diseases. However, EV-D68 infection is also associated with extra-respiratory complications, including polio-like paralysis. It is unclear how EV-D68 spreads systemically and infects other organs. We hypothesized that immune cells could play a role in the extra-respiratory spread of EV-D68. We showed that EV-D68 can infect and replicate in specific immune cells, that is, B cells and dendritic cells (DCs), and that virus could be transferred from DCs to B cells. Our data reveal a potential role of immune cells in the pathogenesis of EV-D68 infection. Intervention strategies that prevent EV-D68 infection of immune cells will therefore potentially prevent systemic spread of virus and thereby severe extra-respiratory complications.

Development of an IFNγ ELISPOT for the analysis of the human T cell response against mumps virus

In the last decade, mumps virus (MuV) causes outbreaks in highly vaccinated populations. Sub-optimal T cell immunity may play a role in the susceptibility to mumps in vaccinated individuals. T cell responses to mumps virus have been demonstrated, yet the quality of the MuV-specific T cell response has not been analyzed using single cell immunological techniques. Here we developed an IFNγ ELISPOT assay to assess MuV-specific T cell responses in peripheral blood mononuclear cells (PBMC) of healthy (vaccinated) donors and mumps patients. Various in vitro MuV-specific stimulation methods of PBMC were compared, using either live or inactivated MuV alone or MuV-infected autologous antigen presenting cells, i.e. Epstein Barr Virus-transformed B lymphoblastoid cell lines (EBV-BLCL) or (mitogen pre-activated) PBMC, for their ability to recall IFNγ-producing responder cells measured by ELISPOT. For the detection of MuV-specific T cell responses, direct exposure (24h) to live MuV was the preferred stimulation method when assay sensitivity and practical reasons were considered. Notably, flowcytometric confirmation of data revealed that primarily T cells and NK cells produce IFNγ upon live MuV stimulation. Depleting PBMC from CD56+ NK cells prior to stimulation with live MuV led to the enumeration of MuV-specific T cell responses by ELISPOT. Our assay constitutes a tool to evaluate memory MuV-specific T cell responses in MuV vaccinated or infected persons. Furthermore, this study provides evidence that live MuV not only induces IFNγ production by T cells, but also by NK cells.

Comparison of Immunologic Assays for Detecting Immune Responses in HIV Immunotherapeutic Studies: AIDS Clinical Trials Group Trial A5181

This study was designed to evaluate which of several T-cell-specific, immune response assays are the most relevant in measuring the key characteristics of an effective immune response to HIV-1. Using 5 HIV-1 antigens as stimulants, we assessed lymphocyte proliferation, supernatant gamma interferon (IFN-gamma) cytokine production (CP), single-cell IFN-gamma production by enzyme-linked immunospot (ELISPOT) assay, with and without Epstein-Barr virus-transformed B-lymphoblastoid cell lines (B-LCLs), and intracellular cytokine production (ICC) for IFN-gamma and interleukin 2 (IL-2) by flow cytometry. We used these to compare specimens from HIV-1-infected subjects who were virally suppressed with a stable antiretroviral therapy (ART) regimen (group A) with specimens from subjects not on ART but with HIV-1 viremia of <3,000 copies/ml (group B). The lymphocyte proliferation assay (LPA) did not significantly differentiate between the two groups. Using fresh peripheral blood mononuclear cells (PBMCs), the CP and ELISPOT assays for IFN-gamma detected the greatest differences between the two groups, specific for three of the five HIV-1 antigens, whereas significant differences were seen only in response to one antigen when cryopreserved cells were used. The strongest correlations were seen between the CP and ELISPOT assays. The ELISPOT B-LCL assay showed a cell concentration-dependent increase in IFN-gamma production compared to that shown by the standard ELISPOT assay but did not differentiate between the groups. In the ICC assay, greater numbers of IFN-gamma-producing T cells were seen in group B, and little or no detectable IL-2 production was seen in both groups. These studies highlight complexities of immunologic monitoring of T-cell responses in multisite clinical trials in HIV infection and outline considerations for optimizing these efforts.

Burkitt lymphoma-associated molecules impair HLA class II activation of CD4+ T cells (100.20)

Abstract Burkitt Lymphoma (BL) is a very fast growing form of non-Hodgkin's lymphoma. The common characteristic of all BL is the translocation and constitutive activation of the c-myc oncogene. Our present study suggests that BL and Epstein-Barr Virus-transformed B-lymphoblastoid cell lines (B-LCL) constitutively express HLA class II proteins and bind peptide ligands. However, unlike B-LCL, BL cells were not capable of activating CD4+ T cells. While BL and B-LCL possessed similar levels of HLA class II, invariant chain (Ii) and HLA-DM, BL cells expressed lower levels of co-stimulatory molecules (CD80/CD86). Biochemical and functional assays showed that addition of external co-stimulatory signals to BL cells failed to functionally deliver HLA class II antigens to CD4+ T cells. Although both BL and B-LCL were capable of binding peptides at neutral and acidic pH, BL cells were only able to activate T cells at low pH (5.5), suggesting that BL-associated inhibitory molecules were eluted at the acidic pH. BL conditioned medium, as well as acid eluent from BL cells and primary B-cell tumors, inhibited functional class II presentation by both B-LCL and mature dendritic cells, while those eluent from B-LCL and primary B-cells did not. No characteristic inhibitory cytokines were identified in the acid eluent from BL or primary B-cell tumors. Taken together, these results suggest that BL-associated molecules may inhibit HLA class II activation of CD4+ T cells.

Stable Isotope Tagging of Epitopes

Identification of peptides presented in major histocompatibility complex (MHC) class I molecules after viral infection is of strategic importance for vaccine development. Until recently, mass spectrometric identification of virus-induced peptides was based on comparative analysis of peptide pools isolated from uninfected and virus-infected cells. Here we report on a powerful strategy aiming at the rapid, unambiguous identification of naturally processed MHC class I-associated peptides, which are induced by viral infection. The methodology, stable isotope tagging of epitopes (SITE), is based on metabolic labeling of endogenously synthesized proteins during infection. This is accomplished by culturing virus-infected cells with stable isotope-labeled amino acids that are expected to be anchor residues (i.e. residues of the peptide that have amino acid side chains that bind into pockets lining the peptide-binding groove of the MHC class I molecule) for the human leukocyte antigen allele of interest. Subsequently these cells are mixed with an equal number of non-infected cells, which are cultured in normal medium. Finally peptides are acid-eluted from immunoprecipitated MHC molecules and subjected to two-dimensional nanoscale LC-MS analysis. Virus-induced peptides are identified through computer-assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules. Using this approach we identified novel measles virus and respiratory syncytial virus epitopes as well as infection-induced self-peptides in several cell types, showing that SITE is a unique and versatile method for unequivocal identification of disease-related MHC class I epitopes.

Long-Term Preservation of Antibody-Dependent Cellular Cytotoxicity (ADCC) of Natural Killer Cells Amplified In Vitro From the Peripheral Blood of Breast Cancer Patients After Chemotherapy

Twenty percent of breast cancer adenocarcinomas overexpress the oncogene c-erb-2 that is recognized by the humanized anti-Her2/neu monoclonal antibody Herceptin. Results from clinical studies suggest that antibody-dependent cellular cytotoxicity (ADCC) is involved in the clinical response of Herceptin-treated patients. The purpose of the current study was to evaluate the possibility of amplifying in vitro the CD3-/CD16+ natural killer (NK) cell subset that mediates ADCC from breast cancer patients after chemotherapy. Peripheral blood mononuclear cells from six breast cancer patients taken 2 months after chemotherapy completion were co-cultured with an autologous irradiated Epstein-Barr virus-transformed B-lymphoblastoid cell line (LCL) in the presence of interleukin-2 (IL-2) for 4-6 weeks. These LCL + IL2 activated cultures (ACs) were tested for ADCC potential, and their CD3/CD16 NK proportion was quantified. Among the ACs, the proportion of CD3-/CD16+ NK cells increased up to 64% over the first 2 weeks of culture and the ACs continued to expand for 1 month thereafter. Control and patient ACs displayed ADCC activity (tested in the presence of Rituximab against the autologous LCL to take into account any possible effect of inhibitory NK receptors) as well as against the MCF-7(Her2/neu) breast cancer cell line in the presence of Herceptin. This ADCC activity was maintained during the entire culture period. In conclusion, chemotherapy in breast cancer patients does not obviate the possibility of amplifying in vitro the NK cell subset that mediates ADCC. Consequently, adoptive transfer of lymphocytes mediating ADCC can be considered using this protocol to test its benefit in patients under Herceptin treatment.

Susceptibility of B lymphocytes to adenovirus type 5 infection is dependent upon both coxsackie–adenovirus receptor and αvβ5 integrin expression

Human lymphocytes are resistant to genetic modification, particularly from recombinant adenoviruses, thus hampering the analysis of gene function using adenoviral vectors. This study engineered an Epstein-Barr virus-transformed B-lymphoblastoid cell line permissive to adenovirus infection and elucidated key roles for both the coxsackie-adenovirus receptor and alphavbeta5 integrin in mediating entry of adenoviruses into these cells. The work identified a strategy for engineering B cells to become susceptible to adenovirus infection and showed that such a strategy could be useful for the introduction of genes to alter lymphoblastoid-cell gene expression.

Immunization against a low‐frequency human platelet alloantigen in fetal alloimmune thrombocytopenia is not a single event: characterization by the combined use of reference DNA and novel allele‐specific cell lines expressing recombinant antigens

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal immunization against a fetal platelet (PLT) alloantigen. In cases of FNAIT attributed to low-frequency PLT alloantigens, the laboratory diagnosis is often hampered by the lack of adequate PLTs. Three families with maternal immunization against fetal PLT antigens were analyzed. In Family 1, previous immunization of another female or woman has been observed. In Families 2 and 3, newborns presented with the typical clinical picture of FNAIT. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism and direct sequencing with reference to DNA from Epstein-Barr virus-transformed B-lymphoblastoid cell lines. Antibodies were characterized by glycoprotein (GP)-specific immunoassay with a panel of stable Chinese hamster ovary cell lines expressing low-frequency alloantigens. In three families, maternal immunization associated with the low-frequency alloantigens human PLT antigen (HPA)-8bw (Sra), HPA-11bw (Groa), and HPA-13bw (Sita) was identified. Maternal serum samples showed positive reactions in an antigen capture assay with cell lines carrying recombinant GP IIb/IIIa (HPA-8bw and -11bw) or GPIa/IIa (HPA-13bw), respectively. These results could be confirmed by genotyping analysis of fathers and newborns. This study demonstrates that cases of FNAIT attributed to low-frequency PLT alloantigens cannot be regarded as single events. The availability of reference DNA and cell lines expressing recombinant PLT alloantigens can facilitate their identification.

Cell surface phenotyping and cytokine production of Epstein–Barr Virus (EBV)-transformed lymphoblastoid cell lines (LCLs)

Epstein–Barr Virus-transformed B lymphoblastoid cell lines (EBV-LCLs) are routinely used for the in vitro expansion of T cells. However, these cell lines are reported to produce the cytokine IL-10, which is inhibitory for T cells. We, therefore, characterized a panel of 37 EBV-LCLs for a variety of cell surface markers, for secretion of various cytokines including IL-10 and for immunoglobulin production. These cell lines were derived from normal donors or patients with nonsmall cell lung cancer, acute myelogenous leukemia, melanoma or colon cancer. Overall, 26 lines were positive for CD19 and CD20, and 11 were negative for both. All of the lines were strongly HLA-DR+, while CD40 expression was variable. Twenty-four (65%) were both CD23+ and secreted immunoglobulin, and 33 expressed κ and/or λ light chains. Additionally, all of the EBV-LCLs were negative for T cell (CD3), NK cell (CD16, CD56), monocyte (CD14) and granulocyte (CD66b) surface markers. Some level of IL-10, IL-6, IL-12p40 and TNF-α cytokine production was detected in 33, 18, 19 and 12 EBV-LCLs, respectively. Together, these data reflect the heterogeneity of EBV-LCLs, which cautions their use nondiscriminately in various immunologic assays.

Flow cytometric determination of intracytoplasmic Wiskott–Aldrich syndrome protein in peripheral blood lymphocyte subpopulations

We have produced a novel monoclonal antibody (mAb) directed against Wiskott–Aldrich syndrome protein (WASP) by immunizing mice with the recombinant protein. The mAb designated 5A5 is highly specific to WASP and suitable for Western blot analysis and immunoprecipitation. A flow cytometric assay using the 5A5 mAb identifies expression of intracytoplasmic WASP in lymphocytes from normal individuals. Double staining analysis with cell surface CD3, CD19, and CD56, and intracytoplasmic molecules revealed WASP expression in each subpopulation. With regard to WASP expression in patients with Wiskott–Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT), peripheral blood mononuclear cells (PBMCs) from nine patients and Epstein–Barr virus-transformed B-lymphoblastoid cell lines from seven patients examined did not show WASP expression by flow cytometric analysis. These results were confirmed by Western blot analysis. We conclude that WASP expression in lymphocyte subpopulations from patients with WAS and XLT can be more precisely evaluated by flow cytometry as compared with Western blot analysis. This flow cytometry method is important as a supplement to Western blots, but even more important as an alternative and powerful assay that can contribute to research on WASP as well as diagnosis in a clinical setting.

Detection of diverse hepatitis C virus (HCV)-specific cytotoxic T lymphocytes in peripheral blood of infected persons by screening for responses to all translated proteins of HCV.

Broadly directed hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) have been identified from liver-infiltrating lymphocytes but have been more difficult to assess in peripheral blood of infected persons. To enhance the detection of CTL from peripheral blood mononuclear cells (PBMC), we cocultured PBMC with autologous Epstein-Barr virus-transformed B-lymphoblastoid cell lines that had been infected with recombinant vaccinia virus constructs so that they expressed the entire translated polyprotein of HCV-H, a type 1a strain. These stimulated cells from HCV-infected as well as exposed seronegative persons were then cloned at limiting dilution and tested for HCV-specific CTL activity using a standard (51)Cr release assay. HCV-specific CTL were detected in PBMC from seven of nine persons with chronic hepatitis, including five of seven in whom CTL had previously been detected from liver biopsy specimens but not PBMC. In a single person with chronic HCV infection, CTL directed against as many as five different epitopes were detected in peripheral blood and were similar in specificity to those detected in liver tissue. This technique was used to evaluate eight subjects identified to be at high risk for HCV exposure due to continued injection drug abuse; no evidence of CTL in PBMC was found. We conclude that CTL can be detected in PBMC from the majority of persons with chronic HCV infection but are present at lower levels or absent in exposed but persistently seronegative persons. The high degree of concordance of HCV epitopes identified from liver and PBMC suggests that this strategy is a reasonable alternative to liver biopsy for characterizing the CTL response to HCV in chronically infected persons.

Efficient class II major histocompatibility complex presentation of endogenously synthesized hepatitis C virus core protein by Epstein-Barr virus-transformed B-lymphoblastoid cell lines to CD4(+) T cells.

The induction of an efficient CD4(+) T-cell response against hepatitis C virus (HCV) is critical for control of the chronicity of HCV infection. The ability of HCV structural protein endogenously expressed in an antigen-presenting cell (APC) to be presented by class II major histocompatibility complex molecules to CD4(+) T cells was investigated by in vitro culture analyses using HCV core-specific T-cell lines and autologous Epstein-Barr virus-transformed B-lymphoblastoid cell lines (B-LCLs) expressing structural HCV antigens. The T- and B-cell lines were generated from peripheral blood mononuclear cells derived from HCV-infected patients. Expression and intracellular localization of core protein in transfected cells were determined by immunoblotting and immunofluorescence. By stimulation with autologous B-LCLs expressing viral antigens, strong T-cell proliferative responses were induced in two of three patients, while no substantial stimulatory effects were produced by B-LCLs expressing a control protein (chloramphenicol acetyltransferase) or by B-LCLs alone. The results showed that transfected B cells presented mainly endogenously synthesized core peptides. Presentation of secreted antigens from adjacent antigen-expressing cells was not enough to stimulate a core-specific T-cell response. Only weak T-cell proliferative responses were generated by stimulation with B-LCLs that had been pulsed beforehand with at least a 10-fold-higher amount of transfected COS cells in the form of cell lysate, suggesting that presentation of antigens released from dead cells in the B-LCL cultures had a minimal role. Titrating numbers of APCs, we showed that as few as 10(4) transfected B-LCL APCs were sufficient to stimulate T cells. This presentation pathway was found to be leupeptin sensitive, and it can be blocked by antibody to HLA class II (DR). In addition, expression of a costimulatory signal by B7/BB1 on B cells was essential for T-cell activation.

Human Rh D Monoclonal Antibodies (BRAD-3 and BRAD-5) Cause Accelerated Clearance of Rh D+ Red Blood Cells and Suppression of Rh D Immunization in Rh D- Volunteers

The use of prophylactic anti-D to prevent Rh D immunization in Rh D- women and subsequent hemolytic disease in Rh D+ infants is widespread, but has led to shortages of the anti-D Ig. With the aim of substituting monoclonal anti-D for Rh D prophylaxis, we have compared the abilities of monoclonal and polyclonal anti-D to clear Rh D+ red blood cells (RBCs) infused into Rh D- male volunteers and to suppress Rh D immunization. Two human monoclonal antibodies (MoAbs), BRAD-3 (IgG3) and BRAD-5 (IgG1), produced from stable Epstein-Barr virus-transformed B-lymphoblastoid cell lines, were selected because of their proven in vitro activity in promoting RBC lysis in antibody-dependent cell-mediated cytotoxicity assays. RBC clearance was assessed by intravenous injection of 3 mL of 51chromium-labeled D+ RBCs into 27 volunteers 48 hours after intramuscular injection of monoclonal or polyclonal anti-D. Further 3-mL injections of unlabeled D+ cells were administered at 6 and 9 months to induce immunization. Blood samples were taken throughout the 12-month period of study for the serologic detection of anti-D. The mean half-life (t50%) of RBCs in 7 recipients of 300 micrograms BRAD-5 (5.9 hours) was similar to that in 8 recipients of 500 IU polyclonal anti-D (5.0 hours), whereas D+ cells were cleared more slowly in some of the 8 subjects injected with 300 micrograms BRAD-3 (mean t50% 12.7 hours) and in 1 individual administered 100 micrograms BRAD-3 (t50% 41.0 hours). The rate of RBC clearance in both groups administered 300 micrograms monoclonal anti-D correlated with the amount of antibody bound per cell, determined by flow cytometry. There was no evidence of primary immunization having occurred in any subject after 6 months of follow-up. Five of 24 subjects produced anti-D after one or two further injections of RBCs, confirming that they were responders who had been protected by the monoclonal or polyclonal anti-D administered initially. Four of these responders were recipients of monoclonal anti-D (3 BRAD-3, 1 BRAD-5). One individual who received BRAD-5 produced accelerated clearance of D+ RBCs at the third unprotected RBC challenge but did not seroconvert. This study shows that the human MoAbs BRAD-3 and BRAD-5 can prevent Rh D immunization, and indicates that they may be suitable replacements for the polyclonal anti-D presently used in prophylaxis of Rh D hemolytic disease of the newborn.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of B-Cell Lines Established From Two X-Linked Severe Combined Immunodeficiency Patients: Interleukin-15 Binds to the B Cells But Is Not Internalized Efficiently

X-linked severe combined immunodeficiency (XSCID) is characterized by absent or profoundly reduced numbers of T cells and normal numbers of B cells in the circulation. Affected patients have mutations of the interleukin-2 (IL-2) receptor gamma chain gene. Using Epstein-Barr virus-transformed B-lymphoblastoid cell lines (B-LCLs) established from two unrelated XSCID patients, we could show that neither expressed the IL-2 receptor gamma chain on the cell surface. A novel cytokine IL-15, which has biologic activities similar to those of IL-2, could bind to the XSCID B-LCLs in the absence of the gamma chain, although both the beta and gamma chains of the human IL-2 receptor were previously shown to be required for IL-15 binding by transfected COS cells. Furthermore, a significant reduction and delay of IL-15 internalization by B lymphoblasts from XSCID patients was observed when compared with that of normal control B-LCLs. These results show the existence of a novel IL-15-specific receptor component that contributes to IL-15 binding but is insufficient for IL-15 internalization in the absence of the IL-2 receptor gamma chain.

Heterogeneous PIG-A Mutations in Different Cell Lineages in Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal defect of hematopoietic stem cells in which affected cells are characterized by the lack of glycosylphosphatidylinositol (GPI)-anchored proteins. The lesion in PNH lies in the defective synthesis of N-acetyl-D-glucosaminyl-phosphatidylinositol (GlcNAc-Pl), the first intermediate in GPI biosynthesis. Reintroduction of the PIG-A gene into GPI(-) patient cells reportedly complements this defect. We have analyzed here PIG-A transcripts of six PNH patients. GPI+ and GPI- cell lines from each individual were used, ie, Epstein-Barr virus-transformed B-lymphoblastoid cell lines, T-cell lines, and natural killer cell clones. Reverse transcriptase polymerase chain reaction and sequencing showed three different PIG-A splicing variants in GPI+ cell lines, in which the largest transcript contained the wild-type PIG-A coding region sequence. GPI-deficient cell lines showed abnormal splicing variants. Sequencing of PIG-A complementary DNA and genomic DNA showed heterogeneous mutations ranging from different point mutations to small deletions. Two lymphocyte cell lines (T- and B-cell lines) of one patient presented with the same mutation. For another patient, two different mutations were detected in one natural killer cell line. Therefore, different cell lineages have somatic mutations in PIG-A that lead to PNH.

Three monoclonal antibodies against human LFA-1 alpha and beta chains with different biological activities.

Three murine monoclonal antibodies (MAbs) directed against human lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18), designated as MAY.017, MAY.035, and MAY. 044, were newly generated. The hybridomas were screened for their ability to inhibit a phorbol ester-stimulated aggregation of Epstein-Barr virus-transformed B-lymphoblastoid cell line (B-LCL). The MAbs bound to peripheral blood leukocytes, T cell lines, B cell lines, and some of myeloid/monocytic lines, but not to B-LCL derived from a patient with leukocyte adhesion deficiency. MAY.035 immunoprecipitated a complex of proteins with molecular masses of 155 kDa and 95 kDa, while MAY.017 and MAY.044 did a complex of 130 kDa, 155 kDa and 95 kDa proteins. MAY.035 was shown as to recognize the alpha chain of LFA-1 (CD11a), and both MAY.017 and MAY.044 the beta chain of the beta 2 integrin family (CD18). All the three MAbs inhibited lymphocyte proliferative responses to mitogens or alloantigens. MAY.017 blocked cytolytic activity mediated by natural killer cells.

Human b cells express membrane-bound and soluble forms of the cd 14 myeloid antigen

The expression of the myeloid differentiation antigen CD14 on the B lineage was analyzed. A CD14-specific monoclonal antibody was used to isolate the antigen from normal B, B-type chronic lymphocytic leukemia cells, and a representative Epstein-Barr virus-transformed B lymphoblastoid cell line (EBVLCL). A soluble form of this protein was detected in the culture supernatant of all the B cell types tested. The molecule expressed in the normal B and B-type chronic lymphocytic leukemia cells was identical in size to the 52,000 mol. wt monocyte-isolated CD14 glycoprotein. A 64,000 mol. wt antigen was isolated from the lymphoblastoid cell line. Similar 2-D gel electrophoretic patterns to that of the monocyte-derived CD 14 were obtained from the normal B and B-type chronic lymphocytic leukemia cell-isolated molecules. These similarities were reflected in minor isoelectric point (p I) differences between the polypeptide spots (p I4.8), in the first dimension, and identical molecular weight (52,000) in the second dimension. The EBVLCL-isolated polypeptide, when analyzed by 2-D gel electrophoresis, showed a p I identical to that of the myeloid antigen (p I 4.6). The isolated soluble form was of smaller (47,000 mol. wt, normal B and B-type chronic lymphocytic leukemia cells) or similar size (64,000 mol. wt, lymphoblastoid cell line) compared with their corresponding membrane-bound forms. Interestingly, two-colour immunofluorescence analysis showed that only two out of four CD14-speciflc mAb tested bound to the B cells. We conclude that the CD14 antigen is, in fact, expressed in the B lineage. Its cell surface expression and serum level in the prognosis of B-type chronic lymphocytic leukemia patients needs to be evaluated.

Polymorphism of HLA‐DR2,DQwl haplotypes in Asian Indians

We examined the polymorphism of DR2,DQw1 haplotypes in Epstein-Barr virus-transformed B-lymphoblastoid cell lines (HTCs) and unrelated (32 Canadian Caucasians and 24 Asian Indians) individuals by restriction fragment length polymorphism (RFLP) and oligonucleotide typing. The data demonstrate that three subtypes of DR2,DQw1 haplotypes, DRw15(B1.1501).DQw6a(A1.0102,B1.0602),DRw15(B1.1502). DQw6b(A1.0103,B1.0601), DRw16(B1.1601).DQw5(A1.0102,B1.0502) are present in HTCs and Canadian Caucasians. Of these, DRw15(B1.1501).DQw6a (A1.0102,B1.0602) haplotype was present in majority (81.3%) of Caucasians. Among Asian Indians, this haplotype was present only in one DR2,DQw1-positive individual. In addition, three new haplotypes representing different combinations of DRB1, DQA1 and DQB1 genes were demonstrable in Asian Indians. These new haplotypes are DRw15(B1.1501).DQw6b(A1.0103,B1.0601),DRw15(B1.1501). DQw5(A1.0102,B1.0502), and DRw15(B1.1501).DQw6c(A1.0102, B1.0601). The most frequent haplotypes among Asian Indians were DRw15(B1.1502).DQw6b(A1.0103,B1.0601) and DRw15(B1.1501). DQw6b(A1.0103,B1.0601). The distribution of subtypes of DR2,DQw1 haplotypes in Asian Indians was significantly different from that in Canadian Caucasians. The results in the present study have important implications for HLA and for HLA-disease associations.

Restriction of Epstein-Barr virus-specific cytotoxic T cells by HLA-A, -B, and -C molecules

HLA-loss variants of an Epstein-Barr virus-transformed B-lymphoblastoid cell line (EBV-LCL) 721 were used as target cells to identify HLA molecules utilized by EBV-LCL-specific cytotoxic T cells. Split culture analysis of cytotoxic T cells plated at limiting dilution showed killing of HLA-loss variants bearing either HLA-A2 or -B5 molecules, with 10 times higher frequency of cytotoxic T cells restricted by the HLA-B5 molecule. Clonal analysis confirmed the restriction by HLA-A2 or -B5 of some cytotoxic T-cell clones and identified cytotoxic T-cell clones cytolytic for target cells which do not express HLA-A or -B but do express the HLA-C determinant. Thus, our results show immunodominance of the HLA-B5 restriction determinant for EBV-induced antigens in the donor of the HLA-loss variants and provide evidence that the HLA-C molecule can also serve as restriction determinant for EBV-LCL-specific cytotoxic T cells.

Assessment of human allospecific IL-2-secreting helper T lymphocytes in limiting dilution cultures using restimulation and split well analysis

A limiting dilution (LD) culture was established which allows the detection of allospecific interleukin-2 (IL-2)-secreting helper T lymphocyte precursors (HTL-p) among human peripheral blood mononuclear cells (PBMNC). HTL-p stimulated with allogeneic Epstein-Barr virus-transformed B lymphoblastoid cell lines (EBV-LCL) in the presence of exogenous recombinant IL-2 (r-IL_2) clonally developed into IL-2 secreting effector cells when restimulated against the original stimulating alloantigen. Split well cultures were performed prior to restimulation to assess the antigen specificity of the response. Frequencies of alloreactive IL-2-secreting HTL-p ranged from | 1 100 to 1 800 . Allospecificity of effector T cells was determined by a strong decline of frequencies obtained after restimulation against third-party antigens. In (clonal) segregation analyses the vast majority of IL-2-secreting progenies (80%) were specific for the original stimulating alloantigen. Allele specificity was disclosed by using class II MHC related third-party restimulator cells. By comparison with LD short-term cultures it became evident that exogenous r-IL-2 in the initial culture period was required to reveal optimal precursor frequencies of IL-2-producing T cells. Furthermore, successful antigenic restimulation was strictly confined to these culture conditions.