EBV-transformed B-cell Lines Research Articles

Protein studies of human non-Hodgkin's B-lymphoma: appraisal by two-dimensional gel electrophoresis.

We have utilized two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) coupled with silver stain to identify cellular proteins in human non-Hodgkin's B-lymphoma (NHL). Five cell lines (SKDHL2B, WSU-DLCL2, WSU-NHL, WSU-FSCCL and SKLN1), representing four different NHL maturational stages and a normal Epstein-Barr virus (EBV)-transformed line of B-cell origin (SKLN1) were studied. The NHL lines were immunophenotyped using flow cytometry with lineage associated monoclonal antibodies. Whole cell lysates of the cell lines were subjected to 2-D PAGE analyses. The gels were analyzed with an image scanning computer and the qualitative differences of protein patterns were studied. Results revealed great similarities in patterns of the NHL lines. A master map containing common NHL-protein spots was constructed. When the map of each tumor line was compared to the master map, several protein spots were associated with each NHL-grade. Search for these proteins in the normal EBV-transformed B-cell line showed that only one of the proteins (S3; M(r)/pI 19/5.9) was present. Proteins that were detected in malignant NHL, but not in the normal EBV-line, could provide important information regarding the human NHL B-lymphocyte data-bases. Whether or not these proteins are definite malignant markers to distinguish between different NHL maturational stages needs further exploration through electroblotting and microsequencing.

Effect of mycophenolic acid on Epstein-Barr virus infection of human B lymphocytes.

The effect of mycophenolic acid (MPA) on the growth of Epstein-Barr virus (EBV)-transformed B cells and on EBV lytic-cycle gene expression was investigated. MPA inhibited the proliferation of newly infected or established EBV-transformed B-cell lines but was not able to block cell growth transformation or the viral lytic cycle. These results suggest that MPA might reduce transformed-cell proliferation in transplant patients and possibly decrease the risk of development of EBV-related lymphoproliferative disorders.

Identification of colon‐tumor‐associated antigens by T‐cell lines derived from tumor‐infiltrating lymphocytes and peripheral‐blood lymphocytes from patients immunized with an autologous tumor‐cell/bacillus calmette‐Guérin vaccine

Tumor immunity developing as a response to an autologous colon-tumor/bacillus Calmette-Guérin (BCG) vaccine appears to be associated with induction of CD4+ helper T cells, implied by the observation that vaccine efficacy is associated with major histocompatibility complex class-II molecule expression on the vaccine tumor cells. Therefore, in an attempt to identify colon-tumor-associated antigens responsible for conferring immunity, we examined and compared the proliferative responses of peripheral-blood lymphocytes (PBL) from patients immunized with the autologous tumor/BCG vaccine to T-cell lines cloned expanded from colon-tumor-infiltrating lymphocytes to 5 antigens isolated on the basis of their reactivity by colon-tumor-reactive human monoclonal antibodies. Enzymatically dissociated colon tumors provided a source for establishment of cloned T-cell lines, tumor cell lines propagated in vitro or in vivo as nude-mouse xenografts and EBV-transformed B-cell lines used as antigen-presenting cells. Of 104 different T-cell lines tested, only 3 proliferated in response to CTAA 28A32-46K, and I to the CTAA28A32-32K antigen. In contrast, PBL from 64% of patients immunized with the autologous colon-tumor/BCG vaccine responded to the CTAA 28A32-32K antigen. This antigen is related to a family of calcium- and phospholipid-binding placental proteins termed annexins. Since proliferative responses developed to this antigen after vaccination in 64% of individuals, this antigen may be an important common colon-tumor-associated rejection antigen.

CD30‐antigen‐specific targeting and activation of T cells via murine bispecific monoclonal antibodies against CD3 and CD28: Potential use for the treatment of hodgkin's lymphoma

Cross-linking of specific tumor antigens with the T-cell-associated CD3 and CD28 antigens can increase IL-2 secretion, proliferation and antigen-specific cytotoxicity in resting T cells. This cross-linking can be achieved effectively by bispecific monoclonal antibodies (BiMAb) with specificity for both the tumor antigen and CD3 or CD28 antigen, respectively. To take advantage of the enhanced activation of CD3 pre-activated T cells by additional activation via the CD28 antigen, BiMAb OKT3/HRS-3 with reactivity to both CD3 and the Hodgkin's-lymphoma-associated CD30 antigen and the BiMAb 15E8/HRS-3 with reactivity to both CD28 and CD30 antigen were generated by hybridoma fusion. Resting T cells, represented by Jurkat cells (CD3+/CD28+) were specifically activated to produce IL-2 by co-cultivation with an EBV-transformed B-cell line (LAZ509, CD30+/CD19+) only in the presence of the CD30/CD28 cross-linking BiMAb and an additional cross-linking anti-CD3/CD19 BiMAb (OKT3/6A4). Neither the cross-linking BiMAbs alone nor any combination of the monospecific parental MAbs induced a comparable IL-2 production by Jurkat cells in the presence of LAZ509. In addition, using a combination of these BiMAbs, an antigen-dependent cytotoxicity was induced by targeting APC-depleted peripheral blood lymphocytes to CD30+ L540 cells. T cells, previously specifically activated by CD3/CD30 in the presence of CD30 antigen, were cytotoxic to CD30+ cell lines only after incubation with BiMAb anti-CD28/CD30. Neither of the BiMAbs nor any of the parental antibodies induced a comparable effect. Our results indicate that such BiMAbs may offer a new approach for specific immunotherapy of Hodgkin's lymphoma, which takes advantage of cytokine secretion and cytotoxicity of activated T cells.

Restoration of superoxide generation to a chronic granulomatous disease- derived B-cell line by retrovirus mediated gene transfer

Failure of a superoxide generating system, the NADPH oxidase, present in neutrophils and other phagocytes gives rise to chronic granulomatous disease (CGD), a group of single-gene inherited disorders all characterized by an extreme susceptibility to pyogenic infection, with potentially fatal consequences. About 30% of CGD cases are caused by an autosomally inherited deficiency of a 47-Kd cytoplasmic component of the oxidase (p47-phox). Epstein-Barr virus (EBV) immortalized B-lymphocyte lines established from these CGD patients also express this NADPH oxidase defect and consequently are rendered incapable of generating superoxide on stimulation. We have used a p47-phox-deficient EBV-transformed B-cell line as a recipient for retroviral transfer of a functional p47-phox cDNA. The presence and activity of the retrovirally encoded p47-phox in the transduced cells is demonstrated and we show that this restores their capacity to generate superoxide.

Restoration of Superoxide Generation to a Chronic Granulomatous Disease-Derived B-Cell Line by Retrovirus Mediated Gene Transfer

Abstract Failure of a superoxide generating system, the NADPH oxidase, present in neutrophils and other phagocytes gives rise to chronic granulomatous disease (CGD), a group of single-gene inherited disorders all characterized by an extreme susceptibility to pyogenic infection, with potentially fatal consequences. About 30% of CGD cases are caused by an autosomally inherited deficiency of a 47-Kd cytoplasmic component of the oxidase (p47-phox). Epstein-Barr virus (EBV) immortalized B- lymphocyte lines established from these CGD patients also express this NADPH oxidase defect and consequently are rendered incapable of generating superoxide on stimulation. We have used a p47-phox-deficient EBV-transformed B-cell line as a recipient for retroviral transfer of a functional p47-phox cDNA. The presence and activity of the retrovirally encoded p47-phox in the transduced cells is demonstrated and we show that this restores their capacity to generate superoxide.

Retardation in tumor growth and metastasis demonstrated in a human CLL B-cell line after the acquisition of an extra chromosome 11

In this study we described the isolation of a subclonal EBV-transformed human CLL B-cell line with retarded growth rate and metastatic potential from its clonal parent (D10-1), which was karyotyped: 46,XY,dup(1)(q11→q32). The subclone, designated D10-1C, was isolated by limiting dilution of D10-1 following selection in 8-azaguanine-supplemented medium. Chromosome analysis of D10-1C revealed a constitution of 47,XY,+ 11,dup(1)(q11→q32). This is the first demonstration that partial trisomy 1q-associated growth advantage in human cancer cells can be retarded by the presence of an additional dose of chromosome 11. Human chromosome 11 had been shown to be responsible for the suppression of tumor development. Whether the same suppressor genes are involved in D10-1C remains to be elucidated. The procedure described here for the enrichment and isolation of a growth-retarded mutant from D10-1 may be applicable in other malignant cell systems.

Marked cell-type-specific differences in glycosylation of human interleukin-6

Human interleukin-6 (IL-6) secreted by cytokine- or endotoxin-induced fibroblasts, monocytes, keratinocytes, endometrial stromal cells, and endothelial cells, when analyzed under denaturing and reducing conditions, consists of a set of differentially modified phosphoglyco-proteins of molecular mass in the range from 23 to 30 kD (a set of at least three O-glycosylated 23- to 25-kD species and a set of at least three N- and O-glycosylated 28- to 30-kD species). The 23- to 25-kD and 28- to 30-kD fibroblast-derived IL-6 species have been separately purified to homogeneity with the use of a combination of lectin and immunoaffinity chromatography. Glycosidase digestion experiments on such purified preparations confirmed that almost all human fibroblast-derived IL-6 species were O-glycosylated; additionally, the 28- to 30-kD species were N-glycosylated. Amino acid sequencing revealed that the major amino terminus in the fibroblast-derived 23- to 25-kD O-glycosylated IL-6 was at Ala 28 whereas the major amino terminus in the 28- to 30-kD N- and O-glycosylated IL-6 was at Val 30, suggesting that targeting of newly synthesized IL-6 polypeptides into the two different processing pathways in fibroblasts may be keyed to differences in the signal peptide cleavage site. Unexpectedly, IL-6 “constitutively” secreted by the Epstein-Barr virus (EBV)-infected human and primate (tamarin) B-cell lines designated sfBJAB and sfBT, respectively, consisted of a major apparently unglycosylated 21-kD species and a minor 25-kD N-glycosylated species. Similarly, human IL-6 secreted by Sf9 insect cells infected with a baculovirus expression vector for human IL-6 consisted of a 21-kD unglycosylated species (amino terminus at Ala 28) and a 25-kD N-glycosylated species. In contrast, IL-6 secreted by the EBV-transformed human B-cell line KH consisted exclusively of the 28- to 30-kD N- and O-glycosylated species. Taken together, the present data highlight the marked differences in the glycosylation patterns of IL-6 polypeptides secreted by different human cell types and help reconcile previous disparate descriptions of natural human IL-6 proteins.

Detection of cytotoxic T lymphocytes specific for synthetic peptides of gp160 in HIV-seropositive individuals.

Four synthetic peptides corresponding to the IIIB sequence of gp160 of HIV were recently reported to stimulate Th cell function by PBL from HIV-infected, asymptomatic patients. In the present report, we used these same peptides to demonstrate CTL activity in a similar patient population. EBV-transformed B-cell lines from asymptomatic, HIV seropositive and seronegative control donors were pre-incubated with the peptides. Fresh PBL from 19 (76%) of 25 HIV seropositive donors lysed autologous targets pulsed with at least one of the four peptides. Autologous targets pulsed with two non-immunogenic peptides were not lysed. PBL from none of the eight HIV seronegative controls lysed peptide-preincubated autologous targets. The CTL activity was mediated by T cells, was predominantly MHC class I restricted, and was increased by in vitro restimulation of PBL with the peptides. HLA A-2 was identified as a restricting element for all four peptides in different patients, and for three of the peptides in the same donor. HLA-A1 or -B8 may also present some of the peptides. Thus, the same peptides can be recognized by human Th cells and class I MHC-restricted CTL.

Target lysis by human lak cells is critically dependent upon target binding properties, but LFA‐1, LFA‐3 AND ICAM‐1 are not the major adhesion ligands on targets

The cytotoxicity mediated by the CD2+ CD3- lymphocyte subset, either NK or LAK, is puzzling since no specific antigen recognition structures, equivalent to the CD3-associated heterodimer T-cell receptor, have been recognized on these cells so far. The possibility exists that the CD3- cytotoxic effectors recognize their targets through non-specific adhesion mechanisms. The goal of this study was: (a) to examine the correlation between binding properties and susceptibility to lysis of 6 informative target cell lines; (b) to evaluate the role, as ligands on these targets, of adhesion molecules such as LFA-1, LFA-3 and ICAM-1. The effectors used in this study were IL-2-activated LGL, predominantly CD3-, or highly purified CD3- lymphocytes from normal human donors. The 6 target lines studied included 2 pairs of EBV-transformed B-cell lines (721 LCL vs. 721.134, and MM vs. MM-10F2) in which the parental lines were resistant to lysis while HLA variants were susceptible. A third pair was the Daudi Burkitt cell line, susceptible to LAK lysis, and an HLA-positive transfected Daudi line which was more resistant to lysis. The binding properties of these targets to LAK effectors (conjugate formation) were evaluated using a sensitive double fluorescence flow cytometry method. In each pair examined, the susceptible targets formed more conjugates and were surrounded by more cytotoxic LAK effectors than their resistant counterparts, indicating that the conjugation properties of targets are closely correlated with their susceptibility to LAK lysis. The expression of adhesion molecules on the informative targets was examined by indirect immunofluorescence and their role was evaluated by inhibition of lysis after pre-coating the targets with the relevant antibodies. The differences in the expression of the classical cell-cell adhesion molecules LFA-1, LFA-3 and ICAM-1 on the target surfaces were only marginal, insufficient to explain the striking differences in susceptibility to lysis and in binding properties. Coating the target cells with antibodies directed against these adhesion determinants had no effects on the lysis of susceptible target cells. The same antibodies reacting with the LAK effectors did inhibit lysis. Taken together, these results suggest that, on the targets, presently undefined membrane adhesion structures may have a major role in conjugate formation between target and CD3- effectors and determine the susceptibility of the targets to lysis.

The MHC class II deficiency syndrome: Heterogeneity at the level of the response to 5-azadeoxycytidine

The involvement of DNA methylation in aberrant MHC class II gene expression in EBV-transformed B-cell lines from 2 patients (THF and DGN) with the MHC class II deficiency syndrome (bare lymphocyte syndrome) was investigated. Incubation of the cells in the presence of various concentrations of 5-azadeoxycytidine resulted in the induction of expression of HLA DR genes in the DGN cell line, whereas, in the THF cell line, no effect of 5-azadeoxycytidine treatment on the expression of the HLA DR genes could be detected. Subsequent Southern blot analysis using methylation-sensitive restriction enzymes ( ApyI, EcolII and HhaI) and 5-azadeoxcycytidine-treated and untreated genomic DNA, indicated that the lack of HLA DR-A expression in the DGN cell line is not caused by hypermethylation of the 5′ region of the HLA DR-A gene. These results indicate that 5-azadeoxycytidine treatment of the DGN cell line leads to activation of a methylation-sensitive factor that is involved in the regulation of transcription of the DR-A gene. In cell line THF, however, demethylation does not restore the activity of this factor. The lack of MHC class II expression in this cell line is caused by some other defect. The results of our analysis indicate that at least two different factors are involved in regulation of MHC class II gene expression.

Properties and heterogeneity of human fetal pre-B cells transformed by EBV.

A series of 75 EBV-transformed pre-B and B-cell lines from fetal bone marrow at 14 to 18 wk of gestation was cloned for phenotypic, functional and molecular genetic studies. B-cell type volume regulation in response to hypotonic stress, low level CD9 (BA2), and, perhaps biased by our use of EBV, functional EBV receptors with expression of CD21 (B2) determinants characterized the most immature cells detected. These "B-progenitors" contained EBV genomes, maintained Ig H and L chain (as well as TCR) constant region genes in germ-line configuration and did not express other B, T, or myeloid lineage-associated surface markers including CD20 and MHC class II determinants. Such cells may represent B progenitor cells preceding classical pre-B-lymphocytes in pathways of B cell differentiation. Reminiscent of Abelson virus-induced transformation of immature murine B cells, EBV transformability together with the above properties may be the earliest markers of B lineage commitment in man. The expression of MHC class II Ag, CD20, C mu-H and then L chain rearrangements and expression followed in less immature pre-B lymphocytes and permitted a classification of lines into discrete subgroups of increasing maturity. The physical organization of the H chain locus in a given line was a stable characteristic. However, fetal pre-B cell lines showed considerable intraclonal heterogeneity with respect to H chain gene expression and that of differentiation markers such as CD20. Subcloning experiments indicated that this heterogeneity reflected clonally stable expression patterns distributed among subclones in an all-or-none fashion. The induction, by IL-6, of L chain expression in some but not all of these clones was linked to the presence of C mu transcription products, consistent with a possible regulatory role of mu protein in L chain rearrangement and expression. Although pre-B cell differentiation likely follows inherent programming, external signals seem able to hasten development along prescribed, hierarchical differentiation pathways.

Frequency of V H-gene utilization in human EBV-transformed B-cell lines: The most J H-proximal V H segment encodes autoantibodies

We have studied V H-gene utilization in a collection of 187 IgM-secreting EBV-transformed cell lines and have begun to correlate V H-gene family expression with binding properties of the secreted immunoglobulins. The results of these studies demonstrate that (1) frequency of V H-gene utilization in fetal and adult tissue-derived cell lines correlates with the complexity of the family and (2) the single-membered most J H-proximal V H-6 family encodes autoantibodies reminiscent of autoantibodies found in the sera of patients with systemic lupus erythematosus. Nucleotide sequence analysis of V H-6-expressing clones revealed that each clone utilizes a short D H segment, resulting in a CDR3 region of conserved length. Our data suggest that EBV does not selectively transform human B cells on the basis of V H-gene family expression and that the V H-6 family encodes polyspecific autoantibodies that may serve an important regulatory function in the immune system.

Glycosylation of human leukocyte locus A molecules is dependent on the cell type

Peripheral blood monocytes and B cells were isolated from a normal donor, and a portion of the B cells was transformed by the Epstein-Barr virus (EBV). Human leukocyte locus A (HLA) class-I and class-II molecules were immunoprecipitated by specific monoclonal antibodies after cell labeling with [3H]mannose. Glycopeptides of HLA molecules were obtained by pronase digestion and were analysed by lectin-affinity chromatography. Complex structures were hydrazinolysed, and their sialic acid content was analysed by ion-exchange chromatography, whereas the high-mannose structures were separated by HPLC. In normal cells, class-I antigens bear principally fucosylated biantennary structures while HLA-DR class-II antigens bear bi-, tri- and tetra-antennary structures and high-mannose structures. HLA antigens are more sialylated on normal B cells than on normal monocytes. An EBV cell line had a very different pattern of HLA-antigen glycosylation when compared with the original B cells. In the transformed cells, the fractions containing biantennary structures are largely decreased. In contrast, an increase of the tri- and tetra-antennary structure fractions is noticed, particularly in class-II molecules, while both triantennary and high-mannose structures are increased in class-I molecules. Moreover, when compared to normal B cells, the complex structures of class-I antigens in the EBV-transformed B-cell line are undersialylated while they are oversialylated in the case of the class-II molecules.

β-1,4-mannosyl-glycoprotein β-1,4-N-acetylglucosaminyltransferase III activity in human B and T lymphocyte lines and in tonsillar B and T lymphocytes

beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase III (GlcNAc-T III) catalyzes the incorporation of a "bisecting" N-acetylglucosamine (GlcNAc) residue in beta 1-4 linkage to the beta-linked mannose of the core of asparagine linked-protein bound oligosaccharides (N-glycans). The activity of GlcNAc-T III was determined in Triton X-100 extracts of four human Epstein-Barr virus (EBV)-infected B-cell lines, in four T-cell lines originally established from lymphocytes of patients with acute lymphatic leukemia, and in human tonsillar B and T lymphocytes. The four EBV-transformed B-cell lines showed appreciable GlcNAc-T III activities (ranging from 3.4 to 19.0 nmol.h-1.mg protein-1), while the tonsillar resting B lymphocytes had much less activity (0.68 nmol.h-1.mg protein-1). The four T-cell lines and the tonsillar T lymphocytes had negligible GlcNAc-T III activities (ranging from 0.02 to 0.25 nmol.h-1.mg protein-1). Enzyme product was identified by high resolution proton nuclear magnetic resonance spectroscopy and methylation analysis. This is the first demonstration of GlcNAc-T III activity in human lymphocytes. The presence of GlcNAc-T III in B-cell lines correlates with the reported occurrence of bisecting GlcNAc residues in the oligosaccharides of human immunoglobulins G, A1, M, and D, tonsillar class II antigens, and membrane glycoproteins from B lymphocytes. The negligible GlcNAc-T III activity of the four human T-cell lines and of tonsillar T lymphocytes agrees with the reported absence of bisected structures in N-glycans from human T lymphocyte membrane glycoproteins.

Comparative biochemical and genetic characterization of clonally related human B-cell lines secreting pathogenic anti-Pr 2 cold agglutinins

To study the biology of cold autoimmune hemolytic anemia, Epstein-Barr virus (EBV)-transformed B-cell clones were established from a patient with splenic lymphoma associated with immune hemolysis due to an anti-Pr 2 cold autoantibody. Studies were performed comparing the cold autoantibody present in culture supernatants of these cell lines to the pathogenic cold autoantibodies present in the patient's plasma. Cytogenetic studies of splenic lymphocytes demonstrated an abnormal karyotype (51XX,+3,+9,+12,+13,+18). After EBV transformation, eight clones secreting IgM,κ anti-Pr were isolated; each clone had the same abnormal karyotype as above. DNA isolated from the clones and spleen was analyzed by Southern blot hybridization with J H, C μ, and C κ probes; identical gene rearrangements were seen in each case. Anti-Pr antibodies, isolated from culture supernatant and serum were compared by isoelectric focusing (IEF) and demonstrated similar banding patterns. Distinctive banding patterns, however, were observed in 2 8 clones, suggesting structural differences. Adsorption studies with red blood cells further showed that the observed IEF banding patterns were solely due to anti-Pr cold autoantibody. With a thin-layer chromatography method, the biochemical determinants recognized by the cold autoantibodies were defined as glycolipids containing Neu Acα2-3Galβ1-4Glc sequences. The data demonstrate that the autoantibodies of the EBV-transformed B-cell lines were similar to the pathogenic monoclonal serum autoantibody in both structure and specificity. These clonal cell lines may thus serve to further study the biology of human B-cell lymphomas with defined autoantibody specificity.

A major 50-kDa human B-cell growth factor-II induces both Tac antigen expression and proliferation by several types of lymphocytes

Many cytokines have been documented to have a multiplicity of biological effects by acting on a variety of cells. In order to determine whether human BCGF-II acts on any cells in addition to normal B cells, the effect of human BCGF-II on murine thymocytes, human peripheral blood T cells, a human natural killer-like cell line, YT, and Epstein-Barr virus (EBV)-transformed B-cell lines was further examined. BCGF-II augmented incorporation of [ 3H]thymidine by murine thymocytes in combination with suboptimal doses (0.5 μg/ml) of concanavalin A (Con A) but not at lower doses (0.1 μg/ml) of Con A, a concentration usually used for interleukin 1 (IL-1) assays. BCGF-II could not induce proliferation or Tac antigen (Ag) expression on normal peripheral blood T cells stimulated with OKT3 antibody. Both proliferation and Tac Ag expression on YT cells were also augmented by BCGF-II. BCGF-II induced both high- and low-affinity IL-2 receptor (IL-2R) on YT cells as determined by 125I-IL-2-binding assay. Two of seven EBV-transformed B-cell lines tested (ORSON and AUM cells) in response to BCGF-II exhibited augmentation of proliferation and cell surface Tac Ag expression. BCGF-II in the presence of low doses (0.1 μg/ml) of phorbol myristate acetate (PMA) also induced Tac Ag mRNA (3.5 and 1.5 kb) in these B-cell lines. The IL-2R induced on these B-cell lines, however, consisted mostly of low-affinity receptors. Both Tac Ag and its mRNA in these B-cell lines were not induced by Forskolin but by PMA, suggesting that this induction may involve protein kinase C. The present study shows that human BCGF-II can stimulate YT cells, murine thymocytes, and some EBV-transformed B-cell lines but not peripheral blood T cells. Consequently, BCGF-II can induce the growth and differentiation of a number of cell types in addition to normal B cells.

Presence of in vivo-activated T-cells expressing HLA-DR molecules and IL-2 receptors in peripheral blood of patients with nasopharyngeal carcinoma.

Epstein-Barr Virus (EBV) is associated with two malignant diseases, African Burkitt's Lymphoma (BL) and Undifferentiated Nasopharyngeal Carcinoma (UNPC). North Africa is a geographical area with a high incidence of NPC. Our purpose in this study was to explore cell-mediated immunity of peripheral blood lymphocytes (PBL) from patients with UNPC and DNPC. We found an elevated percentage of OKT8 cells and of large granular lymphocytes (LGL) (30-35% HNK-I-positive cells) compared to PBL from healthy matched individuals. PBL from NPC patients contained 35% HLA-DR-positive and 30% Interleukin-2 (IL-2) receptor-positive circulating lymphocytes. PBL from NPC patients exhibited a normal proliferative response to phytohemagglutinin (PHA) and Concanavalin A (Con A) and an increased response to pokeweed mitogen (PWM). Natural killer (NK) activity towards K562 cells was low in our patients who, in addition, exhibited no lytic activity against HLA-matched EBV-transformed B cells. This lack of cytotoxicity against an EBV-transformed B-cell line cannot be explained by an impairment of IL-2 secretion, and is probably a result of the presence of high numbers of OKT8 suppressor T cells.

Polymorphisms within the HLA-DRw6 haplotype. III. DQα and DQβ polymorphism associated with HLA-D

We have studied HLA-DQ encoded antigens from HLA-DRw6 homozygous cells and analyzed the DQ region at the DNA level. HLA-DQ molecules were isolated from EBV transformed B-cell lines and analyzed for DQα and DQβ polymorphisms. From the same set of cells, DNA was isolated and analyzed for RFLP. Polymorphism could be detected by both techniques, i.e., on the protein level and on the DNA. The variation in pI of the DQα and β chains correlated with the polymorphism as detected by HTC typing, as did the variation in molecular weight of the bands hybridizing to DQ specific cDNA probes; identical patterns were detected for cells of one HLA-D specificity and different patterns for different HLA-D types. Additionally, DQ reactive PLT reagents were raised against DRw6 positive cells. Panel studies revealed that these DQ reactive proliferative T cells can discriminate between the polymorphic DQ antigens on cells with different HLA-D specificities.

Production of IL-1α and IL-1β by clones of EBV transformed, human B cells

An EBV transformed B-cell line was derived from peripheral blood cells from a single donor. B-cell clones were isolated from that line and analyzed for transcription of IL-1 genes, production of biologically detectable IL-1 and antigen presenting capabilities. One clone was found to secrete IL-1 activity into tissue culture medium, and to express the gene for IL-1a. Three other clones were positive for cell surface IL-1 activity but no activity could be detected in culture supernatants. Those clones express the gene for IL-1β but not IL-1α. All clones were approximately equal in their ability to present antigen to resting and primed T cells.