The effect of x-rays on the precursors of antibody forming cells (B cells) as measured with the in vitro limiting dilution assay

The effect of X-irradiation upon murine antibody-forming cell precursors (B cells) was established in cultures of spleen cells stimulated with the B cell mitogen lipopolysaccharide (LPS). At day 5 and 7 the numbers of IgM- and IgG2-secreting cells were determined in cultures of irradiated and nonirradiated spleen cells. From these numbers a Do of 0.6-1.2 Gy for the IgM, and of 0.9-2.1 Gy for the IgG2 response was calculated. Similar Do values were obtained under limiting dilution culture conditions. In the limiting dilution assay the effect of irradiation upon the size of the IgM-producing clones could also be determined. It was found that irradiation reduced the number of LPS-reactive B cells without affecting the size of the clones produced by the surviving cells.     

Fine specificity of HLA-B27 cellular allorecognition. HLA-B27f is a functional variant distinguishable by cytolytic T cell clones

Three HLA-B27 allospecific cytolytic T lymphocyte (CTL) clones were isolated by limiting dilution of HLA-B27-negative responder cells stimulated with HLA-B27.1-positive lymphoblastoid cells. These clones displayed three distinct reaction patterns when tested for their lytic ability against target cells expressing various structurally defined HLA-B27 subtypes. One of the clones was specific for HLA-B27.1; a second CTL clone reacted only with B27.1 and, less efficiently, with B27.2; the third clone recognized both B27.1 and B27f targets but not cells expressing any other B27 subtype. These results indicate that HLA-B27f is a functional variant amenable to differential recognition by alloreactive CTL. A correlation of the structure of the HLA-B27 subtypes with the reactivity of these clones revealed that multiple B27-specific alloreactive CTL are activated against epitopes of the HLA-B27.1 molecule sharing common structural features. This illustrates the complexity and fine specificity of the allogeneic CTL response against class I HLA antigens and suggests that their immunodominant regions are those which are capable of eliciting a diverse polyclonal response against each of these regions, rather than inducing the selective expansion of a single T cell clone.     

Comparison of membrane IgM expression in the murine B cell lymphoma 70Z/3 treated with LPS or supernatant containing T cell factors

The cultured murine B cell lymphoma, 70Z /3, can be induced to express membrane IgM ( mIgM ) after exposure to lipopolysaccharide (LPS) or T cell-derived factors. The kinetics and magnitude of the responses have been compared in wild type 70Z /3 cells and three variants by using flow cytometric analysis and immunoprecipitation. Wild type 70Z /3 cells respond to LPS more quickly and with twofold greater mIgM than to concanavalin A-induced spleen cell supernatant (CAS). Variants were selected for their abnormal mIgM expression in response to LPS, but individual variants also showed normal, aberrant, or no response to CAS. When cells were induced with suboptimal amounts of LPS and CAS, a synergistic effect on the magnitude of mIgM expression was seen in wild type and variant cells. This suggests that both inducing agents are utilizing some part of a common inductive mechanism. The different responses of the variant cell lines will allow further genetic dissection and comparison of the mIgM expression pathways used in response to LPS and CAS.     

Isolation and characterization of Chinese hamster ovary cell variants deficient in the expression of fibronectin receptor

Chinese hamster ovary cell populations were enriched for cells displaying low surface expression of the 140-kD integrin fibronectin receptor (FnR) by means of fluorescence-activated cell sorting using monoclonal anti-FnR antibodies. Selected cells were cloned by limiting dilution, and the resulting clones were screened for low cell surface FnR expression by ELISA. Two multiply sorted populations gave rise to variant clones possessing approximately 20 or 2% FnR expression, respectively, compared with wild-type cells. Growth rates of the "20%" and "2%" clones on serum-coated plastic dishes were similar to that of wild-type cells. Variant cells expressing 20% FnR could attach and spread on substrata coated with purified fibronectin, although somewhat more slowly than wild-type cells, while cells expressing 2% FnR could not attach or spread. Cells from all variant clones attached normally to vitronectin substrata, but some of the 2% clones displayed altered morphology on this type of substratum. Motility assays in blind well chambers showed a correlation of movement with level of expression of FnR. The number of cells migrating in response to fibronectin was greatly reduced compared with wild-type cells for the 20% FnR variant clones, while variant clones with 2% FnR showed virtually no migratory activity. Surface labeling with 125I and immunoaffinity purification of FnR showed reduced levels of intact FnR on the plasma membranes of variants with 20% FnR, while none was detected in variants expressing 2% FnR. Nevertheless, beta subunits were detected on the surfaces of all variant clones. Immunoblots of cell lysates from wild-type cells and from both types of variant clones showed substantial amounts of FnR beta chain as well as enhanced amounts of a pre-beta moiety in the variants. alpha chain was markedly reduced in the 20% variants and essentially absent in the 2% variants, indicating that failure to assemble intact FnR in these variants was due to deficiencies of alpha chain production. Dot blots of total mRNA from a representative clone expressing 20% FnR showed reduced levels of material hybridizing to an 0.97-kb hamster FnR alpha chain cDNA probe as compared with wild type, while mRNA from a representative clone expressing 2% FnR had no detectable hybridizable RNA; this seems to agree well with the results obtained by immunoblotting. Thus, the defect in the variant clones seems to be due to reduced levels of alpha chain mRNA leading to a deficit of mature FnR and consequent alterations in cell adhesion and motility on fibronectin substrata.     

Aberrant trafficking of the B cell receptor Ig-alpha beta subunit in a B lymphoma cell line

The B cell Ag receptor (BCR) has two important functions: first, it binds and takes up Ag for presentation to T lymphocytes; and second, it transmits signals that regulate B cell development. Normal expression of the BCR requires the association of the Ag binding subunit, membrane IgM (mIgM), with the signaling component, the Ig-alpha beta heterodimer. After assembly in the endoplasmic reticulum, the intact BCR travels through the secretory pathway to the cell surface. In this paper, we report two variants of the B lymphoma cell lines, WEHI 279 and WEHI 231, that have both lost the ability to express mu heavy chain and consequently do not express mIgM. However, these variants do express the Ig-alpha beta heterodimer. In one variant, WEHI 279*, the Ig-alpha beta remained trapped intracellularly in the absence of mIgM. The other variant, 303.1.5.LM, expressed an aberrantly glycosylated Ig-alpha beta on the cell surface that was capable of signaling after cross-linking with anti-Ig-beta Abs. Further characterization uncovered a point mutation in the 303.1.5.LM mb1 gene that would change a proline for a leucine in the extracellular domain of Ig-alpha. The 303.1.5.LM Ig-alpha beta could not associate with a wild-type mIgM after mu heavy chain was reconstituted by DNA transfection. Thus, this mutation could define a region of the Ig-alpha polypeptide that is important for recognition by the endoplasmic reticulum quality control system, for association with glycosylating enzymes, and for the association of Ig-alpha beta subunits with mIgM subunits to create a complete BCR complex.     

Analysis of cellular immune response to EBV by using cloned T cell lines

Eight cloned T cell lines specific for Epstein Barr virus-transformed B lymphocytes were derived. In the presence of the autologous virus-infected B cells, the T cell lines show HLA-restricted cytotoxic activity and also secrete alpha-interferon in sufficient amounts to inhibit infection and transformation. Four of these clones showed restriction to a single HLA locus (two for A3, and two for B7) and three showed exquisite self-restriction lysing only autologous targets. These seven clones expressed the classical cell surface phenotype of cytotoxic T cells being T3, 8, 11, and la-positive and T4-negative. An eighth clone that lacked the T8 surface marker appeared to recognize both B7 and BW51. HLA restriction was confirmed: 1) by the ability of a monoclonal antibody against an HLA-A,B,C framework antigen (W6-32) to block the cytotoxicity; 2) the failure of the clones to lyse Daudi, an EBV-positive, HLA-A,B, C-negative cell line; and 3) successful competition of the cytotoxicity by autologous but not allogeneic cold targets. The cloned T cells do not kill EBV-negative targets such as autologous pokeweed mitogen blasts and cell lines including CEM and the natural killer cell target K562. The results suggest T cell clones may be generated against an EBV-associated membrane antigen on transformed B cells, perhaps equivalent to the lymphocyte-determined membrane antigen, and that the recognition is restricted by a single HLA determinant. We propose that single T cells can play multiple roles in controlling EBV infection in vitro and in vivo including the elimination of transformed cells by cytotoxicity and the prevention by secreted interferon of further re-infection and transformation.     

Different phenotypic variants of the mouse B cell tumor A20/2J are selected by antigen- and mitogen-triggered cytotoxicity of L3T4-positive, I-A-restricted T cell clones

The L3T4+, Lyt-2-, cloned BALB/c T cell lines 5.9.24 and 5.8.6 are cytotoxic for the BALB/c B cell tumor line A20/2J. The T cell cytotoxicity against A20/2J cells could be triggered either by the specific antigen ovalbumin (OVA), which is recognized by the T cell clones in association with I-Ad determinants, or by the T cell mitogens Con A and rabbit anti-mouse brain (RaMBr) antiserum. Repeated exposure of A20/2J cells to 5.9.24 and 5.8.6 T cell cytotoxicity selected variant cell lines that had developed resistance to cytotoxicity. The variant lines could be classified into four different variant phenotypes of which three were stably maintained in vitro. The type of variant obtained appeared to be related to the nature of the ligand used to trigger T cell cytotoxicity during selection. Cytotoxicity triggered by the antigen OVA generated type 1 variants that expressed abnormally low levels of I-Ad determinants at the cell surface. Type 1 variants were resistant to OVA-triggered 5.9.24 T cell cytotoxicity, but were fully susceptible to cytotoxicity triggered by Con A or RaMBr antiserum. RaMBr-triggered cytotoxicity generated two unique types of variant cell lines: type 3 variants that were deficient in cell surface Fc receptors and resistant to 5.9.24 cytotoxicity only when triggered by RaMBr antiserum, and type 4 variants that were resistant to cytotoxicity triggered by all three ligands. One type 4 variant, the IC-1 cell line, appeared to be resistant to soluble cytotoxic factors released by 5.9.24 T cells after activation by antigen. All of these variant lines retained sensitivity to cytotoxicity by classic Lyt-2+ cytotoxic T lymphocytes (CTL), a finding that indicates that L3T4a+ T cells and Lyt-2+ CTL use different molecules to attack their target cells. The variant phenotypes were inherited by clones derived from the original cell lines. Because the variants were generated without mutagenesis, they are thought to have been derived by the immunoselection of pre-existing variant cells that arose spontaneously in the parental A20/2J cell line. It is postulated that inheritable variation of A20/2J cells may represent changes that normally occur during B cell differentiation in response to T cell signals. The variant A20/2J cell lines described here provide material for the investigation of B cell surface structures that may regulate T-B cell interactions.     

In vitro generated human monoclonal trinitrophenyl-specific B cell lines. Evidence that human and murine anti-trinitrophenyl monoclonal antibodies cross-react with Escherichia coli beta-galactosidase

Stable human antigen-specific monoclonal B cell lines were established without prior in vivo immunization. This was accomplished by expanding the anti-trinitrophenyl (TNP) B cells in vitro with the antigen TNP-Brucella abortus and then immortalizing them with Epstein-Barr virus. Five anti-TNP clones were selected by sequential limiting dilution. All five anti-TNP clones secreted IgM kappa antibodies. When tested against a panel of self and environmental antigens, all five anti-TNP clones exhibited cross-reactivity with an Escherichia coli-derived beta-galactosidase. To determine whether this was a more general phenomenon, a panel of murine monoclonals were tested and found to bind to beta-galactosidase. It is therefore possible that human and murine anti-TNP beta cell responses reflect reactivity against an environmental antigen, namely an epitope present on E. coli-derived beta-galactosidase. This approach of expanding human antigen-specific B cells by antigen stimulation in vitro, with a T-independent hapten-carrier conjugate before Epstein-Barr virus transformation, may prove useful in the development of human monoclonals for therapeutic purposes.     

Human B lymphocytes activated by Epstein-Barr virus (EBV) or by mitogens suppress mitogen-induced immunoglobulin production

Polyclonal activation of human B lymphocytes by LPS or protein A, alone or in combination or by Epstein-Barr virus (EBV), generates suppressive conditions that inhibit the response of human B lymphocytes to pokeweed mitogen (PWM), measured by the induction of immunoglobulin-secreting cells (PFC). Moreover, EBV-transformed B cell lines of normal or neoplastic (Burkitt lymphoma) origin also suppressed the PWM-induced immunoglobulin production of normal B cells. Cell separation experiments have shown that mitogen activated autologous B cells stimulate suppressor T cells in a similar way as B cell-derived lymphoblastoid cell lines. The significance of this phenomenon is considered in relation to the escape of the activating microorganism or virus from immune control and the occurrence of network interactions within the immune system.     

Differential recognition by human cytotoxic T cell clones of human M1 fibroblasts transfected with an HLA-B7 gene (JY150) suggests the existence of two different HLA-B7 alleles in the cell line JY (HLA-A2,2;B7,7;Cw-,-;DR4,w6)

We have used a panel of human HLA-B7-specific CTL clones to identify an HLA-B7 gene (JY150) transfected into human M1 fibroblasts (M1/B7). Only a subset of the CTL clones recognized the M1/B7 cells, whereas all CTL clones recognized the donor of the B7 gene, the cell line JY (HLA-A2,2;B7,7;Cw-,-;DR4,w6). Analysis of the fine specificity of these CTL clones was performed by testing the reactivity on M1 cells transfected with an HLA-B27K gene and on a panel of cell lines typed for HLA-B7 subtypes (variants). These results, combined with one-dimensional IEF analysis of the M1/B7 cells and the B7 subtypes, indicated that the differential recognition by the CTL clones of the transfected gene was not caused by aberrant expression of the gene itself or due to the absence of critical accessory molecules on the M1 fibroblast cells. Our data suggest that the widely used HLA-B7 reference cell line JY is not homozygous at the HLA-B locus, but contains two different B7 alleles encoding the B7.2 and B7.4 subtypes.     

A new tyrosinase epitope recognized in the HLA-B*4002 context by CTL from melanoma patients

Melanoma reactive CTL were obtained by stimulating PBL from a melanoma patient in remission since 1994 following adjuvant TIL immunotherapy, with the autologous melanoma cell line. They were cloned by limiting dilution. One CTL clone recognized melanoma cell lines expressing tyrosinase and the B*4002 molecule, either spontaneously or upon transfection. We demonstrated that this clone recognizes the tyrosinase-derived nonapeptide 316-324 (ADVEFCLSL) and the overlapping decapeptide 315–324 (SADVEFCLSL). We derived two distinct additional specific CTL clones from this same patient that were also reactive against B*4002 melanoma cell lines, suggesting a relative diversity of this specific repertoire in this patient. Stimulating PBMC derived from four additional B*4002 melanoma patients with the tyrosinase 316–324 nonapeptide induced the growth of specific cells for two of the patients, demonstrating the immunogenicity of this new epitope. Our data show that this nonapeptide is a new tool that could be used to generate melanoma-specific T cells for adoptive immunotherapy or serve as a peptide vaccine for HLA-B*4002 melanoma patients.     

Idiotype variants emerging after anti-idiotype monoclonal antibody therapy of a murine B cell lymphoma

One of the difficulties encountered with the treatment of human B cell malignancies with anti-Id antibodies is the emergence of Id variants. The current study was designed to investigate this phenomenon further by using the murine B cell lymphoma model 38C13. Tumors were harvested that developed despite treatment with the anti-Id antibody S1C5 in mice inoculated with 38C13 cells and evaluated by immunofluorescence. Various phenotypes were found among escaping tumor cells. Some cells continued to react with S1C5 whereas others lost S1C5 reactivity. Among these latter cells, some continued to express surface IgM kappa, whereas others no longer expressed surface mu or kappa. After Id variant cell lines were established, immunofluorescence and ELISA of cell lysates from the surface IgM kappa- lines revealed persistent intracellular mu H chain but no detectable kappa. Surface IgM kappa+ lines were fused with myeloma cells and the Ig proteins secreted by the resultant hybridomas analyzed. The apparent m.w. of the mu-chains of these rescued Ig was the same as wild-type 38C13, whereas the kappa-chains were either the same or different in m.w. from the wild type. The IgM kappa of the variant line, T3C, weakly reacted with S1C5 and did not react with other anti-Id antibodies. The IgM kappa of the other variants were nonreactive with all the antibodies. Immunofluorescence of these surface Ig+ variants confirmed this finding. Some of the surface Ig+ and Ig- variant lines grew identically to wild-type tumor in vivo, but only the weakly S1C5-reactive variant T3C was inhibited in its growth by S1C5. Moreover, T3C was the only one of these lines capable of being lysed in vitro with S1C5 by antibody-dependent cellular cytotoxicity. Further studies revealed that surface Ig+ and Ig- variants emerge in escaping tumors with similar frequency and that these variants represent a major mode of tumor escape from anti-Id treatment in this model.     

The frequency of LPS-responsive B cells to autologous and heterologous thyroglobulin

The frequency of precursors within the mouse splenic B cell pool, reactive with mouse thyroglobulin (mTg) was estimated using a limiting dilution assay system. The mean frequency was found to be 1/3900 B cells. The results provide a minimal estimate of the frequency of mTg-reactive B cells. The frequency of mTg-reactive B cells was not influenced by the MHC locus, as both high- and low-responder strains showed similar frequencies. While the frequency of B cells reactive to human Tg was found to be similar to that reactive to mTg, only 20% of the mTg-reactive clones also cross-react with human Tg. Similarly, only 30% of huTg reactive clones were found to react with mTg. Therefore, a large proportion of Tg-reactive antibodies are restricted to self-determinants and not determinants to conserved regions of the Tg molecule.     

Functional heterogeneity in allospecific cytotoxic T lymphocyte clones. II. Development of syngeneic cytotoxicity in the absence of specific antigenic stimulation

Two out of four long-term murine allospecific cytotoxic T lymphocyte (CTL) clones tested could develop high levels of cytotoxicity against syngeneic target cells when cultured under appropriate conditions. All CTL clones maintained strict allospecificity so long as they were cultured with both appropriate allogeneic stimulator cells and growth factor (supernatant from secondary mixed lymphocyte cultures). In two of the clones, syngeneic reactivity rapidly developed when the allogeneic stimulator cells were replaced with syngeneic or third party stimulator cells, and when the supernatant from EL4 thymoma cells stimulated with phorbol ester was used as growth factor. In addition to killing the appropriate allogeneic target, clones with syngeneic reactivity could kill both syngeneic C57BL/6 targets and H-2-congenic BALB.B targets but not third party unrelated targets, suggesting that the self structure recognized was coded for within the major histocompatibility complex. Such clones did not kill the natural killer (NK) target YAC. The results obtained from cold target inhibition and from subcloning at limiting dilution of clones with syngeneic reactivity suggested that both allogeneic and syngeneic reactivity could be expressed by the same individual cell in the CTL clone. The specificity for syngeneic H-2 as opposed to third party H-2 and NK-sensitive target cells, and the observation that both allospecific and syngeneic killing could be partially blocked by anti-Lyt-2 antibody treatment of the CTL, strongly suggested that different recognition structures are involved in CTL-mediated syngeneic cytotoxicity and NK cytotoxicity.     

Expression of retrovirus-related, cytotoxic T lymphocyte- and transplantation-defined antigens in NIH/3T3 transfectants after a single passage in nude mice

Transfection of T24c-Ha-ras oncogene into NIH/3T3 fibroblasts resulted in the establishment of a transformed cell line (pT) that was tumorigenic when injected s.c. both into Swiss outbred nude mice and normal NIH inbred mice. The passage into nude mice, however, led to the development of a tumor variant (pT-nude) able to subsequently grow into sublethally x-irradiated but not into immunocompetent NIH mice. NIH mice immunized with this tumor variant developed a strong specific CTL response against the immunizing cell line, whereas the parental transformed pT cell line was not lysed. Clones were derived by limiting dilution from anti-pT-nude bulk population and were tested on a panel of transformed NIH/3T3 lines before and after their growth as tumor into nude mice. All of these lines were lysed by the Lyt-2+ CTL clones as a sole consequence of one in vivo passage into nude mice. The cross-reactive Ag were shown to be related to endogenous retroviral products as assessed by 1) immunoprecipitations of gp70, p15E, and p30 viral proteins in the nude variants but not in parental lines, and 2) by the ability of retroviruses from irradiated pT-nude cells to infect NIH/3T3 or pT lines making them susceptible to lysis by anti-pT-nude CTL clones. These results show that a single passage in nude mice can induce retrovirus-related, cell-surface Ag in transplanted neoplastic cells.     

Allospecific human T cell lines and clones which mediate HLA-DR restricted helper activity

In order to analyze the immunoregulatory activity of allospecific human T cells, we have combined the techniques of limiting dilution culture and IL-2 dependent T cell growth to generate cloned alloreactive T cell lines (TCL). These TCL have been expanded in continuous culture for greater than 8 mo with retention of stable phenotypic and functional characteristics. For example, phenotypic analysis of alloproliferative TCL, utilizing a panel of monoclonal antibodies, demonstrate that these cultures are comprised of T cells belonging exclusively to the T3+ T4+ T8- "helper" or "inducer" T cell subset. Coculture of cloned alloproliferative TCL cells with a panel of allogeneic stimulators reveals that these clones are specifically reactive against the HLA-DRw1 determinant. Of greater interest, coculture of selected alloproliferative TCL cells with DRw1+ but not DRw1- B cells results in a vigorous polyclonal response as measured by the reverse hemolytic plaque assay. Although major histocompatibility complex restriction of this T-B interaction operates at the inductive level, help is at least partially unrestricted at the effector level as alloantigen-activated TCL cells provide demonstrable, although not maximal, help for DRw1- B cells.     

Human T cell receptor-gamma delta-expressing T-cell lines recognize MHC-controlled elements on autologous EBV-LCL that are not HLA-A, -B, -C, -DR, -DQ, or -DP.

HLA-loss variants of an EBV-transformed B lymphoblastoid cell line (EBV-LCL) 721 were used to investigate whether human MHC molecules other than known class I or class II were involved in autologous T cell responses. Bulk lymphocyte cultures of purified T cells primed to an autologous variant EBV-LCL that fails to express HLA-class II and has reduced cell surface HLA-class I expression, and oligoclonal TCR-gamma delta-bearing lines derived from them, could lyse both this EBV-LCL and an independently derived, class II expressing autologous variant EBV-LCL that bears no HLA-A, -B, or -C, suggesting the presence of additional HLA-like restriction elements. Cold target inhibition of cytolysis mediated by these lines indicated that a shared or cross-reactive MHC controlled restriction element other than the known MHC determinants was retained by the EBV-LCL variants. Single-cell derived clones from these T cell lines which expressed only the TCR-gamma delta showed this same target cell specificity pattern, proving recognition of MHC-controlled determinants by autologous gamma delta T cells. Anti-gamma delta antibody could inhibit cytolysis by the gamma delta-expressing lines, suggesting that the TCR-gamma delta was involved in recognition of the EBV-LCL targets. Flow cytometric analysis with separate HLA-reactive antibodies indicated that the restriction element for these cytolytic responses is a molecule serologically cross-reactive with HLA-B and -C Ag, yet is a determinant that cannot be HLA-A, -B, -C, -DR, -DQ or -DP.     

Differential regulation of activation, clonal expansion, and antibody secretion in human B cells

Limiting dilution analysis, hemolytic plaque assay, and ELISA procedures were used to study the recruitment, clonal expansion, and antibody secretion in human TNP-specific B cells activated in the presence of TNP-ovalbumin (TNP-OA), pokeweed mitogen (PWM), or regulatory T cells. TNP-OA-responsive, hapten-specific PFC precursor cells occupy approximately 0.5% of all sIgM+/sIgD+ B cells in cord blood, bone marrow, peripheral blood, and tonsil. The PWM-responsive, hapten-specific PFC precursor pool is 70 to 90% smaller and does not express sIgD. Antigen-reactive B cells go through a minimum of three divisions in culture (six to nine PFC per clone), and antibody secretory rates of about 10(4) molecules IgM/cell/hr are achieved. In contrast, PWM-induced clone sizes were at least 60 PFC per clone, with antibody secretory rates of approximately 6 to 7 X 10(4) molecules IgM/cell/hr. Addition of high-dose carrier-primed suppressor T cells to limit dilution cultures reduced PFC precursor cell recruitment by up to 99%. However, in the few clones escaping from suppression, both clonal expansion and antibody secretory rates were much higher than in suppressor cell-free cultures, generating 30 to 60% of the antibody secreted in controls but with consequently much more restricted clonal diversity. When limiting dilution cultures were compared with standard microcultures of 2 X 10(5) cells, both clonal expansion and antibody secretory rates were much lower than expected, with a culture efficiency calculated to be 10 to 20% of that in low-density cultures. Our data suggest that the B cell subsets activated by antigen and by mitogen differ in their abilities for clonal expansion and antibody secretion. The hapten-specific and -responsive B cell family is expressed early in ontogeny, and in adults it is distributed evenly throughout the body. These limiting dilution experiments revealed that the primary effect of regulatory T cells is a drastic reduction in clonal diversity, and much less a mere reduction in overall response magnitude.     

Two distinct human cloned T cell lines that exhibit natural killer-like and anti-human effector activities

Cloned T cell lines from mixed lymphocyte cultures stimulated with autologous Epstein Barr virus- (EBV) transformed lymphoblastoid cell line (LCL) cells were established using a limiting dilution technique in the presence of T cell growth factor (TCGF). The T cell lines included two distinct clones of cytotoxic T cells (Tc) in addition to EBV-specific Tc. A cytotoxic profile of one cloned line was similar to that of endogenous NK cells in peripheral blood. The other cloned Tc line showed an anti-human cytotoxicity. The susceptible targets for this latter Tc line were various human cells, including autologous LCL and peripheral blood lymphocytes (PBL), stimulated with pokeweed mitogen, along with NK-sensitive and NK-resistant cell lines. Weak cytotoxic activity was detected against various xenogeneic cell lines. Furthermore, autologous and allogeneic cloned T cell lines were resistant to killing by the anti-human effector clone. These t wo distinct cloned Tc lines expressed the Leu-1 and Leu-2a antigens, which are markers of suppressor/cytotoxic T cells.     

Establishment of Autoantibody-Producing Cell Lines from Peripheral Blood Lymphocytes of Patients with Systemic Lupus Erythematosus

Autoantibody-producing B cell lines were established from peripheral blood lymphocytes of patients with systemic lupus erythematosus. Peripheral blood lymphocytes obtained from five of seven patients were successfully transformed by Epstein-Barr virus. Two of four established B lymphoblastoid cell lines examined in this study produced anti-nuclear factor antibodies and one of them produced anti-single-stranded DNA and anti-double-stranded DNA antibodies. These results indicate that B cell clones committed to self antigens are transformed by Epstein-Barr virus and continue to produce autoantibodies. In order to establish a monoclonal autoantibody-producing B cell line, the cells were cloned by a limiting dilution method. The data suggest that it is possible to establish a monoclonal autoantibody-producing B cell line by the combination of transformation of B cells by Epstein-Barr virus and extensive cloning.