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Biomedical subjects

R Asofsky

Publications and source records attributed to R Asofsky.

At least 37 records · Page 2Linked to original sources
PubMed1984

Functional studies on B cell hybridomas with B cell surface antigens. IV. Direct effects of cytochalasin B on differentiation.

We previously established B cell hybridomas between M12.4.1 B lymphoma of BALB/c mice and normal B cell of C57BL/6 (B6) mice. These hybridomas express Iab, Iad, and IgM molecules on the cell membrane, and can induce the generation of IgM secretion when treated with purified goat anti-mouse-mu antibody (anti-mu) without T cell factors. In this study, TH2.54, a subclone of a B cell hybridoma, was treated with cytochalasin B (CB), a fungal product that disrupts microfilaments, and the direct effect of CB on the proliferation and differentiation of TH2.54 was examined. CB considerably suppressed the spontaneous proliferation of hybrid cells. This product, however, did not inhibit the generation of IgM secretion by TH2.54 treated with anti-mu. Surprisingly, CB could directly induce the development of IgM-secreting cells by TH2.54 at a relatively high frequency. Among cytochalasins, dihydrocytochalasin B (H2CB), cytochalasin C (CC), and cytochalasin D (CD) showed marked effects on the induction of IgM secretion as well as CB. In addition, the differentiative effect of CB was greatly inhibited by N6, O2-dibutyryladenosine 3':5'-cyclic monophosphate (dbc-AMP), but not by N2, O2-dibutyrylguanosine 3':5'-cyclic monophosphate (dbc-GMP). Analysis by flow microfluorometry (FMF), cytotoxicity assays, and quantitative absorption tests demonstrated that CB treatment of TH2.54 resulted in a significant decrease in the expression of Iab, Iad, and IgM molecules on the cell membrane. In contrast, parental M12.4.1 neither generated any IgM secretion nor changed Iad expression on the cell membrane under the same conditions. The present study suggests very strongly that microfilament-microtubule systems are not involved in the differentiative process of TH2.54 induced by anti-mu. The results also indicate that CB can provide the initiative signal for differentiation of TH2.54 into the maturation lineage; this is followed by a significant change in the expression of Ia and IgM molecules on the cell membrane.

Animals↗
PubMed1983

Role of B cells in the stimulation of syngeneic mixed lymphocyte responses.

Two different approaches were used to examine the role of B cells in the stimulation of syngeneic MLR. The relative inability of spleen and peritoneal exudate cells from B cell deficient mice, treated with anti-mu from birth, to serve as stimulator cells in SMLR was previously shown in SJL mice and confirmed in BALB/c mice in the present studies. Preincubation of cells from anti-mu treated mice with serum Ig does not enhance their ability to stimulate. Upon stimulation with normal spleen cells responses from anti-mu treated mouse T cells are not deficient. Responses of thymus cells from neonates and from adult cortisone-treated mice are also much higher when the splenic stimulator cells come from normal rather than from anti-mu treated mice. The deficiency of the stimulator cells from anti-mu treated mice is in the high density cell population as obtained after BSA gradient fractionation of collagenase treated lymph node or spleen fragments. Low density populations from anti-mu treated and normal mice stimulate equally well. Addition of exogenous IL-2 to the cultures enhances the syngeneic MLR to all stimulator populations and allows stimulation by spleen cells from anti-mu treated mice. It is concluded that, while B cells represent the major stimulator cell population in whole spleen cell suspensions, other accessory cells (dendritic cells?) are more efficient, possibly synergize with B cells by producing IL-1, but usually represent only a minor subpopulation. The other approach concerns the effectiveness of SMLR stimulation by several tissue culture, cloned B lymphoma cell lines, and by a transplantable BALB/c B lymphoma. Of the five stimulating lymphoma cell lines only one stimulates approximately as well in the absence as in the presence of polyethylene glycol. These latter cells (A20.1.11) can also stimulate T cell proliferation and IL-2 production in the absence of Ia+ accessory cells in the responding population and, therefore, either produce their own IL-1 or are able to bypass the requirement for this lymphokine.

Animals↗
PubMed1983

IA mutant functional antigen-presenting cell lines.

We describe a protocol for the selection of mutant cells with an altered pattern of Ia antigenic determinants and antigen-presenting properties from a homogeneous population of functional antigen-presenting cells (APC). The APC line used in this work was obtained by fusing lipopolysaccharide-stimulated B cells from (BALB/c x A/J)F1 donors with cells from the M12.4.1 BALB/c B lymphoma cell line. The resulting hybridomas, including TA3, retained the potent antigen-presenting activity of the parental B lymphoma line and expressed Ia antigens and immune response gene-determined antigen-presenting properties of the A/J type. Mutants of TA3 were obtained by subjecting the cells to negative immunoselection with one monoclonal anti-(alpha) 1-Ak antibody and complement followed by positive immunoselection via electronic cell sorting with a second monoclonal alpha I-Ak or alpha I-Ek antibody. Two types of mutants were obtained. One, A8, appeared to have undergone a fairly limited alteration, since it lost only some of the I-Ak antigenic determinants; the second type appeared to have lost the entire I-Ak molecule but to have retained the I-E molecule. Functional studies with the A8 mutant demonstrated that the loss of a limited number of I-Ak determinants correlated with the loss of a specific I-Ak-encoded restriction element, since A8 failed to present a specific antigen, hen egg lysozyme (HEL), to a HEL-specific I-Ak-restricted T cell hybridoma but retained some capacity to present a second antigen, poly(Glu60Ala30Tyr10) (GAT), to a GAT-specific I-Ak-restricted T cell hybridoma. These results indicate that Ia antigens are the products of immune response gene loci. The availability of such mutants should allow an examination of the relationship between the structure of an Ia molecule and the antigens with which it is co-recognized by T cells.

Animals↗
PubMed1983

Functional studies on B cell hybridomas with B cell surface antigens. I. Effects of anti-immunoglobulin antibodies on proliferation and differentiation.

B cell hybridomas with Ia and IgM molecules on the cell membrane were treated with either purified goat anti-mouse mu antibody (anti-mu) or monoclonal rat anti-mouse IgM antibody (anti-IgM). The spontaneous uptake of [3H] thymidine by these cells was markedly inhibited by both reagents. These hybrid cells could be induced to differentiate into IgM-secreting cells in the presence of these reagents at high frequency. Furthermore, the induction of IgM secretion by B cell hybridomas treated with these antibodies was completely T cell independent, and cell division was not required for the differentiative response to anti-mu. In addition, F(ab')2 fragments of anti-mu showed more effects on proliferation and differentiation of these cells than intact anti-mu. Interestingly, TH2.54, a subline of B cell hybridomas, could generate IgG2a production as well as IgM when incubated with anti-mu. These findings suggest very strongly that the interaction of either goat anti-mu or monoclonal rat anti-IgM with surface IgM molecules on the cell membrane of the B cell hybridomas inhibits in vitro spontaneous proliferation, and results in providing signals for differentiation into Ig-secreting cells without T cell factors.

Animals↗
PubMed1983

Functional studies on B cell hybridomas with B cell surface antigens. II. Ia molecules on the cell membrane and the differentiative response to anti-mu antibody.

TH 2.52, a subline of the B cell hybridoma with Iab, Iad, and IgM molecules on the cell membrane, was treated with F(ab')2 fragments of purified goat anti-mouse mu antibody (anti-mu), and the change in the expression of surface Ia molecules was determined by microcytotoxicity assays, quantitative absorption tests, and analyses of flow microfluorometery (FMF). We have previously reported that TH 2.52 cells can markedly generate IgM after stimulation with anti-mu without T cell factors. In the present studies, it was shown that the expression of surface Iab molecules on TH 2.52, originated from normal B cells of C57BL/6 (B6) mice, significantly decreased after treatment with anti-mu. In contrast, Iad molecules derived from M12.4.1 lymphomas did not change under the same conditions. These results indicate that cross-linking of anti-mu with surface IgM molecules on TH 2.52 provides signals for differentiation into IgM-secreting cells; this is followed by a decrease in the expression of Iab molecules on the cell membrane. Furthermore, monoclonal anti-Ia antibody (anti-Ia) did not inhibit the generation of IgM-secreting cells by TH 2.52 cells treated with anti-mu. In addition, la- sublines of TH 2.52 obtained after mutagenesis with ethyl methanesulfonate (EMS), as well as the original TH2.52, could differentiate into IgM-secreting cells in the presence of anti-mu. These findings suggest very strongly that la molecules on the cell membrane are not required for the induction of IgM secretion by B cells treated with anti-mu.

Animals↗
PubMed1982

I region-restricted antigen presentation by B cell-B lymphoma hybridomas.

The activation of T lymphocytes for many responses requires interaction with specialized antigen presenting cells (APC) which express I region associated (Ia) antigens. Cells of the monocyte-macrophage lineage and the recently described dendritic cell have been shown to be effective APC. Antigen-specific T cells appear to recognize a complex of antigen and Ia molecules on the surface of the APC and such joint recognition is the basis of the restriction of T-cell activation due to the major histocompatibility complex. The Ia molecules appear to be Ir gene products. However the biochemical nature of antigen presentation has been difficult to approach largely because of problems in obtaining sufficient numbers of highly purified APC. Although several Ia antigen-positive B lymphoma cell lines, which have proved very effective APC, have been recently described, they have all been of the same H-2d haplotype. We report here on hybridomas produced by fusion of normal C57BL/6 (B6) B cells (H-2b) to a hypoxanthine guanosine phosphoribosyl transferase (HGPRT) deficient variant of one of these lymphoma cell lines. The hybridomas appear to express functional Ia molecules of both parental cell haplotypes. Such cloned somatic cell hybrids should prove useful in studying the properties of Ir genes and Ia molecules in antigen presentation.

Animals↗
PubMed1982

Frequency of B lymphocytes responsive to anti-immunoglobulin.

The frequency of murine B lymphocytes that respond to antibodies directed against membrane IgM was measured. These anti-mu antibodies induced all, or almost all, resting B cells to enlarge over the first 24 h of stimulation. This probably represents the transition from the resting state (G0) to active transit through the cell cycle. In contrast, only a fraction of these cells, approximately 60% for BDF1 mice, continued through the cell cycle into S phase. This is consistent with previous experiments that had suggested there were some types of B cells that did not proliferate in response to anti-mu. The results presented here demonstrate that many, perhaps all, of these nonresponding B cells, both from normal mice and from mice with the xid defect, actually do respond to the presence of anti-mu by going through early parts of the cell cycle. These cells appear to become blocked at some point before the beginning of S phase, perhaps requiring a signal from a T cell or a macrophage to continue through the cell cycle. Thus, the role of antigen may be to prepare all B cells for proliferation. Different subpopulations of B cells may then require different regulatory signals before actually proliferating or before differentiating into antibody-secreting cells.

Animals↗
PubMed1982

Positive selection of T-cell subsets. I. Proliferative responses of Lyt 2 separated thymocytes and splenic T cells.

Flow microfluorometry analysis of peanut lectin non-agglutinable (PNA-) thymocytes (ThC) reveals the existence of 30%-50% Lyt 1,2,3+ and 50%-70% Lyt 1+,2,3- subpopulations. Using positive selection on anti-immunoglobulin-coated (Mage) plates, we selected PNA- Lyt 2+ and PNA- Lyt 2- ThC as well as their peripheral counterparts in the spleen. These populations were tested in parallel for their ability to respond to concanavalin A (Con A) and phytohaemagglutinin (PHA), to respond to allogeneic stimulation in the mixed lymphocyte reaction (MLR); ThC subpopulations were also tested for their ability to provide synergy with lymph node cells (LNC) in the MLR. It was found that (a) Lyt 2- cells of both thymic and splenic origin responded to all doses of Con A or PHA; (b) PNA- Lyt 2+ ThC were unresponsive to Con A or PHA, whereas splenic Lyt 2+ T cells responded to low doses of mitogens; and (c) PNA- ThC of both Lyt phenotypes responded in a MLR and provided synergy with LNC in the MLR. These data support the notion that Lyt 2+ cells of either PNA- or PNA+ subpopulations must undergo post-thymic maturation before becoming responsive to low doses of T-cell mitogens.

Animals↗
PubMed1982

Regulatory mechanisms in the mixed lymphocyte reaction: evidence for the requirement of two T cells to interact in the generation of effective suppression.

The cellular requirements for suppression of a mixed lymphocyte reaction (MLR) by in vivo alloantigen-activated spleen cells were investigated. We found that the suppression was mitomycin-resistant at days 3 and 5 after antigen injection. By contrast, it was radiation-sensitive on day 3 but not on day 5. Cell fractionation studies using a plate separation technique monitored by flow microfluorometry were performed. We found that neither purified Lyt-2- cells nor purified Lyt-2+ cells independently could suppress an MLR. By contrast, the reconstituted T population was still suppressive. It also was found that culture supernatant factors from allogeneically in vitro restimulated Lyt-2- cells could suppress an MLR if Lyt-2+ cells were present in the responding population. These data obtained by positive selection and reconstitution show that at least two surface immunoglobulin-negative cells interact for the generation of effective MLR suppression. The possibility that we are dealing with a suppressor-inducer, suppressor-effector system is discussed.

Absorption↗
PubMed1982

Mechanisms of protective immunity against Schistosoma mansoni infection in mice vaccinated with irradiated cercariae. II. Analysis of immunity in hosts deficient in T lymphocytes, B lymphocytes, or complement.

Laboratory mice with genetic or induced immunodeficiencies were vaccinated with irradiated cercariae of Schistosoma mansoni and assayed 4 wk later for their resistance to challenge infection. Athymic nude mice failed to develop immunity to challenge or detectable antibody responses to schistosomula, whereas heterozygote (nu/+) controls and nude mice with thymus grafts displayed highly significant levels of resistance and anti-schistosomulum antibody. Similarly, no resistance or antibody production as observed in vaccinated mice that had been made deficient in B lymphocytes by the injection of anti-mu-chain antisera from birth. In contrast, normal levels of vaccine-induced resistance were observed in mice genetically deficient in the fifth component of complement (C5) and in mice decomplemented before challenge by treatment with cobra venom factor. These results indicate that the resistance to challenge infection induced by irradiated cercariae is both thymus and B lymphocyte dependent and therefore is likely to result from specific immune responses directed against schistosome worms. The data also argue against a role for complement in the effector mechanism of vaccine-induced immunity.

Animals↗
PubMed1982

Establishment of B cell hybridomas with B cell surface antigens.

M12.4, one of several B lymphomas derived from BALB/c mice, was mutagenized with ethyl methanesulfonate in vitro. M12.4.1, a subline of the mutant cells, was sensitive to hypoxanthine-aminopterin-thymidine selective medium and was fused with normal splenic B lymphocytes of C57BL/6ByJ mice. The hybridomas obtained from this fusion were shown to express mu heavy chain, H-2KbDb, Iad, and Iab on the cell surface by analyses of flow microfluorometry and a cytotoxicity assay, although parental M12.4.1 lacked mu heavy chain on the cell membrane. These results demonstrate that the B cell hybridomas with B cell surface antigens have been established in vitro. IgM molecules on the cell surface of the hybridomas were shown to originate from normal B cells of C57BL/6ByJ mice by flow microfluorometry analyses after staining with fluorescein-labeled Bet 1, a monoclonal rat antibody that recognizes Igh-6.5, a mouse IgM allotypic determinant. These hybridomas could generate IgM-secreting cells at the high frequency (more than 10% of the cultured cells) when stimulated with bacterial lipopolysaccharide. On the other hand, parental M12.4.1 did not develop any IgM-secreting cells under the same conditions. These findings suggest that these B cell hybridomas with B cell surface antigens may be a good model for the study of B cell differentiation.

Animals↗
PubMed1982

Thymic responses to antigen challenge. I. Tumor cells induced phenotypic and functional changes in vivo.

Thymocytes from mice bearing a footpad injection of 2 x 10(6) syngeneic tumor cells exhibited increased in vitro responsiveness to concanavalin A and allogeneic cells in the mixed lymphocyte reaction. A higher proportion of Thy 1 dull cells (assayed by flow microfluorometry) was seen among thymocytes taken from normal, or control injected mice, relative to tumor injected mice. Injection of culture supernatant from the tumor cells induced no phenotypic or functional shifts among thymocytes, nor was any evidence of viral gp70 antigen expression found in the thymocyte preparation. These findings support the notion that viruses or viral products are not involved in induction of these shifts; however, lymphokine production by host peripheral cells has not been ruled out.

Animals↗
PubMed1982

Modification of thymocytes subsets during pregnancy analysed by flow microfluorometry: role of the alloantigenic status of the conceptus.

Modifications of thymus subsets during pregnancy were monitored using flow microfluorometry and fluorescein-labelled lectins or antibodies. Essentially, it was found that the percentage of peanut lectin-negative (PNA-) cells increased significantly during pregnancy. More interestingly, in the strains tested, the percentage of PNA- cells was more increased in allogeneic than syngeneic pregnancy. This PNA- increase could be mimicked by extracts from syngeneic placenta, but the largest increase in PNA- cells was always observed by injection of allogeneic placental extract, or syngeneic placental extract plus allogeneic cells. These findings suggest that the alloantigenic status of the conceptus does play a role in induction/maintenance of thymus involution, suggesting that it is not a mere hormonal phenomenon and that it could play a role in the events leading to the acceptance of the allogeneic conceptus by the maternal immune system.

Animals↗