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I Lefkovits

Publications and source records attributed to I Lefkovits.

At least 55 records · Page 3Linked to original sources
PubMed1988

Prospective partition analysis of independently assorted sets. Accessory elements supporting clonal lymphoid activation by mitogens.

Accessory cells for mitogen-induced clonal proliferation of lymphocytes can be found after in vitro culture of murine spleen cells. Mitogenic activation of T cells (concanavalin A) depends on such accessory cells. When the cultures in which the accessory cells developed are partitioned, assortment of the accessory cells in cultures is found. When small numbers (1-10) of responder cells, T cells, are dispensed into the above cultures, clonal growth of the T cells is achieved. Neither the dose-response profile of accessory cells nor that of responding cells shows single hit kinetics. Re-categorizing the data set reveals that only cultures with over 20 adherent cells were likely to promote T cell growth and single hit kinetics for the responding cell clone and the accessory cell clone were obtained.

Animals↗
PubMed1988

Immunoglobulin diversification in embryonic chicken bursae and in individual bursal follicles.

Previous studies have shown that the same immunoglobulin (Ig) V lambda gene (V lambda 1) is rearranged in all chicken B cells, and that extensive sequence diversification of this gene occurs during B cell development in the bursa of Fabricius. We used two-dimensional gel electrophoresis to compare the heterogeneity of Ig lambda light chains produced by B cells at different stages of bursal development. Somatically diversified light chains were observed in Ig molecules produced by bursal cells as early as 15 days of embryonic incubation. The two principal species of light chain observed probably represent glycosylated and nonglycosylated forms of lambda chain encoded by alleles of a single lambda gene. Extensive diversification was observed during late embryogenesis. We also studied lambda light chain diversity in cyclophosphamide-treated birds repopulated with normal bursal cells. In these birds, individual bursal follicles are repopulated by single B cell precursors. Follicular cells derived from single B cell precursors were able to produce a spectrum of light chains almost as diverse as that of the total bursal cell population. We used two monoclonal anti-idiotype antibodies to study idiotype expression in individual normal or reconstituted follicles. About 30% of follicles contained 0.1% to 5% of lymphocytes which reacted with one or both of the antibodies. The results indicate that within individual bursal follicles bursa stem cells undergo Ig hyperdiversification.

Animals↗
PubMed1988

Toward an objective classification of cells in the immune system.

The relative abundance of individual proteins shared among clones of lymphocytes provides a meaningful basis for cellular classification. Twelve clones of T cells (obtained by limiting dilution) were analyzed by two-dimensional gel electrophoresis for polypeptide content and then evaluated by the computational technique known as principal component analysis. As a result, relatedness of the clones was established and expressed in terms of taxonomic distances. The data show that a comprehensive and objective classification of the cells involved in the immune system can be approached.

Animals↗
PubMed1988

Increased helper cell activity of NZB mice against H-2-identical allogeneic cells.

The T cells of NZB mice become hyperreactive after stimulation with minor histocompatibility (MIH) antigens. This hyperreactivity has previously been demonstrated only for cytotoxic T cells of NZB, although there was some evidence for an increase of their T-helper cell activity facilitating the response. Here we report a quantitative analysis of T-cell help and help of T-cell subpopulations against autologous, MIH, and H-2 antigens in a limiting dilution assay. After stimulation of NZB T cells with autologous and H-2 antigens, the T-helper cell frequencies did not differ from that of normal mice. After stimulation with MIH antigens however, Lyt 1+2+ T-cells of NZB showed a higher response than those of BALB/c origin. The same difference was seen after prestimulation with ConA or the specific antigen. This demonstrates that NZB-helper cells are more easily activated by weak antigenic differences, and it is possible that this contributes to the prevalence of autoimmune disease in this strain.

Animals↗
PubMed1987

In vivo priming of helper and suppressor T cells by alloantigens. Frequency analysis with the use of an in vitro limiting dilution assay.

Earlier studies have demonstrated that T cells activated in mixed lymphocyte reactions can exert positive as well as negative allogeneic effects on B cells expressing the appropriate alloantigens on their surface. We investigated the effect of in vivo priming of T cells with alloantigens on their capacity to help or suppress allogeneic B cell cultures against sheep erythrocytes. We used immunization protocols that have been shown to be optimal for induction of alloantigen-specific delayed-type hypersensitivity (DTH) and alloantigen-specific suppressor T (Ts) cells for DTH. The results show that in vivo stimulation with alloantigens, depending on the immunization route and the lymphoid organ studied, can be as effective as in vitro stimulation in increasing the frequency of alloantigen-specific helper T (Th) cells and Ts cells. Subcutaneous immunization induced a 10-fold frequency raise of Th cells as well as of Ts cells in the lymph nodes. In the spleen the Th cell population was hardly affected by s.c. immunization, whereas the Ts cell population increased by at least a factor 20. Intravenous immunization, on the other hand, selectively expanded the Th cell population in the spleen, whereas the splenic Ts cell population and the Th and Ts cells in the lymph nodes were not affected. Comparison of these results with our previous data concerning characteristics and the requirements of in vivo activation of alloantigen-specific DTH reactive T cells and of alloantigen-specific Ts cells suggest that different Ts cell populations are involved in suppression of alloantigen-specific DTH in vivo and of allogeneic suppression of in vitro induced sheep erythrocytes specific antibody formation.

Animals↗
PubMed1986

Mitogenic activation of B cells in vitro: the properties of adherent accessory cells as revealed by partition analysis.

The requirement of B cells activated by mitogen (dextran sulfate plus lipopolysaccharide) for accessory cells was studied by partition analysis. Small numbers of splenic B cells were activated to clonal growth, as determined by visual inspection, and to immunoglobulin (Ig) synthesis, as determined by release of Ig into the culture fluid. By placing irradiated adherent cells in the periphery of the microculture wells and forcing responding cells to different areas of the well (slant experiments), it was observed that no cell contact was necessary for B cell activation, and that "promoted" contact ("Rock and Roll" experiments) does not increase the efficiency of activation. Sequential microcultures suggest that only some irradiated adherent cells act as accessory cells, but they can perform this function to more than one B cell. Attempts to perform limiting dilution analysis by varying irradiated adherent cell input showed non-single-hit behavior. When the data were rearranged, taking into account the distribution of irradiated adherent cells, then single-hit behavior with about 1 to 5% of irradiated adherent cells acting as an accessory cells for B cell clonal activation was observed. The evidence suggests that an uncommon irradiated adherent cell releases a soluble factor necessary for B cell activation and/or clonal proliferation.

Animals↗
PubMed1986

Parameters of the labeling of mitogen-activated murine lymphocytes by [35S]methionine for two-dimensional gel electrophoresis. I. Effect of culture conditions.

Labeling with [35S]methionine at a high specific activity is essential to the facile preparation of 2-dimensional gel electrophoretograms with the analytical 2-dimensional charge-size separation procedure (Anderson's ISODALT system). Mitogen-activated T and B lymphocytes subjected to low methionine concentrations would not proceed through cell cycle. In the case of activated B lymphocytes, the use of fetal bovine serum (FBS), dialyzed to lower endogenous methionine concentrations, prevented B cell growth even in the presence of otherwise satisfactory levels of methionine. High concentrations of [35S]methionine (greater than 300 mCi/1) induced B cell death, apparently by radiation damage. Despite these problems, good radioautograms and radiofluorograms of 2D electrophoretograms could be prepared by labeling activated B or T cells in bulk (10(6) cells/ml) with high specific activity [35S]methionine. The polypeptides labeled may be a biased sample since lymphoid cells do not proceed through cell cycle under these conditions. Small numbers (10(3] of activated T cells also yielded satisfactory samples but labeling of small numbers of activated B cells was not possible.

Animals↗
PubMed1986

Analysis of the activation of lymphoid cells by mitogens in vitro with limiting dilution methods: adherent peritoneal cells suppress B-cell activation and synergize with WEHI-3 in the activation of T cells by Con A.

Adherent peritoneal cells (APC) have often been used as a pure and effective macrophage population. Using partition analysis and small numbers of lymphoid cells activated by mitogens (concanavalin A for T cells (in the presence of TCGF) and LPS + DxS for B cells) we found that APC were accessory cells for T cell activation and growth but were not effective for B cells. Although APC were effective in assisting T-cell mitogenesis, they were not especially efficient. However, when APC were mixed with irradiated WEHI-3 cells (a tissue culture line previously shown to exhibit accessory cell activity in vitro for mitogenic activation T and B cells), the APC and WEHI-3 showed apparent synergy. One reason for failure of APC to assist B-cell mitogenesis was traced to the presence of a suppressor cell population which overcame the accessory cell help given by irradiated WEHI-3 cells to LPS-DxS stimulated murine B cells. It is thus possible to find "helper" effects (synergy of APC and WEHI-3 assisting the mitogenesis of T cells), as well as suppressor effects within the range of cells found in adherent accessory cells.

Animals↗
PubMed1986

Self and non-self discrimination by "restriction proteases".

I propose that an organism possesses a set of specific enzymes ("restriction proteases") that cleave self proteins at defined amino acid sequences unless these sequences are rendered inaccessible by glycosylation. Intracellular proteins are degraded by restriction proteases when cells die. In this way, intracellular proteins remain undetected by the immune system. I propose that some autoimmune diseases are caused by the absence of a specific restriction protease.

Autoimmune Diseases↗
PubMed1986

Complete two-dimensional gel electrophoresis pattern of de novo synthesized acute phase reactants.

The early stages of inflammatory responses are characterized by the rapid synthesis of a heterogenous group of plasma proteins known as acute phase reactants. We show that the complex changes in the serum protein composition of mice in response to infections can easily be analysed by two-dimensional (2D) gel electrophoresis, which allows simultaneous analysis of numerous proteins from small volume samples. While changes in the protein composition can be visualized by silver staining, radiofluorography of gels loaded with serum samples from mice that had been labelled in vivo with 35S-methionine allows an analysis of the changes in the pattern of protein synthesis. Thus, these techniques should allow the evaluation of the relative contributions of alterations of protein synthesis and catabolism to the changes in the overall concentration of individual acute phase reactants. Identification of proteins in the 2 D gel pattern can be easily accomplished by co-electrophoresing small serum samples together with immunoprecipitates obtained from in-vivo labelled serum. Using this approach we were able to identify some of the major acute phase reactants of mice. Some of these proteins, like haptoglobin and haemopexin, show concentration increases that are characteristic for type III reactants like C-reactive protein (CRP) or serum amyloid A component (SAA) in man. Results obtained with serum from healthy and infected human newborns indicate that 2D gel electrophoresis could be used to analyse changes in human plasma protein profiles, which would make it a valuable tool for diagnosis and management in certain clinical situations.

Acute-Phase Proteins↗
PubMed1985

Activation/suppression of the immune response in vitro by antigen and dextran sulfate. I. Analysis by limiting dilution methods.

Spleen cells of normal mice which were stimulated with either T-dependent or T-independent antigens and dextran sulfate (DxS) in microcultures at limiting dilution show at low cell densities an increased frequency of responding units (B cells) with both types of antigens. At higher cell densities, DxS induces a suppression of the immune response as assessed by a declining frequency of expressed B cells. When T cells were decreased in number, the stimulating effect of DxS on T-independent responses remained unaltered while the suppressive effect (dependent on cell density) was eliminated. It is suggested that DxS lowers the threshold of activation for regulatory T cells, i.e. of cells which under standard in vitro conditions remain unactivated, and which in the presence of DxS become activated. We conclude that the regulatory cells are rare and that they are not present in most low cell density cultures. The net effect is a potent activation in dilute cultures and lack of response in dense cultures.

Animals↗
PubMed1985

Expression of antibody-forming cell clones in two-stage cultures: an attempt to separate proliferation and maturation events.

Peripheral blood lymphocytes (PBL) from non-immunized rabbits or from rabbits immunized several months previously with a single dose of sheep red blood cells (SRBC) were cultured at several different cell densities in the presence of antigen for 5 days. The cells were then distributed in 10 microliter microcultures at different cell densities to estimate the frequency of responding units. It was found that a shift up in cell concentration from first to second stage allows a more efficient expression of antibody-forming cell clones. We conclude that, during the first stage at low cell density, the precursor cells proliferate and, when they are partitioned in microcultures and the cell concentration is raised, maturation to antibody-forming cells occurs. This approach allows a separation of the proliferation event from the maturation event and it may prove to be a useful tool in the study of B-cell differentiation.

Animals↗
PubMed1984

Activation/suppression of the immune response in vitro by antigen and dextran sulfate. 2. The role of T-cell subsets determined by cell separation and partition analysis.

Culture of spleen cells with dextran sulfate (DxS) and antigen at various different cell densities revealed a T-cell-dependent regulatory pathway not observed in conventional culture. This finding can be explained by the frequent presence in the cultures of a helper cell and the less frequent presence of a suppressor cell, both activated by antigen and DxS. The classic, radioresistant, antigen-specific, helper T cell was not regulated by this newly revealed pathway. The highly frequent, DxS-dependent helper T cell is Lyt-1+2-. The suppressive effect is mediated by a Lyt-1+2+ population consisting of helpers and latent suppressors that can be made active by DxS or Lyt-1+ cells. The specificity of the Lyt-1+ helper cells was not established, but the high frequency observed implies a nonspecific mechanism. The specificity of the suppressor effect was not determined by these experiments. This regulatory mechanism is similar to the phenomena exhibited by polyclonally activated T-cell populations.

Animals↗
PubMed1984

Frequency of expressed immunoglobulin light chain genes in lipopolysaccharide-stimulated BALB/c spleen cells.

Variants of four hybridoma lines secreting antibodies specific for either trinitrophenyl (TNP) or sheep erythrocytes (SRBC) were isolated which have lost either the specific heavy (H) chain or the specific light (L) chain. They were fused to lipopolysaccharide-stimulated mouse spleen cells and the resulting secondary hybridomas were screened for the restoration of the original antibody specificity. Antibody activity was 37 times more frequently restored with fusion lines donating H chain than with those donating L chain. We obtained a variety of different (spleen cell derived) L chains in association with one H chain and one specificity. We found that those L chains originally associated with a given H chain were rescued most often. Their frequencies were 1:74 and 1:490 for an anti-SRBC- and an anti-TNP-restoring kappa L chain, respectively. Two most commonly observed V-J kappa combinations in one anti-SRBC complementation group were detected with a 5- and 30-fold reduced frequency in two anti-TNP groups, indicating somatic diversification of kappa chains. It is shown that the Sp7/HK line resumes anti-TNP activity with a mutated, non-germline lambda 1 chain, which was found in 30% of lambda 1-expressing hybridomas.

Animals↗
PubMed1984

The abnormal function of T cells in chronically anti-mu-treated mice with no mature B lymphocytes.

T cells from anti-mu-treated mice, normal goat serum ( NGS )-treated mice or untreated control mice were compared with respect to their surface antigenic phenotypes, T cell mitogenic responses, helper function and precursor frequency of helper T cells. Anti-mu treatment arrested the development of B cells at an immature stage, as determined by flow microfluorometry; it resulted in no serum IgM, but detectable levels of IgG by solid-phase radioimmunoassay. Proliferative responses to phytohemagglutinin and concanavalin A were significantly decreased in T cells obtained from mu-suppressed C57BL/6 mice, but not from control mice. When T cells from anti-mu-treated mice were tested in vitro for their helper activity to collaborate with B cells from nu/nu C57BL/6 mice to give plaque-forming cells to sheep red blood cells, they provided far less help than did T cells from control mice. The frequency of T helper cells, as measured by limiting dilution analysis, was much lower in the anti-mu- than in the NGS -treated mice. Cell mixing experiments provided evidence for active suppression of T helper function among splenocytes taken from mu-suppressed mice.

Animals↗
PubMed1984

An attempt to assess the overall diversity of murine T cells using two-dimensional gel electrophoresis.

The technique of two-dimensional gel electrophoresis has been applied to study the changes in protein patterns occurring in murine T lymphocytes after activation by a T cell mitogen. The protein maps of individual T cell clones obtained by limiting dilution were also compared. Whole cell lysates from [35S]methionine-labeled cell clones or cell populations were analyzed and radiofluorographs of gels compared. We found that, during the events of differentiation and clonal proliferation, a T cell population undergoes dramatic changes resulting in pronounced changes in the protein pattern. In a typical gel with about 1000 detectable spots, there were about 50 changes from one day to the next. Certain regions of gels were compared using highly exposed films; in these, a much higher proportion of spots was found to vary (47/255 spots). Furthermore, we found that T cell clones obtained by limiting dilution yield a protein pattern that contains both "stable" and "diverse" regions. In the molecular weight region from about 35 000-43 000, there are 0 to 5 differences when one clone is compared with another. Due to a relatively small number of clones analyzed [16], it is probable that the revealed changes are only a fraction of the diversity which remains to be uncovered.

Animals↗