Studies on non-H-2-linked lymphocyte-activating determinants. IV. Ontogeny of the Mls product on murine B cells.

The minor lymphocyte stimulating (Mls) locus codes for lymphocyte activating determinants (LADs) on murine B lymphocytes, but not T lymphocytes. This observation was strengthened by a series of techniques which allow deletion and addition of T and B cells. These included the use of cytotoxic antisera such as anti-Thy 1.2, anti-MTLA, anti-MBLA, and complement, and the use of a goat anti-μ antisera, and finally the use of a fluorescence activated cell sorter (FACS).The studies in this report document the organ distribution and the ontogenetic appearance of the surface LADs on the surface of B lymphocytes from DBA/2N (H-2^d, Mls^a) and CBA/J (H-2^k, Mls^d) mice. Adult-like ability to stimulate H-2 identical BALB/c (H-2^d, Mls^b) and C3H/He (H-2^k, Mls^c) responder cells appeared at about 4–5 weeks of age. Inability of neonatal cells to induce an Mls-defined MLC was found not to be due to a low frequency of B lymphocytes or to the presence of suppressor cells, but due to the absence of the Mls-coded LADs on their surface. These data support the concept that the Mls-coded LADs are present on adult B lymphocytes and are specific markers of B-cell differentiation, which is preceded by membrane IgM and the δ homologue of human IgD, Ia, and the receptor for the third component of complement.     

Cell-mediated lymphocytotoxicity following in vitro sensitization of frozen stored human mononuclear cells in heterologous serum

Human peripheral mononuclear cells were subjected to controlled-rate freezing and stored at−196 °C. Following rapid thawing and slow removal of DMSO, the cells exhibited normal immune responsiveness to phytohemagglutinin and in mixed lymphocyte culture. After in vitro sensitization to frozen allogeneic cells in heterologous serum they developed cell-mediated lymphocytotoxicity demonstrated by ⁵¹Crrelease from specific targets isogeneic with the sensitizing cells.     

A cytokinetic model for heterogeneous mammalian cell populations. III. Tritiated thymidine studies: Correlations among multiple kinetic parameters in human tumors

A computer model for the simulation of radioautographic studies in growing mammalian cell populations was used to study multiple tritiated thymidine (³HTdR) associated parameters in model populations whose properties are comparable to those found in human tumors. Several different DNA synthesis rate patterns are distinguishable, designated type I, intermediate, and type II. Correlations among percent labelled mitosis (PLM) curves, interphase cell labelling patterns, and continuous ³HTdR labelling studies suggest a type I pattern in human breast cancer, an intermediate pattern in human melanoma, and a type II pattern in human adult leukemia. Detailed simulation studies were carried out in human adult acute leukemia and human melanoma. It was possible to fit all available kinetic data in leukemia and melanoma, both with respect to low threshold data, and with respect to radioautographic labelling intensity, provided that simulated experimental conditions and simulated radioautographic conditions corresponded to those actually employed. Kinetic differences between leukemia and melanoma were demonstrated which are in keeping with the natural histories and clinical drug response behavior of these two malignancies.     

A continuous-flow system for the growth of RNA tumor viruses.

By continuously pumping tissue culture medium into roller bottles containing infected cells and continuously harvesting the spent medium, RNA tumor virus can be harvested conveniently within 20 min of its release from the infected cells. An apparatus used to collect Moloney mouse leukemia virus in such a way yielded up to 390 μg protein in purified virus (about 1 mg virus) from each liter of tissue culture fluid.     

Studies of the T-lymphocytes resident in the spleens of thymectomized, irradiated, bone marrow-reconstituted (TXB) mice.

The T-lymphocytes resident in the spleens of thymectomized, lethally irradiated mice that had been reconstituted with syngeneic bone marrow (TXB) were characterized. Both recently reconstituted N-TXB, (approximately 3 weeks after bone marrow injection) and aged (>6 months after reconstitution) A-TXB animals were studied. The T-lymphocytes from spleens of recently reconstituted N-TXB donors did not respond to PHA but did react significantly to Concanavalin A (Con A). The lack of PHA sensitivity was not due to dilution of reactive cells by other cell types. Removal of adherent cells, likewise, did not restore N-TXB spleen cell PHA responsiveness. N-TXB splenic T-cells were cortisone resistant. N-TXB spleen cells by themselves did not cause a graft vs host response. However, N-TXB spleen cells amplified the graft vs host response of normal lymph node cells but not N-TXB lymph node cells. Addition of cyclic GMP enhanced [³H]thymidine uptake of N-TXB spleen cells caused by Con A. N-TXB spleen cells were exclusively spleen seeking. The Con A reactive cell within N-TXB spleens was demonstrated to be of donor origin. Fetal liver as well as syngeneic bone marrow contained cells capable of reconstituting the Con A response. Spleen cells from aged. (>6 months) A-TXB were found to be PHA sensitive. Competitive inhibition assays measuring θ expression in A-TXB spleen cells indicate a significant increase in the θ positive lymphocyte population occurred with time. The data indicate that considerable reconstitution of θ positive cells had occurred in A-TXB donors. The results also suggest that the T-lymphocyte population of the TXB spleen may be a unique subpopulation of T-lymphocytes that resides exclusively in spleen and bone marrow.     

Localization of the human alpha-globin structural gene to chromosome 16 in somatic cell hybrids by molecular hybridization assay

We have used 16 human × mouse somatic cell hybrids containing a variable number of human chromosomes to demonstrate that the human α-globin gene is on chromosome 16. Globin gene sequences were detected by annealing purified human α-globin complementary DNA to DNA extracted from hybrid cells. Human and mouse chromosomes were distinguished by Hoechst fluorescent centromeric banding, and the individual human chromosomes were identified in the same spreads by Giemsa trypsin banding. Isozyme markers for 17 different human chromosomes were also tested in the 16 clones which have been characterized. The absence of chromosomal translocation in all hybrid clones strongly positive for the α-globin gene was established by differential staining of mouse and human chromosomes with Giemsa 11 staining. The presence of human chromosomes in hybrid cell clones which were devoid of human α-globin genes served to exclude all human chromosomes except 6, 9, 14 and 16. Among the clones negative for human α-globin sequences, one contained chromosome 2 (JFA 14a 5), three contained chromosome 4 (AHA 16E, AHA 3D and WAV R4D) and two contained chromosome 5 (AHA 16E and JFA14a 13 5) in >10% of metaphase spreads. These data excluded human chromosomes 2, 4 and 5 which had been suggested by other investigators to contain human globin genes. Only chromosome 16 was present in each one of the three hybrid cell clones found to be strongly positive for the human α-globin gene. Two clones (WAIV A and WAV) positive for the human α-globin gene and chromosome 16 were counter-selected in medium which kills cells retaining chromosome 16. In each case, the resulting hybrid populations lacked both human chromosome 16 and the α-globin gene. These studies establish the localization of the human α-globin gene to chromosome 16 and represent the first assignment of a nonexpressed unique gene by direct detection of its DNA sequences in somatic cell hybrids.     

SDS gel analysis of muscle proteins in embryonic cells

Myogenic and premyogenic (epithelial) somites, primitive streak, lateral plate, area rhomboidalis, and premyogenic wing buds from chick embryos contain polypeptides which comigrate with standard muscle actin and myosin on SDS polyacrylamide gels. The relative percentages of actin and myosin present in these tissues is similar. Ultrastructural examination revealed thin (50 Å) filaments in all these tissues, but thick (130 Å) filaments and myofibrils differentiated only in myotome. Because of their widespread occurrence, we conclude that actin and myosin can no longer be considered “luxury molecules” that identify differentiating muscle.     

Treatment of glutathione synthetase deficient fibroblasts by inhibiting gamma-glutamyl transpeptidase activity with serine and borate.

Glutathione synthetase deficiency results in decreased cellular glutathione content and consequent overproduction of 5-oxoproline. L-serine in borate buffer inhibits γ-glutamyl transpeptidase, the major catabolic enzyme for glutathione. Treatment of glutathione synthetase deficient fibroblasts with 40mM serine and borate for 24 hours produced more than a 2-fold increase in cellular glutathione content. L-serine alone led to a smaller increase in glutathione level, and borate alone was without effect. On exposure to serine and borate, 5-oxoproline formation from L-glutamate was decreased to normal levels in glutathione synthetase deficient fibroblasts, presumably secondary to feedback inhibition of γ-glutamylcysteine synthetase by the increased intracellular glutathione concentration. Cellular free amino acid content was generally unaffected by such exposure although increases were observed in serine and phosphoserine. This model system suggests that γ-glutamyl transpeptidase inhibition may be a rational approach to alleviating the effects of glutathione synthetase deficiency.     

Ability of dialysates containing transfer factor to induce lymphocyte activating factor by human mononuclear cells.

Dialysates of human leukocyte lysates containing transfer factor (TF_d) stimulated human mononuclear cells (MNL) to produce lymphocyte activating factor (LAF). Both unfractionated and adherent MNL cultures were stimulated by TF_d to produce a factor which was mitogenic for murine thymocytes and had the biochemical characteristics of LAF as determined by Bio-Gel P-100, DEAE cellulose, and hydroxylapatite chromatography. Fractionation of TF_d on Sephadex G-25 showed that the specific transfer factor activity of converting in vivo skin tests was present in the major uv-absorbing peak, whereas the substance(s) that induced LAF activity was present in a number of the other fractions. Therefore, the capacity of TF_d to induce monocytes to produce LAF is not a measure of classical transfer factor activity. However, this effect of TF_d may instead participate in the nonspecific immunoenhancing effects of TF_d.     

Secretion of proteoglycans by chondrocytes. Influence of colchicine, cytochalasin B, and beta-D-xyloside.

Chondrocytes obtained from epiphyseal cartilage of fetal guinea pigs or ear cartilage of young rabbits were cultured in monolayer. The influence of colchicine, cytochalasin B, and p-nitrophenyl-β-d-xylopyranoside on secretion of proteoglycans was investigated. Radioactive sulfate was used as a precursor. As observed previously in other systems, β-d-xylosides initiated the synthesis of free chondroitin sulfate chains, competing with the endogenous proteoglycan core protein acceptor. The molecular weights of the chondroitin sulfate chains synthesized both on the xyloside and on the core-protein acceptor in maximally stimulated cells were similar and significantly lower than in proteoglycans synthesized in the absence of xyloside. The size of the chondroitin sulfate chains synthesized on the xyloside was inversely related to the concentration of this compound. This finding suggests that the chain length is dependent on the ratio between available acceptor and chain-lengthening enzymes or precursors. Cytochalasin B, a microfilament-modifying agent, inhibited proteoglycan synthesis, without any effect on secretion. Cells treated with cytochalasin B could be stimulated with β-d-xyloside to synthesize free chondroitin sulfate chains to the same relative degree as cells with intact microfilaments. Colchicine, an antimicrotubular agent, partially inhibited synthesis and secretion of proteoglycan. However, cells treated with colchicine could be stimulated with β-d-xyloside to synthesize and secrete free chondroitin sulfate chains to about the same relative degree as cells with intact microtubules. The data suggest that microtubules may have a facilitatory rather than an obligatory role in the secretion of proteoglycans and that at least part of the effect of colchicine is located at or after the site of glycosaminoglycan synthesis.     

Cytolytic T lymphocyte responses to metabolically inactivated stimulator cells. II. Effect of a soluble "costimulator" factor(s) in primary and secondary mixed leukocyte culture

The ability of a concanavalin A-stimulated spleen cell supernatant (“costimulator”) to overcome the effects of impaired CD and LD antigen presentation by metabolically inactivated stimulator spleen cells was examined in the primary and secondary cytolytic T lymphocyte (T_c) response. (i) Cells inactivated by ultraviolet irradiation or mild glutaraldehyde treatment, which were unable to stimulate primary cytolytic activity on their own, generated near maximal responses in the presence of costimulator. The 30-fold lower efficiency of splenic membrane fragments as antigen in primary MLC with the supernatant indicated that the damage to immunogenicity caused by membrane isolation was not equivalent to that caused by uv light and glutaraldehyde, as has previously been assumed. (ii) Comparison of the relative effects of antigen and costimulator demonstrated that costimulator played the dominant regulatory role in primary MLC, increasing sensitivity to suboptimal antigen doses 10- to 30-fold; neither antigen nor the supernatant appeared preferentially to control the strength of the secondary response. (iii) Metabolically inactivated adult and untreated neonatal spleen cells failed to release costimulator activity in response to concanavalin A. However, the ability of the neonatal cells to induce a primary cytolytic response suggested that costimulator production by the stimulator cells themselves is not essential for primary T_c activation, and supports the hypothesis that the lack of primary immunogenicity of inactivated spleen cells reflects their failure to induce costimulator production by the responder population.     

Ly-4.2: a cell membrane alloantigen of murine B lymphocytes. III. In vitro studies

The alloantigenic specificity Ly-4.2 is present on a restricted population of murine lymphocytes which have previously been shown to have some of the properties generally ascribed to B lymphocytes, both with regard to distribution and function. In the study reported herein, the effect of anti-Ly-4.2 and anti-Thy-1.2 (θ) antisera have been examined in various in vitro systems. (a) T cell-mediated lysis of ⁵¹Cr-labeled P815-X2 target cells by immune allogeneic peritoneal exudate cells is inhibited by anti-Thy-1.2, but not affected by the anti-B (Ly-4.2) reagent. (b) Antibody-dependent lymphocyte-mediated lysis of ⁵¹Cr-labeled sheep red cells was only slightly inhibited by anti-Ly-4.2 and anti-Ig antisera, and not at all by anti-Thy-1.2 antisera, indicating that this type of cell lysis is mediated by neither T (Thy-l⁺) nor B (Ly-4.2⁺,Ig⁺) cells. (c) The response of lymph node lymphocytes to various mitogens was affected thus: PHA, completely inhibited by anti-Thy-1.2 but not by anti-Ly-4.2; Con A, largely inhibited by anti-Thy-1.2, and slightly by anti-Ly-4.2; PWM (pokeweed mitogen), partially inhibited by both antisera; E. coli endotoxin lipopolysaccharide, greatly inhibited by anti-Ly-4.2 but only slightly by anti-Thy-1.2. The findings demonstrate that anti-Thy-1.2 reacts predominantly with T cells and anti-Ly-4.2 with B cells.     

Activation of human and murine B cells by anti-immunoglobulin antibody: dependence of the response on the preexisting level of B-cell activation

Previous reports of the response of B lymphocytes to soluble anti-immunoglobulin (anti-Ig) antibodies have yielded conflicting data. While most studies show activation of B cells, others have shown inhibitory effects. In the assay reported in this report, we were able to obtain widely diverse responses of human B-cell populations to anti-Ig antibody. An explanation of this variability was established by resort to an animal (murine) model. Mice maintained in a pathogen-free environment failed to respond or responded only weakly to anti-Ig antibody. Mice which had previously received heavy antigenic stimulation, but at the time of the experiment were not undergoing any known challenge, showed a marked positive response. Mice deliberately challenged with lipopolysaccharide (LPS) 24 hr prior to anti-Ig antibody exposure showed a high background mitogenesis in control cultures, which was inhibited by anti-Ig antibody. This preliminary study suggests that response to anti-Ig antibody differs in each phase of B-cell differentiation. In future studies it is hoped that this variability in response can be used to characterize different subsets of B-cell differentiation separated by physical or phenotypic parameters.     

Inhibition of the mixed lymphocyte culture by peritoneal exudate cells.

Inhibition of mixed lymphocyte cultures (MLC) by macrophages and by supernatants of short term cultured macrophages was assessed by incorporation of ³H-thymidine (TdRH3) and also by blast cell counts and by determination of cellmediated lympholysis. Peritoneal exudate cells (PEC) induced by thioglycollate, at concentrations >10%, inhibited all three parameters of MLC. Lower concentrations of PEC, and supernatants from cultured PEC, inhibited TdRH3 incorporation, but had no significant effect on blast cell counts or on generation of cytotoxic effector cells. Inhibition by the supernatants could be reversed by dialysis or by use of low specific activity TdRH3. These data indicate that macrophages can inhibit proliferative responses in MLC, but that this must be carefully distinguished from selective inhibition of TdRH3 incorporation.     

Transferrin binding by human lymphoblastoid cell lines and other transformed cells

Viable cells of 18 human cell lines, including 15 transformed cell lines of malignant and lymphoblastoid origin, were examined by an indirect immunofluorescence method for their ability to bind purified transferrin and transferrin in normal human serum. The specificity of the reaction was investigated by study of the binding reactions of several other serum proteins, including albumin, α-1-antitrypsin, and α-2-macroglobulin. Membrane binding of human transferrin was demonstrated in less than 5% of normal peripheral blood mononuclear cells or cultured diploid fibroblasts, but in more than 80% of the cells from 13 of the transformed lines, and the data obtained indicated that this binding reaction reflected the presence of specific receptors for transferrin.     

Proteins synthesized in lymphoid cells early after activation by phytohemagglutinin

A double-isotope labeling approach has been employed in an attempt to identify the proteins synthesized by lymphocytes early after stimulation by phytohemagglutinin (PHA). The earliest effect of PHA, within the first hour, was the induction of large aggregates of cellular proteins, which were not dissociated by 1% sodium dodecyl sulfate (SDS) in the absence of β-mercaptoethanol. These aggregates were composed of proteins of molecular weight approximately 70,000, but they did not include PHA. The aggregates were made up of preexisting as well as newly synthesized cellular proteins. Subsequently, within the first 2 hr after the addition of PHA, there was a nonspecific stimulation of protein synthesis. This was followed by the preferential synthesis of several classes of proteins including at least one group of nuclear proteins. The structural changes described here are among the earliest events known to occur within the lymphoid cell after its interaction with PHA.     

Properties of reticulum cell sarcomas in SJL/J mice. VII. Nature of normal lymphoid cells proliferating in response to tumor cells.

Treatment of SJL lymph node, spleen, and thymus cells with anti-Ly 1.2 serum and C caused a marked reduction in the usually observed T-cell proliferation in response to syngeneic X-irradiated transplantable reticulum cell sarcoma cells. By contrast, treatment of lymphoid cells with anti-Ly 2.2 serum and C either failed to affect or increased the proliferative response. It is concluded that the Ly 1 cell is the major T-cell subpopulation which proliferates in response to RCS.     

The influence upon mitogenic and cellular immunologic reactive systems in vitro by poly(I:C) and BCG murine interferons induced in vivo.

We have previously reported that lymphocytes from W/Fu rats immunized with syngeneic (C58NT)D tumor cells were cytotoxic against these cells in a 4-hr ⁵¹Cr release assay. We have investigated the feasibility of cryopreserving lymphocytes and target cells and have selected freezing conditions which provide good yields of viable cells and functional activity. Lymphocytes from different animals had a recovery of 60–80% viability which resulted in a corresponding 55–75% recovery of cytotoxic activity. Repeated testing of lymphocyte cytotoxicity from a pool of frozen spleen cells against either fresh or frozen (C58NT)D cells gave reproducible cytotoxicity. In addition, recovery of high levels of lymphocyte function was also demonstrated when cryopreserved cells were employed in long-term cytotoxic assays, i.e., ³H-proline and ¹²⁵IUdR release assays, in the lymphoproliferative response to mitogens (PHA and Con A)³ or tumor cells (MLTI) as measured by ³H-thymidine incorporation, and in the in vitro generation of secondary cytotoxicity.By employing these cryoprotective techniques it is possible to have: 1) a population of lymphoid cells with known functional activity and 2) a pool of target cells with known susceptibility to lysis and antigenic content. Furthermore, the use of frozen cells as internal standards in each test also permits the analysis of assay variation as well as the study of variation in various cell types.     

Urease catalysis and structure: X. Alternate bonding-site isozymes of jackbean urease

Three previously uncharacterized, nongenetic urease isozymes have been analyzed by sucrose density gradient sedimentation, gel electrophoresis, and chemical reactivity. The full complement of isozymes could be reliably generated by choosing appropriate levels of NaCl, pH, and ethylene glycol, and was stable for several days in dilute solution. The three forms of interest were found to be quaternary isomers of other isozymes, but differed from them qualitatively in their bonding sites, with disulfide bonds being substituted for noncovalent bonds. The separation of these isomer-pairs during sedimentation and electrophoresis cannot be readily explained by differences in size or charge, but must rather arise from a difference in shape. A simple two-dimensional model can provide the appropriate molecular architecture to satisfy these requirements: Only one of the two half-units in each α-urease molecule undergoes disulfide bonding during polymerization, and it does so with two adjacent molecules, thus producing asymmetric polymers from symmetric starting components.     

Development of choline acetyltransferase activity in chick cranial neural crest cells in culture

The types of mouse parthenogenones obtained in a medium modified with respect to Ca²⁺ and/or Mg²⁺ ions were investigated in “spontaneously” activating eggs after culturing cumulus masses in vitro for 5 hr. The second meiotic division was affected in eggs cultured in medium lacking Ca²⁺ and Mg²⁺ or Ca²⁺ alone, resulting in suppression of second polar body extrusion in a high proportion of cases, giving rise to two pronuclear eggs or eggs that underwent immediate cleavage. Extrusion of the second polar body occurred normally when the cumulus mass was cultured in complete medium and, in a high proportion of eggs, when Mg²⁺ alone was lacking in the medium. The results are discussed with reference to the second meiotic division. The method provides an efficient way for obtaining a large number of different types of parthenogenetic embryos.