bluuebunny/crossref_metadata_2025 huggingface dataset:
Single Mouse Cells Producing Two Antibody Molecules and Giving Rise to Antigen-Driven Intraclonal Variation after Immunization with Two Unrelated Antigens
Authors: Couderc Jacques , Bleux Christian , Ventura Michel , Liacopoulos Panayotis
Published: December 2022

Hemolytic plaque-forming cells (PFC) simultaneously lysing two different hapten-red cell indicators (double PFC) appear regularly along with PFC lysing only one indicator, on the 4th to 5th day of secondary response in mice primed 2 to 4 months before with hapten-protein conjugates mixed in Freund's complete adjuvant. Addition of soluble-specific inhibitors, i.e., the same haptens conjugated to different protein-carriers, results in an inhibition of the corresponding specific lytic activity and the disappearance of the double PFC. Micromanipulation of these cells from the initial medium into a second medium, containing both indicator RBC and one specific inhibitor, inhibited the corresponding specific lysis in 80% of double cells without affecting the unrelated lytic activity. Subsequent micromanipulation into a third inhibitor-free medium resulted in a recovery of the double lysis in more than 50% of them. This evidence for the synthesis of two different antibody molecules by double PFC was further strengthened by individual cell culture experiments; most of these cells divided and engendered daughter PFC-secreting antibody of only one of the two specificities, thus showing first, the transient nature of double production, and second, that each lytic activity can be inherited separately.

Individual cell cultures also showed occurrence of clonal variation in progeny of single PFC. Among PFC from mice immunized with two antigens to progeny variation occurred in 24 out of 46 (0.656) parental cells as against only 1 out of 40 (0.025) observed among individual PFC from animals immunized with one antigen. Moreover, this variation was antigen driven since absence for 48 hr of the one antigen resulted in discontinuance of generation of daughter PFC of the corresponding specificity. A similar degree of antigen-driven variation was observed when spontaneous PFC or PFC arising early after immunization (2nd or 3rd day) with one antigen were individually cultured for 48 hr with a different antigen; one part of parental PFC (0.545) yielded variant daughters of the specificity of the latter antigen, whereas the other part (0.455) yielded progeny of the same as the parental PFC specificity in a clonal manner. These results suggest that differentiation of B cells during the early phase after antigenic stimulation would involve a process of intracellular selection and/or rearrangement of V genes.
The Effect of Nucleosides and Deoxycoformycin on Adenosine and Deoxyadenosine Inhibition of Human Lymphocyte Activation
Authors: Uberti Joseph , Lightbody James J. , Johnson Robert M.
Published: December 2022

Micromolar deoxyadenosine inhibits leucine uptake during the 1st day of proliferation in mitogen-stimulated lymphocytes if adenosine deaminase is inhibited. This inhibition occurs before DNA synthesis begins, suggesting that deoxyadenosine can affect mitogenesis by mechanisms that do not involve ribonucleotide reductase inhibition. If deoxyadenosine addition to mitogen-stimulated lymphocytes is delayed to the 2nd or 3rd day post-stimulation, inhibition of proliferation is markedly reduced. Although the time dependence of deoxyadenosine toxicity resembles that of adenosine, these compounds appear to inhibit early protein synthesis by different mechanisms: 1) deoxycoformycin markedly potentiates deoxyadenosine but not adenosine; 2) deoxycytidine and thymidine reverse deoxyadenosine toxicity but do not alter adenosine toxicity.
Immunoglobulin E in Coccidioidomycosis
Authors: Cox Rebecca A. , Arnold Douglas R.
Published: December 2022

Serum immunoglobulin (Ig) levels of M, G, A, and E were quantitatively measured in 22 patients with active coccidioidomycosis (Group I), 15 patients in clinical remission (Group II), 15 healthy, coccidioidin (CDN) skin-test positive persons (Group III), and 26 healthy, CDN skin-test negative persons (Group IV). Serum IgM levels were comparable in the four study groups. Serum IgG was significantly elevated in patients with active disease (p < 0.001) and those in clinical remission (p < 0.05) and correlated with serum complement-fixing (CF) antibody titers. Serum IgA was elevated in 5 (42%) of 12 patients with active pulmonary disease, 2 (20%) of 10 patients with disseminated coccidioidomycosis, and 1 (7%) of 15 patients in clinical remission.

Thirteen (59%) of the 22 patients in Group I and 5 (33%) of those in Group II had an elevated IgE serum level (>200 units/ml). In contrast, 5 (12%) of the 41 healthy subjects in Groups III and IV showed an elevated IgE level. Total serum IgE strongly correlated (p < 0.001) with anti-CDN IgE which, in turn, correlated (p < 0.01) with immediate wheal and flare responses to CDN. There was not, however, a correlation between serum IgE levels (either total or anti-CDN) and disease severity or duration of clinical disease. In addition to anti-CDN IgE, reaginic antibodies were demonstrable to allergens unrelated to Coccidioides immitis. Hyperproduction of IgE in 49% of 37 patients studied may be attributed to a T lymphocyte defect, presumably a T suppressor cell population(s).
Allotypes of C3 in Laboratory and Wild Mouse Distinguished by Alloantisera
Authors: Natsuume-Sakai Shunnosuke , Moriwaki Kazuo , Amano Shigetoyo , Hayakawa Jun-Ichiro , Kaidoh Toshio , Takahashi Morinobu
Published: December 2022

Structural variations of murine complement C3 are controlled by a single codominant locus. Previous report from this laboratory described production of the alloantiserum directed to one of the C3 allotypes. The second alloantiserum, specific for the gene product of the C3-1b allele, was produced in NC mice by injecting partially purified C3 of BALB/c mouse. This antiserum reacted with C3-1 B, C3-1 AB, or C3-1 BC phenotype, but it did not react with either C3-1 A or C3-1 C phenotype. By the use of this antiserum it was found that C3 in mouse plasma or serum of all of 27 inbred strains previously typed as C3-1 B by IEF are not distinguished from each other antigenically. This implies that C3 in these mice is the gene product coded for by the same allele, C3-1b. With two different alloantisera, one directed to C3-1 B, the other directed to C3-1 A, we phenotyped 106 wild mice collected from four countries in Asia (Afghanistan, Pakistan, the Philippines, and Japan). All mice trapped outside Japan, which belong to either Mus musculus bactrianus or Mus musculus castaneus, had C3-1 B phenotype. On the other hand, mice that were trapped in eight out of nine places of Japan and belong to Mus musculus molossinus subspecies are polymorphic for C3. Three C3 phenotypes were found among Japanese wild mice: C3-1 A, C3-1 B, and C3-1 AB. Based on the reactivity with two alloantisera and IEF analysis, it is now possible to identify all of C3 allotypes in mice of four subspecies of Mus musculus.
Structural Studies on Induced Antibodies with Defined Idiotypic Specificities
Authors: Capra J. Donald , Nisonoff Alfred
Published: December 2022

This paper reports the complete amino acid sequence of the variable region of heavy chains derived from A/J anti-p-azophenylarsenate antibodies bearing a cross-reactive idiotype. The structure of this induced idiotypically defined antibody is homogeneous and provides the first direct evidence that heritable idiotypes are defined chemical entities. There are certain similarities between the structure of this murine antibody and the human myeloma protein Eu as well as guinea pig anti-p-azophenylarsonate antibodies. The amino acid sequence of approximately 15% of the constant region of this IgG1 molecule is also described. Combined with our studies of the variable region of the light chains of these molecules, this study represents the first complete V domain structure of an induced idiotypically defined antibody with heritable characteristics.
The LT System in Experimental Animals
Authors: Hiserodt John C. , Tiangco Gerry J. , Granger Gale A.
Published: December 2022

High levels of cytotoxic activity (lymphotoxin-like (LT)) detectable on L-929 cells was obtained in serum-free culture supernatants when non-adherent murine splenic lymphocytes or nylon wool-enriched T cells were cultured on monolayers of mitogen- (PHA) coated allogeneic or xenogeneic stimulator cells in vitro. Levels of lytic activity were lower in supernatants obtained from splenocyte cultures containing glass-adherent cell populations. Although release of lytic activity was very rapid, reaching maximal levels by 6 to 10 hr, this activity was very unstable. The levels of toxic activity in 8-hr supernatants were 20 to 50 times the levels obtained when lymphocytes were cultured with various dosages of mitogen (PHA-P, Con A) alone, even after 5 days of incubation. This phenomenon was not unique to murine lymphoid cells, for similarly high levels of LT activity were found in supernatants from lymphoid cells obtained from several animal species activated in a similar fashion. These results indicate that lymphoid cells from several animal species are capable of rapidly releasing high levels of cell-lytic activity in vitro not previously noted, and provide a means for obtaining highly active supernatants for biochemical studies. Furthermore, the data suggest that rapid release of LT may depend upon the nature of the cellular activating stimulus involving interaction of the lymphocyte with both lectin and target cell surface.
The LT System in Experimental Animals
Authors: Ross Monica W. , Tiangco Gerry J. , Horn Peter , Hiserodt John C. , Granger Gale A.
Published: December 2022

High levels of material with lymphotoxin- (LT) like activity, cytolytic for L-929 cells in vitro, were rapidly released by lymphoid cells obtained from rat, guinea pig, hamster, and rabbit after co-culture for 7 to 10 hr with lectin-coated L-929 cells in vitro. These supernatants were all rapidly fractionated under similar conditions by molecular sieving, ion exchange chromatography, and polyacrylamide gel electrophoresis, and the results were compared to those previously obtained for human and murine LT molecules. The activity in all supernatants was separated by gel filtration into multiple m.w. classes that were strikingly similar to those previously assigned to human and murine LT molecules, i.e. complex (>200,000 d), α (70 to 150,000 d), β (35 to 50,000 d), and γ (12 to 20,000 d). The α m.w. class was resolved on Ultrogel AcA 44 into two distinct m.w. forms, termed α heavy (αH) (110 to 150,000 d), and α light (αL) (70 to 100,000 d). All supernatants contained LT forms of the Cx, αH, and αL m.w. classes. The smaller β and γ m.w. forms were not as evident in supernatants induced in this fashion. The percentage of activity in each m.w. class varied, but the majority of activity was generally due to αH and αL forms. However, the relative percentage of activity in any given m.w. class was dependent upon the ionic strength of the separating column buffer. The actual molecular dimensions of materials within the α m.w. class varied somewhat between species, however, they did not vary within the smaller m.w. β and γ forms when detected. The αH and αL m.w. forms could each be further resolved into multiple charge subclasses. Although the αH and αL forms in guinea pig and rabbit have been shown by immunologic and/or physical data to be related forms, they can be distinguished by charge from one another. This evidence supports the concept that LT molecules detected in supernatants from experimental animals, although heterogeneous, represent a system of related subunits similar to that defined for the human and mouse. Moreover, this system of cell toxins appears to be conserved in evolution and has common features in each of the animals examined in this study.
Naturally Occurring Antibodies to Liposomes
Authors: Strejan G. H. , Smith Patricia M. , Grant C. W. , Surlan D.
Published: December 2022

Liposomes were prepared from a mixture of sphingomyelin, cholesterol, and dicetylphosphate or L-α-dimyristoyl phosphatidylcholine, cholesterol, and dicetylphosphate, in the presence of glucose. The amount of trapped glucose released from these liposomes was monitored after incubation with a variety of normal and immune sera in the presence of guinea pig complement.

All normal rabbit sera tested were found to release, in the presence of complement, detectable amounts of trapped glucose from sphingomyelin-containing liposomes. After immunization with a variety of unrelated antigens, the anti-sphingomyelin liposome activity increased significantly and in direct proportion to the number of injections, despite the fact that the liposomes used in the assay did not contain the relevant antigen used for immunization. Liposomes prepared from dimyristoyl phosphatidylcholine showed only marginal release of their trapped marker when assayed with the same rabbit sera and complement. These liposomes, however, were fully reactive when the appropriate antigen was inserted in their bilayer structure. The antiliposome activity was associated mainly with the IgM antibody class.

These results raise the interesting possibility that antigenic stimulation may trigger the activation of lymphocyte clones directed against autologous cell-membrane components that cross-react with artificial model membranes containing sphingomyelin.
Allogeneic Marrow Transplantation after Total Lymphoid Irradiation (TLI): Effect of Dose/Fraction, Thymic Irradiation, Delayed Marrow Infusion, and Presensitization
Authors: Gottlieb Michael , Strober Samuel , Kaplan Henry S.
Published: December 2022

BALB/c mice infused with 30 × 106 C57BL/Ka bone marrow (BM) cells 1 day after treatment with fractionated total lymphoid irradiation (TLI) (17 fractions of 200 rads each) became stable mixed chimeras without clinical graft-vs-host disease (GVHD). Mice given 18 fractions of 100, 50, or 25 rads each followed 1 day later by C57BL/Ka BM did not become chimeric, indicating that a critical cumulative radiation dose is required for this effect. Animals given TLI with lead shielding placed over the thymus also developed stable chimerism without GVHD. Thus susceptibility to tolerance induction and protection from GVHD after TLI and allogeneic BM transplantation is not due to alteration of the thymic microenvironment by fractionated irradiation. A delay of 7 or 21 days between completion of TLI and BM administration resulted in a high incidence of graft rejection. Sensitization to minor histocompatibility antigens of the BM donor strain by blood transfusion either before or during TLI resulted in marrow graft rejection in a high percentage of animals.
Composition and Biologic Properties of Soluble IgG-Anti-IgG Immune Complexes: Effects of Variations in the Specificity of Rabbit Antibodies to Different Structural Components of Human IgG
Authors: Arend William P. , Sturge Jerrilyn C.
Published: December 2022

Rabbit antibodies specific for antigenic determinants on the Fab fragments, Cγ2 domain, or Cγ3 domain of human IgG were purified by affinity chromatography. The interaction of these purified IgG antibodies with whole human IgG as antigen was examined. Antigen and antibody preparations were utilized that possessed either intact or reduced and alkylated interchain disulfide bonds. Quantitative precipitin analyses showed that the degree of precipitation, when antibodies were combined with antigen at equivalence, varied between the three antibody preparations. The Fab-specific antibodies gave the highest precipitability with the Cγ2-specific antibodies being intermediate and the Cγ3-specific antibodies exhibiting the least precipitation. In general, reduction and alkylation of either the antigen or antibodies, or both, led to a decrease in the amounts of antibodies precipitated at equivalence. In spite of the differences in precipitability at equivalence, soluble complexes prepared in 3-fold antigen excess possessed comparable amounts of larger-latticed structures (>Ag2Ab2) in each antigen-antibody system. However, the relative amounts of small-latticed complexes present were greater with the Fab-specific antibodies.

The biologic properties of soluble IgG-anti-IgG complexes were evaluated with the antibody preparations of different specificities. The human IgG antigen in the complexes containing Fab-specific antibodies contributed to the ability of the complexes to activate complement and to bind to macrophage Fc receptors. In contrast, the IgG antigen in the complexes containing Cγ2- or Cγ3-specific antibodies was inhibited from contributing to these biologic activities.

It was concluded that the specificity of antibodies to IgG has significant effects on the interaction with antigen and on the biologic properties of soluble IgG-anti-IgG complexes.
The Induction of Cytolytic T Lymphocytes with Syngeneic Trinitrophenyl-Coupled Membranes
Authors: Sherman Linda , Mescher Matthew F. , Burakoff Steven J.
Published: December 2022

Evidence is presented that trinitrophenyl-coupled tumor membranes are able to induce cytolytic T lymphocytes (CTL) when co-cultured with syngeneic spleen cells. These haptenated membranes stimulate spleen cells from naive and immune mice. The specificity of these CTL is determined by the H-2 antigens of the membranes used for stimulation.
The Alternative Pathway C3/C5 Convertase: Chemical Basis of Factor B Activation
Authors: Lesavre Philippe H. , Hugli Tony E. , Esser Alfred F. , Müller-Eberhard Hans J.
Published: December 2022

The structural basis of activation of the alternative pathway C3 convertase was explored. For this purpose a modified isolation procedure of the activating enzyme, Factor D, was elaborated. The procedure affords a 70,000-fold purification of the enzyme with a 20% yield. A simple assay was designed for the quantitation of both Factor D and Factor B activity. On the basis of activity measurements and amino acid analysis, Factor D concentration in plasma was estimated to be 1 µg/ml. Highly purified Factor D was used to activate Factor B in the presence of C3b and Mg++. The resulting fragments, Ba and Bb, were characterized with respect to their circular dichroism spectra, amino acid compositions, reactive sulfhydryl groups, and partial amino- and carboxy-terminal sequences. The results indicate that the BA fragment constitutes the amino-terminal region and the Bb fragment the carboxy-terminal region of Factor B. The bond in Factor B that is cleaved by Factor D is proposed to be an arginyl-lysine bond.
Demonstration of Fcγ Receptors on Human Basophil Granulocytes
Authors: Ishizaka Teruko , Sterk Alan R. , Ishizaka Kimishige
Published: December 2022

Fcγ receptors were detected on human basophil granulocytes. The mononuclear cell fraction of human peripheral blood was incubated with heat-aggregated human IgG (HGG) followed by 125I-anti-HGG. Autoradiography of the cells showed that the majority of basophil granulocytes gave a significant number of grains. Basophils were not labeled by preincubation of the same cells with monomeric HGG followed by 125I-anti-HGG. However, the binding of aggregated HGG to basophils was inhibited by the presence of a high concentration of monomeric HGG or its Fc fragment but not by the Fab fragment. Evidence was obtained that Fcγ receptors are distinct from IgE receptors on the same cells: i) Saturation of basophils with IgE did not affect the binding of aggregated HGG to the cells. ii) Preincubation with and the presence of aggregated HGG failed to affect the binding of 125I-IgE to basophils, or to block passive sensitization of the cells with IgE antibodies. iii) The Fcγ receptors did not co-cap with IgE receptors. Aggregated HGG failed to induce histamine release from basophils even in the presence of D2O. It was also found that the presence of aggregated HGG on basophils did not modulate IgE-mediated histamine release from the cells.
Effect of Glucan on Natural Killer (NK) Cells: Further Comparison between NK Cell and Bone Marrow Effector Cell Activities
Authors: Lotzová Eva , Gutterman Jordan U.
Published: December 2022

We have studied the effect of glucan, a potent immunomodulating agent with immunotherapeutic potential for man, on murine natural killer (NK) cells. The results of our experiments have shown that a single i.v. injection of 4 mg of glucan caused significant depression of NK cell activities residing in the bone marrow and spleens of B6DF1 mice. The depression of NK cell cytotoxicity was expressed 24 hr after glucan treatment and lasted as long as the mice were tested, i.e., 15 days after treatment. Impairment of NK cell functions caused by glucan could not be mediated via mature T cell functions since the congenitally athymic BALB/c mice were also hyporeactive after glucan injection. The negative effect of glucan on NK cells is disturbing if one considers NK cells as an important component in immunosurveillance and in immunity to malignancies.

In order to test the relationship between NK cell and bone marrow effector (BM-E) cells, which can represent the same cell population(s) or subpopulation(s), we have studied also the effect of glucan on bone marrow graft rejection potential in B6DF1 mice. Bone marrow transplantation studies have shown close correlation between NK cell and BM-E cell activities. Specifically, at the time when glucan caused depression of NK cell anti-tumor activities (i.e., on days 2, 6, 11, and 15 after injection), there was also a prevention of rejection of histo-incompatible bone marrow transplants, and vice versa, histoincompatible bone marrow grafts were always rejected by untreated B6DF1 mice expressing high degree of NK cell anti-tumor activities. These results indicate, further, the similarity between NK cell and BM-E cells and, moreover, suggest possible benefits of glucan in bone marrow transplantation.
Ia-Bearing Bone Marrow-Cultured Macrophages Induce Antigen-Specific Helper T Cells for Antibody Synthesis
Authors: Stern Angelika C. , Erb Peter , Gisler Roland H.
Published: December 2022

In bone marrow cell (BMC) cultures supplemented with colony-stimulating factor (CSF), accessory cells develop that are capable of inducing specific helper T cells. These accessory cells become effective after 4 days in culture and can be found not only in the adherent but also in the nonadherent cell population. On the other hand, very few accessory cells with helper cell-inducing capacity are obtained in BMC cultures without CSF. The active BMC-derived cell type has been shown to carry Ia surface antigen, since pretreatment with anti-Ia serum and complement abolished the capacity of these cells to function like macrophages in helper T cell induction. Moreover, the appearance of functional accessory cells in these cultures coincided with the presence of Ia-bearing cells.
IgG Recruiting Component (GRC): B Cell-Derived Signal for IgG Antibody Synthesis
Authors: Hinchman S. M. , Battisto J. R.
Published: December 2022

The B cell-derived soluble factor that has been described as an IgG-recruiting component (GRC) was investigated to: a) ascertain whether it is governed by genetic constraints, b) determine what triggers its synthesis, and c) identify its cellular target.

GRC has been shown to be unrestricted by histocompatibility barriers since it enhanced IgG antibody production in mice of diverse genetic backgrounds. Further, we report that eliminating IgG-bearing cells from B cells to be immunized in vitro allows T cells-replacing factor (TRF) to increase the number of IgM but not IgG PFC. Thus, TRF appears to act on IgM-bearing cells by expanding the IgM PFC number. Adding GRC 48 hr after the addition of TRF to such IgG-depleted cells caused expression of IgG PFC. Hence, B cells lacking IgG but possessing IgM surface immunoglobulins appear to be those that are acted upon by GRC. These data indicate that in whole splenic cell populations, GRC is derived from IgG-bearing B cells that are stimulated by antigen and a component in TRF.
Cyclic Nucleotide Phosphodiesterase Activity in Human Peripheral Blood Lymphocytes and Monocytes
Authors: Wedner H. James , Chan Bruno Y. , Parker Charles S. , Parker Charles W.
Published: December 2022

cAMP and cGMP phosphodiesterase (PDE) activity was assayed in human peripheral blood lymphocytes purified by isopycnic centrifugation as well as in lymphocyte preparations further purified to remove contaminating platelets and monocytes. The 16,000 × G supernatant from sonicates of each of these cell preparations contained two hydrolytic activities for cAMP with apparent Km of 1.1 to 2.5 µM and 33 to 66 µM, and a single hydrolytic activity for cGMP with an apparent Km of 6 to 25 µM. When lymphocytes were disrupted by Dounce homogenization, there was only a single, low Km cAMP PDE activity in the homogenate; however, the 16,000 × G supernatant demonstrated 2 Km similar to that seen in sonicated lymphocytes. Treatment of the Dounce preparations with 0.5% Triton X-100 or 1.0% NP-40 converted these preparations to activities similar to those seen in sonicated preparations. cGMP hydrolytic activity was low or absent in the Dounce preparations and was not altered by centrifugation; however, it was markedly enhanced by detergent extraction. These data indicate that human peripheral blood lymphocytes and monocytes have PDE activities similar to those seen in other tissues.
Tumor-Specific Immunity Induced by Somatic Hybrids
Authors: Kim Byung S.
Published: December 2022

Hybrid cells derived from fusion of a BALB/c plasmacytoma (TEPC-15) and L cells (C3H origin) were used to stimulate tumor-specific immunity against the parental plasmacytoma cells. Live hybrid cells induced tumor-specific immunity against TEPC-15 more effectively than mitomycin-treated hybrid or TEPC-15 tumor cells. Adoptive transfer of immunity with spleen cells of mice immunized with hybrid cells was also more effective than that with mitomycin-treated tumor cells. The immunity induced by the hybrid cells was specific to the TEPC-15 tumor because the mice that received immune spleen cells were not protected against challenge with either HOPC-8 or McPC-603 plasmacytomas. T cell populations were primarily responsible for the transfer of specific immunity based on the sensitivity of immune cells to anti-Thy 1.2 and complement. Mice that had established solid tumors were treated with 5 × 107 spleen cells to evaluate the therapeutic value of the hybrid-induced immune cells. Tumors in the mice that received immune cells gradually regressed over a 40-day period, whereas tumors on the control mice continued to grow. These results suggest that a rearrangement of tumor-specific antigens on allogeneic hybrid cells can enhance their immunogenicity.
Cyclic AMP-Dependent Protein Kinase Activation and the Induction of Ornithine Decarboxylase during Lymphocyte Mitogenesis
Authors: Klimpel Gary R. , Byus Craig V. , Russell Diane Haddock , Lucas David O.
Published: December 2022

Cyclic AMP-dependent protein kinase activation and the induction of ornithine decarboxylase (ODC) were studied along with RNA and DNA synthesis in human peripheral blood lymphocytes following in vitro culture with either mitogens, nonmitogenic agglutinins, or dibutyryl cyclic AMP (DBcAMP). The mitogens phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) all resulted in activation of soluble cyclic AMP-dependent protein kinase, the induction of increased levels of ODC activity, and increases in the rate of incorporation of (3H)-thymidine (DNA synthesis) and (3H)-uridine (RNA synthesis) into acid-insoluble material. High concentrations of PHA or Con A, which resulted in maximal cyclic AMP-dependent protein kinase activation, were only slightly mitogenic. The time at which maximum activation of protein kinase occurred differed for each mitogen but, in each case, always preceded ODC induction and increased RNA and DNA synthesis. In Con A-stimulated cultures, cyclic AMP-dependent protein kinase activation was maximal at 4 hr of culture and remained elevated at 24 and 48 hr. In these cultures increased ODC activity was first observed at 6 to 8 hr of culture and preceded increased RNA synthesis which was detectable at 12 hr of culture. DNA synthesis in all mitogen-stimulated cultures was first observed at 48 hr with maximum (3H)-thymidine incorporation being at 72 hr of culture.

Cyclic AMP-dependent protein kinase activation, induction of ODC, and increased RNA and DNA synthesis were all shown to be dependent upon lymphocyte-Con A binding and were blocked by early addition of α-methyl mannoside. After 12 hr of culture, α-methyl mannoside addition had little effect on Con A induction of ODC or on RNA and DNA synthesis measured at 24 to 72 hr. In contrast, cyclic AMP-dependent protein kinase activation, measured at 24 hr, could be completely inhibited when α-methyl mannoside was added as late as 12 or 20 hr to Con A-stimulated cultures. The nonmitogenic agglutinins, wheat germ agglutinin (WGA) and Agaricus bisporus lectin (ABL), failed to increase any of the parameters tested. DBcAMP also did not induce ODC or cause any increased RNA or DNA synthesis. However, DBcAMP (10-3 M) did cause activation of cyclic AMP-dependent protein kinase as early as 15 min of culture. DBcAMP activation of protein kinase was concentration-dependent, maximal at 12 hr, and remained elevated through 48 hr of culture. The addition of DBcAMP (10-3 M) to Con A-stimulated cultures caused a 90 to 95% inhibition of ODC activity and RNA and DNA synthesis. This inhibition was concentration-dependent and paralleled the degree of activation of cyclic AMP-dependent protein kinase elicited by each concentration.

These results suggest that the selective activation of cyclic AMP-dependent protein kinase and induction of increased ODC activity are early integral events linked to and regulating lymphocyte mitogenesis.
Suppressor T Cells Regulate the Cytolytic T Lymphocyte Response to Syngeneic Tumors Induced by Murine Sarcoma Virus (MSV) in the Mouse
Authors: Plata Fernando , MacDonald H. Robson , Shain Benjamin
Published: December 2022

Studies were designed to analyze the immune activities of spleen cells from mice previously injected with murine sarcoma virus (MSV) and undergoing the processes of MSV tumor growth and rejection. Fractionation of MSV-primed spleen cells according to cell size by velocity sedimentation at unit gravity showed that MSV-specific cytolytic T lymphocytes (CTL) generated in vivo underwent an apparent transition in size from large to small cells as the tumor regressed. The majority of CTL precursors, however, were invariably recovered among small to medium-sized MSV-immune cells, as revealed by CTL generation in vitro in secondary mixed leukocyte-tumor cell cultures (MLTC). Evidence was obtained for the existence in MSV-immune spleens of two suppressor cell populations capable of inhibiting CTL generation in vitro: one population probably consisted of macrophages and could be removed by treatment with carbonyl iron; the second population was comparised of T cells and inhibited the differentiation of tumor-immune CTL precursors in a selective manner. These results provide a preliminary overview of the mechanisms regulating the generation, differentiation, and activity of tumor-specific CTL in a syngeneic model system.
Observations of IgG1 Anti-DNP Hybridoma-Mediated ADCC and the Failure of Three IgM Anti-DNP Hybridomas to Mediate ADCC
Authors: Greenberg Arnold H. , Lydyard Peter M.
Published: December 2022

Murine monoclonal IgG1 and IgM anti-DNP hybridoma antibodies were examined for their ability to mediate ADCC by murine and human effector cells. Adherent and nonadherent BALB/c mouse spleen cells, as well as null cells obtained from nu-nu (nude) mouse spleen, were able to kill SRBC-TNP50 via IgG1 anti-DNP hybridoma. The cytotoxicity was inhibited by IgG, but not IgM or IgA myeloma proteins, and IgG2a was more effective than IgG1. ADCC of SRBC-TNP mediated by IgG1 hybridoma was highly dependent on the TNP substitution ratio. SRBC-TNP50 and SRBC-TNP20 were readily lysed, but SRBC-TNP10 to SRBC-TNP0.5 target cells were increasingly insensitive to cytolysis. IgG1 hybridoma-mediated ADCC was also observed with human peripheral blood lymphocytes, as well as T lymphocytes isolated by E-rosetting. Depletion of FcG-bearing peripheral blood T cells by EAG-rosetting eliminated the IgG1-mediated ADCC.

Sera from mice bearing three different IgM anti-DNP hybridomas with hemolytic titers of 1/60,000 to 1/400,000 and up to 600 µg/ml of antibody were unable to mediate ADCC by all populations of mouse effector cells examined. In addition, human effector cells enriched for FcM-bearing cells by depletion of EAG-rosetting cells were unable to kill SRBC-TNP50 in the presence of IgM anti-DNP hybridoma.

Contaminating heterophile antibodies capable of mediating ADCC to SRBC and CRBC were found in the sera of IgM hybridoma-bearing mice. The activity was directed against erythrocyte membrane determinants, and not TNP determinants, and could be completely removed by absorption with erythrocytes. This heterophile antibody-mediated ADCC could be inhibited by both IgG2a-and IgM-aggregated myeloma protein.
Idiotypic Analysis of Anti-GAT Antibodies
Authors: Ju Shyr-Te , Cramer Donald V. , Dorf Martin E.
Published: December 2022

The specificity and distribution of an interspecties cross-reactive idiotype are described in 13 inbred strains of rats. This interspecies cross-reactive idiotype was defined by the ability of rat anti-GAT antisera to inhibit the binding of the murine CGAT idiotype to the corresponding anti-CGAT idiotypic antiserum. High levels of the interspecies cross-reactive idiotype were detected in 9 of 13 rat strains; all individuals of these 9 strains produced detectable levels of the idiotype. The idiotype was always associated with anti-GAT antibodies. Following immunization with GAT, all individuals of the other four rat strains produced anti-GAT antibodies with low or undetectable levels of the interspecies cross-reactive idiotype, although the anti-GAT antibodies from these latter strains had fine specificity patterns similar to that of the idiotype-positive strains. Immunization of the low idiotype producer LEW and F344 strains with GAT conjugated to the highly immunogenic fowl γ-globulin carrier also failed to induce detectable levels of the interspecies cross-reactive idiotype. Prolonged immunization schedules were used to determine the persistence of this idiotype in rats; the data demonstrated that the ability (or inability) to produce the interspecies cross-reactive idiotype is a stable trait and lasts over 174 days. There was no apparent association of the idiotypic marker with the known rat immunoglobulin heavy and light chain allotypic specificities. Finally, the rat interspecies idiotype is cross-reactive, but not identical with the mouse CGAT idiotype.
IgD Is Present on the Cell Surface of Murine Lymphocytes in Two Forms: δ2L2 and δL
Authors: Eidels Leon
Published: December 2022

Murine cell surface immunoglobulin (Ig) isotypes were analyzed by lactoperoxidase-catalyzed radioiodination, immunoprecipitation, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE analysis revealed three radioactive peaks: I) IgM (µ2L2; 224,000 daltons), II) IgD (δ2L2; 185,000 daltons), and III) IgD half-molecules (δL; 105,000 daltons). The size of the δ-chain obtained from both the δ2L2 and the δL molecules was the same (68,000 daltons). The δL molecules are apparently not the result of a sulfhydryl-catalyzed reduction or disulfide interchange and are probably not generated by proteolysis, since addition of alkylating agents and protease inhibitors did not decrease the amount of δL observed. Addition of sulfhydryl-oxidizing agents also did not decrease the amount of δL, suggesting that the δL molecules cannot be reoxidized to yield δ2L2 molecules. The percentage of IgD in the δL form present on lymph node and spleen cells of A/J mice and on spleen cells of BALB/c mice was the same (∼55%). The percentage of δL on splenocytes from BDF/1 mice (2 to 7 months old) was ∼42%. Spleen cells from strains that are “IgD-deficient,” CBA/N and NZB, and spleen cells from 2-week-old BDF/1 mice, which have a low δ/µ ratio, displayed a coordinate decrease in the levels of both δ2L2 and δL. This coordinate decrease resulted in no alternation in the percentage of IgD present in the δL form (∼42%). These results, taken together, indicate that indeed the cell surface Ig of murine lymphoid cell populations consists of monomeric IgM, monomeric IgD, and IgD half-molecules.
Membrane Immunoglobulin Is Present on Thymic and Splenic Lymphocytes of the Trout Salmo Gairdneri
Authors: Warr Gregory W. , Deluca Dominick , Griffin Billy R.
Published: December 2022

Three rabbit antisera raised to trout IgM showed positive immunofluorescent reactions with lymphocytes of trout spleen and thymus. These antisera showed no detectable cross-reactivity with keyhole limpet hemocyanin, as determined by direct radioimmune precipitation and adsorption, and did not appeare to react significantly with carbohydrate moieties on trout IgM, as determined by the failure of proteolytic digests of trout IgM to inhibit in radioimmunoassay against intact trout IgM. Membrane immunoglobulin determinants were detectable on the membrane of both thymocytes and splenocytes with the lactoperoxidase-catalyzed iodination reaction. Radioimmunoassay of lysates of lymphocytes confirmed the association of immunoglobulin with trout thymocytes. By radioimmunoassay, lysates of thymic and splenic lymphocytes contained similar amounts of immunoglobulin, equivalent to approximately 8 × 104 molecules of IgM per cell (calculated as the monomer µ2L2).
Potentiation of IgE Response in Vitro by T Cells from Rats Infected with Nippostrongylus Brasiliensis
Authors: Suemura Masaki , Ishizaka Kimishige
Published: December 2022

The effect of T cells from rats infected with Nippostrongylus brasiliensis (Nb) on the IgE response was studied by using an in vitro system. Mesenteric lymph node cells from DNP-OA (dinitrophenyl derivatives of ovalbumin)-primed rats were cultured with DNP-OA or DNP-HSA in the presence of T or B cells from Nb-infected rats. It was found that T cells from the infected rat, but not normal T cells, selectively enhanced the IgE-forming cell response of DNP-OA primed cells to homologous antigen without affecting the IgG-forming cell response. The enhancement of the IgE response requires carrier-primed T cells; T cells from Nb-infected animals failed to enhance the Ig-forming cell response of DNP-OA primed cells to DNP-HSA. Culture supernatants of T cells from Nb-infected rats selectively enhanced the IgE response of DNP-OA primed cells to homologous antigen without affecting IgG response. The IgE-potentiating factor present in the unstimulated cultures is different from T cell-replacing factor(s), which was obtained by stimulation of antigen-primed T cells with antigen. When T cells from Nb-infected rats were incubated with Nb antigen, culture supernatant of the cells enhanced both IgE and IgG responses of DNP-OA primed cells to DNP-OA or DNP-HSA. It was also found that the molecular size of IgE-potentiating factor was significantly smaller than T cell-replacing factor. The results suggested that an optimal IgE response may require two subsets of T cells; one is carrier-specific T cells, which are common for all isotypes, and another subset is cells forming IgE-potentiating factor.
B Lymphocyte: Activation by Insoluble Anti-Immunoglobulin: Induction of Immunoglobulin Secretion by a T Cell-Dependent Soluble Factor
Authors: Parker David C. , Fothergill John J. , Wadsworth Daniel C.
Published: December 2022

Purified rabbit antibodies to mouse κ chain or Fab become strongly mitogenic for mouse splenic B lymphocytes when covalently attached to the surface of polyacrylamide beads. The same antibody preparations are weakly if at all mitogenic in soluble form. The proliferative response to these anti-Ig beads is not dependent on T lymphocytes, adherent cells, serum supplement, or Fc receptor interactions. The response to anti-Ig beads can be blocked by soluble F(ab′)2 anti-Ig at concentrations that modulate surface Ig, or by excess soluble mouse IgG. Hence, the response results from direct interaction of the beads with B cells via surface Ig.

In the presence of a supernatant of Con A-treated spleen cells (Con A supernatant), B lymphocytes respond to anti-Ig beads by sustained proliferation and differentiation to polyclonal IgM and IgG secretion. The response is comparable in magnitude to the lipopolysaccharide response. Ig secretion is completely dependent on the Con A supernatant, which by itself has only a small, primarily differentiative effect on isolated B lymphocytes. It can be added 22 hr after the start of culture without changing the kinetics of the response; addition at later times up to 66 hr delays the appearance of secreting cells, but not the size of the peak response. Cells from 10-day-old mice and adult male (CBA/N♂ × BALB/c♀) F1 mice fail to respond.

These experiments suggest that redistribution of surface Ig by the anti-Ig beads delivers a limited proliferative signal to a subset of B lymphocytes, but that sustained cell division and differentiation to Ig secretion by anti-Ig activated B lymphocytes require soluble substances produced by activated T lymphocytes and/or T lymphocyte-activated accessory cells.
Variations in the Responses of Mouse Strains to Rat Xenografts
Authors: Burdick James F. , Jooste Somarie V. , Winn Henry J.
Published: December 2022

There is strong histoincompatibility between the rat and the mouse such that grafts of rat skin (1) or primarily vascularized hearts (2) placed in mice undergo acute rejection about 5 to 9 days after transplantation. In the case of skin grafts, which have been more thoroughly studied, this prompt rejection has been shown to occur in all of a large number of mouse strains that have been tested (3). If, however, the recipients of skin grafts receive a brief course of treatment with anti-lymphocyte serum (ALS)4 at the time of grafting, survival of the grafts is prolonged and the extent of prolongation varies among the recipient strains to a surprising degree (3). That this latter phenomenon is not restricted to skin grafts is demonstrated by the results obtained with primarily vascularized xenografts of hearts as described in this report.

Mice were purchased from the Jackson Laboratory, Bar Harbor, Maine.
Depletion of NK by Cellular Immunoadsorption
Authors: Jensen Pamela J. , Koren Hillel S.
Published: December 2022

The binding of human natural killer (NK) cells to their tumor cell targets was investigated by using monolayers of sensitive target cell lines. Monolayers of K562 and HSB, a myeloid and T cell line, respectively, were prepared on poly-L-lysine-coated plastic tissue culture dishes and briefly fixed with 0.2% formaldehyde. Freshly isolated peripheral blood lymphocytes (PBL) were incubated on the monolayers. Nonadherent PBL were then removed, after gentle agitation, by decanting and gently washing the monolayer. They were tested, along with unseparated controls, for NK activity in a short-term 51Cr release assay. PBL that were nonadherent to a tested monolayer had only 20 to 60% of the control cytotoxic activity. Our results suggest that NK recognition sites on the effector lymphocytes were able to interact with reciprocal determinants on the target cell monolayers, resulting in selective loss of NK effector cells from the PBL population. The specificity of the NK effector-target interaction was investigated by testing the ability of each monolayer to remove activity against both targets. These data imply heterogeneity with regard to recognition structure within the NK effector population as well as among the target cells.
Role of Lipids in the Immune Response
Authors: Wolf Raoul L. , Merler Ezio
Published: December 2022

By further study, a previously described inhibitor of lymphocyte proliferation and immunoglobulin synthesis, soluble inhibitory factor (SIF), has been shown to comprise two moieties; a nondialyzable protein, apparently inert with regard to inhibition of proliferation, and a polar lipid bound to the protein fraction, presumably by hydrophobic interaction. The lipid contained the activity responsible for inhibition of PHA-induced DNA synthesis. It was tentatively identified as a lipid with an as yet unidentified polar group, on the basis of mobility on thin layer chromatographic plates and behavior on silicic acid.
The Pathology and Homing of a Transplantable Murine B Cell Leukemia (BCL1)
Authors: Warnke Roger A. , Slavin Shimon , Coffman Robert L. , Butcher Eugene C. , Knapp Michael R. , Strobert Samuel , Weissman Irving L.
Published: December 2022

The pathology and homing characteristics of a murine B cell leukemia are described. Experiments utilizing autoradiography to determine the early homing pattern of the leukemic cells revealed a pronounced localization of the labeled cells to the spleen. The cells that were seen in the white pulp showed preferential localization to the follicles or B cell domains. Tissue section immunofluorescence with antibodies to κ- and λ-light chains was used to study the initial mouse with this disease as well as to study the mice that were injected with in vivo passaged cells. These mice also showed predominant involvement of the spleen. Although the initial mouse with this disease had 200,000 λ-bearing B lymphocytes per mm3 in the peripheral blood and closely resembled a human chronic lymphocytic leukemia patient, the studies described suggest that this murine B cell neoplasm is a lymphoma with a striking predilection for splenic involvement. The other organs including the bone marrow as well as the peripheral blood appeared to be involved secondarily. This unusual spontaneously occurring murine B cell disease provides a useful model for the investigation of certain commonly occurring human lymphomas and leukemias.
Suppression of Cell-Mediated Tumor Cell Lysis and Complement-Induced Cytotoxicity by Trypan Blue
Authors: Scornik Juan C. , Ruiz Phillip , Hoffman Edward M.
Published: December 2022

Different forms of cell-mediated cytotoxicity were suppressed in the presence of trypan blue. The systems affected included lysis of antibody-coated tumor cells by normal and C. parvum-stimulated mouse peritoneal cells and lysis of allogeneic targets by immune effector cells. The inhibition, measured in a 4-hr 51Cr release assay, was reversible and did not occur in the presence of 30% fetal calf serum or albumin. Binding between effector and target cells through Fc receptors was not affected, and lysis of allogeneic cells was inhibited at the lytic step rather than at the binding step. In contrast, lysis of sensitized erythrocytes was not inhibited by trypan blue, suggesting that lysis of these targets may not involve the steps required in tumor cell lysis. Trypan blue blocked the function of antibody before binding to target cells and also suppressed complement-induced cytolysis. Most individual complement components were susceptible to the inhibitory action of trypan blue. These results reveal an affinity of trypan blue for proteins in general that may be responsible for many of its biologic actions.
Potentiation of Antibody-Dependent Cell-Mediated Cytotoxicity by Target Cell-Bound C3b
Authors: Ghebrehiwet Berhane , Medicus Rudolf G. , Müller-Eberhard Hans J.
Published: December 2022

Antibody-dependent cytolytic effector lymphocytes are known to possess, in part, receptors for activated C3. Employing a model system consisting of 51Cr-labeled chicken erythrocytes and purified human peripheral lymphocytes, we investigated the effect of target cell bound C3b on antibody-dependent cellular cytotoxicity (ADCC). At concentrations of anti-target cell antibody too low to cause effective ADCC, target cell bound C3b cooperated with antibody to produce marked target cell lysis. In the presence of a 1/6.25 × 106 dilution of antichicken erythrocyte rabbit IgG, cell lysis increased from 20% to 65% by the attachment of 18,000 C3b molecules per cell. C3b-dependent enhancement of ADCC was dose dependent. It was augmented by attachment of activated properdin (P̄) to the C3b-bearing target cells.