bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Properties of Human Lymphocyte Activating Factor (LAF)
Authors: Blyden G. , Handschumacher R. E.
Published: December 2022

Lymphocyte-activating factor (LAF) has been shown to be produced by LPS-stimulated human adherent cells (monocytes) and peripheral leukocytes, but many non-macrophage cell lines failed to produce LAF. Other macrophage activators including latex microspheres, antigen-antibody complexes, and barium sulfate induce the production of LAF. There is a delay of 6 hr before significant amounts of LAF activity appear in the supernatant medium and maximum activity is found after 12 to 24 hr.

Chromatography of concentrated crude supernatant fractions containing LAF activity on Sephadex G-100 gave two peaks of activity (approximately 85,000 and 13,000 daltons). The latter constitutes the major activity and has been purified at least 500-fold with Sephadex G-100, anion exchange, and adsorption chromatography. Optimal stimulation with LAF induces mitosis in 10% of murine thymocytes. The purified activity is sensitive to chymotrypsin and is not affected by treatment with sodium periodate, sulfhydryl reagents, and phenylmethanesulfonylfluoride.

The response of thymocytes to LAF decreases with age after 10 weeks and thymocytes obtained from animals injected with cortisone or tumor-bearing animals have an increased responsiveness to LAF.
Specific Binding of T Lymphocytes to Macrophages
Authors: Lipscomb Mary F. , Ben-Sasson Shlomo Z. , Uhr Jonathan W.
Published: December 2022

Peritoneal exudate lymphocytes obtained from immune guinea pigs and cultured for 1 week on antigen-pulsed autologous macrophages were tested for their ability to bind to fresh antigen-pulsed autologous macrophages or to macrophages pulsed with an irrelevant antigen. Up to 30% of the lymphocytes bound to macrophages bearing the relevant antigen whereas only 2 to 5% remained nonspecifically bound to macrophages after vigorous washing. Specific binding was observed in cultures as early as 1 hr. Analysis of the kinetics of binding suggests that the observed nonspecific binding is not a step in specific binding. The possibility that weaker antigen-independent association between lymphocytes and macrophages precedes specific binding cannot be excluded. No evidence was obtained that serum antibody adsorbed to the macrophage or T cell plays a role in this cell interaction or that the T cell can bind antigen directly. We suggest that the observed specific binding represents the initial event in stimulation of T lymphocytes by antigen.
Disulfide Bonding of Secretary Component to a Single Monomer Subunit in Human Secretory IgA
Authors: Underdown Brian J. , De Rose Joseph , Plaut Andrew
Published: December 2022

The arrangement of disulfide bonds joining secretory component (SC) to the α chains in secretory IgA was studied by determining the molecular size of the principal fragments resulting from CNBr digestion of secretory dimeric Fc fragments from IgA ((Fc)2α fragments). In vitro complexes formed by incubating 125I-free SC and myeloma 131I-(Fc)2α fragments were isolated by gel filtration and subsequently digested with cyanogen bromide. The CNBr digests of SC·(Fc)2α fragments were analyzed by gel filtration in 5 M guanidine. Two principal fragments were obtained, one containing a monomeric Fc fragment from IgA (Fcα) associated with SC (m.w. ≃ 110,000) and a second containing the second Fcα monomer (m.w. ≃ 50,000) from the dimeric SC·(Fc)2α. Similar results were obtained when secretory (Fc)2α fragments isolated from native secretory IgA dimer were subjected to CNBr digestion.

The data indicate that SC is disulfide bonded to a single monomer subunit in secretory IgA dimer.
Evidence for A B Lymphocyte Defect Underlying the Anti-X Anti-Erythrocyte Autoantibody Response of NZB Mice
Authors: DeHeer David H. , Edgington Thomas S.
Published: December 2022

The autoimmune hemolytic anemia of NZB mice is pathogenetically mediated by a genetically prescribed anti-erythrocyte autoantibody response directed to the X erythrocyte autoantigen. The cellular locus of the immunoregulatory defect underlying the anti-X response was explored by adoptively transferring bone marrow cells (BMC) from NZB mice to lethally irradiated histocompatible recipients. Before adoptive transfer, BMC from donor mice were assayed for antigen-binding lymphocytes with receptors for the X autoantigen (X-ABL) by immunocytoadherence assays and for anti-X autoantibody-secreting cells (X-PFC) by plaque-forming cell assays. Twelve weeks after adoptive transfer, splenic lymphocytes from recipient mice were assayed for X-PFC and humoral anti-X autoantibody by Coombs' tests. Transfer of 15 to 30 × 106 BMC containing 6 to 12 × 103 X-ABL but no X-PFC from 6- to 8-week-old NZB mice to lethally irradiated BALB/c, B10.D2, C57BL/Ks, and DBA/2 mice produced X-PFC in 70% of the recipients. Development of X-PFC was not simply dependent upon available X-ABL since transfer of 15–30 × 106 BMC, containing comparable numbers of X-ABL, from BALB/c, B10.D2, C57BL/Ks. or DBA/2 mice to NZB or syngeneic recipients did not produce X-PFC. Transfer of BMC from NZB mice to BALB/c, B10.D2, and DBA/2 mice with weekly administrations of AKR anti-θ antiserum had no effect on the development of X-PFC; T lymphocyte ablation was evidenced by the absence of θ+ spleen cells. These results suggest that the pathogenetic anti-X response is not genetically prescribed at the level of macrophages, humoral factors, or T cells, but rather appears to be a phenotypic expression of a primary B lymphocyte defect permitting or promoting differentiation of NZB X-ABL.
Regulation of Immunoglobulin Production in Human Peripheral Blood Leukocytes: Cellular Interactions
Authors: Saxon Andrew , Stevens Ronald H. , Ashman Robert F.
Published: December 2022

The intercellular influences regulating immunoglobulin (Ig) synthesis by normal human peripheral blood leukocytes (PBL) were investigated in cells stimulated by pokeweed mitogen (PWM). This ststem was shown to be totally T lymphocyte dependent as purified B lymphocytes (≤1% T lymphocytes) failed to make significant amounts of Ig. No evidence was obtained for an Ig class switch as all classes of Ig (IgM, IgG, IgA) were shown to be produced in increasing amounts over a 6-day time period.

T lymphocytes demonstrated maximum helper effect when mixed with equal numbers of B cells. This helper effect was mediated through the dual mechanisms of increasing the number of B lymphocytes containing cytoplasmic Ig and by increasing the maturity of these B lymphocytes as demonstrated by an increasing Ig production per B lymphocyte. When present in higher numbers, T lymphocytes were also capable of suppressing Ig production. This T-mediated suppression was first evident as a decrease in the Ig produced per B lymphocyte (decreased maturity). With maximum T suppression Ig-containing B lymphocyte numbers were also diminished.

T lymphocyte help was relatively independent of macrophages (phagocytic cells) and did not require DNA synthesis for expression. Both T help and suppression were shown to cross allogeneic barriers. Immature T lymphocytes (thymocytes) were incapable of mediating either activity. Normal human PBL contain T lymphocytes capable of mediating both T help and suppression and the Ig produced by PBL was shown to be the balance of these activities. This balance probably represent the participation of distinct T lymphocyte subpopulations analogous to the T helper (Ly 1+) and T suppressor (Ly 2+, 3+) populatins in the mouse.
Immunologic Tolerance to HGG in Mice
Authors: Jones Tom B. , Kaplan Alan M.
Published: December 2022

Adoptive transfer of spleen cells or spleen cell lysates from mice tolerant to human-γ-globulin (HGG) specifically suppressed the response of normal syngeneic recipients to HGG. The suppressive activity could be transferred for over 100 days after tolerance induction. The suppression induced by both spleen cells and spleen cell lysate was found to be specific as evidenced by a normal response to a challenge with turkey-γ-globulin or goat erythrocytes. The activity of the suppressive lysate could be removed by passing the material through an HGG immunoadsorbent column but not by passing it through an anti-HGG column or a BSA column. These results indicated that the factor had antigen specificity and was probably not antigen-antibody complexes. That this suppression was not due to a shifting of the kinetics of the antibody response has also been demonstrated. The antigen-specific suppressor factor in the tolerant spleen cell lysates was a protein with a m.w. of approximately 45,000 daltons. The kinetics of the appearance of both suppressor cells and suppressor factor were consistent with a mechanism of active suppression functioning in the maintenance of tolerance to HGG.
Immune Adjuvancy of Lipopolysaccharide and a Nontoxic Hydrolytic Product Demonstrating Oscillating Effects with Time
Authors: Behling Ulrich H. , Nowotny Alois
Published: December 2022

Since the first reports of immune adjuvancy of endotoxic lipopolysaccharides (LPS) (1, 2), large numbers of such preparations have proven to be effective adjuvants for both soluble and cellular immunogens in a host of laboratory animals (for review, see Neter (3)). Kind and Johnson (4) as well as Franzl and McMaster (5) investigated the effect of time of LPS treatment on the primary immune response.

In earlier investigations on radioprotective properties of LPS, we observed a unique time-dependent survival pattern of lethally irradiated mice pretreated with LPS (6). It was established that two optimal time intervals, peaking at -1 to -3 and -8 to -12 days, existed. A closely parallel time-dependent radioprotective profile of the nontoxic, polysaccharide-rich fraction (PS)3 of LPS could also be established. Quite recently a similar time-dependent, LPS-induced protection against TA3-Ha tumors in mice was also observed by us (7).
Specific Restoration of Delayed Hypersensitivity by Lymphoid Tissue Extracts
Authors: Green Jon A. , Williams Juliann , Levy Hilton B.
Published: December 2022

Mice lose demonstrable delayed hypersensitivity (DH) to DNFB, picryl chloride, or sheep red blood cells. Reconstitution of immune responsiveness can be accomplished by administration of cell-free lysates of spleens from mice with active DH to structurally related, but not to unrelated antigens. Peritoneal exudate cell lysates from mice with active DNFB-DH also restore DH to this antigen. Sera from sensitized mice, and sera and lymphoid tissue extracts from unsensitized mice are without activity. The restorative property of splenic lysates from DNFB-sensitized mice is unstable at 56°C, not sedimented at 90,000 × G and inactivated by trypsin or magnesium ions. The presence of unexpressed, restorable DH may provide a biologic basis for the so called “transfer factor” phenomenon.
Effect of LDL-In, a Normal Immunoregulatory Human Serum Low Density Lipoprotein, on the Interaction of Macrophages with Lymphocytes Proliferating in Response to Mitogen and Allogeneic Stimulation
Authors: Curtiss Linda K. , Edgington Thomas S.
Published: December 2022

The immunoregulatory properties of LDL-In, a normal species of human serum low density lipoprotein which suppresses inductive events involved in triggering of lymphoid cells by lectins and antigens, were analyzed in order to distinguish between a primary effect on macrophages and lymphocytes. LDL-In was found to be equally effective in suppression of the response of human lymphocytes to PHA at concentrations of lectin demonstrated to impart apparent macrophage-independence or macrophage-dependence to the culture system. Exposure of only the macrophages to LDL-In was shown to be without effect on subsequent in vitro lymphocyte responses to either PHA or allogeneic cells, whereas exposure of only the lymphocytes to LDL-In was fully effective. The cellular locus was further identified by demonstrating that the responder lymphocytes, but not the stimulator lymphocytes, were the target of the suppressive activity in mixed lymphocyte reactions. These data considered in conjunction with previous studies suggest that the primary untriggered lymphocyte is the most probable cellular target for the bioregulatory effects of LDL-In.
Two Migration Inhibitory Factors with Different Chromatographic Behavior and Isoelectric Points
Authors: Remold Heinz G. , Mednis Alma D.
Published: December 2022

Migration inhibitory factor (MIF), produced by stimulation of guinea pig lymph node cells with concanavalin A, was fractionated by Sephadex G-100 gel filtration, sucrose density gradient electrophoresis, and isoelectrofocussing. Two distinct species were identified and separated. One, pH 3-MIF, has an isoelectric point of 3.0 to 4.5 and elutes from Sephadex G-75 columns with molecules having an apparent m.w. of 65,000 (Kd of 0.05 to 0.12). The other, pH 5-MIF, has an isoelectric point of 5.0 to 5.5 and elutes with molecules having an apparent m.w. between 25,000 and 43,000 (Kd of 0.15 to 0.23).
Studies on the Generation of B Lymphocytes in Fetal Liver and Bone Marrow
Authors: Owen John J. T. , Wright Donald E. , Habu Sonoko , Raff Martin C. , Cooper Max D.
Published: December 2022

With the use of immunofluorescence techniques, cells containing cytoplasmic IgM (cIgM+), but lacking detectable surface IgM (sIgM+), have been identified in mouse fetal liver and adult bone marrow as a distinct cell population to sIgM+ B lymphocytes. We have shown that there is a considerable difference in the rate of entry of cIgM+ and sIgM+ cells into DNA synthesis in these locations. Moreover, within the cIgM+ population, the largest cells are the main group entering DNA synthesis. Our results are compatible with the notion that a pool of rapidly proliferating, large cIgM+ cells is present in fetal liver and adult bone marrow and that these cells give rise to populations of smaller cIgM+ cells, which move out of cell cycle, and convert to sIgM+ B lymphocytes. However, we recognize that this interpretation is speculative.

Finally, we have shown that fetal bone marrow is a site of generation of sIgM+ B lymphocytes, but the question as to whether these cells are derived from Ig- precursors within marrow itself remains open.
Specific T Cell-Dependent, Antigen-Antibody Mediated Suppression of Macrophages: Abrogation by Nonspecifically Stimulated T Cells
Authors: Rao V. Srinivasa , Mokyr Margalit Birnbaum , Gershon Richard K. , Mitchell Malcolm S.
Published: December 2022

The injection of hyperimmune antibody to leukemia L1210 into allogeneic mice before challenge with L1210 suppressed the ability of their peritoneal macrophages to take up L1210 cells in the presence of specific cytophilic antibody. This suppression has previously been shown to be thymus-dependent. Although the adoptive transfer of syngeneic normal thymocytes i.v. failed to overcome the suppression of macrophages, administration of an unrelated alloantigen, leukemia EL4, together with syngeneic thymocytes, did reverse suppression. The transfer of allogeneic thymocytes, without further antigen, also overcame suppression thereby enabling them to attach L1210 normally. Syngeneic peripheral (splenic) T cells with alloantigen, or allogeneic peripheral T cells, were somewhat less effective than thymocytes at reversal, and non-T cells were completely ineffective. Pretreatment of the donors of syngeneic thymocytes with unrelated alloantigen i.p. from a minimum of 4 hr to 14 days before transfer also restored functional competence to the macrophages. Nonspecific antigenic stimulation of a population of naïve thymocytes appears to prevent their recruitment as suppressor T cells, perhaps by changing the individual cell, by altering the cellular composition of the thymus gland, or by both. These data point up similarities between the “enhancement” phenomenon (immunosuppression caused by antibody and antigen) and “infectious immunologic tolerance,” in both of which T cells are intimately involved. The data also suggest that the macrophage may be a cellular site of specific immunologic unresponsiveness.
Multiple Polysaccharide Antigens of Group B Streptococcus, Type Ia: Emphasis on a Sialic Acid Typespecific Polysaccharide
Authors: Kane Judith A. , Karakawa Walter W.
Published: December 2022

Group B streptococci, type Ia (strain 090/14/4), were subjected to sequential extraction procedures and the various extracts were fractionated by a combination of DEAE-cellulose chromatography and gel-filtration. Three major antigens were isolated: the conventional group-specific and type-specific carbohydrates and an acidic polysaccharide consisting of galactose, glucose, glucosamine, and sialic acid. Immunochemical data suggest that the acidic antigen, with the exception of the immunodominant sialic acid, is structurally similar to the conventional pH 2.0 extracted type-specific antigen. Removal of the terminal sialic acid residues from the acidic antigen by mild acid hydrolysis resulted in a residual polymer which was chemically and immunologically analogous to the type-specific carbohydrate. Precipitin analysis focused attention to the cross-reactivity between this acidic antigen and anti-types Ib, Ic, and III sera.
Macrophage Function in Tumor-Bearing Mice: Tumoricidal and Chemotactic Responses of Macrophages Activated by Infection with Mycobacterium Bovis, Strain BCG
Authors: Meltzer Monte S. , Stevenson Mary M.
Published: December 2022

Peritoneal macrophages from mice with syngeneic transplantable footpad tumors were less responsive in vitro to chemotactic stimuli than were macrophages from mice without tumors. The macrophage chemotactic defect was about half the normal response and was evident in mice with nonpalpable 3 to 5-day tumors. Depressed macrophage chemotaxis persisted until the death of the mouse at 6 to 8 weeks. The number of peritoneal exudate cells elicited in tumor-bearing mice 8 to 14 days after intraperitoneal infection with BCG was also depressed. This inflammatory defect was detected 1 week after tumor transplantation and persisted for more than 5 weeks. The yield of peritoneal exudate macrophages induced by BCG infection in mice with tumors was about half the normal yield. In contrast to resident peritoneal macrophages, peritoneal exudate macrophages induced by BCG in tumor-bearing mice were as responsive to chemotactic stimuli as BCG-activated macrophages from control mice. BCG-activated macrophages from tumor-bearing or control mice were cytotoxic to tumor cells in vitro; no differences in tumoricidal responses of these BCG-activated macrophages were evident. The number of BCG organisms necessary to induce activated tumoricidal macrophages in mice with or without tumors was also identical. Peritoneal exudate macrophages from tumor-bearing mice infected with BCG at a dose that did not induce tumoricidal cells showed chemotactic defects similar to those of the resident macrophages. These results suggest that the response to BCG infection in tumor-bearing mice was defective. The defect was quantitative: a sequela of abnormal inflammatory responses. BCG-activated macrophages from tumor-bearing and control mice were functionally indistinguishable.
Quantitation of Measles Virus-Specific Immunoglobulins in Serum, CSF, and Brain Extract from Patients with Subacute Sclerosing Panencephalitis
Authors: Mehta P. D. , Kane A. , Thormar H.
Published: December 2022

The amount of measles-specific IgG in sera, CSF, and brain extracts from patients with SSPE was determined by radial immunodiffusion method with the use of a number of rabbit antisera specific for human γ-chain, after each sample was abosrbed suitably with a lyophilized preparation of measles-precipitating antigen (MPA) made in Vero cells infected with Edmonston strain of measles virus. The measles-specific IgG contents of the sera ranged from approximately 10 to 20% and those of CSF and brain extracts from 25 to 75% of the total IgG, indicating a synthesis of measlesspecific IgG within the CNS. By employing the same method, a significant amount of measles-specific IgM was demonstrated in sera of three out of six SSPE patients. Sera, CSF, and brain extracts from controls when absorbed with MPA and quantitated by the same method did not show significant amounts of measles-specific IgG. When the agarose gel electrophoretic patterns of unabsorbed and MPA-absorbed SSPE brain extracts were compared, the absorbed extracts were found to lack homogeneous bands in a γ-globulin region in contrast to the presence of two to three homogeneous IgG bands seen in the unabsorbed brain extracts. On the other hand, the patterns of unabsorbed and absorbed SSPE sera did not show such a significant difference in γ-globulin region. These data strongly support the concept that measles virus is responsible for the formation of homogeneous bands within the CNS in SSPE.
On the Mechanism of Cell Membrane Damage by Complement: Evidence on Insertion of Polypeptide Chains from C8 and C9 into the Lipid Bilayer of Erythrocytes
Authors: Hammer Carl H. , Shin Moon L. , Abramovitz Aaron S. , Mayer Manfred M.
Published: December 2022

The preceding paper (Hammer, C. H., A. Nicholson, and M. M. Mayer, 1975, Proc. Natl. Acad. Sci., 72:5076) presented evidence on insertion of polypeptide chains from the C5b and C7 subunits of C5b, 6, 7 complex into the phospholipid bilayer of erythrocyte membranes. In the present study, EAC1–8 and EAC1–9 (sheep erythrocytes carrying rabbit antibody and complement proteins C1 through C8 or C9, respectively), prepared with either 125I-C8 or 125I-C9, were incubated with trypsin or chymotrypsin and the release of 125I was measured. Only 9 to 19% of the specifically bound radioactivity was released. In addition, elution experiments were performed with 0.02 M EDTA-1.0 M NaCl. This solution did not elute C9 from EAC1–9. By contrast, cellbound C9 was recovered from erythrocyte membranes with sodium dodecyl sulfate (SDS). Thus, enzymatic stripping and elution experiments indicate that cellbound C9 behaves like an integral membrane protein, presumably due to insertion into the lipid bilayer.

EAC1–9 membranes that had been subjected to extended digestion with trypsin or chymotrypsin were extracted with SDS to recover the enzyme-resistant part of the C9 molecule from the membrane. Even though this domain of C9 carried 90% of the radioiodine associated with native C9, its m.w. was found to be only 18,000 daltons by analysis on SDS-PAGE. This represents one-quarter of the native C9 molecule.
Human T Lymphocyte Receptors for Sheep Red Blood Cells and Specific Antigens: Are They Indentical Sites on the Cell Membrane?
Authors: Galili U. , Klein E. , Schlesinger M.
Published: December 2022

The cell surface of human T lymphocytes is characterized by the presence of specific antigens and by E-receptors for sheep red blood cells (SRBC). The exposure of human T cells to specific anti-T sera inhibits the formation of E-rosettes. The relationship between E-receptors and specific T cell antigens was investigated in a number of human cell lines and in enzyme-treated lymphocytes. Cells of the Molt-4 line were found to be susceptible to the cytotoxic effect of rabbit anti-human thymus serum (RAHT) rendered specific for human T cells by absorptions. The line of Molt-4 tested failed to form E-rosettes with either untreated or neuraminidase-treated SRBC. Thus, Molt-4 cells seemed to possess T cell-specific antigens in the absence of detectable E-receptors. A second line, K-562, formed E-rosettes with neuraminidase-treated SRBC. Although K-562 cells seemed to possess E-receptors similar to those on PBL, they were resistant to the cytotoxic effect of RAHT, and exposure to RAHT failed to inhibit the formation of E-rosettes by K-562 cells. Trypsin treatment of either PBL or thymus cells totally eliminated their ability to form E-rosettes, but did not significantly alter their susceptibility to the cytotoxic effect of RAHT. Although E-receptors may be located in close proximity to T cell-specific antigens on the membrane of T lymphocytes, the present findings indicate that these two surface markers constitute distinct entities.
Depressed Mononuclear Leukocyte Chemotaxis in Thermally Injured Patients
Authors: Altman Leonard C. , Furukawa Clifton T. , Klebanoff Seymour J.
Published: December 2022

Microbial infection is the leading cause of morbidity and mortality in thermally injured patients. Since normal mononuclear leukocytes (MNL) function is essential in host defense, the chemotactic response of MNL from 25 thermally injured patients was evaluated in vitro. Patients with burns involving <20% total body surface had normal MNL chemotaxis, while MNL from patients with burns ≽20% had significantly depressed chemotaxis. This defect was most pronounced in patients with burns ≽40% and in those who died. Analysis of the data as a function of time revealed that patients who died generally had depressed chemotaxis immediately postburn and that their cells showed no functional improvement with time. In contrast, surviving patients with burns ≽40% initially had normal MNL chemotaxis which decreased significantly at approximately day 15 post burn, reached a nadir about day 45, and gradually returned to normal. Since the average burn size of these two groups was very similar, these data suggest that the early chemotactic response of MNL after a burn may be a predictive index of clinical outcome. Thirteen of the 25 patients developed infections and in all but one case depressed MNL chemotaxis preceded the septic event. Sera from patients with depressed MNL chemotaxis contained a cell-directed chemotactic inhibitor which was heat stable (56°C, 30 min), non-dialyzable and distinct from the sulfadiazine used for topical therapy. These studies document that depressed MNL chemotaxis occurs as a consequence of serious thermal trauma, that this defect may be due to a serum inhibitor and that abnormal MNL chemotaxis may contribute to septic morbidity and mortality in severely burned patients.
Modification of Fetal Immune System by Maternal Anti-DNA Antibody
Authors: Sasaki Takeshi , Ono Yasushi , Ishida Nakao
Published: December 2022

The offspring mice of C3H/He strain whose mothers had been immunized with DNA or passively administered anti-DNA antibody showed enhanced ability to respond to DNA stimulation (3-fold increase in the amount of serum anti-DNA antibody and in the number of anti-DNA antibody-forming cells). The acquisition of the enhanced immune responsiveness to DNA in the offspring was most likely mediated by passively transmitted maternal antibody, not by such other factors as concomitantly transmitted antigen, carrier protein, and adjuvant. The effect of maternal antibody to carrier protein and the possible involvement of maternal lymphocytes transmitted to the progeny were also excluded. This phenomenon was observed in the mice after the transmitted antibody had been removed from them, distinguishing the effect of antibody in fetal and/or neonatal period from the regulatory effect of antibody in adult animals. The effect of maternal antibody was antigen specific. Maternal antibody might in some way modify the clonal development of fetal immune-responding cells.
Regulation of Cytotoxic Lymphocyte Responses in Vitro by Alloantigen-Activated Spleen Cells
Authors: Truitt Gary A. , Rich Robert R. , Rich Susan Solliday
Published: December 2022

Spleen cells obtained from BALB/c mice 4 days after allosensitization to C57BL/6 spleen cells via footpad injection suppressed the in vitro generation of BALB/c cytotoxic lymphocytes (CL) against C57BL/6 spleen cells in mixed leukocyte cultures (MLC). Suppressor activity was demonstrated by spleen cells at 4 and 7 days but not at 2, 10, or 14 days after allosensitization and was abolished by treatment with anti-Thy-1.2 serum and complement. A weak and transient cytotoxic response directed against the sensitizing alloantigen was associated with suppressor spleen cell populations, but was dissociated from suppressor function by two experimental approaches. First, increasing stimulatory cell concentration in MLC did not competitively diminish the suppressor activity; rather, the magnitude of suppression increased as the stimulatory cell concentration was increased. Second. BALB/c suppressor cells generated in vivo by either H-2b or H-2k alloantigens suppressed CL responses generated simultaneously against both alloantigens in vitro. CL responses generated against one or the other H-2 haplotype in vitro were suppressed only by suppressor cells activated by that haplotype. Therefore, splenic suppressor cells activated by alloantigen in vivo required antigen-specific restimulation in vitro; thereafter, responder cells syngeneic with the suppressor cell were rendered hyporesponsive to alloantigens by an antigen-nonspecific mechanism.
Neonatally Induced Tolerance to HGG: Duration in B Cells and Absence of Specific Suppressor Cells
Authors: Benjamin D. C.
Published: December 2022

A specific, long lasting, tolerant state to human γ-globulin (HGG) was established in neonatal A/J mice. These suckling mice received the tolerogen in the colostrum of their mother who had been injected with DHGG. The tolerant state could not be accounted for by “factors” other than HGG in the colostrum. The duration of this tolerance in the intact animal and in the B cell population was 16 to 18 weeks. Naturally occuring nonspecific suppressor cells were evident but specific suppressor cells could not be demonstrated. These results are discussed in relation to possible mechanisms of the induction of tolerance to self.
Soluble Factors in Tolerance and Contact Sensitivity to 2,4-Dinitrofluorobenzene in Mice
Authors: Moorhead John W.
Published: December 2022

Tolerance to 2,4-dinitrofluorobenzene (DNFB) contact sensitivity is in part mediated by suppressor thymus-derived cells (T cells) which are induced by pretreatment with the hapten 2,4-dinitrobenzenesulfonate. If lymph node cell suspensions containing suppressor cells are cultured in vitro, soluble suppressor factor (SSF) is released into the supernatant. When DNFB-immune lymph node cells are incubated with SSF, their ability to transfer contact sensitivity to normal recipients is suppressed. In order for SSF to be produced and/or released, it was necessary to paint the tolerant animals with DNFB 16 to 20 hr before the lymph node cells were cultured, suggesting that SSF was made in response to antigen stimulation. Specificity studies showed that SSF was both antigen specific and strain specific in its action. In addition, it was found that SSF could be absorbed by 2,4-dinitrophenyl-keyhold limpet hemocyanin (DNP-KLH) (not by KLH alone) and by anti-H-2 antibodies but not by trinitrophenyl-KLH, anti-immunoglobulin, or anti-DNP antibodies. Taken together, these results indicate that SSF is a product of the major histocompatibility complex which, although not antibody, has affinity and specificity for the hapten DNP. Furthermore, in order for SSF to suppress DNFB sensitivity, identity is required among genes in the H-2 complex between the donor of SSF and the immune lymph node cells.
Fc Receptors on Mouse Effector Cells Mediating Natural Cytotoxicity Against Tumor Cells
Authors: Herberman Ronald B. , Bartram Scott , Haskill J. Stephen , Nunn Myrthel , Holden Howard T. , West William H.
Published: December 2022

Mouse effector cells mediating natural cytotoxicity against tumor cells have been previously thought to be lymphocytes that lack any detectable cell surface markers. The present study presents evidence for receptors for the Fc portion of IgG on these cells. By adsorption of cytotoxic spleen cells on monolayers of sheep erythrocytes (E) plus IgG antibodies to sheep erythrocytes (EA), 50 to 96% of the total cytotoxic reactivity could be removed. Parallel adsorption of cells on E monolayers or on EA monolayers coated with protein A, to block the Fc portion of IgG, resulted in little or no depletion of cytotoxic activity. The presence of Fc receptors on the NK cells was confirmed by combining EA rosette formation with velocity sedimentation at unit gravity. Peak cytotoxicity occurred at the same sedimentation velocity as the peak of Fc-positive cells. After EA rosette formation, there was a shift to a higher sedimentation velocity in the Fc-positive cells and in the natural cytotoxic activity. The increase in sedimentation velocity of NK activity that was observed in these experiments indicated that most of the cells had only bound a small number (three or four) of antibody-coated erythrocytes. Together, these data indicate that cells with Fc receptors account for most of the total lytic activity of normal mouse spleen cells.
Reactivity to SV40 T Antigen in Athymic (Nude), Anti-Thymocyte Serum-Treated, and Normal Mice
Authors: Knowles Barbara B. , Ford Sharon R. , Aden David P.
Published: December 2022

Antisera prepared in syngeneic mice by hyperimmunization with intact SV40-transformed mouse cells or with somatic cell hybrids between SV40-transformed human and normal mouse cells exhibit anti-SV40 tumor (T) antigen reactivity. Athymic mice bearing tumors formed by SV40-transformed mouse, human or mouse-human hybrids were not reactive with SV40 T antigen. Anti-thymocyte serum (ATS)-treated mice also lacked T antigen reactivity during suppressive treatment but developed antibody to T antigen after discontinuing ATS treatment and tumor regression. We conclude that the presence of growing tumors in the mouse is not necessary for the production of anti-SV40 T antigen antibodies but that helper thymus-derived cells are essential for the humoral response.
The in vitro Antibody Response to Cell Surface Antigens
Authors: Levy Ronald , Dilley Jeanette
Published: December 2022

An in vitro spleen fragment culture system has been developed for the production and analysis of xenogeneic antibody responses to cell surface antigens. Depending on the methods of immunization and in vitro stimulation employed, mouse spleen fragments can produce antibody of both IgG and IgM classes directed against human cell surface antigens for more than 30 days in culture.

A saturation binding analysis of the antibody products indicates that their range of specificities was more restricted than that of serum antibody. Approximately 5% of the in vitro antibody products raised against a homogeneous population of human leukemia cells could distinguish between the antigens present on the leukemia cells and those present on normal human lymphocytes. Methods previously employed to influence the range of serum antibodies expressed against complex immunogens, such as suppression of certain responses by passive administration of antibody at the time of immunization, were tested in the in vitro spleen culture system and resulted in successful modulation of the antibody response patterns observed.
The in vitro Antibody Response to Cell Surface Antigens
Authors: Levy Ronald , Dilley Jeanette
Published: December 2022

A method has been developed for the production of monoclonal mouse antibody responses in vitro against human cell surface antigens. Limiting numbers of immune spleen cells were transferred to syngeneic, irradiated recipients whose spleen fragments were then cultured in vitro and stimulated to produce antibody. The majority of the antibody from any one fragment culture was likely to be the product of a single donor B cell and thus monoclonal. Evidence for this included a linear relationship between donor cells transferred and spleen fragments producing antibody, extremely restricted isoelectric focusing patterns of the individual antibody products, and unique reactivity patterns of these antibodies against a panel of human lymphoid cells. Different human B leukemia cells were seen as immunogenically distinct by the mouse. By using the monoclonal mouse antibodies as probes, a fine analysis of cell surface antigens is now possible.
Distribution of Terminal Deoxynucleotidyl Transferase in Bovine Serum Albumin Gradient-Fractionated Thymocytes and Bone Marrow Cells of Normal and Leukemic Mice
Authors: Pazmin̄o Nelson H. , McEwan Robert N. , Ihle James N.
Published: December 2022

The distribution of terminal deoxynucleotidyl transferase (TdT) peaks I and II, in single cell suspensions of thymuses, bone marrow, and peripheral lymphoid organs fractionated in discontinuous bovine serum albumin gradients, was examined in a variety of mouse strains and Fischer 344 rats to relate the normal patterns of thymocyte differentiation to the leukemic process.

TdT peaks I and II were found in fractions A (10 to 23%), B (23 to 26%), and C (26 to 29%) of the thymus of both normal and leukemic C57BL/6 mice, whereas only peak I was found in the same fractions of AKR mice. TdT in bone marrow was found mainly in fraction A in both normal and leukemic mice. The specific activity of TdT in this fraction, which comprises only 1 to 5% of the total bone marrow cell population, was similar to that of the thymus. The cell population of fraction A of the bone marrow was found to increase (10 to 15-fold) in leukemic mice. Only low levels of TdT activity were found in either whole or fractionated bone marrow of athymic NIH Swiss (nu/nu) mice.
Human T Lymphocyte Subpopulations: Correlation Between E-Rosette-Forming Affinity and Expression of the Fc Receptor
Authors: West William H. , Payne Susan M. , Weese James L. , Herberman Ronald B.
Published: December 2022

E-rosette-forming cells (E-RFC) in human peripheral blood can be subdivided into two fractions on the basis of their relative affinity for sheep red blood cells (SRBC). “High-affinity” E-RFC constituting 54 ± 5% of normal mononuclear cell suspensions are capable of rosette formation, despite elevated temperatures of incubation (29°C) with limited numbers of SRBC. “Low-affinity” E-RFC constituting 23 ± 4% of peripheral blood mononuclear cells require cool temperatures of incubation with an excess of SRBC to form rosettes. The following evidence indicates that low-affinity E-RFC bear surface receptors for the Fc portion of IgG, whereas high-affinity E-RFC do not. a) When High-affinity E-RFC were isolated from mononuclear cell suspensions, very few (2 ± 1%) of these lymphocytes formed rosettes with IgG-sensitized chick red blood cells (EA). When the remaining low-affinity E-RFC were then isolated, up to 85% formed rosettes with chick EA. b) When lymphocytes were incubated with erythrocyte monolayers sensitized with high concentrations of IgG, low-affinity E-RFC adhered to these EA monolayers. High-affinity E-RFC failed to attach to immune complexes. c) Virtually all E-RFC in thymus, tonsil, and lymph node formed high-affinity E rosettes and failed to bind chick EA. Spleen cell suspensions, on the other hand, contained a low-affinity E-RFC subset and E-RFC capable of binding chick EA. d) The proportion of low-affinity E-RFC in the peripheral blood of normal subjects correlated closely with the proportion of Fc-positive E-RFC among the mononuclear cells of those individuals. These observations suggest a close association between the presence of the Fc receptor on human T cells and the affinity with which those cells bind SRBC. Modified E rosette procedures permit rapid identification or isolation of high-affinity, Fc-negative and low-affinity, Fc-positive E-RFC subpopulations.
Generation of Cytotoxic T Lymphocytes during Coxsackievirus B-3 Infection
Authors: Wong Catherine Y. , Woodruff Judith J. , Woodruff Jack F.
Published: December 2022

The generation of cytotoxic reactivity in the spleens of adult male and female BALB/c mice infected with Coxsackievirus B-3 was compared. Cytotoxicity was measured by using an in vitro 51Cr release assay employing syngeneic virus-infected and uninfected fibroblasts as target cells. Males exhibited a stronger response and significant levels of cytotoxicity were evident by day 3 after infection. Generally activity detected at this time was not viral specific and both infected and uninfected target cells were lysed. Treatment of day 3 male spleen cells with anti-thy-1.2 + C abolished both reactivities indicating that each was mediated by cytotoxic T cells. Male cytotoxicity peaked on day 7 and effector cells obtained at this time were virus specific. In marked contrast, day 7 female immune cells caused little or no target cell lysis. Females, however, exhibited significant levels of cytotoxicity at earlier intervals, primarily between days 3 and 6. Although this activity then faded, a second wave of reactivity was observed in some between the 10th and 17th day. At all intervals female cytotoxicity was not viral specific and uninfected target cells were lysed as well or better than viral infected cells. Treatment of day 3 female spleen cells with anti-thy-1.2 serum + C significantly diminished cytotoxicity against infected targets but had no effect on activity directed against uninfected fibroblasts. The results indicate that the female response was composed of a mixture of different effector cell populations with T cells responsible for only a portion of the activity. These sex-related differences were specific for Coxsackievirus infection and were not observed in animals infected with vaccinia virus. The cytotoxic response of Coxsackievirus infected females was correlated with the finding that viral clearance was more rapid and early antibody production more vigorous than that observed in males.
Physicochemical Consequences of Opsonization: Perturbation of Liposomal Membranes by Salmonella typhimurium 395 MS Opsonized with IgG Antibodies
Authors: Tagesson Christer , Magnusson Karl-Eric , Stendahl Olle
Published: December 2022

When phagocytosis-resistant Salmonella typhimurium 395 MS bacteria sensitized with anti-MS IgG antibodies were incubated with liposomes composed of phosphatidylcholine, cholesterol, and dicetylphosphate, a previously sequestered liposomal marker (4-methylumbelliferylphosphate) was released. Unsensitized or F(ab′)2-sensitized bacteria had no such effect. This perturbation was neither dependent upon negatively charged dicetylphosphate nor upon cholesterol. It was further evident that palmitoyl-poly(ethyleneglycol) caused released of the trapped marker in a similar way as sensitized bacteria. These findings demonstrate a similarity between sensitized bacteria and a hydrophobic probe and lend support to the hypothesis that the perturbation was brought about by hydrophobic interaction. The observations indicate that liposomes, like phagocytes, possess “receptor sites” for the activated part of IgG and raise the possibility that phagocytic effectors can operate in a relatively nonspecific manner.
Humoral Immunostimulation
Authors: Shearer William T. , Gottlieb Charlene , Kornfeld Stuart
Published: December 2022

Variant cell lines (LC1, LC2) obtained by growth of mouse L cells (L) in cytostimulatory and cytotoxic doses, respectively, of rabbit anti-L cell antiserum (AL) were found previously to be altered in many ways relative to the parent cell line. A major change was the reduction of those surface membrane antigens that AL recognizes. These cell variants have now been found to have increased membrane sialic acid relative to L. Treatment of intact variant cells with neuraminidase (50 units/ml, 37°C, 1 hr) greatly restored the susceptibility of LC1 to lysis with AL. In the presence of 1/100 dilution of AL and 5% complement the viability indices (1.00 = no cell kill) of untreated and neuraminidase-treated cells were respectively: L 0.10 and 0.03 and LC1 0.91 and 0.40. Neuraminidase-treated LC2 cells retained their resistance to AL. Parallel studies with 125I-ALIgG showed increased binding to neuraminidase-treated LC1 relative to native LC1. These findings suggest that the altered membrane sialic acid content affects the immunologic behavior of this cell variant by masking the original cell surface antibody-binding sites. This represents a possible mechanism for tumors to escape immunologic control.
Resistance of H-2 Heterozygous Mice to Parental Tumors
Authors: Clark Edward A. , Harmon Richard C. , Wicker Linda S.
Published: December 2022

Hybrid resistance to the C57BL lymphoma EL-4 and to two 3-methylcholanthrene-induced B10.A(4R) fibrosarcomas of recent origin was studied in H-2 heterozygous mice. Histocompatible tumor recipients were derived from mating C57BL/10, B10.A, and H-2a/H-2b intra-H-2 recombinant mice in such a way as to provide heterozygosity at defined portions of the H-2 complex. Hybrid resistance was assessed by comparing the mean survival times and tumor incidence of H-2 heterozygotes with those of homozygous 4R (sarcomas) or B10 (EL-4) mice. The results reveal that heterozygosity at the D end of the H-2 complex controls hybrid resistance to fibrosarcomas as well as to lymphomas. Evidence is discussed which suggests that the locus controlling hybrid resistance, Hh-1, is located within the H-2 complex between the S and D regions. Hh-1-controlled hybrid resistance was clearly demonstrable when tumor cells were inoculated by the intraperitoneal route but was not detected if tumor cells were injected subcutaneously. Both Hh-1-controlled hybrid resistance and its in vitro correlate, natural cytotoxicity, appear to be most strongly expressed in the peritoneum. Pretreatment of Hh-1 heterozygous mice with Hh-1-H-2Db parental spleen cells consistently reduced tumor resistance compared to mice pretreated with reciprocal parental Hh-1-H-2Dd splenocytes. This suggests that Hh-1 codes for or regulates the expression of cell-surface antigens found on normal H-2b parental cells. In addition, we have found that other H-2 genes, probably I region immune response genes, also influence fibrosarcoma growth in F1 hybrid hosts. Thus, several loci within the H-2 complex appear to play a role in the polygenic control of the immune response to fibrosarcomas.
Immune Response to a Mammary Adenocarcinoma
Authors: Huber S. A. , Lucas Z. J.
Published: December 2022

Sera from Fischer 344 rats bearing a 13762A ascites mammary adenocarcinoma (MTA) blocked cell-mediated cytotoxicity (CMC) by spleen cells in vitro and enhanced tumor growth in vivo. Blocking (or enhancing) factors were detected in serum 3 days after i.p. immunization; peak activity occurred on days 5 and 7 and decreased sharply thereafter. Both CMC and the blocking of CMC were immunologically specific for mammary adenocarcinoma; normal breast cells did not inhibit lysis of tumor targets. Blocking factor in sera 3 and 5 days after immunization was present only in fractions coeluting with IgG on Sephadex G-200 chromatography. The blocking factors adsorbed to and quantitatively eluted from anti-rat IgG immunoadsorbant columns. In contrast, the Sephadex G-200 distribution of blocking factors of sera 7 and 10 days after immunization was polydisperse; although major activity was eluted in the IgG area, both void volume and lower m.w. areas also blocked CMC. The blocking activities of void volume and IgG fractions of all immune sera were removed by adsorption to immune lymphocytes but not by tumor cells, nonimmune lymphocytes, or lymphocytes immunized to unrelated antigens. Pretreatment of the cytolytic cells, but not the tumor cells, with the IgG fractions of immune sera inhibited CMC as well as if they had been continuously present in the assay. Collectively, these data suggest that blocking occurs at the level of the cytolytic lymphocyte, mediated either by tumor antigen-antibody to tumor antigen complexes, anti-receptor site antibodies, or both.