bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytotoxic Lymphocytes Induced by Soluble Factors Derived from the Medium of Leukocyte Cultures
Authors: Kasakura Shinpei
Published: December 2022

Studies were undertaken to evaluate the cytotoxic capacity of human peripheral blood lymphocytes activated by either supernatants (CFM) derived from lymphocyte cultures or lymphocytes treated for 60 min at 45°C. The effect of the addition of heat-treated cells on the cytotoxic activity of CFM-induced effector cells was also studied. CFM from either unmixed or mixed cultures of lymphocytes was capable of activating cytotoxic effector cells. These effector cells could kill any allogeneic target cells but failed to effect cytotoxicity on the target cells autologous to the responding cells. Both the heat-treated cells and CFM from cultures of these cells also activated lymphocytes to cytotoxic effector cells having specific receptors for nonself antigens. The question of whether heat-treated cells activate cytotoxic cells by themselves or through secreted soluble factor cannot yet be clearly answered.

The findings of the present investigation suggest that expression of cytotoxicity induced in MLC is not necessarily restricted to the target cells syngeneic to the stimulator cells, but can be extended to any allogeneic target cells by the indirect effect of soluble factor secreted from stimulated cells that causes a polyclonal activation of cytotoxic precursors in the responding cell populations. The present findings also emphasize the need for caution in the use of heat-treated lymphocytes as innocent-bystander cells in MLC to provide additional cytotoxic specificities in the responder cells, since heat-treated cells alone can activate lymphocytes to cytotoxic effector cells that kill any allogeneic target cells.
Hapten-Specific IgE Antibody Responses in Mice
Authors: Chiorazzi Nicholas , Fox David A. , Katz David H.
Published: December 2022

Mice of the inbred strains SJL (H-2s) and AKR (H-2k) are “non-responders” and “low-responders,” respectively, in terms of their capacity to develop antibody responses of the IgE class when immunized with conventional proteins and hapten-protein conjugates under conditions optimal for eliciting IgE responses in “high-responder” mice, such as BALB/c (H-2d), to these same antigens. For example, BALB/c mice preimmunized with ASC and then challenged 7 days later with DNP-ASC develop peak augmented primary IgE anti-DNP antibody responses of 320 PCA units, whereas SJL and AKR mice develop responses which are 16-fold and 4-fold lower, respectively. However, pretreatment of the latter two strains with appropriate doses of either x-irradiation (150 R), cyclophosphamide (100 mg/kg) or ALS (150 µl) before carrier-preimmunization strikingly enhances the magnitude of IgE antibody responses in such mice to levels as high as 64-fold above those of untreated control mice of the same strains.

Evidence obtained in these experiments indicates that the capacity of such maneuvers to convert poor IgE responders to high responder status reflects elimination of nonantigen-specific suppressor T lymphocytes which are naturally present and normally function to suppress or “dampen” the IgE antibody response in a relatively selective manner. It appears that these cells modulate IgE responses by acting at least at two distinct points: 1) The most effective activity seems to be at the level of induction of carrier-specific helper T cells; 2) A second locus of inhibitory activity is more distal in the response, either impeding helper T cell-B cell cooperative interactions or suppressing B cell differentiation and/or function directly. Taken collectively, these observations demonstrate that the state of poor responsiveness of the SJL and AKR strains for the IgE antibody class is not a reflection of a genetic inability to develop IgE responses but rather a manifestation of a genetic capability to actively inhibit IgE antibody synthesis.
A Disaccharide Hapten from Streptococcal Group C Carbohydrate that Cross-Reacts with the Forssman Glycolipid
Authors: Coligan John E. , Fraser Blair A. , Kindt Thomas J.
Published: December 2022

A disaccharide hapten was isolated in approximately 20% yield from an acid hydrolysate of the streptococcal Group C carbohydrate. This disaccharide was assigned the structure 3-0-α-N-acetylgalactosaminosyl-N-acetylgalactosamine on the basis of chemical and immunochemical data. The ability of the hapten to inhibit completely the binding between Group C carbohydrate and most Group C antibodies indicated that this disaccharide is the immunodominant feature of the Group C carbohydrate. Fractionation of antisera on an α-N-acetylgalactosamine immunoadsorbent column yielded several populations of anti-Group C antibodies. The reaction between labeled Group C carbohydrate and antibodies which strongly bound the immunoadsorbent column was 50% inhibitable by the disaccharide hapten when the hapten was added in approximately 15-fold excess (w/w) of the labeled antigen. On the other hand, the binding of those antibodies which did not bind to the immunoadsorbent column was poorly inhibited under these conditions. The Forssman glycosphingolipid, which has a common terminal digalactosamine unit, was shown to likewise inhibit Group C carbohydrate binding reactions.
Regulatory Factors Produced by Lymphocytes
Authors: Lee Sheng-Chung , Lucas Zoltan J.
Published: December 2022

Supernatants of phytohemagglutinin-stimulated human tonsil cells contain two growth inhibitory factors. These factors, called inhibitors of DNA synthesis (IDS), reduce 3H-thymidine incorporation into mitogen-stimulated lymphocytes and into growing HeLa cells. By Sephadex chromatography, these factors have volumes of distribution corresponding to about 80,000 and 40,000 daltons. Both factors inhibit the activity of calf thymus DNA polymerase α in cell-free assays (termed inhibitor of DNA polymerase, IDP). The larger factor, which is chromatographically separable from α-lymphotoxin (α-LT), is completely inactivated by heating at 70°C for 15 min. This treatment does not destroy α-LT. Using supernatants from PHA-stimulated tonsil cells cultured for 5 days in serum-free medium, we attained a 150-fold purification with a succession of molecular sieving, ion exchange, and adsorption chromatographic procedures. Although not purified to homogeneity, the extensive copurification of IDS and IDP activities and their identical heat inactivation profiles suggest that they are the same entity. IDP separated free of α-LT inhibits thymidine incorporation into HeLa cells without causing cell death. α-LT purified free of IDS does not inhibit thymidine incorporation into HeLa cells, not even at concentrations 7000 times that necessary to kill 50% of growth-inhibited L cell cultures.
Murine and Rat IgE: Relationships in Terms of Binding to Cell Receptors and to Antibodies against Rat Epsilon Chain
Authors: Lang Glen M. , Conrad Dan H. , Kelly Ken A. , Carter Brian G. , Froese Arnold , Sehon Alec H.
Published: December 2022

The similarity between murine and rat IgE was examined in terms of their fixation to target cells and interaction with monospecific antibodies to rat ε-chain (anti-ε). Purified rat monoclonal IgE (IgEr) was found to block the fixation of murine reagin (IgEm) to mouse and rat skin and to rat basophilic leukemia (RBL) cells. The capacities of mouse reaginic serum (MRS), rat reaginic serum, and IgEr to inhibit the binding of radiolabeled 125I-IgEr to RBL cells were shown to be similar. These results suggest that the binding of IgE of either species occurs on the same or on adjacent receptor sites of mast cells and RBL cells. The antigenic cross-reactivity between IgEm and IgEr was established by depletion of the reaginic activity from MRS by treatment of MRS with anti-ε. The reaginic activity of MRS could be recovered by the addition of IgEr to anti-ε:IgEm complexes. From these findings it may be inferred that i) IgEm and IgEr share some antigenic determinants and ii) the regions of the immunoglobulins responsible for fixation to receptors on mast cells and RBL cells are identical or similar.
Immune Response to Immunization Via the Anterior Chamber of the Eye
Authors: Kaplan Henry J. , Streilein J. Wayne
Published: December 2022

The immunizing abilities of alloantigens placed within the anterior chamber of the eye have been studied in inbred rats. Although intracameral inoculation of F1 hybrid lymphocytes into parental strain recipients elicited both cell- and antibody-mediated immunity, a delimited interval was identified postinoculation during which the systemic cell-mediated immune response was suppressed as indicated by prolonged acceptance of orthotopic skin allografts. The prompt appearance of hemagglutinating antibodies in the serum of immunized rats followed a time course which coincided with the suppression of cell-mediated immunity and suggested that the two events are causally related. Since exposure to allogeneic antigens on lymphoid cells via the anterior chamber elicits a transient suppression in cell-mediated immunity, whereas humoral immunity is preserved, the phenomenon resembles immune deviation.
Analysis of the Major Histocompatibility Complex in Syrian Hamsters
Authors: Duncan William R. , Streilein J. Wayne
Published: December 2022

Among the genetic loci incorporated into the major histocompatibility complex in every species studied to date have been prominent genes encoding for strong histocompatibility determinants that elicit detectable alloantibody responses and which are the chief antigenic targets of cell-mediated cytotoxicity reactions. The K and D regions of the H-2 complex in the mouse and the A, B, and C regions of the HLA complex in man are representative examples. Syrian hamsters, as described in this report, do not make alloantibodies to antigens of this type and only very poorly do they carry out in vitro cell-mediated cytotoxicity to target cells putatively bearing these antigens. Since hamsters are quite capable of discriminating analogous antigenic differences in xenogeneic species, and xenogeneic sources cannot distinguish immunologically between the antigens encoded by the two hamster major histocompatibility alleles, Hm-1a and Hm-1b, we conclude that the hamster strains we work with are serologically indistinguishable by the methods used here. However, they obviously differ for determinants which elicit T cell-mediated responses, as evidenced by their ability to express acute skin graft rejection, mixed lymphocyte reactivity, graft-vs-host reactions, and cell-mediated cytotoxicity reactions. Such alloreactivity may reflect a mutation at an SD locus, affecting antigenic sites recognized only by T cells, or that the available hamster strains are SD identical, but differ at loci similar to the I region loci in mice. Alternatively, we cannot exclude the possibility that Syrian hamsters somehow fail to express properly the genes coding for SD determinants.
Functional Characteristics of Peyer's Patch Lymphoid Cells
Authors: Kagnoff Martin F.
Published: December 2022

The frequency of B cells in Peyer's patches from normal BDF1 and sheep red blood cell (SRBC)-fed BDF1 mice that could respond to antigenic determinants on SRBC and trinitrophenyl (TNP) was determined using an in vitro system of limiting dilution analysis. In normal mice, one B cell in 1.9 × 104 Peyer's patch cells could be induced to an anti-SRBC response and one B cell in 3.6 × 104 Peyer's patch cells could be induced to an anti-TNP response. The frequency of B cells capable of responding to SRBC in normal mice was similar in Peyer's patches and spleen. However, after feeding mice SRBC for 3 weeks, there was a 6-fold reduction in the frequency of B cells in Peyer's patches capable of responding to SRBC but no change in the frequency of B cells capable of responding to TNP. The average clone size of Peyer's patch B cells responding to SRBC was similar in normal and SRBC-fed mice. Although antigen-feeding does not stimulate Peyer's patch B cells in situ to humoral antibody synthesis, antigen-feeding can markedly alter the reactivity of the antigen-sensitive cell population in Peyer's patches. We previously demonstrated that T cells in Peyer's patches could be specifically carrier primed for helper function by SRBC feeding. We have now demonstrated that antigen-feeding reduced significantly the frequency of B cells in Peyer's patches capable of responding to the fed antigen. Peyer's patches appear to serve an important function as a sampling site for intestinal antigens.
Cyclic Antibody Formation to Polyglycerophosphate in Normal and Injected Rats
Authors: Bolton Ronald W. , Rozmiarek Harry , Chorpenning Frank W.
Published: December 2022

Normal adult Sprague-Dawley rats were bled serially over a 30-week period and their sera were examined for antibodies to polyglycerophosphate (PGP) by a standardized passive hemolysis test. Levels of “natural” antibodies to PGP fluctuated during this period with a majority of animals exhibiting pronounced cycling of serum antibody levels, however, the individual cycles were not synchronized with each other. Feeding of radiolabeled Gram-positive bacilli to these animals and examination of lymphoid tissue by liquid scintillation counting revealed that the antigen persisted mainly in the mesenteric lymph nodes. A second group of rats was injected i.v. with radiolabeled Gram-positive bacilli and tissues were examined for plaque-forming cells (PFC) of PGP specificity, and the sera were examined by passive hemolysis. Cycling of both anti-PGP antibodies and PFC became synchronized in the injected animals with peaks of serum antibody evident at 16 and 28 days post-injection and splenic PFC peaks at 4 and 16 days post-injection. Cycling was also observed in the mesenteric lymph nodes and bone marrow. Examination of lymphoid tissue from the rats injected i.v. revealed that antigen introduced by this route also persisted mainly in the mesenteric lymph nodes. This report demonstrates individual cycling of natural responses to environmental antigen and to the same determinant in secondary responses, indicating its importance as a regulatory mechanism.
Mechanisms of B Cell Tolerance
Authors: Bankert Richard B. , Mayers George L. , Pressman David
Published: December 2022

The bacterial dextran B1355, which is normally a potent thymus-independent immunogen, was made tolerogenic by oxidation. The injection of the oxidized dextran into BALB/c mice before, at the same time, or up to 4 days after the injection of the immunogenic form of the dextran resulted in a marked immunologically specific suppression of the number of anti-dextran antibody-forming cells found in the spleen. This suppression resulted from a direct inactivation of antibody-forming cell precursors rather than from either inhibition of antibody secretion or the exhaustive utilization of precursor B cells that have been observed in other tolerance systems. A substantial degree of tolerance was achieved after only a 1-hr in vivo exposure of the spleen cells to the tolerogen. At a dose of 1 mg of oxidized dextran per mouse, tolerance persisted for at least 3 weeks. A complete recovery was apparent by 10 weeks. The stability of the tolerance was demonstrated by transferring tolerant spleen cells to irradiated recipients. The response in the recipient animals to an immunogenic dextran challenge remained suppressed. It appears that the tolerogenicity of the oxidized dextran is due to its ability to couple covalently with free amino groups in or near the receptor site of the cell membrane via the reactive dialdehyde groups of the dextran.
Inhibition of Polymorphonuclear Leukocyte Capping by A Chemotactic Factor
Authors: Mandell Brian F. , Spilberg Isaias , Lichtman Jeff
Published: December 2022

Incubation of human polymorphonuclear leukocytes with colchicine and fluorescein-concanavalin A leads to the formation of a polarized cap of fluorescence not seen if cells are incubated with fluorescein-Con A alone. When cells are preincubated with a chemotactic factor before colchicine treatment, the capping is inhibited in a dose-related manner. Studies with α-methylmannoside indicate that the caps represent extracellular fluorescein-Con A and are not areas of Con A internalization. Experiments utilizing an irreversible inhibitor of serine esterases suggest that a chemotactic factor-activated enzyme is involved in the inhibition of cap formation in the human neutrophil.
Neutropenia Induced by Systemic Infusion of Chemotactic Factors
Authors: O'Flaherty Joseph T. , Showell Henry J. , Ward Peter A.
Published: December 2022

Autologous plasma, when exposed to complement-activating agents that generate leukotactic activity, induces transient neutropenia after infusion into rabbits. Trypsinized human C5 as well as the chemotactic fragment isolated from zymosan-activated human serum also induces transient neutropenia in rabbits, as do chemotactically active synthetic tripeptides. In each instance the neutropenia-inducing activity parallels the chemotactic activity. Thus, neutropenia-inducing activity and chemotactic activity seem to be related physiologic responses of neutrophils. In vivo margination and/or agglutination of leukocytes may underlie this phenomenon of neutropenia.
Modulation of Lipopolysaccharide (LPS)-Mediated Function by Structural Differences of Two Physically Distinct Fractions of Escherichia Coli K235 LPS
Authors: Goodman Michael G. , Morrison David C. , Weigle William O.
Published: December 2022

Lipopolysaccharide (LPS), extracted from Escherichia coli K235 by the butanol water technique, was fractionated by gel filtration chromatography into high m.w.(LPS I) and low m.w.(LPS II) fractions. These two forms of LPS were characterized by different densities and chemical compositions. Chemical analysis provided evidence for greater amounts of lipid A and lipid A-associated protein (LAP) per unit weight associated with LPS II. The biologic activity of the two LPS preparations was compared over a spectrum of different parameters. LPS II was shown to be a more potent mitogen and toxin than LPS I, whereas the two preparations were demonstrated to be of equal activity as polyclonal B cell activators, immunogens, and adjuvants. A modulatory role for the polysaccharide component of the LPS molecule is discussed.
Regulatory Mechanisms in Cell-Mediated Immune Responses
Authors: Nadler Lee M. , Hodes Richard J.
Published: December 2022

Two antigen-nonspecific T cell-dependent suppressor systems were compared for their effects upon CML and MLR. Suppressor cells generated by an in vitro culture of spleen cells were compared with suppressor cells generated by in vivo priming with alloantigen. Culture-induced suppressor cells were themselves unable to respond in CML or MLR; were able to suppress actively the CML and MLR responses of untreated responding cells; were mitomycin-sensitive; and, produced no easily demonstrable suppressive supernatant. Alloantigen-primed cells were able to respond in CML and MLR; could suppress proliferation in MLR, but were able to suppress CML only after mitomycin treatment; and, produced suppressive supernatants active in suppressing both CML and MLR. In addition to cataloging the differences and similarities between these suppressor populations, the data have been employed to analyze the mechanisms by which suppression occurs in CML and MLR.
Specificity and Structural Analysis of a Guinea Pig Transfer Factor-Like Activity
Authors: Dunnick Wesley A. , Bach Fritz H.
Published: December 2022

A transfer factor-like activity was prepared by Sephadex G-25 chromatography of immune guinea pig leukocyte lysates. This isolated material leads to antigen-dependent migration inhibition and thymidine uptake by nonimmune lymphoid cells. Tests of the “transfer factor” from guinea pigs immunized to either ovalbumin or bovine γ-globulin demonstrated the donor specificity of the in vitro activity. The activity is susceptible to heat (56°C), alkali (0.5 M sodium hydroxide), pronase, and phosphodiesterase. The pronase susceptibility is blocked by traysylol, a protease inhibitor; the phosphodiesterase susceptibility is not blocked by traysylol. The guinea pig factor was purified further by alkaline phosphatase treatment, Sephadex G-25 chromatography, and DEAE-cellulose chromatography. The final product, active in vitro, represents about 0.03% of the cellular material absorbing 260 nm light, and contains polymerized amines and phosphate. Gel electrophoresis of the fluram-reactive components suggests a limited heterogeneity of the DEAE-cellulose-purified material. These data are consistent with the active “transfer factor” molecule including both peptide and phosphate-containing components.
Antibody-Hapten Reaction Kinetics: A Comparison of Hapten Interactions with IgG and Fab Preparations
Authors: Skubitz Keith M. , O'Hara Donald S. , Smith Thomas W.
Published: December 2022

Association and dissociation kinetics for the interaction of hapten with both IgG and Fab preparations from a high affinity rabbit antiserum were determined by recently described techniques by using dextran-coated charcoal for rapid separation of free and antibody-bound hapten. The association and dissociation rate constants were determined for the interactions of 3H-ouabain with an IgG preparation, an Fab preparation obtained by treatment of IgG with papain followed by Sephadex G-100 gel permeation chromatography, and a mixture containing approximately equal concentrations of Fab and IgG-binding sites. These data were then analyzed for evidence of cooperativity of the two binding sites of IgG. Association rate constants for interactions of IgG and Fab with 3H-ouabain were not significantly different [3.43 ± 0.29 (S.D.) × 106 M-1 sec-1 and 3.36 ± 0.26 × 106 M-1 sec-1, respectively]. Dissociation data revealed no significant difference in dissociation kinetics between preparations of IgG, Fab fragments, or a mixture of the two containing approximately equal concentrations of binding sites. The ratios of association and dissociation rate constants were in satisfactory agreement with average intrinsic association constants determined under equilibrium conditions. The results are interpreted as showing no demonstrable cooperativity between the two binding sites of the IgG preparation under study in its interaction with the hapten 3H-ouabain.
Suppressor Lymphocytes in Young and Aged Humans
Authors: Hallgren H. M. , Yunis E. J.
Published: December 2022

Using human peripheral lymphocytes activated by concanavalin A (ConA), we observed suppression of the normal lymphocyte response to mitogens and allogeneic cells. The suppressive effect requires at least a 24 hr activation period of lymphocytes with ConA and is dose dependent. Activation of the suppressor population and stimulation of the responding population with mitogenic doses of ConA caused suppression of the response to mitogens, whereas activation and stimulation with submitogenic doses generally caused enhancement of the response. Responding lymphocytes showed individual variation in their ability to be suppressed by ConA-activated lymphocytes. This variable suppression could be demonstrated in autologous or allogeneic lymphocytes stimulated by ConA and also in the mixed lymphocyte reaction. ConA- activated lymphocytes derived from an aging human population differ from those of young individuals in showing a variable loss of suppressor activity. The decline of this suppressive phenomenon in aged humans may provide an explanation for the increased frequency of autoantibodies seen in the aging human population.
Studies on Demyelination by Activated Lymphocytes in the Rabbit Eye
Authors: Brosnan Celia Fildes , Stoner Gerald L. , Bloom Barry R. , Wisniewski Henryk M.
Published: December 2022

The rabbit eye model has been used to investigate the role of antibody to CNS antigens in effecting primary demyelination. Serum from rabbits immunized with homologous spinal cord in complete Freund's adjuvant was injected into the vitreous of normal rabbits, alone, or admixed with the supernatants of activated lymphocytes which induce a mononuclear cell infiltrate. Neither immune serum alone nor the nonspecific inflammatory response induced by the lymphocyte supernatants was capable of causing primary demyelination. In contrast, injection of products of activated lymphocytes plus anti-CNS antibodies induced focal primary demyelination. The demyelinating activity was found in the serum of animals that had been specifically immunized to CNS antigens and was not found in the serum of normal rabbits or serum from rabbits immunized with homologous lung or kidney in FCA or FCA alone. By ammonium sulfate precipitation the factor was shown to be present in the globulin fraction of the serum. It was removed by an immunoadsorbent column of sheep anti-rabbit IgG, and the adsorbed IgG, upon elution, retained the activity. It was relatively stable to heating at 56°C for 30 min. Light microscopic examination indicated that the primary demyelination occurred in the vicinity of the invading cells, and electron microscopy suggested an active stripping of myelin by these cells. These results establish an antibody-dependent cell-mediated mechanism for primary demyelination.
Development of Cellular Anti-Tumor Immunity in Chickens Bearing Tumors Induced by Rous Sarcoma Virus
Authors: Israël Evelyne , Wainberg Mark A.
Published: December 2022

The technique of peripheral lymphocyte stimulation in response to antigen was used to demonstrate the existence of cell-mediated anti-tumor immunity in chickens bearing tumors induced by avian sarcoma viruses. The expression of this anti-tumor response against virus-containing transformed cell culture supernatant fluids and cell extracts varied among animals in terms of time after inoculation with the oncogenic agent. Animals whose tumors had completely regressed rapidly lost the ability to mount such continuing immunity. In addition, we found evidence that some normal animals express endogenous levels of natural immunity against neoplasms induced by these viruses and/or against the viruses themselves.
The Effect of BCG on Experimental Cutaneous Leishmaniasis in Mice
Authors: Weintraub Johathan , Weinbaum Fredric I.
Published: December 2022

The effect of administration of live Bacillus Calmette-Guerin (BCG), a nonspecific immunostimulant, on the course of experimental cutaneous leishmaniasis in mice was investigated. BALB/c mice were injected in the footpad with Leishmania tropica, NIH S-strain; in mice that were not pretreated with BCG this produced a reproducible fatal infection characterized by local inflammation, regional lymphadenopathy, and dissemination of parasites with hepatosplenomegaly. In mice that were pretreated with BCG and similarly infected with L. tropica there was a reduction in the severity of cutaneous disease and a significant (p < 0.005) decrease in mortality without evidence of visceralization.
Letters to the Editor
Authors: Junger William J.
Published: December 2022

Sir: After reading the interesting report by Noble et al. (1) on thyroid antibodies in spontaneous autoimmune thyroiditis in the Buffalo rat, I was unable to reconcile myself with their statement in the Introduction that “the disease appears first at the age of 4 months” and their statement in the Abstract which read in part “When the rats were sacrificed at 4 months, at a time when spontaneous disease is not seen in untreated animals….” Moreover, as can be seen from the accompanying table, spontaneous thyroiditis has been documented in female Buffalo rats only 3-months old. As well, it is unfortunate that Noble et al. did not distinguish between sexes with regard to their neonatally thymectomized animals.

William J. Junger

Thyroid Research Laboratory

Faculty of Medicine

Memorial University of Newfoundland

St. John's, Newfoundland

Canada A1B 3V6
Functional and Morphologic Characterization of Human T Cells Continuously Grown in Vitro
Authors: Ruscetti Francis W. , Morgan Doris A. , Gallo Robert C.
Published: December 2022

Long-term growth (now over 13 months) of thymus-derived lymphocytes from numerous normal human bone marrow and peripheral blood cell samples was accomplished by using a factor present in media obtained from mitogenstimulated human peripheral blood lymphocytes. This long-term growth could neither be initiated nor maintained by mitogens alone. All cell cultures were greater than 90% E rosette-positive, whereas the tests for B cell markers, surface IgG and IgM, and EAC rosette were routinely negative. There was no evidence for the presence of granulocytes, monocytes, and their precursors in these cultures. The E rosette-positive cells were then tested to see if they had T cell functions. PHA, Con A, and pokeweed mitogens stimulated lymphproliferative responses in these cultures comparable to those of fresh peripheral blood cells. These proliferating cells were also able to release cell mediators, such as interferon and colony-stimulating activity. Further evidence for the T lymphocyte nature of these cultured cells was obtained from one-way mixed leukocyte cultures in which these cells responded to but were unable to stimulate allogeneic cells. The functional and morphologic characteristics of these cultured cells show that these cells are T cells that grow continuously in vitro.
Regulation of Antibody Response in Different Immunoglobulin Classes
Authors: Suemura Masaki , Kishimoto Tadamitsu , Hirai Yoshikatsu , Yamamura Yuichi
Published: December 2022

Selective suppression of anti-DNP IgE antibody response by DNP-Tbc-primed T cells was studied by using the in vitro culture system of murine spleen cells. The addition of DNP-Tbc-primed cells to DNP-OA-primed cells suppressed the in vitro production of anti-DNP IgE antibody response induced with DNP-OA without any effect on anti-DNP IgG antibody response. The addition of DNP-MGG-, DNP-KLH-, or DNP-Asc-primed cells to DNP-OA-primed cells did not show any suppressive effect on anti-DNP IgE antibody response. Depletion of T cells by anti-ϑ treatment abolished the suppressor activity of DNP-Tbc-primed cells, whereas depletion of B cells by anti-Ig-column did not affect the suppressor activity. The suppressor activity of DNP-Tbc-primed cells was dose-dependent and radiosensitive. Cell-free supernatants (CFS) from the culture of DNP-Tbc-primed cells with DNP-HSA-pulsed macrophages or DNP-HSA-coupled Sepharose beads showed the suppressor activity on anti-DNP IgE antibody response of DNP-OA-primed cells. CFS did not show any effect on IgG response. The suppressor activity of CFS was not absorbed with DNP-HSA- or anti-Ig-coupled Sepharose immunoadsorbent, indicating that the IgE class-specific suppressor factor is not antigen-specific and does not have Ig-determinants.
Resistance of Macrophages from C3H/HeJ Mice to the in Vitro Cytotoxic Effects of Endotoxin
Authors: Glode L. Michael , Jacques A. , Mergenhagen S. E. , Rosenstreich D. L.
Published: December 2022

Endotoxin (LPS) is toxic for normal mouse macrophages in vitro at concentrations (50 µg/ml) that are fully mitogenic and nontoxic to lymphocytes. Macrophages from LPS-resistant C3H/HeJ mice are not killed by LPS in vitro whereas those from a closely related LPS-responsive strain (C3H/HeN) are killed. Killing is the result of a direct interaction between LPS and the macrophage since this phenomenon is observed in lymphocyte-depleted cultures and since the presence of additional LPS-sensitive spleen cells will not enhance the killing of either C3H/HeN or C3H/HeJ macrophages. Furthermore, C3H/HeJ macrophages remain resistant to LPS-induced killing despite prior immunization with LPS in vivo or incubation with anti-LPS serum in vitro. LPS preparations vary in their cytotoxic effect on macrophages, and Escherichia coli K-235 LPS (phenol-water extracted), which discriminates well between LPS responsive and nonresponsive B cells, also discriminates well between LPS-sensitive and resistant macrophages. However, there is no apparent correlation between the mitogenic potential of LPS preparations and their cytotoxic effect on macrophages. These findings suggest that the C3H/HeJ mouse strain possesses a defect in LPS responsiveness which is intrinsic to its macrophages as well as its B lymphocytes.
Human Factor D̄ of the Alternative Complement Pathway: Purification and Characterization
Authors: Volanakis John E. , Schrohenloher Ralph E. , Stroud Robert M.
Published: December 2022

D̄ was purified to homogeneity from outdated human plasma by successive chromatography on Bio Rex 70, Sephadex G-200, Bio Rex 70, and Sephadex G-75. Column fractions were monitored for D̄ activity by a hemolytic diffusion plate assay. The overall yield was approximately 4% by activity. A m.w. of 22,900 daltons was established by sedimentation equilibrium. Amino acid analyses have been obtained and Isoleucine has been determined as the NH2-terminus. Incubation of D̄ with purified B and CoVF in the presence of Mg++ resulted in cleavage of B, as judged by SDS-polyacrylamide gel electrophoresis and immunoelectrophoresis. D̄ hydrolyzed certain synthetic amino acid esters of arginine, lysine, and tyrosine. Benzoyl-L-arginine methyl ester (BAME) was the most sensitive substrate for D̄ among those tested. The substrate profile of D̄ was distinct when compared to that of C1̄s, C1̄r, plasmin, urokinase, and trypsin. Both the enzymatic and hemolytic activity of D̄ were irreversibly inhibited by treatment with 10 mM DFP as well as by reduction and alkylation.
Release of Phospholipids from Liposomal Model Membrane Damaged by Antibody and Complement
Authors: Kinoshita Taroh , Inoue Kozo , Okada Mariko , Akiyama Yoko
Published: December 2022

When liposomal membranes were attacked by antibody and complement, enzymatic degradation of membrane phospholipids was not observed. However, intact membrane phospholipids were released into the surrounding medium in amounts beyond that expected from nonspecific proteinphospholipid interaction. On the other hand, when liposomal membranes were treated with phospholipase A purified from venom of “Habu” snake (Trimeresurus flavoviridis), trapped glucose marker was easily released, but phospholipids or their degradation products were not released into the medium and remained in the membrane structure.
Chemotaxis of Rat Lymphocytes
Authors: Ward P. A. , Unanue E. R. , Goralnick S. J. , Schreiner G. F.
Published: December 2022

Rat lymphocytes obtained from spleens, lymph nodes, and thymus glands showed migratory responses to a variety of factors including fluids from mixed lymphocyte culture fluids from concanavalin A-stimulated cells, fluids from phagocytizing macrophages, and to anti-rat IgG. Migratory responses to the last factor were bimodal over a dose range of anti-Ig; at high concentrations of anti-Ig, the response appeared to be nonspecific, whereas, at low concentrations, the responses seemed to be chemotactic in character. When lymphocytes from spleens, lymph nodes, and thymic glands were compared, qualitative and quantitative differences on the responses were evident with use of the three attractants. When spleen lymphocytes were separated into T cell- and B cell-enriched fractions, T cells responded to the culture fluids from mixed lymphocyte cultures, whereas B cells seemed to respond poorly, if at all. Only B cells responded to anti-Ig. These findings may explain, at least in part, the accumulation of lymphoid cells at sites of inflammatory stimuli.
Distribution of the Thy-1 Antigen in Cellular and Subcellular Fractions of Adult Mouse Brain
Authors: Stohl William , Gonatas Nicholas K.
Published: December 2022

By using a cytotoxicity inhibition assay employing AKR anti-C3H thymocyte antiserum, we have determined the degree of expression of the thy-1 antigen in fractions of adult mouse brain. As expressed as cytotoxicity inhibitory capacity per mg protein with C3H whole brain arbitrarily assigned a value of 1.0, the following values were found: C3H cerebral cortex, 5.8; C3H cerebral cortex synaptosomes, 2.5: C3H whole brain myelin, 0.65; C3H cerebral cortex neurons, 0.16; and C3H cerebral cortex mitochondria, 0.10. Neither C1300 neuroblastoma cells nor any AKR neural fraction had detectable levels of thy-1.

The findings indicate that the thy-1 antigen is found mainly in mouse cerebral cortex and in synaptosomal fractions, whereas myelin fractions contain lower but perhaps significant amounts of thy-1. Cerebral cortex neurons, isolated by a method requiring a 90-min mild trypsinization at 37°C, did not display significant amounts of the thy-1 antigen.

These results lend themselves to further study in the area of differentiation and development of central nervous system components and in the area of central nervous system immunopathology.
Interference with the Transmission of T Cell-Derived Messages by Macrophage Membranes
Authors: Ptak Wlodzimierz , Naidorf Kenneth F. , Gershon Richard K.
Published: December 2022

Heat-killed macrophages (HK MØ) and/or semi-purified MØ membranes interfere with primary thymus-dependent but not thymus-independent in vitro immune responses. In addition, they can absorb out and render inactive the helper activity found in mixed lymphocyte culture supernatants which augments the primary in vitro response of normal spleen cells. They do not, however, inactivate the helper activity which augments the response of thymus-deprived spleen cells. Both the immunologic interference and the absorbing activity of the HK MØ are eliminated by treatments which inactivate or block Fc receptors. We suggest that HK MØ or their membranes can act as competitive antagonists for live MØ functions and in so doing block communication networks between T cell subclasses. Thus they can serve as useful probes both for studying some of the roles live MØ might play in mediating immunologic functions as well as dissecting out some of the many arcane T cell interactions which normally occur.
Suppression of Immunoglobulin Synthesis by Transplantation of T Cells from Anti-µ Bursectomized Chickens into Normal Recipients
Authors: Kermani-Arab V. , Leslie G. A.
Published: December 2022

In order to study the nature and immunoregulatory capacity of lymphocytes from agammaglobulinemic chickens, line 63 inbred chickens were injected iv with 4 mg of specifically purified goat anti-chicken IgM (µ) antibody on day 14 of embryonation, and surgically bursectomized immediately after hatching. At 12 weeks of age these chickens were agammaglobulinemic and free of immunoglobulin-bearing B cells as assessed by immunofluorescence and lactoperoxidase catalyzed 125I cell surface-labeling techniques. Lymphocytes from these birds lacked the capacity to restore immunoglobulin synthesis in chemically induced agammaglobulinemic birds, whereas, their T cell function was retained as determined by their in vitro responsiveness to phytohemagglutinin (PHA) and concanavalin A (Con A). Of special significance was the observation that the adoptive transplantation of splenic, bone marrow, or thymic lymphocytes from agammaglobulinemic chicks into normal newly hatched syngeneic recipients rendered the recipients severely hypogammaglobulinemic or agammaglobulinemic. In contrast with the agammaglobulinemic donors, the hypogammaglobulinemic recipients possessed B lymphocytes. The ability of effector cells from the anti-µ bursectomized (anti-µ-Bx) donors to transfer agammaglobulin was abrogated by pretreatment with anti-chicken thymocyte antiserum plus complement.
Lymphocyte-Mediated Cytotoxicity: Effects of Ageing, Adult Thymectomy and Thymic Factor
Authors: Bach Marie-Anne
Published: December 2022

Adult thymectomy, as well as ageing, depressed splenic lymphocyte-mediated cytotoxicity (LMC) in the mouse. Ageing depressed significantly LMC as early as 19 weeks of age, independently of the number of cells used for immunization. Thymectomy affected LMC only when suboptimal numbers of immunizing allogeneic cells were used. This effect peaked at 6 to 12 weeks after thymectomy. No difference between thymectomized and normal mice was observed when LMC was tested 16 to 20 weeks after thymectomy, at an age when normal control mice themselves already showed a lowered LMC due to ageing.

The effect of in vivo treatment with a circulating thymic factor (TF), which was shown to disappear with ageing as well as after adult thymectomy, has been tested in adult thymectomized mice and normal young and ageing mice. TF treatment prevented LMC depression in adult thymectomized mice, whereas it depressed paradoxically splenic LMC in normal young and old mice. The possible mechanisms of the effects of adult thymectomy, ageing, and thymic factor on the different T cell subsets involved in allogeneic killer cell generation are discussed.
Tumor Neutralization, Immunotherapy, and Chemoimmunotherapy of a Friend Leukemia with Cells Secondarily Sensitized in vitro
Authors: Cheever Martin A. , Kempf Raymond A. , Fefer Alexander
Published: December 2022

The aim of our study was to induce in vitro a secondary response to a C57BL/6 Friend leukemia (FBL-3) by C57BL/6 lymphoid cells, to document their enhanced anti-tumor cytotoxicity in vitro and to study their anti-tumor effect in vivo by tumor neutralization (Winn assay) and tumor therapy of progressive FBL-3 in C57BL/6 mice. Spleen cells (Ci) from mice immunized once in vivo with FBL-3 were cultured for 5 days with either x-irradiated FBL-3 (CiFx), C57BL/6 spleen cells (CiCx), BALB/c spleen cells (CiBx) or without any x-irradiated cells (Ci-cult.) and their activity subsequently was assayed in vitro and in vivo. CiFx cells were markedly more cytotoxic in vitro by the 51Cr release assay than were CiCx or CiBx and were most effective in tumor neutralization (Winn assay). For adoptive immunotherapy, mice given lethal (104) FBL-3 i.p. on day 0 received the cultured spleen cells on day 1. Treatment with 5 × 106 CiFx cured 14 of 14 mice whereas 19 of 20 mice given 5 × 106 CiCx or CiBx died with tumor. For adoptive chemoimmunotherapy of advanced leukemia, mice were given 107 FBL-3 i.p. on day 0. On day 5, they received cyclophosphamide (CY) plus cultured spleen cells. Untreated mice died by day 14. CY alone prolonged survival but all mice died within 4 weeks. Treatment with CY plus 5 × 106 Ci-cult. or CiBx was no more effective than CY alone—40 of 40 mice died with tumor. However, CY plus 5 × 106 CiFx cured 16 of 22 mice. Thus, cells generated in a secondary response in vitro were not only cytotoxic in vitro and effective in tumor neutralization, but were also curative in immunotherapy of an early leukemia and in chemoimmunotherapy of an advanced leukemia.
Receptors for C3 on Rat Peritoneal Mast Cells
Authors: Sher Alan , McIntyre Susan L.
Published: December 2022

Purified rat peritoneal mast cells adhere to schistosomula of Schistosoma mansoni which have been pre-incubated in fresh normal rat serum. This cytoadherence reaction is dependent on complement and in particular on components of the alternative pathway. Since antibodies to rat C3 but not IgG block the attachment of the cells to the complement-treated larvae, it appears that C3-specific receptors on the mast cell surface are responsible for the adherence phenomenon. These receptors can also be demonstrated by the rosetting of mast cells with rat complement-treated zymosan particles or fluoresceinated bacteria. The key properties of the receptors are their specificity for homologous (rat) complement, their sensitivity to digestion with trypsin, and their functional dependence on Mg+ + ions. Thus, the rat mast cell receptors share many of the characteristics of the C3 receptors previously identified on monocytes, macrophages, and polymorphonuclear leukocytes.
Functional Heterogeneity Among the T-Derived Lymphocytes of the Mouse
Authors: Araneo Barbara A. , Marrack Philippa , Kappler John W.
Published: December 2022

The T1 subpopulation of peripheral T cells was defined in mice by its short half life, insensitivity to anti-thymocyte sera (ATS) in vivo, and slow kinetics of response to antigen. The T2 subpopulation was defined by its long life time, elimination by ATS in vivo, and rapid response to antigen. Mice containing only T1-type T cells were constructed by adult thymectomy (ATx) followed immediately by the elimination of T2 cells by ATS treatment. Immunization of these mice with SRBC led to the production of memory helper cells in the T2 subpopulation. This process depended on the presence of T1 cells and for the most part required SRBC immunization, although a few SRBC-specific T2 cells reappeared in the mice in the absence of antigen. We conclude that T1 cells can give rise to T2 cells in an antigen-driven step and that the two populations correspond to virgin and memory T cells, respectively.
Lymphocyte-Mediated Cytotoxicity Against Human Allogeneic and Autologous Lymphoid Targets After Concanavalin A-Activation of Cytotoxic Effector Cells
Authors: Hunninghake Gary W. , Fauci Anthony S.
Published: December 2022

The present study has demonstrated that suspensions of human peripheral blood lymphocytes can be activated by Con A to become cytotoxic effector cells displaying specificity against a broad range of alloantigen-bearing lymphoid targets. The immunologic nature of the reaction was demonstrated by the fact that autologous targets were not lysed except when an agglutinin (PHA) was present during the cytotoxicity assay. Certain culture conditions were critical for optimal responses including the density of cells, the presence of 2-mercaptoethanol in the culture media, and certain batches of human AB serum which strongly supported the generation of cytotoxic lymphocytes. Cytotoxic cells were generated only after 2 to 3 days of culture and were not present at 24 hr or after 4 days of culture. The optimal concentration of Con A in the culture media was 10 to 20 µg/ml. The target cells employed were human lymphocytes maintained in culture without stimulation for 2 to 3 days. The cytotoxicity assay was a 4-hr assay and relatively high effector-to-target cell ratios (250 to 500:1) were necessary to obtain maximal killing against allogeneic lymphoid targets, whereas lower ratios were adequate against nonlymphoid targets such as Chang liver cells. Con A-generated lymphocytes from particular individuals were consistent in their capacity to mount a high or low cytotoxic response against allogeneic targets. This was independent of the degree of HL-A haplotype similarity or dissimilarity with the target cell. In addition, individuals who were low responders in the Con A-generated cytotoxicity mounted MLC blastogenic responses and MLC-generated alloantigen specific cytotoxic responses as high as those individuals who were high responders in the Con A-generated cytotoxicity assay. Also, lymphocytes from both high and low responders manifested similar degrees of blastogenic responses to Con A. The present study provides a simple and reproducible system for the generation and assay of cytotoxic effector cells from human peripheral blood after polyclonal activation by Con A and provides a model which can be used in studies of the mechanisms and regulatory factors involved in the activation of human cytotoxic lymphocytes.