bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms in T cell leukemogenesis. II. T cell responses of preleukemic BALB/c mice to Moloney leukemia virus antigens.
Authors: Lee J C , Horak I , Ihle J N
Published: December 2022

The T cell responses of Moloney leukemia virus (MoLV)-infected preleukemic BALB/c mice were examined. The major in vitro response detectable was T cell blastogenesis in response to the major viral envelope protein MoLV gp71 and an internal viral protein p12. The majority of the preleukemic mice had readily detectable responses to gp71, whereas the presence of a response to p12 was less consistent. With both antigens, T cell blastogenesis showed typical antigen response characteristics similar to those detected in other immune responses to C-type viruses. Proliferation was dependent on a Thy-1+, Lyt-1+, 2- population and was macrophage-independent. In contrast to most immune responses to C-type viruses, which are temporally restricted, T cell blastogenesis was detectable throughout the preleukemic period of 4 to 16 wk of age. During this period neither gp71-specific T cells nor PHA-responsive T cells were found to express viral antigens. The correlations between T cells responding to gp71 and leukemia were examined. Under conditions in which MoLV inoculation of BALB/c mice does not induce leukemia, no T cell responses were deectable. These results suggest a causal relationship between the presence of antigen-specific T cells and the ability of MoLV to induce leukemia. The results are discussed with reference to the possible role of chronic immune stimulation in virus-induced leukemogenesis.
Characterization of ligand-binding activity of isolated murine Fc gamma receptor.
Authors: Schneider R J , Atkinson J P , Krause V , Kulczycki A
Published: December 2022

Fc gamma-binding macromolecules were isolated from 2 murine macrophage-like cell lines by affinity chromatography using various immunoglobulins coupled to Sepharose. P388D1 and J744.2 cells were radiolabeled with 125I using a modified lactoperoxidase method, washed cells were solubilized using NP-40, and the solubilized cell lysates were incubated at 4 degrees C with immunoadsorbents. Fc gamma receptor-like material was obtained from the IgG-Sepharose columns by elution at 4 degrees C with 0.5 N acetic acid containing 1% NP-40 into buffer for neutralization. Purified material thus obtained retained its ligand-binding activity, since approximately 30 to 60% rebound to IgG-Sepharose. Upon analysis by SDS polyacrylamide gel electrophoresis, putative Fc gamma receptor had an apparent m.w. of 50 to 65,000. Neither a comparable SDS-PAGE band nor receptor activity was isolated from a 3rd murine cell line, which lacks the Fc gamma receptor. Putative receptor obtained by elution from IgG2a-Sepharose rebound equally well to IgG2a-, IgG1- and IgG2b-Sepharose, but did not rebind to IgG3-, F(ab')2-, or BSA-Sepharose. Similarly, Fc gamma-binding macromolecules eluted from IgG2b-Sepharose rebound only to IgG2b-, IgG2a- and IgG1-Sepharose rebound only to IgG2b-, IgG2a- and IgG1-Sepharose. The rebinding of either receptor preparation was inhibited in a dose-dependent manner by both monomeric IgG2a and monomeric IgG2b, and myeloma protein from each subclass inhibited to the same extent. Therefore, the mouse Fc gamma receptor in its isolated state does not appear to discriminate between monomeric IgG2a and IgG2b.
Inactivation of natural killer cell cytotoxic activity after interaction with target cells.
Authors: Perussia B , Trinchieri G
Published: December 2022

Human peripheral blood lymphocytes were cultured with unlabeled target cells or absorbed to K562 cells and then isolated by centrifugation on discontinuous Ficoll-Methrizoate gradient. In both cases, lymphocytes that reacted with these target cells at 37 degrees C were inactivated and were no longer able to kill freshly added 51Cr-labeled target cells that were susceptible to spontaneous killing. This inactivation was time and temperature dependent and did not alter the ability of lymphocytes to lyse antibody-sensitized target cells. The natural killer (NK) cell inactivation that followed the interaction with target cells was not observed when target cells were used that were able to induce interferon (IFN) production when cultured with lymphocytes. This NK inactivation was reversed by incubation of the inactivated effector cell preparations with IFN.
Variation in the synthesis of membrane proteins of human T lymphocytes during mitogenic activation: recognition of a 70,000 dalton protein with anti-p23,30 heteroantiserum and a novel 42,000 dalton protein with anti-p44,12 heteroantiserum.
Authors: Boto W O , Humphreys R E
Published: December 2022

Membrane proteins of [35S]methionine-labeled, human T lymphocytes were analyzed by SDS polyacrylamide gradient slab gel electrophoresis and autoradiography each day during a 6-day period of activation with phytohemagglutinin or with concanavalin A. This process was characterized by the novel appearance and limited duration of synthesis of many proteins, in particular of 30, 35, 48, 50, and 55 kilodalton molecules in the early days of blast transformation and subsequently of 120, 125, 135, and 145 kilodalton proteins. The HLAA-A,-B antigens and beta 2-microglobulin, as recognized by anti-p44,12 serum, were synthesized by both resting and mitogen-activated T cells on each day of culture. But, an additional 42-kilodalton protein was recognized with this same antiserum on days 4 and 5 of activation. A 70-kilodalton protein, immunoprecipitated by anti-p23,30 (anti-HLA-protein, immunoprecipitated by anti-p23,30 (anti-HLA-DR) heteroantiserum, was synthesized principally on days 2 and 4 of mitogenic transformation. This molecule was absent from normal, resting T cells, the T cell line, CCRF-CEM, and the B cell line, Raji. In a parallel test, the same anti-p23,30 serum detected the conventional HLA-DR bimolecular glycoprotein complex of 29 and 34 kilodaltons in nonionic detergent solubilized Raji B cell membrane preparations. This study described in detail the molecular changes in the membrane proteins of activated T lymphocytes and included the definition of novel forms of HLA-A,B and HLA-DR associated molecules.
An abnormality of immune complex kinetics in murine lupus.
Authors: Magilavy D B , Rifai A , Plotz P H
Published: December 2022

In order to understand better the role of immune complex metabolism in the pathogenesis of autoimmune diseases, we have investigated the early stages of immune complex uptake by the liver, the major organ responsible for clearance of soluble complexes in the mouse. Livers were perfused in situ via the portal vein over 3 to 5 min with trace amounts of radiolabeled soluble model immune complexes. In 4 nonautoimmune strains (BALB/c, DBA/2, CAF1, NZW) 60 to 72% of the model complexes perfused were taken up and remained in the liver after 20 min of continuous perfusion with oxygenated Krebs-Henseleit buffer. In NZB and NZB/W F1 female mice at ages 0.5 to 11 mo, 66 to 78% of the model complexes remined in the liver. However, when a dose of heat-aggregated human gamma-globulin sufficient to saturate the reticuloendothelial system was perfused 7 min after radiolabeled complexes, 15.2 +/- 7.2% (mean +/- SD) of the complexes were displaced in the nonautoimmune strains. In contrast, 32.6 +/- 10.5% of the complexes were displaced from the liver in NZB and NZB/W F1 female mice (p < 0.001). Thus, although hepatic uptake of immune complexes in autoimmune mice appears to be normal or even enhanced, there may be impaired phagocytosis by the hepatic RES or weaker binding of complexes to the surface of the Kupffer cells. Such surface-bound immune complexes remaining accessible to the circulation may contribute to the autoimmune process.
Interleukin-2 induction of hapten-specific cytolytic T cells in nude mice.
Authors: Gillis S , Watson J
Published: December 2022

In light of i) increasing evidence that the thymus guides the development and antigen recognition repertoire of cytotoxic T cells (CTL) and ii) the capacity of purified interleukin 2 (IL-2) to allow for the in vitro generation of CTL from nude mice, we investigated the ability of IL-2 to influence the in vitro generation of hapten-specific cytotoxicity from nude mouse spleen cell cultures. We found that stimulation of nude mouse splenocytes with trinitrophenol (TNP)-derivatized autologous or allogeneic spleen cells in the presence of IL-2 generated hapten-specific CTL. EFfector cells harvested from IL-2-supplemented cultures killed only TNP-derivatized target cells; however, the cytotoxicity observed was not restricted to a particular target histocompatibility type. This study confirms our previous observations that IL-2 has the capacity to induce growth of CTL from pre-T cell populations in the total absence of thymic influence. However, it is clear that the generation of H-2 restricted killing (recognition and lysis of cells bearing antigen presented solely in association with "self" H-2) probably requires further processing of pre-killer T cell populations by a thymus-derived differentiation signal. In addition, these studies provide further evidence that a major role of the thymus, in addition to programming effector T cell antigen recognition repertoire, may be its function to provide essential differentiation of the IL-2 producer T cell.
Cell-mediated immunity in herpes simplex virus-infected mice: H-2 mapping of the delayed-type hypersensitivity response and the antiviral T cell response.
Authors: Nash A A , Phelan J , Wildy P
Published: December 2022

An adoptive transfer system was used to investigate the H-2 restriction of delayed-type hypersensitivity (DTH) to herpes simplex virus. A successful DTH transfer was achieved when donor and recipient were compatible at the I-A region, with K and D region compatibility unnecessary. However, the rapid clearance of infectious virus from the inoculation site was found only when the donor and recipients were compatible at H-2K (and presumably D) and I-A regions.
Acid hydrolases and tryptase from secretory granules of dispersed human lung mast cells.
Authors: Schwartz L B , Lewis R A , Seldin D , Austen K F
Published: December 2022

beta-Hexosaminidase, beta-glucuronidase, arylsulfatase, and tryptase were each released along with histamine from dispersed purified human lung mast cells of 40 to 80% purity by rabbit IgG anti-human IgE. The net per cent release ratio of each enzyme to histamine was determined over all doses of antibody employed to activate the mast cells and over all time points after activation, and indicated the per cent of each enzyme stored in secretory granules along with histamine. By multiplying the net per cent release ratio of each enzyme to histamine by total enzyme content in a preparation of 10(6) mast cells, values for secretory granule content per 10(6) mast cells were found to be 3.8 U for beta-hexosaminidase, 0.03 U for beta-glucuronidase, 0.03 U for arylsulfatase, and 0.9 U for tryptase. Subtype analysis of beta-hexosaminidase by diethylaminoethyl- (DEAE) cellulose chromatography revealed that the B isomer predominates in human mast cell secretory granules, whereas the A isomer predominates in secretory granules of the rat mast cell. Tryptase, the predominant neutral protease of the human mast cell secretory granule, has a m.w. of 130,000 by gel filtration chromatography, whereas the major neutral protease of the rat mast cell is chymotryptic and of 25,000 m.w. The presence of acid hydrolases, a tryptase, and histamine in human mast cell secretory granules suggests that the activated mast cell plays a direct role in the production of acute and subacute inflammation.
Single or multicellular origin of human T lymphocyte colonies in vitro: modification by 12-o-tetradecanoylphorbol 13-acetate (TPA).
Authors: Singer J W , Ernst C , Whalen C K , Steinmann L , Fialkow P J
Published: December 2022

The assumption that human T lymphocyte colonies have a unicellular origin has been directly tested with peripheral blood mononuclear cells from 2 women heterozygous for the common X-linked glucose-6-phosphate dehydrogenase (G-6-PD) gene (GdB) and the variant GdA. T cells were cultured in semisolid medium in the presence of phytohemagglutinin (PHA) and T lymphocyte growth factor with or without preincubation in suspension culture with PHA (2-stage and 1-stage assays, respectively). The enzyme type of individual T cell colonies was then determined electrophoretically at the lowest colony density with adequate growth (usually less than 100 colonies/dish). In the 2-stage system, 90 of 97 tested colonies had equal amounts of A and B enzyme activities suggesting multicellular origin of the colonies. Similarly, in the single-stage system, 21 of 31 colonies had both A and B enzymes. Increasing the density of the soft agar did not influence the frequency of A/B colonies. However, when 12-O-tetradecanoylphorbol 13-acetate (TPA), a promoter of T cell colony growth shown in other systems to inhibit metabolic cooperation, was added, a striking decrease in frequency of colonies with both G-6-PD types was found. In the 2-stage culture, 0 of 9 colonies had a double-enzyme type and in the single-stage system, the frequency of A/B colonies declined to 9 of 34 (p less than 0.025). The data suggest that despite the apparent multicellular origin of T cell colonies in cultures with TPA, most colonies do originate from single cells when cultured with TPA at low colony densities. Stimulation of cell growth or inhibition of metabolic cooperation between cells by TPA are possible explanations for these differences.
Plasminogen activator is an apparent lymphocyte mitogen.
Authors: Cohen S D , Israel E , Spiess-Meier B , Wainberg M A
Published: December 2022

Culture fluids of avian sarcoma virus (ASV)-transformed but not normal chicken embryo cells frequently elicited a mitogenic response in normal avian and murine lymphocytes. We examined the possibility that plasminogen activator (PA) might be responsible for the observed mitogenic effect. PA activity, present in culture medium, was correlated positively with lymphocyte mitogenic capacity. Treatment of cells with phorbol myristate acetate, which elevates PA levels, increased mitogenesis. Similar treatment with dexamethasone, which inhibits PA biosynthesis and/or secretion, reduced lymphocyte mitogenic activity. Addition to culture fluids of either benzamidine or diisopropylfluorophosphate, both specific PA inhibitors, blocked lymphocyte proliferative responsiveness to culture fluids. In contrast, neither epsilon-amino-caproic acid nor trasylol, which inhibits plasmin esterase activity but not PA, abrogated lymphocyte responsiveness. Furthermore, purified urokinase, an enzyme of similar substrate specificity to PA, had lymphocyte stimulatory activity. These results strongly suggest that PA can function as a lymphocyte mitogen.
Cell surface glycoproteins of cytotoxic T lymphocytes induced in vivo and in vitro.
Authors: Kaufmann Y , Berke G
Published: December 2022

CTL-blasts, generated in vitro or obtained from educated spleens in vivo, have previously been shown to display high levels of a unique membrane glycoprotein T145 (ca. 145,000 daltons), inferred to be a common CTL marker and implicated in CTL cytotoxicity. As shown here, however, these suggestions do not hold for all cytotoxic T lymphocytes. We have shown that T145 is not expressed on highly active CTL-induced in vivo by a standard alloimmunization protocol. The finding that several CTL populations are T145 positive, whereas others are T145 negative, may be due to the existence of functional CTL in 2 stages of differentiation or may represent different CTL lineages. In addition, no correlation was found between cytolytic activity and expression of T145. The functional significance of T145 on CTL is discussed.
The mechanism of thymus-dependent antibody formation in bone marrow.
Authors: Koch G , Osmond D G , Julius M H , Benner R
Published: December 2022

During the primary immune response of mice to i.v. administered thymus-dependent antigens the spleen is the major site of localization of antibody-producing plaque-forming cells (PFC). During the secondary response, on the other hand, large numbers of PFC not only appear in the spleen, but also in the bone marrow. By inducing B memory cells with a DNP-carrier complex and activating the DNP-specific B memory cells with the same hapten conjugated to a heterologous carrier, we show in this paper that B memory cells, but not necessarily T memory cells, must be present before booster immunization for PFC to appear in the bone marrow. The origin of the PFC that appear in the bone marrow during secondary type immune response was studied in parabiotic mice consisting of members congenic for the Igh-1 locus. From analysis of the allotype of antibodies produced by PFC in the marrow of such pairs of parabionts it appeared that antibody formation in bone marrow is dependent on the immigration into the marrow of B memory cells activated in peripheral lymphoid organs. Consistent with such a migration of activated cells, radioautographic studies in guinea pigs demonstrated an influx of newly formed mononuclear cells into the bone marrow via the blood stream during the first 3 days after intravascular antigen administration.
Role of prostaglandin E2 in the induction of nonspecific T lymphocyte suppressor activity.
Authors: Fischer A , Durandy A , Griscelli C
Published: December 2022

Activated human monocytes and concanavalin A (Con A)-activated T lymphocytes are known to suppress T and B lymphocyte proliferation and B cell maturation into immunoglobulin-producing cells. We have now shown that monocyte suppressive activity is predominantly mediated through release of prostaglandin E2 (PGE2), which is active only in the presence of a "short-lived," radiosensitive T lymphocyte subset. PGE2, at high concentration, can activate T suppressor lymphocytes (TS), which display the same characteristics as Con A-activated TS lymphocytes. Moreover, Con A activation of TS lymphocytes was obtained only in the presence of PGE2, as specific anti-PGE2 antiserum or indomethacin prevented TS activation; this suggested a double signal as a prerequisite for activation of the nonspecific TS cell subset. We propose that TS lymphocytes modified by Con A become sensitive to small amounts of PGE2 produced by monocytes that must be present during the Con A-stimulated activation phase of suppressive cells.
Altered natural killer cell biology in C57BL/6 mice after leukemogenic split-dose irradiation.
Authors: Parkinson D R , Brightman R P , Waksal S D
Published: December 2022

Natural killer (NK) cell activity was examined in the spleens of C57BL/6 mice given leukemogenic split-dose irradiation. The radiation protocol resulted in severe depression of spontaneous NK cell activity; this activity was not fully restored after treatment with the interferon inducer poly I:C. In vitro mixing studies provided no evidence for active suppression in vivo as a mechanism for this decrease in activity. In addition, spontaneous activity was restored towards control levels after bone marrow transfusion from nonirradiated mice. Despite the low NK cell activity, there was no difference between control and irradiated mice in the numbers of target-binding cells (TBC). The results are most compatible with the radiation-induced loss of a cell with normal NK activity from spleen and bone marrow after the split-dose radiation protocol. In addition, a population of cells able to competitively block normal NK cell lysis of YAC-1 tumor cells is found in the bone marrow, spleen, and thymus of the irradiated mice lacking NK cell activity. These findings are considered from the perspective of their implications regarding NK cell ontogeny, and the possible role of the NK cell in radiation leukemogenesis.
A monoclonal antibody that identifies human peripheral blood monocytes recognizes the accessory- cells required for mitogen-induced T lymphocyte proliferation.
Authors: Rosenberg S A , Ligler F S , Ugolini V , Lipsky P E
Published: December 2022

These studies were carried out to evaluate the functional characteristics of the cells identified by a monoclonal anti-human mononuclear phagocyte (M phi) antibody, 63D3. This antibody reacts specifically with all human peripheral blood M phi and not with normal human peripheral blood T or B cells. By using the fluorescence-activated cell sorter, human peripheral blood mononuclear cells (PBM) were sorted into 63D3-positive and 63D3-negative populations. Cell volume analysis indicated that 63D3-negative cells were identical to the non-adherent cells (NAC) in PBM, whereas 63D3-positive cells had a bimodal size distribution comparable to glass-adherent cells (AC). Neither 63D3-negative cells nor NAC were able to proliferate in response to mitogenic stimulation, whereas co-culture with either mitomycin C-treated AC or 63D3-positive cells restored mitogen responsiveness. Thus, the 63D3-positive population contains the relevant accessory cell required for mitogen-induced human lymphocyte proliferation.
The role of subcellular factors in pulmonary immune function: physicochemical characterization of two distinct species of lymphocyte-activating factor produced by rabbit alveolar macrophages.
Authors: Simon P L , Willoughby W F
Published: December 2022

T cell stimulatory factors produced by rabbit alveolar macrophages were investigated. Physicochemical characterization revealed that alveolar macrophages (harvested by bronchopulmonary lavage and stimulated in tissue culture with bacterial lipopolysaccharide) released 2 predominant species of lymphocyte-activating factor (LAF) with isoelectric points of 4.6 and 7.4, and m.w. of 14,400 and 11,600 daltons, respectively, as calculated by the Svedberg equation. Using C3H/HeJ mouse thymocytes (and in some instances nylon wool-purified nonadherent rabbit spleen or lymph node cells) as target cells, rabbit LAF was found to induce proliferative responses directly, as well as enhance proliferative responses to phytomitogens. Both LAF species were inactivated by heating, treatment with trypsin, or at low (2.3) pH. The pI 7.4 LAF was also unstable at high pH (9.0). The thymocyte stimulatory activity of both LAF species was not inhibited by the anti-proteases alpha-1-anti-trypsin, Traysylol (aprotinin), leupeptin, or pepstatin.
Neutralization of vesicular stomatitis virus (VSV) by human complement requires a natural IgM antibody present in human serum.
Authors: Beebe D P , Cooper N R
Published: December 2022

Neutralization of VSV by human serum ws previously shown to involve C1, C2, C3, and C4 of the classical complement (C) pathway. All normal human sera tested were equivalently active in this regard. However, purified C1, C2, C3, and C4 were unable to mediate VSV neutralization. In the present studies an additional factor required for C-mediated neutralization was isolated from normal human serum and identified as a natural IgM antibody specific for a viral encoded antigen. Purified IgM bound to the virus and formed a complex that activated component C1. Normal serum concentrations of purified IgM, C1, C2, C3, C4 neutralized VSV to the same extent as normal serum. Purified IgM did not neutralize VSV alone or in conjunction with C1, C2, and C4. Inclusion of C3 resulted in full neutralization and C3b binding to the virus was demonstrated. Thus, normal human serum contains a natural antibody of the IgM class that is directed toward a viral antigen. The antibody facilitates neutralization by forming an immune complex that activates C1 and thus efficiently initiates the classical pathway at the viral surface. Neutralization occurs with C3b deposition on the viral envelope and probably results from a blanket of C protein that interferes with viral attachment to susceptible cells.
The third component of complement inhibits human lymphocyte blastogenesis.
Authors: Needleman B W , Weiler J M , Feldbush T L
Published: December 2022

Purified human C3 was studied for its ability to regulate human peripheral blood lymphocyte activation in a serum-free tissue culture system. C3 and its fragments formed by trypsin digestion were not mitogenic. However, C3 inhibited both mitogen- and antigen-induced lymphocyte proliferation in a dose-related manner. The observed inhibition by C3 increased with an increase in the dose of mitogen, with 50% inhibition seen at a C3 dose of 2.5 to 25 microgram/ml. Inhibition was induced even when C3 was added at 69 hr in a 99-hr culture, suggesting an effect on late events in activation. The inhibition was not mediated through generalized cytotoxicity, was not an artifact of kinetic alteration and still occurred in macrophage-depleted cultures. A small m.w. fragment of C3 which contained the inhibitory capacity in our C3 preparation was similar to C3a in size, anaphylatoxin activity, heat and acid stability and inhibition by serum. Our results indicate that cleavage products by C3 may play a negative role in lymphocyte activation.
The involvement of herpes simplex virus type 1 glycoproteins in cell-mediated immunity.
Authors: Carter V C , Schaffer P A , Tevethia S S
Published: December 2022

The participation of herpes simplex virus (HSV) glycoproteins in T cell-mediated lysis of HSV-infected syngeneic target cells was examined by using a temperature sensitive (ts) mutant defective in glycoprotein synthesis at the nonpermissive temperature (39 degrees C), and 2-deoxy-D-glucose and tunicamycin, known inhibitors of both HSV replication and glycoprotein synthesis in HSV-infected cells. Lymphocytes cytotoxic for HSV-infected cells lysed C57BL/6 Wt-3 cells infected with the mutant, ts A1, at 39 degrees C less efficiently than at 33 degrees C. Treatment of HSV-infected C57BL/6 Wt-3 cells with 1% 2-deoxy-D-glucose for 14 hr reduced their susceptibility to T cell-mediated lysis by 73% in the 51Cr release assay. Treatment of HSV-infected C57BL/6 Wt-3 cells with 0.2 microgram/ml tunicamycin for 14 hr reduced their susceptibility to T cell-mediated lysis by 78% in the 51Cr release assay. The reduction in T cell-mediated lysis by 2 deoxy-D-glucose and tunicamycin was found not to be due to the effects of the compounds on the H-2 antigens. We conclude that HSV specific glycoproteins are involved in T cell-mediated lysis of HSV-infected cells.
Membranes of EBV-carrying virus nonproducer cells inhibit leukocyte migration of EBV-seropositive but not seronegative donors.
Authors: Szigeti R , Volsky D J , Luka J , Klein G
Published: December 2022

We have previously shown that the leukocytes of healthy EBV-seropositive (but not seronegative) donors respond with migration inhibition (LMI) when confronted with extracts of EBV-carrying (but not EBV negative) cells. In the present study, we have examined whether this EBV-specific LMI response is capable of detecting a membrane antigen on the surface of EBV-carrying virus nonproducer cells. Crude membranes from EBV-genome carrying and EBV-negative cell lines were used as antigen. Contamination with the EBV-determined nuclear antigen (EBNA) was ruled out. Membranes from EBV-genome carrying nonproducer cells inhibited the migration of leukocytes from healthy seropositive donors, whereas membranes from EBV-negative lines had no such effect. Seronegative donors did not show any LMI. The clear difference between the EBV-negative Ramos line and its EBV-converted sublines was particularly conclusive in showing that the membrane component is determined or induced by the viral genome.
Constitutive production of interleukin 2 activity by a T cell hybridoma.
Authors: Stull D , Gillis S
Published: December 2022

Several laboratories have recently confirmed the importance that interleukin 2 (IL-2, formerly referred to as T cell growth factor) plays not only in the maintenance of cultured functional T cell lines but also as a potent regulator of immune responses. Despite the development of preparative IL-2 purification procedures, as well as identification of IL-2 producer tumor cell lines, simple sources of lectin-free IL-2 containing conditioned medium remain undefined. In hopes of establishing such a source of mitogen-free IL-2, we elected to fuse the high titer IL-2 producer tumor cell line, LBRM-33, with a drugmarked T cell lymphoma. In this communication we report the successful isolation of a T cell hybridoma that constitutively synthesizes and secretes biologically active IL-2. Hybridoma conditioned medium contained approximately 10 to 100 times the amount of IL-2 produced by conventional T cell mitogen-stimulated murine splenocyte preparations. In addition to providing a simple means for producing IL-2 containing conditioned medium without the presence of contaminating lectin, we hope that constitutive IL-2 producer hybrid cell lines will prove interesting as vehicles for dissection of the molecular events involved in IL-2 biosynthesis.
Functional analysis of human T cell subsets defined by monoclonal antibodies. II. Collaborative T-T interactions in the generation of TNP-altered-self-reactive cytotoxic T lymphocytes.
Authors: Friedman S M , Hunter S B , Irigoyen O H , Kung P C , Goldstein G , Chess L
Published: December 2022

In previous reports we have demonstrated that human T cells, responding to soluble and alloantigens, release helper factor(s) that amplify primary in vitro hapten-altered-self-reactive CTL responses. In the present studies, we have employed complement-fixing monoclonal antibodies (OKT4 and OKT8) that recognize functionally distinct human T cell subsets to investigate the role of T-T interaction in the generation of these killer cells. In all experiments, purified OKT4+ responder T cells were deficient in cytotoxic activity, whereas responder populations containing OKT8+ T cells generated substantial cytotoxicity; demonstrating that TNP-altered-self-reactive CTL precursors are contained within the OKT8+ T cell subset. Further, optimal cytotoxic responses were obtained from responder populations containing both OKT4+ and OKT8+ T cells, suggesting that cooperative interaction between these subsets may result in an amplification of killer cell activity. This interpretation was supported by the following observations: (1) the amplifying effect of soluble antigen required the presence of both OKT4+ and OKT8+ responders; (2) during MLC, OKT4+ but not OKT8+ responder T cells generate helper factor(s) that amplify TNP-altered-self-reactive CTL responses; (3) helper factor(s) bypass the requirement for direct OKT4-OKT8 T cell interaction, triggering a CTL response that is proportional to the percentage of OKT8+ T cells present within the responder population. In additional studies, we determined that the TNP-altered-self-reactive effector CTL maintain the OKT3+, OKT4-, OKT8+ surface phenotype displayed by the CTL precursor.
Comparison of pyrimidine and purine nucleoside secretion and nucleoside kinase expression in resident and elicited peritoneal macrophages.
Authors: Stadecker M J
Published: December 2022

Thymidine was found in culture supernatants from both normal peritoneal macrophages and macrophages elicited in vivo with LPS under conditions in which there was no detectable TK activity. Abrogation of thymidine secretion was observed only when elicited macrophages were cultured in the presence of macrophage growth factor (MGF), whereas resident macrophages continued secreting under similar conditions. Similarly, TK activity was induced with MGF only in elicited, but not in resident, macrophages; LPS added in vitro failed to render resident macrophages susceptible to stimulation by MGF. Tests for adenosine and deoxyadenosine in macrophage supernatants proved that these nucleosides were not secreted by these cells. Although AK was consistently expressed in both resident and elicited macrophages, no significant dAK levels could be detected in either cell population. In contrast to TK, both purine nucleoside kinases could not be modulated or induced with MGF. The data indicate different reactivity to MGF by resident and elicited macrophages and a difference in pyrimidine vs purine nucleoside secretion as well as nucleoside kinase expression by these cells.
Mechanisms of contraction induced by partially purified slow reacting substance from human polymorphonuclear leukocytes and leukotriene D in guinea pig ileal smooth muscle.
Authors: Findlay S R , Lichtenstein L M , Siegel H , Triggle D J
Published: December 2022

On the basis of the experiments described here, we suggest that the mechanism of action of SRS in guinea pig ileal longitudinal muscle is very similar to that used by other agonists in this preparation, namely interactions at a specific receptor to activate Ca++ channels leading to Ca++ influx and mechanical response. SRS responses differ in 1 important respect from those produced by other agonists. SRS and leukotriene D cause the generation of a slower contraction, which has a greater resistance to reversal by washing. The basis for this unique and prolonged duration of action is unknown but may represent an exceptionally tight, or even irreversible, association with a membrane component. Current studies on the structure of SRS may be helpful in this regard. Although not definitive, this report clearly indicates that a SRS "receptor" is, in fact, present on smooth muscle and that it utilizes a pool of calcium channels common to all smooth muscle agonists.
T lymphocyte-mediated cytotoxicity against autologous EBV-genome-bearing B cells.
Authors: Tsoukas C D , Fox R I , Slovin S F , Carson D A , Pellegrino M , Fong S , Pasquali J L , Ferrone S , Kung P , Vaughan J H
Published: December 2022

We have stimulated human peripheral blood lymphocytes in vitro with autologous EBV-infected or noninfected B cells. A cytotoxic response was obtained only when virally infected cells were used. The activity of the effector cells was restricted by the major histocompatibility complex and was directed against EBV-genome-bearing targets. The highest cytolytic response was obtained when lymphocytes of individuals previously exposed to the virus (EBV-VCA positive) were used. Lymphocytes of noninfected donors (EBV-VCA negative) gave a low response; the relative frequency of their effector cells was at least 4-fold lower. Lymphocytes of newborns did not respond. The cytotoxic activity was mediated by T lymphocytes of the cytotoxic/suppressor subset, as determined by cytofluorographic analysis and antibody plus complement-mediated lysis, using monoclonal antibodies to human lymphocyte surface antigen.
Cell-mediated cytotoxicity to non-MHC alloantigens on mouse epidermal cells. III. Epidermal cell-specific cytotoxic T lymphocytes.
Authors: Tyler J D , Steinmuller D
Published: December 2022

We have previously shown that murine epidermal cells (EC) from 5 H-2 compatible donor strains induce cytotoxic T lymphocytes (CTL) in C3H hosts that preferentially lyse donor EC as opposed to donor lymphoid cells (LC). This preference has been shown to be dependent on a single alloantigen disparity, designated epidermal alloantigen (Epa). In this report we studied the specificity of the CTL to determine whether Epa-antigens are in fact EC specific. Two populations of CTL could be identified by cross-immunization, cold-target inhibition, antigen-driven suicide, and limiting-dilution analyses. A minor CTL population recognizes alloantigens that are expressed by both EC and LC, but this population is necessary only for lysis of LC targets. The major CTL population is specific for Epa-antigens and reacts only with EC targets. At the induction phase of CTL responses, donor LC stimulators can weakly stimulate Epa-specific CTL, indicating LC populations express Epa-antigens in immunogenic form but at very low levels compared with EC. The preferential lysis of EC targets in these strain combinations can be attributed to the major Epa-specific CTL population, whereas lysis of LC targets is due to CTL specific for other alloantigens that are shared by EC and LC. The identification of CTL-activating, EC-specific alloantigens may help to explain the extreme immunogenicity of MHC compatible skin allografts.
Evidence for IgE-dependent cytotoxicity by rat eosinophils.
Authors: Capron M , Bazin H , Joseph M , Capron A
Published: December 2022

Human and rodent eosinophils have been shown previously to act as effector cells against Schistosoma mansoni schistosomula by ADCC mechanisms involving IgG antibodies. The present work brings novel evidence for the existence in rat schistosomiasis of an IgE-eosinophil dependent cytotoxicity mechanism. The role of IgE antibodies present in the rat serum after 6 weeks of infection was clearly established by immunoadsorption and inhibition experiments, whereas the participation of IgG and complement in this system could be ruled out. Mast cell products, including ECF-A tetrapeptides, appear to play an essential role in significantly increasing eosinophil cytotoxicity. A kinetic study of the IgG-dependent cytotoxicity mechanism previously described and of this IgE-mediated mechanism according to rat schistosomiasis revealed the preeminent role played by IgG antibodies in early infection, whereas IgE predominated after 6 wk of infection. The possible significance of IgE-eosinophil cooperation in ADCC mechanisms in parasite and nonparasite models is discussed.
Complement does not facilitate plasmodial infections.
Authors: Ward P A , Sterzel R B , Lucia H L , Campbell G H , Jack R M
Published: December 2022

The influence of the complement (C) system on Plasmodial infections in vivo (Plasmodium berghei in rats) and in vitro (Plasmodium falciparum) has been determined. In rats C3 depletion by treatment of animals with the C3 inactivator isolated from cobra venom factor results in infection that develops more rapidly, reaches a higher peak of parasitemia and is associated with an increased mortality rate (60%), in contrast to a lower degree of parasitemia and lack of mortality in C3-intact rats. The infection of human red cells by P. falciparum is not affected when serum C is inactivated either by heat treatment or by incubation with preformed immune complexes. Furthermore, human sera genetically deficient in C2, C3, C4, or C5 are as effective in facilitating the infection of red cells as is C-intact serum. These data suggest that, in contrast to Babesia sp., the in vitro or the in vivo infection of red cells by Plasmodium sp is not facilitated by the availability of C.
Characterization of genetic defects in macrophage tumoricidal capacity: identification of murine strains with abnormalities in secretion of cytolytic factors and ability to bind neoplastic targets.
Authors: Adams D O , Marino P A , Meltzer M S
Published: December 2022

Peritoneal macrophages from BCG-infected C3H/HeJ or A/J mice, in contrast to BCG-activated macrophages from C3H/HeN mice, were ineffective at lysing adherent 1023 sarcoma targets, nonadherent P815 mastocytoma targets, or nonadherent EL-4 lymphoma targets. The ability of macrophages from BCG-infected C3H/HeJ mice to secret cytolytic factor (CF) into the supernatant medium was markedly impaired. This secretory deficit, however, did not extend to plasminogen activator, secretion of which was augmented. In contrast, the ability of BCG macrophages from A/J mice to secrete CF was comparable to or even slightly higher than that of macrophages from C3H/HeN mice. The ability of BCG-elicited macrophages from A/J mice to bind either of 2 neoplastic targets (the P815 mastocytoma and the EL-4 lymphoma), however, was greatly reduced. The ability of BCG-elicited macrophages from C3H/HeJ mice to bind these targets was comparable to that of macrophages from C3H/HeN mice. The data suggest that the phenotypically-similar deficits in tumoricidal capacity of BCG-elicited macrophages from C3H/HeJ and A/J mice are mediated by mechanistically different defects in macrophage-tumor cell interactions.
Serum-mediated suppression of nonspecific B cell activation. I. Description of an inhibitory capacity in normal mouse serum and characterization of the inhibitory component.
Authors: Walker S M , Sturtevant J E , Weigle W O
Published: December 2022

Normal mouse serum (NMS) in relatively small amounts was demonstrated to inhibit in vitro splenic B cell activation by several B cell mitogens, polyclonal activators, and T-independent antigens. Proliferative and polyclonal responses elicited by bacterial endotoxin (ET), PPD, or Fc fragment of Ig were suppressed routinely 90% or greater when 0.5% homologous NMS was present in the culture medium. Similar suppression of T-independent antibody responses to TNP-ET and TNP-Ficoll was also observed. In contrast, activation by the T cell mitogen, Con A, and a T-dependent antigen response to SRBC was not inhibited by NMS. Suppression of B cell responses by NMS was not dependent on T cells. Preliminary characterization of NMS-In revealed a m.w. of approximately 200,000, an isoelectric point of 5, and solubility in 40% saturated ammonium sulfate. Both functionally and physically, NMS-In appears to be different from other currently relatively well characterized regulatory substances in serum. NMS-In was found in plasma as well as in serum, suggesting that NMS-In may function naturally in vivo to minimize nonspecific activation of B cells.
Lysis of target cells infected with vesicular stomatitis virus (VSV) in the presence of tunicamycin by anti-VSV cytotoxic T lymphocytes.
Authors: Harris D T , Hale A H , Lefrancois L
Published: December 2022

We have analyzed the requirement for the expression of the major surface glycoprotein (G protein) of vesicular stomatitis virus (VSV) on target cells for recognition and lysis by anti-VSV cytotoxic T lymphocytes (CTL). In addition, we have attempted to determine if the carbohydrate moieties on the G protein are required for recognition and lysis by anti-VSV CTL. When VSV (Orsay) is grown at 30 degrees C in the presence of tunicamycin (TM), glycosylation of G protein is inhibited; however, nonglycosylated G protein is found on the surface of the cell and active virus particles are produced. In contrast, VSV (Orsay) grown at 39 degrees C in the presence of TM produces low titers of virus and the presence of G protein on the surface of cells is not detectable. The susceptibility of these target cells to lysis by anti-VSV CTL was analyzed. The results suggest that expression of the G protein is required for target cell lysis by anti-VSV CTL. However, the presence of the carbohydrate moieties on the G protein are nt an absolute requirement for recognition by anti-VSV CTL. VSV-infected target cells incubated in the presence of TM were lysed by anti-VSV CTL up to 50 to 80% of the infected target cell control. This result suggests either that some clones of anti-VSV CTL recognize carbohydrate moieties or that carbohydrate moieties play some as yet undefined nonantigenic role in the recognition of the target antigen by the CTL receptor.
Immunoglobulin class commitment exhibited by B lymphocytes separated according to surface isotype.
Authors: Teale J M , Lafrenz D , Klinman N R , Strober S
Published: December 2022

In order to determine the extent to which B cells exhibit class commitment, the isotype(s) secreted by individual B cells and their progeny was studied in relation to the isotype(s) expressed on the surface of the B cell. Both primary and secondary B cells were separated according to cell surface isotypes and analyzed under optimal stimulation conditions using the T-dependent splenic focus assay. From these studies the following conclusions conclude be made: a) essentially all mature primary B cells appear to express mu on their surface; b) accordingly, the primary B cell population appears to be devoid of B cells expressing solely IgG on their surface; c) the majority of primary B cells in the presence of optimal T cell help have the potential for multiple class production; d) the secretion of IgA coincides with that of IgM and not IgG in its synthesis by immature B cells; e) a substantial proportion of memory B cells bear mu on their surface; f) memory B cells that lack s-mu are unable to yield clones secreting IgM; and g) a substantial proportion of memory B cells have the potential for multiple class production.
The biochemical and biological characterization of lymphocyte regulatory molecules. VI. Generation of a B cell hybridoma whose antibody product inhibits interleukin 2 activity.
Authors: Gillis S , Henney C S
Published: December 2022

In attempts to generate monoclonal antibodies with reactivity directed against the lymphokine Interleukin 2 (IL-2, T cell growth factor), spleen cells harvested from BALB/c mice previously immunized with rat IL-2 were fused with the BALB/c myeloma SP2. Several of the resultant hybrid cell lines secreted a product that significantly neutralized (greater than 50%) IL-2--dependent T cell proliferation. Several lines of evidence suggested that the inhibitory activity was associated with a monoclonal IgG antibody directed against IL-2 determinants. First, passage of cloned hybrid cell culture supernatants through a protein A-coupled Sepharose column yielded purified immunoglobulin G fractions that inhibited mouse, rat, and human IL-2 activity. Secondly, hybridoma-derived IgG, in concert with lyophilized Staphylococcus aureus, was capable of precipitating both "cold" and intrinsically labeled IL-2 activity. Finally, Sepharose conjugated with purified IgG fractions provided on extremely reactive IL-2 absorption matrix. These results suggest that monoclonal antibodies directed against IL-2 determinants may eventually provide new detection assays for IL-2 and allow affinity chromatography to be employed for the isolation of this lymphokine.
Natural cytotoxic cells against solid tumors in mice. III. A comparison of effector cell antigenic phenotype and target cell recognition structures with those of NK cells.
Authors: Lattime E C , Pecoraro G A , Stutman O
Published: December 2022

Antisera directed to antigens present on natural killer (NK) cells such as Qa-2,3, Qa-5, NK-1.2, Ly 11.2, and Asialo GM 1 failed to eliminate natural cytotoxic (NC) cell activity from murine spleen cells, suggesting a heterogeneity of effector populations involved in natural cell-mediated cytotoxicity (NCMC). Cross-cold target inhibition studies demonstrated that certain similar recognition structures are present both on NK and NC susceptible tumor target cells.
Characterization of the T lymphocyte colony-forming cells and evidence for the acquisition of T cell markers in the absence of the thymic microenvironment in man.
Authors: Triebel F , Robinson W A , Hayward A R , de Laforest P G
Published: December 2022

The nature of the T colony-forming cell (T-CFC) in agar is still controversial. We present evidence that blood mononuclear cells depleted of T cells by E-rosetting or lysis with OKT 3 and complement can give rise to E+ OKT 3+ colonies in the presence of supernatants of PHA-stimulated lymphocytes (P-SUP) containing a cell growth factor. Both OKT 4+ (67%) and OKT 8+ (32%) cells were found in the colonies, and less than 10% of these cells were Ia+. We consider that colonies in agar can arise from the proliferation and maturation of a circulating immature progenitor (T colony-forming cell, or T-CFC) that does not need the thymic environment to acquire the OKT 3, 4, and 8 antigens or E receptor. In contrast, Ia+ OKT 3+ cells that are maintained by P-SUP in long-term liquid culture are reportedly derived from mature T cells, and this proliferation might represent the counterpart of the in vivo response to any mitogenic stimulation of the peripheral compartment of mature T cells.