bluuebunny/crossref_metadata_2025 huggingface dataset:
A human NK and K cell subset shares with cytotoxic T cells expression of the antigen recognized by antibody OKT8.
Authors: Perussia B , Fanning V , Trinchieri G
Published: December 2022

The antigen recognized by monoclonal antibody OKT8 is expressed on the cell membrane of 30 to 50% of human NK/K cells. The reactivity of OKT8 with NK/K cells was determined by indirect methods (treatment of the effector cells with OKT8 antibody and complement (C) and separation of OKT8(+) and (-) effector cell populations by fluorescence-activated cell sorting or by rosetting techniques) and, at single cell level, by C-dependent lysis of effector NK cells that bind and kill K562 targets. Analysis by indirect immunofluorescence (flow cytofluorometry) of lymphocyte subpopulations mediating NK/K cytotoxic activity and deprived of OKT8(+) T cells reveals that the NK/K cell subset bears OKT8 antigen at a density lower than that present on cytotoxic T cells. The OKT8 antigen on NK/K cells is trypsin- and pronase-sensitive, but it is resynthesized by the same effector cells during 24 hr of culture at 37 degrees C. OKT8 antibody does not inhibit NK killing, and, on a per cell basis, OKT8(+) cells within the NK/K subset mediate the same level of cytotoxic activity as OKT8(-) NK/K cells. Analogous results were obtained by using anti-Leu-2a, an antibody with the same specificity as OKT8 on cytotoxic/suppressor T cells, but not when OKT5 was used, which might identify a distinct epitope on the same antigenic molecule. The possible significance of these findings in understanding the cell lineage of NK/K cells is discussed.
T cell factor-mediated suppression of immunoglobulin secretion by a human B lymphoblastoid cell line.
Authors: Wilkins J A , Jordan Y , Rutherford W J , Warrington R J
Published: December 2022

The Epstein Barr Virus-transformed B lymphoblastoid cell line JR-2, which spontaneously secretes IgG detectable by a hemolytic plaque technique, is suppressed by the concanavalin A-induced T cell-derived nonspecific suppressor factor T-SF in a dose-dependent manner. This suppression is reversible after 24 hr and is not due to a cytotoxic mechanism. The T-SF does not affect proliferation of JR-2. Suppression by the T cell factor is inhibited or blocked by 2-mercaptoethanol or L-rhamnose and is also induced by gel chromatography fractions of the T cell supernatants containing molecules of 20,000 to 25,000 daltons. The JR-2 line appeared to be uniquely sensitive to the T-SF in that other potential suppressor T cell-derived factors were not active and 15 other B lymphoblastoid cell lines were not significantly inhibited by the T-SF.
T cell hybridomas coexpressing Fc receptors (FcR) for different isotypes. II. IgA-induced formation of suppressive IgA binding factor(s) by a murine T hybridoma bearing Fc gamma R and Fc alpha R.
Authors: Yodoi J , Adachi M , Teshigawara K , Miyamainaba M , Masuda T , Fridman W H
Published: December 2022

T2D4 murine T hybridoma cells have previously been shown to express Fc receptors (FcR) for IgG (Fc gamma R) and for IgA (Fc alpha R) and to produce an IgG binding factor (IgGBF) that suppresses IgG and IgM responses. In the present work we report on the behavior of IgA bound to T2D4 cells and on the production of IgA binding factor (IgABF) and its ability to suppress IgA antibody production. A dose-dependent binding of MOPC315 IgA with anti-TNP activity by T2D4 cells was demonstrated by rosette formation with trinitrophenylated ox red blood cells (TNP-ORBC) and fixation of iodinated DNP-BSA. IgA bound to the cells disappeared after a short-term culture of 3 hr at 37 degrees C, but not at 4 degrees C. Because this phenomenon was inhibited by 0.1% sodium azide and 100 microM dansylcadaverine, a transglutaminase inhibitor, Fc alpha R-IgA complexes seemed to be released by an active process involving receptor movement. In the culture supernatant of IgA-treated T2D4 cells, we detected a factor(s) that binds to IgA-Sepharose and competitively inhibits the binding of IgA to T2D4 cells. The factor (IgABF) failed to inhibit the rosette formation of Fc gamma R(+) cells with IgG-sensitized ORBC (EAox gamma), indicating that it binds specifically to IgA. IgABF was undetectable in the culture supernatants of untreated T2D4 cells of Fc alpha R(-) BW5147 T lymphoma cells used as parent cells for the establishment of the hybridoma. To study the effect of IgABF on antibody formation, culture filtrates of IgA-treated or untreated T2D4 cells were fractionated on IgA-Sepharose beads and were added to BALB/c spleen cells cultured with pokeweed mitogen. By use of a reverse plaque assay, it was shown that the IgA plaque-forming cell (PFC) response was suppressed by the acid eluate but not by the effluent of IgA-Sepharose beads incubated with the filtrates of IgA-treated T2D4 cell cultures. The suppression was IgA specific, because neither IgG nor IgM responses were suppressed by the eluate. As expected, there was no significant IgA suppressive activity in the acid eluates of the beads incubated with the culture filtrate of untreated T2D4 cells or IgA-treated BW5147 cells. IgA-specific suppressive activity proved to be due to IgA binding factor(s), because suppressive activity in the eluate was completely adsorbed by IgA-Sepharose but not by IgG- nor BSA-Sepharose.
Allo-Ia reactive murine T-cell lines. II. Mechanisms of clonal expansion of T cells explored by use of the allo-Ia reactive T cell clones.
Authors: Narimatsu H , Saito K
Published: December 2022

Murine T cell clones, which were retrieved from an A.TH anti-A.TL(lak) T cell line and had been long-term cultured in the medium supplemented with T cell growth factor (TCGF) and mitomycin C(MMC)-treated feeder cells of either Is or Ik haplotype, were found to survive in TCGF-free medium for a long time, quite in contrast to so far reported TCGF-dependent T cell clones. When T cells of these clones at the full growth in the TCGF-medium were transferred to TCGF-free medium, they survived at resting state for a long time, and half-life, i.e., the time when 50% of the transferred cells were still viable, of some clones reached 20 days. The cloned T cells at the resting state retained full responsiveness to the specific lak antigen but lost the responsiveness to TCGF as determined by [3H]thymidine uptake, whereas the same T cells harvested from TCGF-medium did not show the antigen-specific responsiveness. The cloned T cells at the resting state showed marked DNA synthesis in response to the specific antigen but never entered the phase of the cell division. Addition of TCGF to the antigen-activated cloned T cells at their peak DNA synthesis triggered the cell division without time lag. Thus, it was confirmed at a single clone level that two sequential signals, one via the antigen-receptor reacting with specific antigen and another via the TCGF-receptor accepting TCGF, are required for clonal expansion of T cells reacting with antigen. The mitogen-responsiveness among five clones was examined at their resting state; two clones responded to Con A and PHA only in the presence of accessory cells (MMC-treated, T cell-depleted syngeneic spleen cells), and one clone responded well to Con A and PHA in the absence of accessory cells. Thus, most of our clones retained physiologic characteristics of T cells directly collected from mice even after long-term culture in TCGF-medium.
A lymphocyte cell surface molecule that is antigenically related to actin.
Authors: Sanders S K , Craig S W
Published: December 2022

When viable murine lymphocytes are incubated sequentially with a saturating amount of affinity-purified, rabbit anti-actin and highly conjugated FITC-goat anti-rabbit Ig, about 52% of mesenteric lymph node (MLN) lymphocytes and 36% of thymocytes exhibit a faint, but sharply punctate surface fluorescence. Cell surface actin (CSA) can be distinguished from staining of cytoplasmic actin in permeable cells, which are identified by their uptake of ethidium bromide. Staining of actin in ethidium bromide-permeable cells is 10-fold more intense than staining of actin on ethidium bromide-impermeable cells and is seen as uniformly fluorescent rings or crescents at the periphery of the cell and as dimmer, diffuse fluorescence centrally. Binding of rabbit anti-actin and goat anti-rabbit Ig to the lymphocyte cell surface is not mediated by Fc receptors; F(ab')2 fragments of these antibodies detect the same number of positive cells as do the intact molecules, and affinity-purified anti-KLH does not bind significantly. The cell surface stain, measured by flow cytometry or fluorescence microscopy, can be absorbed by pretreatment of the anti-actin with immobilized actin but not with IgG-Sepharose. Double-label experiments show that about 70% of the non-B cells and 30% of the MLN B cells bear detectable CSA. Although we have not ascertained the origin of CSA, we find that the number and brightness of cells exhibiting CSA cannot be increased by preincubating the cells with exogenous native skeletal muscle actin or with supernatant from dissociated MLN, indicating that there are no free binding sites for exogenous actin. The findings imply that either there is a developmentally expressed binding site(s) for actin, or that at various stages of development lymphocytes express a protein antigenically related to actin on their surface.
Measuring leukotrienes of slow reacting substance of anaphylaxis: development of a specific radioimmunoassay.
Authors: Hayes E C , Lombardo D L , Girard Y , Maycock A L , Rokach J , Rosenthal A S , Young R N , Egan R W , Zweerink H J
Published: December 2022

Rabbits were immunized with leukotriene C4 (LTC4) coupled to thiolated keyhole limpet hemocyanin (KLH) by using 6-N-maleimidohexanoic acid as a spacer molecule. Immune serum was obtained with 7.9 nmol of LTC4-specific immunoglobulin per milliliter and a mean association constant of 2.1 X 10(9) M-1. A radioimmunoassay was developed that detected 0.1 pmol of LTC4 per 1-ml sample. LTD4 and LTE4, three isomers of LTC4, the sulfones of LTC4, LTD4, and LTE4, and one isomer of LTD4 reacted to varying degrees in the assay. A number of other structurally related compounds, such as LTB4 and 5-HETE, did not react. Conditions were established to determine LTC4 levels in human plasma without loss of LTC4 during sample preparation and without the need for extraction procedures before the measurement of LTC4.
Respiratory responses to leukotrienes and biogenic amines in normal and hyperreactive rats.
Authors: Brunet G , Piechuta H , Hamel R , Holme G , Ford-Hutchinson A W
Published: December 2022

The effects of leukotriene D4, serotonin, and methacholine were studied on respiratory smooth muscle in vitro and respiratory responses in vivo in three strains of rats. These were an inbred strain of hyperresponsive rats, Sprague Dawley rats, and Fischer rats. Trachea from inbred rats responded in vitro to serotonin and methacholine but not to leukotrienes or histamine. Parenchyma from inbred rats responded to serotonin, methacholine, and leukotrienes. In vivo respiratory responses in inbred rats were observed after aerosol administration of histamine and serotonin, methacholine, and leukotriene D4. When these in vitro and in vivo experiments were repeated in Sprague Dawley and Fischer rats, a clear correlation was observed between the responses of strains of rats to aerosolized antigen and responses to spasmogenic mediators. It is concluded that inbred rats have a nonspecific bronchial hyperreactivity that contributes to their sensitivity to aerosolized antigen and that they may be a useful model for human asthmatic conditions.
Specificity of in vitro cytotoxic T cell clones directed against vesicular stomatitis virus.
Authors: Rosenthal K L , Oldstone M B , Hengartner H , Zinkernagel R M
Published: December 2022

Cytotoxic T lymphocytes (CTL) generated in mice against a particular serotype of vesicular stomatitis virus (VSV) were previously shown to cross-reactively lyse syngeneic target cells infected with serologically distinct types of VSV. To analyze the antigenic basis of this T cell cross-reactivity, we generated CTL against VSV-Indiana (VSV-Ind) and established them by limiting dilution as cloned in vitro cell lines. The cells continuously proliferate in medium containing concanavalin A-induced T cell growth factors. All of the cells are Thy-1.2+ and Lyt-2.2+. Lysis by these cells is H-2Dd-restricted, no natural killer cell activity is detectable, and all the clones cross-reactively lyse target cells infected with either VSV-Ind or VSV-New Jersey (VSV-NJ). In addition, no specific blocking of primary, secondary, or cloned anti-VSV CTL was achieved with the use of several monoclonal antibodies specific for the glycoprotein of VSV and capable of neutralizing either VSV-Ind or VSV-NJ. These results suggest that VSV serotype-specific neutralizing antibodies may recognize immunodominant determinants of VSV glycoprotein that are distinct from those recognized by the majority of VSV-specific CTL.
Monoclonal cytotoxic T lymphocyte hybridomas capable of specific killing activity, antigenic responsiveness, and inducible interleukin secretion.
Authors: Kaufmann Y , Berke G
Published: December 2022

We have recently described the production of cytotoxic T lymphocyte (CTL) hybridomas that grow continuously in culture, exhibiting constitutive, allospecific (anti-H-2b) killing activity. We now report on the response of these monoclonal CTL hybridomas to specific antigen (H-2Db) and to mitogenic lectins. Both specific antigen and T cell mitogens enhance hybridoma-mediated specific target cell killing. In addition, stimulated, but not unstimulated hybridoma cells secrete considerable amounts of IL 2 into the culture medium. Repeated cloning of the hybridomas provides strong evidence that both killing activity and IL 2 secretion can be attributed to one cell. Unfractionated Con A supernatants, containing IL 2 and other factors known to influence T cell responsiveness, or IL 2-containing media of stimulated hybridomas affect neither the growth nor the lytic activity of the hybridomas. Anti-LFA-1 monoclonal antibody, a potent inhibitor of CTL and CTL hybridoma-mediated target cell lysis, abolishes antigen- or mitogen-induced IL 2 secretion by the CTL hybridomas. Involvement of a single hybridoma receptor in antigen recognition (afferent and efferent) and in initiating IL 2 secretion is proposed. The CTL hybridomas displaying retarded killing activity before the antigenic or mitogenic stimulation appear to represent an intermediate stage in CTL differentiation, reminiscent of "memory" CTL.
Transplantation tolerance: Lyt-1+2- helper T cells require a second proliferation signal to overcome Lyt- 1-2+ suppressor T cell activity.
Authors: Chen B P , Splitter G A
Published: December 2022

This study investigated why nude mouse T cell precursors that differentiated in an allogeneic thymus graft were rendered tolerant to alloantigens of the thymus genotype. Our results showed that Lyt- 1-,2+ suppressor T cells inhibited clonal expansion of helper T cells reactive to thymus-donor alloantigens as the mechanism of tolerance. This suppressor T cell activity could be modulated in vitro with either exogenous interleukin 2 (IL 2) or anti-Lyt-1 monoclonal antibody as an IL 2 inducer. Monoclonal antibody stimulation of IL 2 production by primed helper T cells was effective even in the presence of suppressor T cells. These results indicate: i) that alloantigen-specific helper T cells were not defective in their ability to recognize alloantigens of the thymus-donor genotype and to produce IL 2; ii) that Lyt-1-,2+ suppressor T cells prevented Lyt-1+,2- helper T cells specific for the thymus-donor alloantigens from producing IL 2; and iii) that signals delivered through binding of anti-Lyt-1 antibody modulated suppressor activity on alloantigen-primed helper T cells and permitted IL 2 production by these helper T cells. Accordingly, a delicate balance of helper and suppressor T cell activity was required for the maintenance of transplantation tolerance. Furthermore, these results argue against clonal deletion of Lyt-1+,2- helper T cells reactive to alloantigens of the thymic genotype as a mechanism of such transplantation tolerance.
T cell responses induced by the parenteral injection of antigen-modified syngeneic cells. I. Induction, characterization, and regulation of antigen-specific T helper cells involved in delayed-type hypersensitivity responses.
Authors: Miller S D , Butler L D
Published: December 2022

This report presents evidence for the role of antigen-specific helper T cells in augmenting the in vivo development of delayed-type hypersensitivity (DTH) responses to both hapten and protein antigens. The role of these helper T cells in the in vivo induction and regulation of DTH responses was investigated. Mice were primed subcutaneously with optimal numbers (3 X 10(7)) of either protein antigen- or TNP-modified syngeneic spleen cells. Primed spleen or lymph node cells, but not thymocytes or unprimed cells, were found to significantly augment the DTH response of syngeneic recipients injected subcutaneously with suboptimal numbers (1 to 2 X 10(6)) of antigen-modified syngeneic cells. Primed spleen or lymph node cells augmented both in vivo ear swelling reactions and in vitro antigen-induced T cell proliferative responses in recipient animals. The helper effect was found to be mediated by a population of radioresistant, Thy-1+, Lyt-1+2-, I-A+ cells, a phenotype identical to that of antigen-specific Tprlf cells found in primed lymph nodes. In contrast, effector TDH cells were found to be Thy-1+, Lyt-1+2-, I-A- cells. Splenic T cells from TNP-primed mice augmented TNP-specific DTH responses, but not DTH to irrelevant protein antigens, and vice versa. Helper T cell induction correlated with the presence of H-2 I-region determinants on the inducer cells, because antigen-modified spleen cells were the most efficient inducers, modified thymocytes were less efficient, and modified erythrocytes were ineffective. Mapping studies also indicated that I-region identity between the antigen-modified spleen cell immunogen and the Th donors was both necessary and sufficient for DTH Th cell induction. In addition, functional helper T cell activity could be both specifically tolerized and suppressed by the transfer or suppressor T cells raised by the i.v. injection of antigen-modified syngeneic cells.
Genetically determined resistance to foreign bone marrow transplantation in mice: characterization of the effector cells.
Authors: Miller S C
Published: December 2022

Mice of most strains show a genetically determined ability to reject a variety of foreign marrow grafts even after lethal irradiation. The phenomenon is both host strain and donor marrow graft-dependent. To characterize the effector cell responsible for graft rejection, attempts were made to 1) determine to what morphologic subclass it belongs; 2) determine its life span; and 3) establish whether genetically different host environments influence the functioning of the effector cell. Mice of the 129/J strain (normally nonresistant), C57BL/6 strain (made non-resistant), and the homozygous mutants of C57BL/6, i.e., C57BL/6 (bg/bg), were recipients of C57BL/6 marrow or spleen cells. After lethal irradiation, hosts were given marrow or spleen cells from normal, strongly resistant C57BL/6 donors pretreated with a) 950 R whole body irradiation or b) twice daily injections for 4 days of the cell cycle toxic drug hydroxyurea followed by 950 R. In other cases, hosts were recipients of the lymphoid cell-rich fraction of marrow from irradiated C57BL/6 donors or adherent cells taken from cultures of marrow cells of unirradiated C57BL/6 donors. Three hours after receiving C57BL/6 marrow or spleen cells, irradiated hosts were given allogeneic DBA/2 marrow (always strongly rejected by C57BL/6 mice and always accepted by 129/J strain mice). Seven days later, host spleens were removed and the numbers of microscopic colonies were counted from subserial sections. The results demonstrate that 1) mice either normally or rendered nonresistant to a marrow allograft can be made to develop resistance by the administration of either whole spleen cells or marrow lymphoid cells from lethally irradiated strongly resistant donors; 2) adherent cells from cultures of marrow from strongly resistant mice are ineffective in conferring resistance; 3) the cell effective in conferring resistance has a life span greater than 4 but less than 7 days; and 4) the effector cell can function in genetically different environments of nonresistant strains.
Noncovalently bonded subunits of 22 and 28 kd are rapidly internalized by T cells reacted with anti-Leu-4 antibody.
Authors: Kan E A , Wang C Y , Wang L C , Evans R L
Published: December 2022

Using immunofluorescence analysis in a FACS or cytofluorograph, we monitored the density of four different human T cell surface molecules (Leu-1/OKT1, Leu-2/OKT5,8, Leu-3/OKT4, and Leu-4/OKT3) at different intervals of incubation at 37 degrees C with monoclonal antibodies that react with these structures. Leu-1 and Leu-2 are probable human homologues of the murine Lyt-1 and Lyt-2,3 antigens, respectively. Leu-3 is a 55 kd antigen that is restricted to Leu-2- (helper) T cells. Leu-4 is expressed by all peripheral T cells and has at least two components of 22 and 28 kd that have an undefined structural relationship. The ultimate degree of modulation of each antigen varied considerably. Modulation of Leu-4 was particularly striking, because this antigen disappeared completely from the cell membrane within 7 hr of culture with saturating concentrations of alpha Leu-4. After modulation of surface 125I-labeled T cells, Leu-4 was found in the detergent-solubilized cell lysate, but not in the cellfree supernatant, indicating that it was internalized. Our results also indicated that the internalized antigen-antibody complex existed in at least two forms: a) as an antigen-antibody complex having only the lower m.w. (22 kd) component bound to antibody, or b) as free antigen having the 28 kd chain in noncovalent association with the 22 kd moiety. These studies indicate that the Leu-4 structure is composed of at least two distinct chains that are noncovalently associated. The rapid modulation and structural properties of the Leu-4 complex are discussed in the context of its possible functions in T cell immunity.
Blockade of the interleukin-2 receptor by anti-Tac antibody: inhibition of human lymphocyte activation.
Authors: Depper J M , Leonard W J , Robb R J , Waldmann T A , Greene W C
Published: December 2022

We have previously shown that monoclonal anti-Tac antibody inhibits the proliferation of interleukin-2 (IL-2) dependent human continuous T cell lines. Further, we have shown that anti-Tac specifically blocks greater than 95% of the binding of radiolabeled II-2 to a continuous T cell line. In view of these data, we suggested that anti-Tac antibody may bind to and block the human T cell receptor for IL-2. We now report the effects of anti-Tac on the activation of human peripheral blood T lymphocytes. We find that anti-Tac: 1) blocks T cell proliferation induced by soluble antigens (80-90%), autologous antigens (90%) and alloantigens (75-90%); 2) partially inhibits T cell proliferation induced by mitogenic lectins, including Concanavalin A (50-88%), pokeweed mitogen (40-87%), and phytohemagglutinin (20-80%); 3) abrogates (greater than 95%) the generation of cytolytic T lymphocytes in allogeneic cell cocultures, but does not inhibit killing by cytolytic T lymphocytes once formed; 4) and inhibits T cell dependent pokeweed mitogen activated B cell immunoglobulin production (78-95%). We further demonstrate that anti-Tac inhibition of proliferation is not secondary to diminished production of IL-2. Finally, in antigen induced T cell proliferative assays, we demonstrate that the addition of highly purified IL-2 reverses the inhibitory effects of anti-Tac. These data are consistent with the hypothesis that anti-Tac recognizes the human IL-2 receptor and illustrate ways in which this antibody can be used to modulate the human immune response.
Studies on demyelination in vitro: the requirement of membrane attack components of the complement system.
Authors: Liu W T , Vanguri P , Shin M L
Published: December 2022

Anti-spinal cord antibodies (anti-SC) cause demyelination of well myelinated mouse cerebellum cultures in the presence of fresh serum. Heating the serum for 30 min at 56 degrees C abolishes the demyelinating activity. We studied the role of complement (c) in demyelination initiated by anti-SC in well myelinated mouse cerebellum cultures. Demyelination was assessed morphologically. The extent of demyelination was correlated to the dose of whole serum C as well as the dose of antibody. To evaluate the requirement of membrane attack components of C, C5b-C9, sister cultures were treated with antibody + C8 deficient human serum (C8D-HS) with and without purified human C8. Extensive demyelination was observed in C8-reconstituted cultures whereas antibody + C8D-HS did not demyelinate, indicating the essential requirement of C5b-8, and/or C5b-9. Extensively demyelinated cultures remyelinated when fresh medium was supplied, suggesting that the process of antibody and C-mediated demyelination is selective for myelin membrane in this system.
Interferon-gamma induces enhanced expression of Ia and H-2 antigens on B lymphoid, macrophage, and myeloid cell lines.
Authors: Wong G H , Clark-Lewis I , McKimm-Breschkin L , Harris A W , Schrader J W
Published: December 2022

The levels of class II major histocompatibility complex (MHC) antigens (la antigens) on cells of a cultured B lymphoma line (WEHI-279) were significantly increased after 24 hr incubation with medium conditioned by concanavalin A-stimulated mouse or rat spleen cells, or by an azobenzenearsonate- (ABA) specific T cell clone that had been stimulated with ABA-coupled spleen cells or concanavalin A. The levels and properties of the la-inducing activity correlated with those of interferon-gamma (IFN-gamma) measured by inhibition of virus plaque formation. Both the la-inducing activity and the IFN-gamma from the T cell clone had an apparent m.w. of 40,000 determined by gel filtration, were sensitive to treatment with trypsin or exposure to pH 2, but were stable to heat (56 degrees C, 1 hr). The induction of la antigens on WEHI-279 cells was dose-dependent, and the maximum response occurred at a concentration corresponding to 1 to 2 U/ml of antiviral activity. This T cell-derived IFN-gamma-like molecule also increased the expression of cell surface la antigens on another B cell line (WEHI-231), and cell lines of macrophage (J774) and myeloid (WEHI-3B and WEHI-265) origin. Furthermore, in all cases the levels of class I MHC (H-2K or H-2D) antigens were also increased. Similar patterns of induction of Ia and H-2 antigens were obtained with supernatants containing IFN-gamma produced by a monkey cell line (COS) that had been transfected with a plasmid bearing the cloned murine IFN-gamma gene. This activity was sensitive to pH 2 and was not present in the supernatant from COS cells that were not transfected with the murine IFN-gamma gene. These results established that IFN-gamma is the T cell-derived molecule that induces the enhanced expression of Ia and H-2 antigens on B cells and macrophages. A major physiologic role of IFN-gamma may be to regulate immune function through the enhanced expression of MHC antigens.
Coordinate production by a T cell hybridoma of gamma interferon and three other lymphokine activities: multiple activities of a single lymphokine?
Authors: Zlotnik A , Roberts W K , Vasil A , Blumenthal E , Larosa F , Leibson H J , Endres R O , Graham S D , White J , Hill J , Henson P , Klein J R , Bevan M J , Marrack P , Kappler J W
Published: December 2022

The T cell hybridoma FS7-20, produced by the fusion of normal B10.BR T cells to the AKR thymoma BW5147, was found when stimulated with concanavalin A (Con A) to produce the lymphokines: interleukin 2 (IL 2), interferon-gamma (IFN gamma), macrophage-activating factor (MAF), Ia induction factor IaIF), and the B cell helper factor interleukin X (IL X). The clones and subclones of FS7-20 varied dramatically in their ability to produce these lymphokines, presumably because of karyotypic variations. The ability to produce IL 2 segregated independently from the ability to produce the four other lymphokine activities; however, production of the latter activities showed a strong correlation. This coordinate production of IFN gamma, MAF, IaIF, and IL X was also observed with a cloned normal cytotoxic T cell line, cr15. These results suggest either that IFN gamma, MAF, IaIF, and IL X are all manifestations of a single molecular species or that, although these activities are different structurally, their production is controlled by a common genetic mechanism. In support of the first possibility, the IFN gamma, MAF, IaIF, and IL X activity produced by FS7-20 were all found to be equally sensitive to inactivation at pH 2. These results illustrate the usefulness of using T cell hybridomas for the study of lymphokines.
Production and characterization of antibodies to thymosin beta 4.
Authors: Goodall G J , Hempstead J L , Morgan J I
Published: December 2022

Thymosin beta 4 antibodies have been raised in rabbits by conjugating the peptide to keyhole limpet hemocyanin, and a radioimmunoassay for thymosin beta 4 has been established utilizing tritiated thymosin beta 4 as binding ligand. RIA values for thymosin beta 4 in rat tissues reveal levels ranging between 8 and 448 micrograms/g wet weight. Highest levels are observed in spleen, although the peptide is also relatively abundant in thymus, lung, and nasal epithelium. Brain, kidney, liver and testis contain only 10 to 20% the concentration of thymosin beta 4 seen in spleen, whereas heart and muscle contain only 5 to 10% that of spleen. However, thymosin beta 4 probably has a ubiquitous distribution in the rat, being present in all tissues examined. The specificity of the antiserum used in the RIA has been defined. The thymosin beta 4 molecule seemingly bears two major epitopes that lie between amino acid residues 1-8 and 22-32. The antiserum does not cross-react with other unrelated peptides. The antiserum will precipitate biosynthetically labeled beta 4 from cultured rat peritoneal macrophage.
The antigenic structure of HLA-A2: an analysis with competitive binding assays and monoclonal antibodies.
Authors: Ways J P , Parham P
Published: December 2022

Twelve mouse monoclonal antibodies against HLA-A2 were analyzed by competitive binding assays on cells. The A2 molecule was found to have two topologically distinct regions of highly specific alloantigenic determinants. One region encompasses determinants shared with HLA-A28 and the other contains determinants shared with HLA-B17 and a variant, A28*, of HLA-A28. Each of these two alloantigenic regions is more closely associated with a different broadly polymorphic determinant shared by many HLA gene products. The topographic relationship between the two alloantigenic regions, four monomorphic determinants, and the carbohydrate moiety of the HLA heavy chain was also assessed. All the antigenic determinants preferentially associate with one or other of the two alloantigenic regions. An analysis of these results with reference to the amino acid sequences of A2 and A28 suggests a working hypothesis whereby the A2,A28*,B17 region primarily involves residues of the NH2-terminal or first domain, and that the A2,A28 region primarily involves residues of the second domain of the A2 heavy chain.
Evidence that schistosome MHC antigens are not synthesized by the parasite but are acquired from the host as intact glycoproteins.
Authors: Simpson A J , Singer D , McCutchan T F , Sacks D L , Sher A
Published: December 2022

Schistosoma mansoni schistosomula recovered from the lungs of mice have previously been shown to express serologically detectable products of the major histocompatibility complex (MHC). To determine whether these determinants are products of the schistosome genome, DNA from cercariae and adult worms of the parasite was probed with a 32P-cDNA clone encoding a human class I MHC antigen. Although this probe hybridized to mouse DNA, no hybridization was observed with DNA isolated from schistosomes, indicating that there are no DNA sequences homologous to class I MHC antigens in the parasite genome. The class I MHC antigens found on schistosomes were characterized in a further series of experiments. Two monoclonal antibodies known to recognize spatially distinct determinants of H-2Kk both bound to lung-stage schistosomula recovered from mice expressing this haplotype, suggesting that a significant portion of the H-2K molecule is present at the larval surface. Furthermore, both antibodies precipitated from lysates of 125I-lactoperoxidase-labeled lung-stage schistosomula, a molecule of approximately 45,000 daltons similar in mobility to H-2Kk precipitated from 125I-labeled mouse spleen cells. These results support the hypothesis that the class I MHC antigens expressed on S. mansoni are not synthesized by the parasite but are acquired from the host as intact glycoproteins.
Western Blot analysis of the antigens of Toxoplasma gondii recognized by human IgM and IgG antibodies.
Authors: Sharma S D , Mullenax J , Araujo F G , Erlich H A , Remington J S
Published: December 2022

Western Blot analysis revealed that both IgM and IgG antibodies present in the sera of humans infected with Toxoplasma gondii recognize three major antigens with apparent m.w. of 32,000, 22,000, and 6000, respectively. In addition, IgG antibodies recognized at least 17 other antigenic components. After subcellular fractionation, enrichment of the three major antigens recognized by IgM and IgG antibodies by the membrane fraction was observed. Solubilization of membrane-enriched preparations with a mixture of sodium dodecyl sulfate and sodium deoxycholate did not reveal any new antigenic structures that reacted with IgM or IgG antibodies. Treatment of Toxoplasma lysate preparations and various fractions obtained after differential centrifugation with NaIO4 diminished the reactivity of the antigens with both IgM and IgG antibodies. Lipase treatment had no effect on the number or nature of antigens recognized by IgM antibody. Treatment with pronase and trypsin eliminated the 32,000 and 22,000 m.w. antigenic components detected by IgM antibodies, whereas such treatment had no effect on the 6000 m.w. component. Periodic acid-Schiff staining of polyacrylamide gels of Toxoplasma sonicates revealed the presence of three components corresponding to m.w. of 62,000, 45,000, and 6000, respectively. At least 15 components, including the 6000 m.w. component, directly bound concanavalin A.
Clonal analysis of H-2Kb + TNP recognition by T cells with the use of H-2Kbm mutants and H-2Kb-specific monoclonal antibodies.
Authors: Davignon J L , Guimezanes A , Schmitt-Verhulst A M
Published: December 2022

Eleven long-term cytotoxic T lymphocyte (CTL) clones derived from C57BL/10 T cells sensitized in vivo and in vitro with trinitrobenzene sulfonate- (TNBS) treated syngeneic cells were all restricted to the K end of H-2b. The fine specificity of these CTL clones was analyzed by using H-2Kbm mutant target cells and H-2Kb-specific monoclonal antibodies (mAb). Seven distinct patterns of reactivity of the T cell clones could be observed with the use of six H-2Kbm mutant target cells. Further heterogeneity could be detected in terms of the ability of anti-Lyt-2 mAb to inhibit CTL activity. Cross-reactivity between H-2Kb + TNP and H-2Kbm + TNP was observed for all clones tested for bm5 and bm6, but less frequently for bm3 (8/11), bm8 (7/10), bm4 (4/11), and bm1 (3/11). It was further observed that amino acid substitutions located in the first domain only (one clone), or in the second domain only (six clones), or in either the first or the second domain (three clones) of the H-2Kb molecule could affect target cell recognition by a given T cell clone. the latter type of reactivity suggested that some clones recognized "conformational" determinants of the H-2 molecule, or that amino acid substitutions in one domain might influence the structure of the next domain. One H-2Kb + TNP-reactive clone exhibited a heteroclitic behavior with decreasing avidities for target cells expressing H-2Kbm8 + TNP, H-2Kb + TNP, and H-2Kbm8, which further extends the various patterns of T cell cross-reactions observed within a given class of MHC products. The use of H-2Kb-specific mAb in blocking studies as an attempt to define further the H-2Kb epitopes recognized by CTL clones indicated that: a) TNBS treatment may affect the antigenicity of the H-2Kb molecule as assessed by some mAb; and b) that the T cell clone-target cell interaction may or may not be inhibited by a given mAb, depending on structural variations of the H-2Kb molecule (use of H-2Kbm mutants) that do not affect the interaction itself. These results indicate that this type of analysis does not permit correlation of serologic- and T cell-defined epitopes.
Generation of thymocyte subpopulations in organ culture: correlated analysis of Lyt-2 phenotype and cell cycle status by flow microfluorometry.
Authors: Sékaly R P , Ceredig R , MacDonald H R
Published: December 2022

We have investigated the cell cycle kinetics and the proliferative activity of Lyt-2-defined subpopulations in organ-cultured thymic rudiments. Fetal thymuses that were removed at 13 days of gestation and cultured for 4 to 7 days were incubated with bromodeoxyuridine and were then stained with Hoechst 33342 to quantitate the duration of the cell cycle and the number of cycling cells. The total duration of the cell cycle was 30 hr. The sorting of organ culture cells according to their Lyt-2 phenotype and their forward light scatter, followed by Hoechst fluorescence and propidium iodide analysis, indicated that small Lyt-2+ cells that are themselves noncycling were derived through mitosis from another cell population. The combination of the Hoechst-BrdU substitution technique with monoclonal antibodies and the organ culture should provide a powerful tool for the study of lineage pathways in the thymus.
Effects of cytochalasins on lymphocytes: mechanism of inhibition of rosette formation.
Authors: Mookerjee B K , Jung C Y
Published: December 2022

The mechanisms operative in the inhibition by cytochalasins of human peripheral blood T lymphocytic rosette formation with sheep erythrocytes remain obscure in the light of the multiplicity of biologic effects of cytochalasins. Moreover, we have shown the existence of three distinct classes of cytochalasin-binding sites (H-, M-, and L-sites) in such lymphocytes (J. Biol. Chem. 256:1290-1300, 1981). We have, therefore, explored the mechanism of rosette inhibition and present evidence that shows: a) Inhibition of rosetting is not caused by inhibition of glucose transport in lymphocytes; b) cytochalasin binding to the H- and M-sites, both integral plasma membrane proteins, is not involved in the effect; c) nonspecific partitioning of cytochalasins in the plasma membrane lipids of lymphocytes appears unlikely to explain the effect; d) evidence presented in this paper strongly suggests that cytochalasin binding to the actin associated L-site mediates the inhibition of rosetting. We conclude that cytoskeletal microfilaments play a critical role in the normal functioning of cell surface receptors for binding to sheep erythrocytes.
Immunoregulation in the rat: requirements for in vitro B cell responses to classical TI-1 and TI-2 antigens.
Authors: Morisaki I , Eldridge J H , Michalek S M , Hamada S , McGhee J R
Published: December 2022

The presence of suppressor cells and their mediators has made it difficult to induce B cell mitogenic or immune responses in rat spleen cell cultures. In the present study, we have defined culture conditions required for induction of in vitro thymic independent (TI) immune responses in the rat. Rat spleen cell cultures support low responses to various trinitrophenyl (TNP) haptenated antigens including TNP-Brucella abortus (TNP-BA), TNP-lipopolysaccharide [LPS; either phenol (Ph)- or butanol (Bu)-water extracted], TNP-Ficoll, and TNP-dextran. However, all of these antigens induced good splenic anti-TNP PFC responses when given at appropriate doses in vivo. When spleen cells were depleted of adherent cells and cultured with TI antigens in vitro, good anti-TNP PFC responses were seen with TNP-BA, whereas, lower responses were induced by TNP-LPS (Ph or Bu). No responses were observed in cultures incubated with either TNP-Ficoll or TNP-dextran. Purified splenic B cell cultures [prepared by panning on plates coated with anti-rat F (ab')2] supported good responses to TNP-LPS (Ph or Bu) and TNP-BA. The addition of irradiated splenic adherent cells (macrophages, M phi) to either M phi-depleted or purified B cell cultures completely abrogated in vitro responses to TNP-BA or TNP-LPS (Ph or Bu). Purified splenic B cell cultures generally responded poorly to TNP-Ficoll or TNP-dextran. Addition of indomethacin (IM) to spleen cell cultures abrogated suppression and allowed anti-TNP PFC responses to TNP-BA, TNP-LPS (Ph or Bu), TNP-Ficoll, and TNP-dextran. Furthermore, nude spleen cell cultures treated with IM, also allowed significant TNP-Ficoll and TNP-dextran immune responses; however, untreated cultures did not respond to these antigens. Our studies indicate that rat splenic B cell cultures are responsive to TI antigens, and highest responses occur with the murine TI-1 class, e.g., TNP-BA and TNP-LPS. Inhibition of suppression with IM restored splenic B cell responses to the murine TI-2 class, i.e., TNP-Ficoll and TNP-dextran.
Accelerated 86Rb+ (K+) release from the cytotoxic T lymphocyte is a physiologic event associated with delivery of the lethal hit.
Authors: Russell J H , Dobos C B
Published: December 2022

Cloned CTL have been loaded with the K+ analogue 86Rb+. When mixed with unlabeled target cells, accelerated 86Rb+ release occurs from the CTL. The magnitude of accelerated 86Rb+ release is proportional to the efficacy of the target in standard 51Cr release assays. The signal responsible for accelerated 86Rb+ release has been localized to a postbinding event and has the physiologic requirements of delivery of the lethal hit. Thus, accelerated 86Rb+ release from the CTL represents the first direct observation of a physiologic event within the CTL related to a functional CTL-target interaction.
Immunoregulatory human T lymphocytes triggered as a consequence of viral infection: clonal analysis of helper, suppressor inducer and suppressor effector cell populations.
Authors: Meuer S C , Cooper D A , Hodgdon J C , Hussey R E , Morimoto C , Schlossman S F , Reinherz E L
Published: December 2022

The regulatory functions of a series of human T cell clones specific for an autologous Epstein-Barr virus transformed B lymphoblastoid cell line were examined. Two T4+ T cell clones, termed AT4II and AT4IV, and one T8+ clone, AT8III, were maintained in culture for greater than or equal to 9 months and were characterized in detail. Both T4+ clones provided helper function for autologous B cell immunoglobulin production when added to unstimulated peripheral blood mononuclear cells. In addition, these same clones produced soluble inducer factors after specific antigenic stimulation. However, when AT4II, AT4IV and their subclones were tested on pokeweed mitogen stimulated peripheral blood mononuclear cells, it was found that AT4IV provided help for immunoglobulin production whereas AT4II cells were strongly suppressive. This suppression by AT4II was indirect and required the presence of fresh, autologous, unirradiated T8+ cells. In contrast, the T8+ AT8III clone markedly inhibited Ig production by autologous B cells in the absence of any additional T8+ cells from peripheral blood and produced a soluble suppressor factor upon specific antigenic triggering. Thus, after stimulation with autologous Epstein-Barr virus transformed cells, at least three discrete regulatory human T cell populations can be defined at the clonal level: helper, inducer of suppression and suppressor effector clones.
Human lymphocyte function associated antigen-1 (LFA-1): identification of multiple antigenic epitopes and their relationship to CTL-mediated cytotoxicity.
Authors: Ware C F , Sanchez-Madrid F , Krensky A M , Burakoff S J , Strominger J L , Springer T A
Published: December 2022

Human lymphocyte function-associated antigen (LFA)-1, a heterodimeric lymphocyte surface glycoprotein of 177,000 and 95,000 relative molecular weight has been implicated to function in the cytotoxic T lymphocyte (CTL) effector mechanism. Seven mouse hybridoma lines producing monoclonal antibodies (MAb) reactive with this structure were studied. Three unique and 3 partially over-lapping epitopes on human LFA-1 were defined by competitive cross inhibition binding assays using biosynthetically labeled anti-LFA-1 MAb. In contrast, of five rat antimouse LFA-1 MAb, all five recognized a common or shared epitope. An HLA-B7 specific human CTL line expressed 1.1 X 10(5) LFA-1 sites per cell with a direct saturation binding assay. Human CTL expressed two to four times more LFA-1 than peripheral blood lymphocytes or B and T lymphoblastoid cell lines. Titration of each of the anti-LFA-1 MAb in a 51chromium release cytolytic assay revealed quantitative differences in the ability of the different anti-LFA-1 MAb to block cytolysis indicating distinct functional and antigenic epitopes exist on the human LFA-1 molecule. Anti-LFA-1 MAb reversibly inhibited the CTL reaction by slowing the initial rate of cytolysis. These results suggest anti-LFA-1 MAb inhibit CTL function by specific blockade of a functionally relevant molecule.
The use of mutants of Escherichia coli for the identification of human lymphocyte subpopulations in blood smears.
Authors: Bratescu A , Mayer E P , Teodorescu M
Published: December 2022

In previous work we have shown that some bacteria can bind to human lymphocytes and can be used to identify lymphocyte subpopulations in conventionally stained blood smears. These bacteria are of different species or genera, which makes it difficult to study the binding mechanism. Also, the main marker for B cells, Brucella melitensis, is of very small size and highly pathogenic. Here we show that B cells as well as some of the T cell subpopulations can be identified by different mutants obtained from a strain of an Escherichia coli. Two procedures were used to generate mutants. First, E. coli-YS57 (pro-his-trp-) was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine and the binding to mouse spleen cells was used as a selective pressure. Second, phage-resistant mutants of E. coli-YS57 were obtained and tested for the ability to bind to lymphocytes. Out of 10 strains selected by the former procedure, 5 bound to a significant number of human lymphocytes. All four phage-resistant mutants bound to human lymphocytes. Out of the total of nine mutants that bound to lymphocytes, six bound consistently, i.e., they bound to similar percentages of peripheral blood lymphocytes from different normal donors. One phage-resistant mutant, E. coli USC-106, bound only to B cells. The subpopulations of lymphocytes identified by the mutants were essentially the same as those identified by different species or genera of bacteria. We concluded that E. coli mutants can be obtained that identify human lymphocyte subpopulations and that one of these mutants recognizes B cells; these mutants may be used to study the nature of the receptors for bacteria on lymphocytes, which appear to have a lectin-like nature.