bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a membrane surface glycoprotein associated with T-cell activation.
Authors: Sportsman J R , Park M M , Cheresh D A , Fukuda M , Elder J H , Fox R I
Published: January 2023

A cell surface antigen (gp140) was previously shown to exist on T cell subsets as well as on monocytes and macrophages in normal peripheral blood. Elevated expression of this antigen was associated with immune system disorders, acute lymphocytic leukemias, and in vitro activation of T cells. The antigen could be identified with monoclonal antibody (MAb) T305. Gp140 was a biosynthetic product of T cells because it could be labeled with [3H]leucine or [3H] glucosamine. Biochemical studies of gp140 used high performance liquid chromatography with nitrocellulose blotting to isolate aliquots suitable for 125I radiolabeling and immunoprecipitation to demonstrate: a) a reduction in m.w. of gp140 KD to 90 KD after deglycosylation by trifluoromethanesulfonic acid, b) alteration of isoelectric point from 4.1 to 5.7 after neuraminidase treatments, c) absence of N-linked sugars based on resistance to endoglycosidase F, d) resistance to trypsin and chymotrypsin digestion but susceptibility to pronase, and e) presence of sialic acid and lactosaminoglycan as O-linked sugars. Gp140 could be labeled with the periodate/NaB[3H]4 technique, indicating its similarity to a class of sialoglycoproteins previously described on activated T-cells in mouse and man. The antigenic epitope recognized by MAb T305 contains sialic acid linked (2----3) to galactose; however, periodate oxidation of the exocyclic ring of sialic acid did not affect binding by MAb T305. In an attempt to determine the functional role of gp140, we tested the ability of MAb T305 to block: a) proliferation of peripheral blood lymphocytes to mitogens, b) response to interleukin 2 (IL 2) of an IL 2 dependent T cell line, and c) growth of a T-ALL derived cell line. No inhibition of proliferation or growth was noted. Although the function of gp140 remains unknown, its association with lymphocyte activation and certain disease states suggests that it may provide a target for modulation of the immune response. These studies characterize the structural features of gp140 and further define the epitope recognized by MAb T305.
Activation of human B cells and inhibition of their terminal differentiation by monoclonal anti-mu antibodies.
Authors: Maruyama S , Kubagawa H , Cooper M D
Published: January 2023

Anti-mu antibody preparations have been found to exert both positive and negative effects on B cell activation and differentiation. To explore these paradoxical influences of IgM cross-linkage on human B cells, three gamma 1 kappa murine monoclonal antibodies specific for human mu-chains (DA4.4, AB6.4, 145.8) were examined for their comparative effects on activation of B cells and inhibition of terminal plasma cell differentiation. All three antibodies appeared equally efficient in immunoprecipitation of surface IgM molecules; however, fluorescence-activated cell sorter analysis revealed that the DA4.4 and AB6.4 antibodies saturated the B cell surface IgM at slightly lower concentrations than did the 145.8 antibody. When the affinity-purified antibodies were added in varying concentrations to cultures of small resting B cells, all three antibodies induced B cell enlargement and DNA synthesis, but with varying degrees of efficiency (DA4.4 greater than AB6.4 much greater than 145.8). In striking contrast, large B cells isolated either by FACS or density gradient separation were unresponsive. The anti-mu-induced proliferative response of small B cells required relatively high B cell densities, but not T cells or the Fc portion of the antibody molecules. The maximal proliferative response was obtained during the third day of culture, and the response curve suggested that anti-mu induced only one round of B cell replication. All three antibodies were capable of completely inhibiting T cell factor-induced differentiation of large B cells into IgM plasma cells; both F(ab')2 fragments and intact anti-mu antibodies were effective in final concentrations as low as 1 microgram/ml. Significant suppression of IgG and IgA plasma cell differentiation was also achieved, but required higher concentrations of the anti-mu antibodies. For each antibody, there was a close correlation between the efficiency of inducing small B cell proliferation and of inhibiting large B cell differentiation into plasma cells. The results show that the B cell response to cross-linkage of cell surface IgM varies according to the differentiation stage. We postulate that the mature resting B cell represents the only stage in the life history of the B cell during which surface Ig cross-linkage leads to a positive signal, negative signals being the rule at other stages in B cell replication and differentiation.
Immunoactive products of placenta. IV. Impairment by placental cells and their products of CTL function at effector stage.
Authors: Chaouat G , Kolb J P
Published: January 2023

Trophoblast-enriched cell suspensions prepared by collagenase digestion from midterm murine placentae were found resistant to CTL-mediated lysis. Treatment of such cells by trypsin or neuraminidase rendered these cells susceptible to such lytic effectors. Collagenase-prepared cell suspensions could impair CTL action, whereas neuraminidase- or trypsin-treated cells did not retain this property. This effect was also observed with extracts. These results indicate that soluble factors (which we will characterize in another paper) released by trophoblast cells (in fact, spongiotrophoblast) can interfere in a dose-dependent fashion with the action of lytic effectors. We suggest that such active mechanisms are physiologic components of the placental barrier and might be defective in some cases of immunologic abortions.
Human neuroblastoma cell lines are susceptible to lysis by natural killer cells but not by cytotoxic T lymphocytes.
Authors: Main E K , Lampson L A , Hart M K , Kornbluth J , Wilson D B
Published: January 2023

The susceptibility of human neuroblastoma cells to direct cellular cytotoxicity has not been previously established. This is of particular interest because of their aggressive growth and low HLA expression. Neuroblastoma lines CHP 100 and CHP 126 were found to be excellent targets in 4-hr CML assays. Natural killer (NK) cells from fresh PBL and from an NK clone, 3.3, have high lytic activity against both cell lines. We also studied mixed lymphocyte culture-generated cytotoxic lines containing allo-specific cytotoxic T lymphocytes (CTL) directed against HLA antigens present on the neuroblastoma target cell lines. These lines did show excellent lytic activity, but cold target competition studies indicated that all of the lysis resulted from NK activity. This was verified by using inhibition studies with the use of monoclonal antibodies. OKT 3 and anti-HLA antibodies that block CTL function caused no reduction in kill. In contrast, anti-lymphocyte function antigen-1 (anti-LFA-1), which blocks both NK and CTL function, significantly inhibited lysis. These results serve as a functional confirmation of earlier findings of a very weak expression of HLA-A,B,C and beta 2-microglobulin on neuroblastoma cells.
IL 2 receptor induction on human T lymphocytes: role for IL 2 and monocytes.
Authors: Wakasugi H , Bertoglio J , Tursz T , Fradelizi D
Published: January 2023

In this report we studied the requirements for the activation and proliferation of highly purified human T lymphocytes. Purified T cells incubated for 3 days with PHA neither proliferate nor express IL 2 receptors as detected by FACS analysis with the use of anti-Tac antibodies. However, purified T cells incubated with Con A or anti-T3 moAb do not proliferate, albeit 30 to 35% T cells express Tac epitopes. The addition of IL 2, either natural purified or recombinant, resulted in both the appearance of Tac antigen and the proliferation of PHA-activated T cells. Much to our surprise, IL 2 did not induce proliferation of Tac-positive T cells activated by Con A or soluble anti-T3 unless monocytes were added to the cultures. These data suggested that two classes of IL 2 receptors might exist on T cells, one of which was not functionally involved in T cell proliferation. In keeping with this interpretation, we have been able to demonstrate, using a radiolabeled IL 2 binding assay, that anti-T3 moAb induced almost exclusively IL 2 receptors of low affinity (Kd = 30 to 70 X 10(-9) M) and that additional signals, provided by monocytes, are required for the acquisition of high affinity receptors. IL 2 itself can induce high affinity receptors on PHA-stimulated T cells but not on cells activated by Con A or anti-T3. In this latter case the physical presence of monocytes is required and cannot be substituted by IL 1, thus indicating a previously unreported role for monocytes. It is postulated that the contact of monocytes with T cells induces a switch from an inactive low affinity conformation of the IL 2 receptor to a functional high affinity one.
Monoclonal antibodies specific for single chain or two chain GAT-specific suppressor factors: production and analysis of in vitro modulating properties.
Authors: Sorensen C M , Pierce C W
Published: January 2023

Fusion of spleen cells from rats hyperimmunized with T cell hybridoma derived GAT-specific TsF1 or TsF2 suppressor T cell factors has resulted in the generation of hybridomas secreting monoclonal antibodies reactive with the appropriate GAT-TsF used for immunization, and in several cases, reactive with other GAT-TsF1 and TsF2. The monoclonal anti-TsF1 antibodies are capable of modulating in vitro GAT-specific PFC response in a GAT-specific manner; some suppress responses to GAT directly, whereas others reverse GAT-TsF1-mediated suppression of responses. The monoclonal anti-TsF2 antibodies all reverse suppression but are reactive with combinatorial determinants, I-J+ chains or antigen-binding chains of the GAT-TsF2. The data are discussed in terms of the nature of the determinants recognized by these antibodies as well as the potential uses of these reagents for studying the suppressor T cell pathway and potential relationships between Ts1, Ts2, and T helper cells.
A Yersinia enterocolitica serotype 0:3 lipopolysaccharide-specific monoclonal antibody reacts more strongly with bacteria cultured at room temperature than those cultured at 37 degrees C.
Authors: Ogasawara M , Granfors K , Kono D H , Hill J L , Yu D T
Published: January 2023

It has been suggested that the O-side chains of the lipopolysaccharide (LPS) of serotype 0:3 strains of Yersinia enterocolitica vary quantitatively and qualitatively depending on whether they are cultured at 37 degrees C or 25 degrees C. It is uncertain whether this affects the expression of the serotype-specific antigens that are probably carried on the LPS. We studied this question with a serotype 0:3-specific monoclonal antibody, 2C1. This monoclonal antibody immunoprecipitated a 39K major protein from detergent-solubilized 125I-labeled Yersinia preparation. However, results of Western blot experiments demonstrated that the antigens reactive with 2C1 were not actually the 39K protein but the O-side chains of the LPS. Significantly, reactivity could be detected whether the bacteria were cultured at room temperature or at 37 degrees C. However, absorption experiments confirmed that there were less accessible antigenic determinants on the 37 degrees C-LPS. The LPS preparations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. These SDS-PAGE profiles showed that less O-side chains were present in the 37 degrees C-LPS. Hence, the most likely explanation for our observation is that the 37 degrees C incubation condition induced a partial smooth to rough transition of the Yersinia LPS with a decrease in the amount of 2C1-reactive antigen.
An exoglycosidase-sensitive triggering site on NK cells which is coupled to transmethylation of membrane phospholipids.
Authors: Kiyohara T , Dennis J W , Boegman R J , Roder J C
Published: January 2023

Glycosidic enzymes were used as probes to analyze the mechanism of NK cell-mediated cytotoxicity. Pretreatment of nylon wool-enriched CBA/J spleen cells, a murine NK clone, or human peripheral blood lymphocytes (PBL) with alpha-mannosidase, an exoglycosidase, led to a marked dose-dependent inhibition of NK lytic activity against YAC-1.2 or K562 tumor cells. Maximal inhibition occurred after a 60-min pretreatment of murine effectors at 37 degrees C, and the kinetics of NK inhibition by alpha-mannosidase was similar to the reported kinetics for enzymatic activity. Released hexose was detected chemically in the supernatant of mouse spleen cells treated with NK inhibitory dose of alpha-mannosidase, and inactivation of enzymatic function with EDTA reversed the NK inhibitory effect. These results suggest that alpha-mannosidase inhibited NK function by virtue of its enzymatic action. Culture of human PBL for 20-hr after treatment with this enzyme led to a greater than 70% recovery in NK lytic function. Recovery was blocked by incorporating tunicamycin, a glycosylation inhibitor of asparagine-linked glycoproteins, into the culture medium. These results suggest that the alpha-mannosidase-sensitive site may be de novo synthesized glycoprotein. Neuraminidase, beta-galactosidase, endo-beta-N-acetylglucosaminidase-D and H, and peptide-N-glycosidase treatments did not inhibit human NK cell lysis of K562 cells. Pretreatment of nylon wool-enriched CBA/J spleen cells or Percoll-enriched human LGL with alpha-mannosidase did not influence their capacity to bind YAC 1.2 target cells or K562 target cells, respectively, Ca++ pulse experiments revealed that the alpha-mannosidase-sensitive site on the NK cells was involved after target-effector binding but before the Ca++ influx. Pretreatment of effector cells with this enzyme which normally occurs after effector-target cell interaction. These results suggest that the phospholipid methylation reaction is coupled to the alpha-mannosidase-sensitive site on the NK cells. By analogy to other physiologic systems, such as histamine release in mast cells, the triggering of phospholipid methylation in the NK cells may serve as a mechanism for signal transduction across the plasma membrane.
Separate control of B lymphocyte early activation and proliferation in response to anti-IgM antibodies.
Authors: DeFranco A L , Raveche E S , Paul W E
Published: January 2023

Resting B cells enter and progress through the G1 phase of the cell cycle in response to low concentrations (1 to 5 micrograms/ml) of anti-IgM antibodies. Commitment to enter S phase requires the presence of a fivefold to 50-fold higher concentration of anti-IgM. These and other results strongly suggest that two separately controlled events are involved in B cell activation. The current studies demonstrate that B cells incubated with high concentrations of anti-IgM from the initiation of culture become independent of additional anti-IgM approximately 10 hr before entry into S phase. We have designated this anti-IgM independent portion of the G1 phase of the cell cycle as G1 beta, whereas the earlier phase is referred to as G1 alpha. Furthermore, low concentrations of anti-IgM are sufficient for progress through early portions of G1 alpha, but high concentrations are required for the last 4 to 8 hr (G1 alpha') if the cells are to go through the rest of the cell cycle. Removal of anti-IgM at any time during G1 alpha causes prompt cessation of the size enlargement that accompanies progress through G1. Such cells retain their size and their relative place in G1 for periods of at least 17 hr and recommence movement through G1 alpha phase when anti-IgM is readded. Thus, B cells may exist in states of partial activation and must possess a mechanism to integrate the amount of stimulatory signal they have received; they enter a commitment period for S phase only when that signal passes some threshold value.
Immunoregulation by T cells. I. Characterization of the IEk-specific Lbd self-reactive T cell clone that helps, suppresses and contrasupresses B cell responses.
Authors: Quintáns J , Suzuki H , Sosman J A , Shah P D
Published: January 2023

This paper describes an L3T4+, Lyt-2- cloned T cell line of CBA/N origin that is specific for IEk-encoded products. Splenic dendritic cells are the predominant stimulatory populations for Lbd cells, being approximately 100 times more effective than large activated B cells. Small B cells and T cells are nonstimulatory. Lbd cells help large B cells to make polyclonal antibody responses, but are unable to activate small B cells and to replace antigen-specific helper T cells in T cell-dependent responses to phosphorylcholine or sheep red blood cells. In the presence of antigen-specific helper T cells, Lbd can amplify, suppress, or contrasuppress T cell function, depending on the type and relative ratios of antigen-reactive cells. We explain our findings as a consequence of the self reactivity among helper T cells.
The major histocompatibility complex-restricted antigen receptor on T cells. IX. Role of accessory molecules in recognition of antigen plus isolated IA.
Authors: Gay D , Coeshott C , Golde W , Kappler J , Marrack P
Published: January 2023

Antibody inhibition studies were done to determine which molecules on the surface of the T cell hybridomas other than their receptors for antigen plus IAd were involved in interaction with antigen-presenting B cells, with artificial IAd membranes on glass beads, or with anti-receptor antibodies coupled to Sepharose beads. We found that T cell LFA-1 was only involved when B cells were used to present antigen plus IAd, whereas T cell L3T4 was involved in the response of T cells to antigen plus IAd either on cells or in artificial membranes, but not if anti-receptor antibodies were used to stimulate the T cells. From these results we concluded that LFA-1 may be involved in the recognition of a ligand on cells that was not present in artificial membranes, but that L3T4 might interact with a nonpolymorphic portion of class II molecules present in both intact antigen-presenting cells and the antigen-presenting artificial membranes.
Monoclonal antibodies that inhibit activation and proliferation of lymphocytes. I. Expression of the antigen on monocytes and activated lymphocytes.
Authors: Yagita H , Masuko T , Takahashi N , Hashimoto Y
Published: January 2023

It has been shown previously that HBJ127 and HBJ98 monoclonal antibodies raised against a human bladder cancer cell line, and B3 monoclonal antibody against a rat bladder cancer cell line recognized unique cell surface antigens abundant in proliferating cells of the corresponding species. Distribution of the antigens and kinetics of the appearance on human and rat lymphoid cells were examined by means of flow cytometry. Rat macrophages and human peripheral blood monocytes were stained strongly with the B3 and HBJ127 monoclonal antibodies, respectively. With regard to lymphocytes, the expression of the B3-defined antigen on rat lymphocytes was found to have a negative correlation with the maturation of the lymphocytes; the antigen was most abundant in bone marrow cells, less abundant in thymocytes, and least abundant in spleen, lymph node, and peripheral blood lymphocytes. Similarly, the HBJ127-defined antigen on human peripheral lymphocytes was negligible. On activation with Con A or alloantigens, however, both rat and human T lymphocytes did strongly express these antigens. Activation of human or rat B cells with lipopolysaccharide also resulted in the augmented expression of these antigens. Kinetics studies revealed that the antigen expression was readily manifested within 12 hr on activation of rat or human T cells with Con A, was augmented progressively with culture time, and reached a plateau within 36 hr. This somewhat earlier appearance of these antigens apparently preceded the manifestations of the IL 2 receptor (Tac antigen) and the augmented DNA synthesis. The B3-defined antigen on Con A-stimulated T cells was more rich on the lymphocytes in S and G2/M phases than those in G1 phase, and the expression was not significantly affected by the addition of hydroxyurea, but was moderately inhibited by the addition of sodium butylate. These results suggest that the appearance and expression of the B3-defined antigen and probably also those of the HBJ127/HBJ98-defined antigen are correlated with lymphocyte activation and subsequent progression through the cell cycle.
Detection and characterization of cytotoxic T lymphocyte precursors in the murine intestinal intraepithelial leukocyte population.
Authors: Ernst P B , Clark D A , Rosenthal K L , Befus A D , Bienenstock J
Published: January 2023

Highly purified preparations of intraepithelial leukocytes (IEL) were obtained from the small intestinal mucosa. Leukocytes from the lamina propria (LPL) were isolated and phenotypically compared with IEL to verify that IEL were minimally contaminated by LPL. Because approximately 80% of IEL expressed the Lyt-2 antigen usually associated with cytotoxic/suppressor T lymphocytes, we wished to determine if precursors for cytotoxic T cells were present in this population. In order to generate cytotoxic cells, IEL and spleen cells from CBA/J mice (H-2k) were co-cultured with irradiated allogeneic spleen cells (H-2d or H-2b) in a one-way mixed leukocyte reaction (MLR). Four to six days later, the cultured cells were assayed against 51Cr-labeled H-2d or H-2b tumor or Con A-stimulated lymphoblast target cells, and the specificity of alloantigen-stimulated IEL and spleen cells was compared. The cytotoxic cells derived from both tissues displayed antigen-specific lysis of the allogeneic targets. Treatment of effector cells, generated from intraepithelial or splenic precursors, with monoclonal antibodies against Thy-1.2, Lyt-1.1, or Lyt-2.1 antigens plus complement, decreased cytotoxicity 85 to 100%, even though only 20 to 50% of the cells were lysed. The alloantigen specificity and surface antigen phenotype of the cultured IEL cells were identical to those of spleen cells and allowed us to conclude that IEL contained a cytotoxic T lymphocyte precursor (CTLp). Further characterization showed that, like spleen, the intraepithelial CTLp was Thy-1+ and Lyt-1+ and their sedimentation velocity was the same but differed from intraepithelial natural killer cells. Although 80% of IEL were Lyt-2+, the frequency of CTLp in the IEL population was estimated to be threefold to fivefold lower than in spleen, and the Lyt-2+ cells were shown not to be an enriched source of CTLp. Thus, the function of the majority of the IEL is still not known. However, there exists within this population CTLp, which may be capable of being stimulated with luminal antigens.
Characteristics of human basophil sulfidopeptide leukotriene release: releasability defined as the ability of the basophil to respond to dimeric cross-links.
Authors: MacGlashan D W , Peters S P , Warner J , Lichtenstein L M
Published: January 2023

Human basophils release approximately 90 pmol of LTC4/micrograms histamine when challenged with anti-IgE antibody, but donor to donor variation produces a 1000-fold range of response. There is little conversion to LTC4 to LTE4 in purified preparations of basophils, but conversion to LTE4 does occur if cell densities are high during incubation. Like histamine release, leukotriene release is calcium and temperature dependent and is complete in 20 min, with a t1/2 of approximately 8 min. The process of desensitization also ablates leukotriene release, but there is a distinct two phase process where leukotriene release is enhanced after 5 min of desensitization, whereas histamine release is inhibited and total ablation of leukotriene release occurs only after 45 min of desensitization. Human basophils respond well to stimulation with covalently cross-linked trimeric IgE myeloma but respond poorly to dimeric IgE. This differential sensitivity to the two forms of cross-linked IgE is most exaggerated in the context of leukotriene release, where dimer is 30-fold less efficacious and 100- to 1000-fold less potent than trimer on some donors' basophils. This dichotomy of response is also observed in antigen-challenged cells, where the bivalent hapten, BPO2, also poorly induces leukotriene release in accord with the fact that it predominantly induces dimeric cross-links of penicillin-specific IgE. Anti-IgE dose-response curves reveal a region of dimeric cross-link dominance that may explain the peculiar differences observed in pharmacologic studies of basophil release induced with antigen vs anti-IgE. In addition, there is a continuum of "releasability," where some donors' basophils display no response (histamine or leukotriene release) to dimeric IgE, and others' basophils are essentially equally responsive to both dimeric and trimeric IgE. This releasability difference manifests itself by conferring increased sensitivity to antigenic challenge in those donors' basophils capable of responding to dimeric cross-links such that these donors' basophils are capable of releasing histamine upon antigen challenge while possessing only 50 molecules of cell surface antigen-specific IgE; other dimer-insensitive donors' basophils require 6 to 10-fold greater IgE densities for equal histamine release.
Enhancement of selective tumor cell binding by activated murine macrophages in response to phorbol myristate acetate.
Authors: Somers S D , Adams D O
Published: January 2023

The fundamental biology of how stable cell-cell bonds develop between activated macrophages and tumor cells, although essential to lysis of the neoplastic targets, remains poorly understood. To investigate whether this phenomenon could be pharmacologically manipulated, we analyzed the effect of phorbol diesters on tumor cell binding by macrophages. Activated murine peritoneal macrophages, treated in vitro with as little as 1 ng/ml of phorbol myristate acetate (PMA), bound significantly more tumor cells than did untreated macrophages. The effect was induced rapidly by PMA (i.e., maximum enhancement was seen within 15 min) and resulted in an average approximately twofold increase in the number of targets bound. The interaction between PMA-treated activated macrophages and tumor cells was completed much more rapidly than by untreated macrophages. The enhanced binding was seen only in macrophages treated with biologically active phorbol esters. Only the selective interaction between activated macrophages and tumor cells was affected (i.e., PMA treatment had no effect on nonselective interactions between activated macrophages and non-neoplastic targets or between nonactivated macrophages and any type of target). Pretreatment of activated macrophages with PMA apparently altered the requirements for microfilaments and microtubules in establishing binding, because cytochalasin B and colchicine, which inhibited control binding, as well as phagocytosis, had no effect on PMA-enhanced binding. PMA treatment did not alter energy requirements for binding, however, because low temperature (4 degrees C) or inhibitors of glycolysis and oxidative phosphorylation blocked both control and PMA-enhanced binding. The enhancement of binding apparently was not due to large quantities of secreted oxygen metabolites but did correlate closely with increased spreading and surface area of the macrophages. PMA treatment resulted in enhanced expression of trypsin-sensitive tumor-cell binding sites on the macrophage surface. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of macrophage membrane proteins labeled with 125I by the lactoperoxidase method revealed at least four trypsin-sensitive cell surface proteins that were re-expressed after PMA treatment. The data suggest that rearrangement and/or induced expression of surface binding sites may be an important step in the binding of tumor cells and indicate that PMA is a useful pharmacologic probe in dissecting the establishment of such binding into discrete steps.
S152 (CD27). A modulating disulfide-linked T cell activation antigen.
Authors: Bigler R D , Bushkin Y , Chiorazzi N
Published: January 2023

A large subset of normal resting peripheral blood T cells express a protein at low density defined by the murine mAb S152. Reactive T cells are present in both the CD4+ and CD8+ subpopulations. This determinant, however, is not expressed on growth factor-independent T cell lines. After activation of mononuclear cells by either Con A or PHA, greater than 80% of the cells stained at a mean fluorescence intensity that was more than six times that seen on resting cells. When PWM- or mixed lymphocyte reaction-activated cultures were studied, 7 to 22% of S152+ cells stained at high intensity whereas most cells stained at the baseline low intensity. The increased fraction of S152+ cells staining at high intensity after activation paralleled both the increased percentage of anti-Tac+ and 5E9+ cells and cellular proliferation measured by thymidine incorporation. Modulation of the S152 Ag was induced when either Con A- or PHA-activated mononuclear cells were placed into secondary cultures containing S152 mAb. Expression of the S152 Ag began to decrease after 2 h and reached a minimum after 6 h. Resting T cells, however, did not appear to modulate when cultured with S152 mAb. Immunoprecipitation and gel electrophoresis analysis revealed the S152 molecule to have a mobility of 120 kDa before reduction. After reduction the molecule was shown to be composed of two 55-kDa molecules with an isoelectric point of 5 to 6 indicating that the S152 Ag is a disulfide-linked homodimer. These studies confirm that the S152 mAb reacts with the newly defined CD27 molecule. The presence of the S152 Ag on resting and activated cells, its parallel increase with the Tac and 5E9 Ag, and its ability to modulate on activated cells suggest that this molecule may play a functional role during T cell activation.
Monoclonal antibodies to scorpion toxins. Characterization and molecular mechanisms of neutralization.
Authors: Bahraoui E , Pichon J , Muller J M , Darbon H , Elayeb M , Granier C , Marvaldi J , Rochat H
Published: January 2023

Two mAb specific for the potent toxin II of the scorpion Androctonus australis Hector have been produced. One of them shows both high affinity binding to the toxin (Kd) = 0.8 nM) and in vivo and in vitro neutralizing properties. The mechanism by which the antibody neutralizes toxin binding to its receptor was shown to be of the competitive type, the epitope overlapping or being close to the receptor-binding region of the toxin. Several residues of the toxin clustered in the C-terminal region were shown likely to be part of the discontinuous epitope recognized by the antibody. The positive charge of the N epsilon-Lys-58 seems to play a pivotal role in the binding of the toxin to both the mAb and the sodium channel receptor.
Recombinant tumor necrosis factor enhances macrophage destruction of Trypanosoma cruzi in the presence of bacterial endotoxin.
Authors: Wirth J J , Kierszenbaum F
Published: January 2023

We examined in this work whether rTNF inhibits the capacity of Trypanosoma cruzi to multiply within murine macrophages or enhances the ability of the phagocytic host cells to destroy internalized parasites. We found that rTNF would not alter the fate of the trypanosomes within macrophages over a 48-h incubation period unless the latter cells were also treated with 1 ng/ml bacterial endotoxin (LPS). Treatment of macrophages with rTNF plus LPS, but not separate treatment with either rTNF or LPS, resulted in a significant decrease in the number of organisms per 100 macrophages with respect to values obtained with mock-treated macrophages. In addition, there was a significant reduction in the proportion of infected macrophages over the 48-h incubation period, indicating parasite clearance by the host cells. The combined effects of rTNF and LPS were seen when macrophages from CBA/J were used but not with LPS-insensitive macrophages from C3H/HeJ mice. Increased trypanosome killing by CBA/J macrophages treated with rTNF plus LPS was not seen when catalase was present in the culture medium, indicating a role for hydrogen peroxide in the cytotoxic effect. These results show that rTNF can affect the fate of T. cruzi within macrophages if LPS is present and point to destruction of internalized organisms rather than inhibition of parasite multiplication as the most likely explanation.
Production of T-T hybrids from T cell clones. Direct comparison between cloned T cells and T hybridoma cells derived from them.
Authors: Ozaki S , Durum S K , Muegge K , York-Jolley J , Berzofsky J A
Published: January 2023

It has been assumed, without direct evidence, that T cell hybridomas and non-transformed T cell clones are both good models of normal Ag-specific T cells. To compare directly the difference in activation of cloned normal T cells and T hybridoma cells with the same TCR, cloned T hybridoma cells were obtained by fusing pre-established, myoglobin-specific, Iad-restricted T cell clones (14.5 and 9.27) with BW5147 cells. T cell clones were pre-activated with IL-2 as well as specific Ag before fusion. Cloned T hybridoma A3.4C6 was derived from Lys 140-specific and I-Ed-restricted clone 14.5. The other cloned T hybridoma, C7R14, was a fusion product of Glu 109-specific and I-Ad-restricted clone 9.27. Both T hybridomas showed the same Ag specificity and Ia restriction as the parental cloned T cells. However, C7R14 showed higher apparent affinity and broader cross-reactivity than 9.27. Clone 14.5, but not hybridoma A3.4C6, appeared to stimulate splenic cells to secrete cytokines inhibiting HT-2A cell proliferation. The most striking difference between the clones and hybridomas was that both clones, but neither of the matched hybridomas, were induced to synthesize IL-1 on stimulation with Ag. Finally, both cloned T cells and T hybridomas killed Ag-pulsed Iad-bearing B lymphoma target cells. This evidence suggests that killing function can be inherited from clones to hybridomas. However, the clones were much more efficient at killing than the hybridomas, and the hybridomas were more efficient at IL-2 production than the clones. Thus, matched pairs of clones and hybridomas differ in their capacity to mediate the two functions or may tend to be selected differently during cloning. Thus, although our results generally support the validity of T cell hybridomas as faithful models of the corresponding T cell clones, a number of subtle and not-so-subtle differences indicate that caution must be used in such an extrapolation.
Membrane Ig-cytoskeletal interactions. III. Receptor cross-linking results in the formation of extensive filamentous arrays of vimentin.
Authors: Albrecht D L , Mills J W , Noelle R J
Published: January 2023

The proposed function of intermediate filaments is to provide a cell type-specific structural framework that maintains cell shape and organelle distribution and mediates signal transduction through its connections with the plasma membrane and the nucleus. Vimentin is the intermediate filament protein expressed in B lymphocytes. Immunocytochemical analysis of the high salt-stable cytoskeletons from B cells stimulated with anti-Ig revealed an increased accumulation of vimentin in the cytoskeleton compared to nontreated controls. This increased accumulation of vimentin in the cytoskeleton was manifested by the organization of vimentin into extensive filamentous arrays (EFA) as viewed in the fluorescent microscope. In contrast to the effects of anti-Ig, activation of B cells with LPS did not induce the organization of vimentin into EFA. This suggested that signals unique to anti-Ig directed EFA formation. Immunocytochemical results were verified by biochemical analysis showing that vimentin was more abundant in isolated cytoskeletons from anti-Ig activated B cells, than cytoskeletons isolated from LPS-activated B cells. These observations established a relationship between increased content of vimentin in the cytoskeleton and the formation of EFA. By testing a wide variety of activating agents, we were able to correlate increased vimentin expression in the cytoskeleton to activating agents that cross-link membrane Ig. It appeared that treatment of B cells with LPS prohibited the induction of EFA by anti-Ig because cotreatment with both anti-Ig and LPS resulted in decreased vimentin accumulation in the cytoskeleton to a level less than that in resting cells. The significance of these results with regard to B cell biology is discussed.
Effect of cyclosporin A on the ontogeny of different T cell sublineages in chickens.
Authors: Bucy R P , Li J M , Xu X Y , Char D , Chen C L
Published: January 2023

We have used a panel of murine mAb against chicken TCR and associated molecules to study the effect of cyclosporin A (CsA) on the ontogeny of the different sublineages of T cells. After injection of CsA (20 mg/kg/day from day 0 to 20) we observed a significant suppression of the normal maturation of the TCR2 (alpha beta TCR) cells in their transition from cortical CD4+CD8+ thymocytes to the mature single positive cells in the thymus medulla. The TCR3 subpopulation, a distinct form of alpha beta-like TCR in chickens, was inhibited from initially developing within the cortex by CsA, indicating that the TCR3 subpopulation is functionally distinct from the TCR2+ cells. In contrast, the maturation and peripheral emigration of TCR1 (gamma delta TCR) cells was unaffected by CsA treatment. Mature splenic T cells sorted for either TCR1+ or TCR2+ subsets were equally sensitive to CsA blockade of Con A-stimulated mitogenesis, indicating that there is no inherent difference in CsA sensitivity between these sublineages. Furthermore, no difference was detected in the expression of class II MHC Ag in thymi of birds treated with olive oil vs CsA. Inasmuch as the mechanism of CsA action appears to involve inhibition of TCR initiated signal transduction for lymphokine synthesis, these data indicate that a similar signaling is involved in thymic repertoire selection for TCR2. The lack of an effect on TCR1 cell maturation suggests that the TCR1 repertoire may not undergo selection in the thymus as do TCR2+ cells.
Differential effects of cytokines on proliferative response of human CD4+ T lymphocyte subsets stimulated via T cell receptor-CD3 complex.
Authors: Wasik M A , Morimoto C
Published: January 2023

We report that the subsets of CD4+ T cells characterized by differential expression of CD45RA (2H4) Ag showed significant differences in proliferative response to immobilized anti-CD3 antibody (Ab) and cytokines: IL-1, IL-2, IL-4, and IL-6. Most strikingly, CD4+/45RA+ but not CD4+/45RA- T cells responded to anti-CD3 Ab and IL-4. Similar difference in response to IL-4 occurred when the subsets were stimulated by two "alternative" T cell activation pathways via CD2 and GD3 Ag. The response of CD4+/45RA+ cells to anti-CD3 Ab and IL-4 was enhanced by the two monokines: IL-1 and IL-6. Further differences between the subsets included the preferential response of the CD4+/45RA+ cells to enhancing effect of IL-6 on proliferation mediated by the anti-CD3 Ab and IL-2. In contrast to IL-6, IL-1 was unable to increase this proliferation significantly. In turn, the CD4+/45RA- cells responded preferentially to a weak stimulation mediated by anti-CD3 Ab either alone, or together with IL-1 and IL-6. Existence of these significant differences in the response of CD4+ T cell subsets costimulatory effects of the cytokines, suggests that the in vivo events resulting in an accumulation of the cytokines in particular combinations may lead to selective activation of one of the CD4+ T cell subsets during the immune response.
Structural requirement for autoreactivity on human pyruvate dehydrogenase-E2, the major autoantigen of primary biliary cirrhosis. Implication for a conformational autoepitope.
Authors: Surh C D , Coppel R , Gershwin M E
Published: January 2023

The E2 component (acetyltransferase) of the pyruvate dehydrogenase (PDH) complex is the major mitochondrial autoantigen recognized by autoantibodies in patients with primary biliary cirrhosis (PBC). Previous work, using only a partial length rat liver cDNA clone of PDH-E2, demonstrated that the immunodominant epitope was localized to the lipoic acid binding site. Human PDH-E2, in contrast to rat PDH-E2, has two lipoic acid binding sites. By using a full length human cDNA for PDH-E2, and by preparation of multiple overlapping recombinant fragments, we have determined that three autoreactive determinants are present on human PDH-E2: two cross-reactive lipoyl domains, and an area surrounding the E1/E3 binding region. The dominant epitope was localized to the inner lipoyl domain whereas the outer lipoyl domain only showed a weak cross-reactivity, and only 1/26 PBC sera reacted weakly to the E1/E3 binding region area. By probing recombinant fusion proteins expressed from small restriction fragments of the inner lipoyl domain, we have found that a minimum of 75 amino acids (residues 146-221) were required for detectable autoantibody binding, and that 93 amino acids (residues 128-221) were necessary for characteristically strong antimitochondrial autoantibody recognition. Such a requirement for a large region suggests the possibility that a conformational autoepitope may be recognized. In addition, we have found that absorption of PBC sera with the purified mammalian PDH complex does not remove reactivity against Escherichia coli Ag. The possible implications for such results are discussed.
Regulation of macrophage activation by IL-3. I. IL-3 functions as a macrophage-activating factor with unique properties, inducing Ia and lymphocyte function-associated antigen-1 but not cytotoxicity.
Authors: Frendl G , Beller D I
Published: January 2023

Here we report that IL-3 (also referred to as multi-CSF because of its colony-stimulating activity on a variety of hemopoietic cell lineages) can function as a macrophage-activating factor (MAF). IL-3 was able to regulate the expression of class II MHC Ag and the cellular interaction molecule lymphocyte function-associated Ag-1 on the surface of murine peritoneal exudate cells. The kinetics of IL-3-induced Ia expression appeared to be distinct from that induced by either IFN-gamma, IL-4, or granulocyte-macrophage-CSF. IL-3 was also distinguished from these factors by the finding that it did not induce macrophage tumoricidal activity. In addition to its inherent MAF activities, IL-3 also showed a marked synergy with low doses of LPS (0.05 to 0.5 ng/ml) as well as IFN-gamma in Ia induction. When lymphocyte function-associated Ag-1 expression was evaluated, the effects of these stimuli appeared to be only additive. Although LPS has been shown to inhibit IFN-gamma-induced Ia expression, in our experiments this property of LPS is manifest only when present at doses greater than or equal to 50 ng/ml. At lower concentrations, LPS potentiated both IL-3- and IFN-gamma-induced class II MHC Ag expression. Data presented here also suggest that the synergistic interactions between low doses of LPS and IL-3 are not mediated by known LPS-inducible cytokines of macrophage origin, because rIL-1, TNF-alpha, or IL-6 did not enhance the response to IL-3. Because IL-3 can also participate in the regulation of IL-1 expression, it appears that IL-3 can function as a MAF which selectively regulates the accessory cell characteristics required for Ag presentation, as opposed to the cytolytic functions of the macrophage.
Selective activation of human monocytes by the platelet-activating factor analog 1-O-hexadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine.
Authors: Rose J K , Debs R A , Philip R , Ruis N M , Valone F H
Published: January 2023

The capacity of platelet-activating factor (PAF) and its 2-O-methyl analog (methoxy-PAF) to activate human monocytes, neutrophils and platelets were compared. Both PAF and methoxy-PAF increased monocyte cytotoxicity toward WEHI 164 cells with a maximal increase in cell killing at 100 pM to 1 nM. Methoxy-PAF was slightly, but significantly, more potent than PAF for increasing cytotoxicity. PAF and methoxy-PAF increased monocyte release of TNF two- to three-fold above control release with no difference in their potency. Methoxy-PAF increased cell-associated TNF maximally after 2 to 3 h of incubation and increased TNF release maximally after 5 to 18 h of incubation. PAF induced release of the neutrophil granule enzyme beta-glucuronidase with maximal net release of 15 to 20% at 100 nM PAF whereas methoxy-PAF did not induce release of beta-glucuronidase. Similarly, 10 nM PAF induced 30% platelet aggregation whereas methoxy-PAF induced aggregation only at 1000-fold higher concentrations. Analysis of PAF and methoxy-PAF metabolism by monocyte and serum acylhydrolases indicates that methoxy-PAF is substantially more resistant than PAF to degradation by these enzymes. These observations indicate that methoxy-PAF activates monocytes selectively and suggest that this phospholipid or a related compound could be used for in vivo immunotherapy.
Inhibition of phospholipase activity in human monocytes by IFN-gamma blocks endogenous prostaglandin E2-dependent collagenase production.
Authors: Wahl L M , Corcoran M E , Mergenhagen S E , Finbloom D S
Published: January 2023

Previous studies from our laboratory have demonstrated that exposure of human monocytes to a stimulant, such as Con A, results in the production of the enzyme collagenase through PGE2-dependent pathway. Inasmuch as rIFN-gamma has been shown to modulate monocyte/macrophage PG synthesis, we examined the effect of rIFN-gamma on the activation sequence leading to collagenase production. The addition of rIFN-gamma (10 to 1000 U/ml) to Con A-stimulated monocytes resulted in a dose-dependent inhibition of PGE2 and collagenase synthesis. The suppression of collagenase production by rIFN-gamma was related to its ability to reduce PGE2 levels as demonstrated by the restoration of collagenase activity by the addition of PGE2. HPLC analysis of the arachidonic acid (AA) metabolites released by monocytes showed that rIFN-gamma caused a reduction in the release of AA and products of the cyclooxygenase and lipoxygenase pathways. These data indicated that rIFN-gamma decreased eicosanoid production by inhibiting the release of AA from phospholipids. This conclusion was supported by the reduction in membrane bound phospholipase activity in rIFN-gamma-treated monocytes. Moreover, the inhibition by rIFN-gamma of PGE2 and collagenase was reversed by the addition of phospholipase A2. Our findings demonstrate that rIFN-gamma inhibits phospholipase activity in activated monocytes and as a result blocks PGE2-dependent collagenase synthesis.