bluuebunny/crossref_metadata_2025 huggingface dataset:
Myelin basic protein and proteolipid protein reactivity of brain- and cerebrospinal fluid-derived T cell clones in multiple sclerosis and postinfectious encephalomyelitis.
Authors: Hafler D A , Benjamin D S , Burks J , Weiner H L
Published: December 2022

T cells were directly cloned from autopsied MS brain plaque tissue and reactivity was measured with the major encephalitogenic neuroantigens, myelin basic protein (MBP), and proteolipid protein (PLP). Control clones were simultaneously derived from the blood. The proportion of T4+ and T8+ T cell clones from the brain tissue differed from that of peripheral blood T cell clones derived at the same time, suggesting that the clones were not derived from the peripheral blood. None of 57 brain-derived T cell clones proliferated to either MBP or PLP, although they responded well to PHA and IL 2. An additional 235 clones derived from the cerebrospinal fluid and 126 clones from the peripheral blood of other subjects with multiple sclerosis also did not proliferate to MBP or PLP. In contrast, five of nine T4+ clones from the CSF of a subject with postinfectious encephalomyelitis exhibited low but clear reactivity to human MBP, supporting the possible role of MBP as the target antigen in this disease. These studies, the first to clone T cells directly from MS plaque tissue, suggest that the lack of consistent T cell reactivity to MBP or PLP in the peripheral blood of MS patients does not appear to be secondary to the sequestration of a large number of these cells in the brain.
Allogeneic matings and immunization have different effects on nulliparous and multiparous mice.
Authors: Chavez D J , McIntyre J A , Colliver J A , Faulk W P
Published: December 2022

CBA/J female mice have a high rate of spontaneous fetal loss when mated with DBA/2 males. We have confirmed that this fetal resorption rate can be significantly reduced by immunizing the female with C57BL rather than DBA leukocytes. The current studies have been extended to show the effect of continued immunization into second pregnancies. Three new findings emerge: second pregnancies in unimmunized CBA/J females X DBA/2 males proceed with a low percentage of spontaneous resorptions; continued immunization of multigravid, multiparous mice is associated with a high percentage of late onset fetal resorptions; and comparison of sex ratios between treated and untreated pregnancies showed no significant shift. These results suggest that untreated CBA/J females mated to DBA/2 males can be immunized to trophoblast antigens during first pregnancies, and that such immunization leads to "normal" reproductive outcome in subsequent untreated pregnancies. Persistent immunization with leukocytes appears to increase the percentage of fetal wastage in subsequent pregnancies regardless of whether the mother receives paternal or nonpaternal leukocytes. Intentional chronic immunization with male leukocytes does not influence the sex ratio of viable offspring. These data are discussed with reference to immunotherapy for women who suffer unexplained, recurrent spontaneous abortions.
The influence of valence on the functional activities of monoclonal anti-L3T4 antibodies. Discrimination of signaling from other effects.
Authors: Haque S , Saizawa K , Rojo J , Janeway C A
Published: December 2022

Anti-L3T4 antibodies have different functional effects on different ligands that activate a cloned, L3T4+ helper T cell line. It is reported here that some of these effects involve positive or negative signaling induced by cross-linking L3T4 molecules, because such effects are not observed using the Fab fragment of anti-L3T4. However, the Fab fragment does inhibit responses to antigen:self class II major histocompatibility complex (MHC) and to anti-T cell receptor monoclonal antibodies directed at a particular V region epitope. The finding that the Fab fragment of anti-L3T4, which does not block binding of anti-T cell receptor V region antibodies and does not negative signal can still block activation by such antibodies suggests an intimate association of L3T4 with the T3: alpha: beta T cell receptor complex. This association may normally be induced by interaction of both structures with antigen:self class II MHC complexes. The data also support the hypothesis that cross-linking the L3T4 molecule in the absence of engagement of the T3: alpha: beta complex generates negative or inhibitory signals. Thus, L3T4 plays a central role in the process of class II MHC-restricted T cell antigen recognition and activation.
Modification of the cytotoxic T cell repertoire in neonatal tolerance. Evidence for preferential survival of cells with low avidity for tolerogen.
Authors: Wood P J , Socarras S , Streilein J W
Published: December 2022

Clonal deletion of developing lymphocytes with potential reactivity for self is thought to play a crucial role in the establishment of self tolerance. One prediction of the clonal deletion hypothesis is that cells bearing receptors with high affinity for self are more likely than cells with low affinity receptors to be deleted from the repertoire. Experimental models of B cell tolerance have provided evidence for the preferential survival of low affinity cells with specificity for tolerogen in tolerant animals, but no comparable evidence exists for T cells. To examine this issue in T cells, cytotoxic T cell lines specific for the Kb mutant class I H-2 molecule, bm1, were generated from C57BL/6 mice rendered neonatally tolerant of bm1 and compared with anti-bm1 lines generated from normal mice. Compared with normal lines, those from tolerant mice differed in five ways: 1) they grew more slowly; 2) they were less efficient at lysing bm1 targets; 3) they showed different patterns of lysis against a panel of third party targets; 4) their cytotoxic activity against bm1 could be increased in the presence of leukoagglutinin, whereas the activity of normal lines was not increased by leukoagglutinin; and 5) their cytotoxic activity was more susceptible to inhibition by anti-Lyt-2 antibody. Taken together, these results demonstrate that the repertoire of the remaining tolerogen-specific cytotoxic T cells in neonatally tolerant mice is different from the normal C57BL/6 anti-bm1 repertoire, and the results are consistent with the idea that the remaining tolerogen-specific cells are low avidity cells that have preferentially escaped the clonal deletion process.
Role of B cell stimulatory factor 1/interleukin 4 in clonal proliferation of B cells.
Authors: Peschel C , Green I , Ohara J , Paul W E
Published: December 2022

B cell stimulatory factor 1 (BSF-1)/interleukin 4 (IL-4) has striking effects on colony formation in soft agar by small resting B lymphocytes. BSF-1 alone induces colony formation in this cell population, presumably in costimulation with a mitogenic substance present in bacto-agar. In costimulation with anti-IgM antibodies, BSF-1 caused initial proliferation of 8 to 10% of B cells, resulting in a large number of cell clusters (10 to 40 cells/clone) after 3 to 4 days of incubation. However, substantial number of colonies (greater than 40 cells/clone) developed only from these clusters when IL-1 was added to the cultures. Using a modified immunoperoxidase staining technique for the determination of IgM allotype, evidence was obtained that B cell colonies stimulated with BSF-1 are derived from a single progenitor cell. Neutralization of BSF-1 with 11B11 after a culture period of 1 to 4 days inhibits further proliferation of B cell colonies, indicating that the action of BSF-1 extends for several cell generations beyond initial stimulation from the resting state. Furthermore, it is demonstrated that the synergistic action of IL-1 with BSF-1 is confined to the late culture period, indicating a growth-promoting effect by IL-1 for activated B cells.
Interferon-gamma is a major regulator of C1-inhibitor synthesis by human blood monocytes.
Authors: Lotz M , Zuraw B L
Published: December 2022

C1 inhibitor (C1INH) is the major control factor for the activation of the classical pathway of complement and for contact system activation. Hepatocytes and blood monocytes are known to synthesize this protease inhibitor. We studied the regulation of monocyte C1INH production by mediators that are generated during inflammatory responses. Purified blood monocytes spontaneously synthesized and secreted C1INH only after prolonged culture. In the presence of interferon (IFN)-gamma, C1INH was detectable within 24 hr and continued to be released at high levels throughout an 8-day culture period. Monocyte C1INH was newly synthesized and was functionally active as determined by forming stable complex with C1s. Other monocyte stimuli were either less potent (IFN-alpha, IFN-beta) or not capable of increasing C1INH release (lipopolysaccharide, interleukin 1, and tumor necrosis factor). The second component of complement, C2, was induced by IFN-gamma to a similar extent as C1INH. These findings demonstrate that IFN-gamma is a major regulator of monocyte C1INH production and may warrant consideration of IFN-gamma in the treatment of C1INH deficiency states.
Mapping of C2, Bf, and C4 genes to the swine major histocompatibility complex (swine leukocyte antigen).
Authors: Lie W R , Rothschild M F , Warner C M
Published: December 2022

Three miniature swine lines, inbred for swine leukocyte antigen (SLA) haplotypes, a, c, and d, and a recombinant line, haplotype g, were analyzed for possible restriction fragment length polymorphisms (RFLP) by Southern blot hybridization with human C2, factor B (Bf), and C4 specific probes. The search for RFLP by using a human C2 probe failed to reveal any variants. However, a Taq I polymorphism was identified with the human Bf probe and Bam HI and Pvu II polymorphisms were identified with the human C4 probe. Overlapping restriction fragments were found with the C2 and Bf probes, which strongly suggests close linkage of C2 and Bf genes in swine. Segregation analyses of the Bf and C4 polymorphisms indicated that the polymorphic fragments followed a Mendelian pattern of inheritance. The recombinant haplotype g, which expresses class I genes of haplotype c and class II genes of haplotype d, was shown to produce an identical RFLP pattern, by using the Bf and C4 probes, as haplotype d, but different from that of haplotype c. This indicates that there is a close association of [C4-Bf-C2] and class II genes in miniature swine. Although these data do not show conclusively the location of the [C4-Bf-C2] genes, it is hypothesized that swine [C4-Bf-C2] genes are located between the class II and class I genes, as has been demonstrated in mouse and man.
A human hybrid hybridoma.
Authors: Tiebout R F , van Boxtel-Oosterhof F , Stricker E A , Zeijlemaker W P
Published: December 2022

Hybrid hybridomas are obtained by fusion of two cells, each producing its own antibody. Several authors have reported the construction of murine hybrid hybridomas with the aim to obtain bispecific monoclonal antibodies. We have investigated, in a model system, the feasibility of constructing a human hybrid hybridoma. We fused two monoclonal cell lines: an ouabain-sensitive and azaserine/hypoxanthine-resistant Epstein-Barr virus-transformed human cell line that produces an IgG1 kappa antibody directed against tetanus toxoid and an azaserine/hypoxanthine-sensitive and ouabain-resistant human-mouse xenohybrid cell line that produces a human IgG1 lambda antibody directed against hepatitis-B surface antigen. Hybrid hybridoma cells were selected in culture medium containing azaserine/hypoxanthine and ouabain. The hybrid nature of the secreted antibodies was analyzed by means of two antigen-specific immunoassays. Our results show that it is possible, with the combined use of transformation and xenohybridization techniques, to construct human hybrid hybridomas that produce bispecific antibodies.
Kinetics and mechanisms of recombinant human interleukin 1 and tumor necrosis factor-alpha-induced changes in circulating numbers of neutrophils and lymphocytes.
Authors: Ulich T R , del Castillo J , Keys M , Granger G A , Ni R X
Published: December 2022

Human recombinant interleukins 1 alpha and 1 beta (rIL-1 alpha and -1 beta) both induced monophasic peripheral neutrophilia and lymphopenia in Lewis rats 1.5 hr after i.v. injection. The kinetics of rIL-1 alpha- and -1 beta-induced neutrophilia were similar to those induced by human monocyte-derived IL-1, IL-1 alpha, and IL-1 beta, and the peripheral neutrophilia was accompanied by a marked decrease in marrow neutrophils. Arachidonic acid metabolites are implicated as biochemical intermediates in the production of the neutrophilia but not lymphopenia, since indomethacin and dexamethasone both completely abrogated IL-1-induced neutrophilia but did not affect the IL-1-induced lymphopenia. Acetylsalicylic acid, a cyclooxygenase inhibitor, did not inhibit IL-1-induced neutrophilia, suggesting that products of the lipoxygenase rather than the cyclooxygenase pathway of arachidonate metabolism may contribute to the neutrophilia. Human recombinant tumor necrosis factor-alpha (rTNF) administered i.v. to Lewis rats induced peripheral neutropenia, two peaks of neutrophilia, and lymphopenia. A wide range of doses of rTNF resulted in an initial neutropenia at 0.5 hr after injection followed by a first peak of neutrophilia at 1.5 hr and a second peak of neutrophilia at 6 hr. The initial neutropenia and the first peak of neutrophilia were not inhibited by pretreatment of rats with dexamethasone, indomethacin, or aspirin. The second peak of neutrophilia was inhibited by both dexamethasone and indomethacin, but was not at all inhibited by aspirin, suggesting that the second peak of neutrophilia is mediated by the release of endogenous cytokines, especially by IL-1, since exogenous IL-1-induced neutrophilia is also completely inhibited by dexamethasone and indomethacin but not by aspirin. The TNF-induced peripheral neutrophilia is also accompanied by a significant depletion of bone marrow neutrophils, indicating that the source of increased circulating neutrophils is, at least in part, via recruitment of marrow neutrophils. Systemic blood pressure was not affected by IL-1 or rTNF at the dosages employed, showing that the changes in circulating leukocyte subsets were not attributable to hemodynamic changes nor to the hemodynamic change-related release of adrenal hormones. Adrenalectomy did not alter the IL-1- or rTNF-induced neutrophilia or lymphopenia, also demonstrating that neither monokine mediates its hematologic effects on peripheral blood leukocytes via the release of adrenal hormones.(ABSTRACT TRUNCATED AT 400 WORDS)
Effects of recombinant human granulocyte and macrophage colony-stimulating factors on signal transduction pathways in human granulocytes.
Authors: Sullivan R , Griffin J D , Simons E R , Schafer A I , Meshulam T , Fredette J P , Maas A K , Gadenne A S , Leavitt J L , Melnick D A
Published: December 2022

We studied the ability of the recombinant human-active hemopoietic growth factors granulocyte-macrophage colony-stimulating factor (GM-CSFrh) and granulocyte colony-stimulating factor (G-CSFrh) to activate receptor-mediated transduction pathways which have been implicated in the stimulation of cytotoxic functions in granulocytes. With the use of a panel of fluorescent probes, we found that these two growth factors exerted no detectable immediate effect on the resting transmembrane electrical potential, the intracellular concentration of free calcium ions, or the cytosolic pH of isolated, mature granulocytes. However, when granulocytes were "primed" by preincubation for 90 min with GM-CSFrh or G-CSFrh, the rate of membrane depolarization induced by 10(-7) M N-formyl-methionyl-leucyl-phenylalanine, but not the rate of rise in free calcium ions, was greatly accelerated. In examining potential mechanisms to account for the priming effect of these growth factors, we found that although they did not induce translocation of protein kinase C or stimulate significant degranulation, they each directly caused prompt release of arachidonic acid from plasma membrane phospholipids. Our data indicate that although GM-CSFrh and G-CSFrh do not activate the transduction signals that have most clearly been implicated in receptor-mediated activation of cytotoxic functions in granulocytes--namely, those coupled to membrane depolarization or release of intracellular calcium ions--they appear directly to induce the release of arachidonic acid esterified to membrane phospholipids, an event which may represent the receptor-mediated activation of membrane phospholipases and which may contribute to the "priming" of the cells for enhancement of their functional responsiveness.
Mouse bone marrow-derived mast cells cocultured with fibroblasts. Morphology and stimulation-induced release of histamine, leukotriene B4, leukotriene C4, and prostaglandin D2.
Authors: Levi-Schaffer F , Dayton E T , Austen K F , Hein A , Caulfield J P , Gravallese P M , Liu F T , Stevens R L
Published: December 2022

Mouse bone marrow-derived mast cells (BMMC), cultured for 2, 7, or 14 days in WEHI-3 conditioned medium in the absence or presence of mouse 3T3 fibroblasts, were examined morphologically and for their functional responses to IgE-Fc-mediated and calcium ionophore-mediated activation. The 7- and 14-day fibroblast-adherent and non-fibroblast-adherent populations of cocultured BMMC had more granules per cell and the granule contents were more electron dense than non-cocultured BMMC or BMMC cocultured for only 2 days. The adherent cocultured BMMC were usually located within multiple layers of fibroblasts, but did not form junctions with the fibroblasts. When activated immunologically, the adherent cocultured mast cells generally discharged their granules singly, but compound exocytosis was occasionally seen. Both the non-adherent cocultured BMMC and the BMMC that were cultured in the absence of fibroblasts were similar to one another in that they exocytosed 9 to 11% of their histamine when sensitized with anti-dinitrophenyl IgE and challenged with dinitrophenyl-bovine serum albumin and 27 to 29% of their histamine when challenged with calcium ionophore. In contrast, adherent cocultured BMMC exocytosed 27 and 61% of their histamine upon immunologic and calcium ionophore activation, respectively, representing a significant two- to three-fold increase relative to that obtained from the other populations of BMMC. When activated immunologically, BMMC cultured in WEHI-3 conditioned medium alone generated a mean of 12 ng of immunoreactive C-6-sulfidopeptide leukotrienes, 1.6 ng of leukotriene B4 (LTB4), and 1.0 ng of prostaglandin D2 (PGD2)/10(6) cells. The immunologic response of the nonadherent 7-day cocultured BMMC was similar. Fibroblast-adherent cocultured BMMC, on the other hand, generated 56 ng of immunoreactive C-6-sulfidopeptide leukotrienes, 6.4 ng of LTB4, and 5.6 ng of PGD2/10(6) mast cells, representing a significant increase for each product. When calcium ionophore was used as the agonist, the adherent cocultured mast cells also generated significantly more arachidonic acid metabolites than nonadherent cocultured BMMC or BMMC cultured in the absence of fibroblasts. Retention times on high performance liquid chromatography confirmed that the generated immunoreactive products were LTB4, PGD2, and LTC4. Thus, coculture of BMMC with fibroblasts induces an alteration in the composition of the secretory granules of the mast cells, as well as an augmentation of the activation-secretion response of the BMMC.(ABSTRACT TRUNCATED AT 400 WORDS)
N-formylpeptide-receptor dynamics, cytoskeletal activation, and intracellular calcium response in human neutrophil cytoplasts.
Authors: Omann G M , Swann W N , Oades Z G , Parkos C A , Jesaitis A J , Sklar L A
Published: December 2022

Cytoplasts (enucleated neutrophils which are depleted of dense granules) were prepared from human neutrophils with a modified procedure which employed dihydrocytochalasin B instead of cytochalasin B. These cytoplasts retained an activatable cytoskeletal network similar to cells in that filamentous actin polymerization in response to an N-formylpeptide (fluoresceinated N-formyl-nle-leu-phe-nle-tyr-lys, FLPEP) occurred with similar dose-response characteristics and was inhibitable by cytochalasin B and dihydrocytochalasin B. Cytoplasts had the same number of receptors per surface area as cells and binding constants and dissociation kinetics were the same for cells and cytoplasts. The conversion of receptors from a rapidly dissociating form to a slowly dissociating form was comparable in cells and cytoplasts. This conversion was not inhibited by cytochalasins and thus did not require actin polymerization. Cytoplasts were capable of internalizing 30% of bound FLPEP after 3 min of binding. Cytochalasins did not block this internalization which thus did not appear to require actin polymerization. After 5 min of binding, [3H]-N-formyl-met-leu-phe cosedimented with the Golgi marker enzymes when cytoplasts were fractionated on sucrose density gradients after N2 cavitation. These results indicate that the internalization mechanism is functional in cytoplasts. The Indo-1-detectable calcium response in cytoplasts had a ED50 similar to cells, though the maximum increase in Ca2+ concentration was about one-half that of cells. The response recovered with time after stimulation and the calcium detected was primarily from intracellular stores. The decay of responses after addition of formylpeptide antagonists was parallel for cells and cytoplasts, and leukotriene B4-induced responses in both cells and cytoplasts. Thus the regulation of the responses in cells and cytoplasts was analogous.
Aluminum fluoride induces phosphatidylinositol turnover, elevation of cytoplasmic free calcium, and phosphorylation of the T cell antigen receptor in murine T cells.
Authors: O'Shea J J , Urdahl K B , Luong H T , Chused T M , Samelson L E , Klausner R D
Published: December 2022

Antigen activation of murine T lymphocytes leads to phosphorylation of three subunits of the murine T cell antigen receptor (L.E. Samelson, M.D. Patel, A.M. Weissman, J.B. Harford, and R.D. Klausner. 1986. Cell 46:1083). Two kinases are activated in this process: protein kinase C which leads to phosphorylation of the gamma and, to a lesser extent, the epsilon subunits on serine residues and a tyrosine kinase which phosphorylates the p21 subunit (M.D. Patel, L.E. Samelson, and R.D. Klausner. 1987. J. Biol Chem. 262:5831). We sought to determine whether treatment of these cells with NaF could simulate any of these antigen-induced events. Indeed NaF treatment resulted in breakdown of polyphosphoinositides and production of phosphoinositols. This treatment also resulted in a rise in cytosolic free Ca2+. EGTA failed to block this rise suggesting that NaF liberated intracellular stores of Ca2+. Finally NaF treatment resulted in phosphorylation of the gamma and epsilon chains of the T cell receptor indistinguishable from the effects of phorbol esters. The NaF effect was potentiated by addition of A1Cl3 consistent with the view that the active moiety is A1F4-. The A1F4--induced phosphorylations were abolished in cells in which protein kinase C was depleted by prior treatment with phorbol myristate acetate. All of these observations are compatible with the interpretation that the A1F4- phosphorylation is mediated by protein kinase C. Antigen and anti-receptor antibody-induced receptor serine phosphorylation and phophatidylinositol turnover are blocked by raising intracellular levels of cyclic adenosine monophosphate. In contrast, A1F4--induced effects were insensitive to cyclic adenosine monophosphate.
Molecular basis of elevated c-myb expression in the abnormal L3T4-, Lyt-2- T lymphocytes of autoimmune mice.
Authors: Evans J L , Boyle W J , Ting J P
Published: December 2022

The presence of the lpr/lpr genotype on a number of murine genetic backgrounds results in a systemic lupus erythematosus-like disease and lymphadenopathy. The T lymphocytes of these mice exhibit a variety of abnormalities; most pertinent to the present report is an abnormally high level of c-myb proto-oncogene mRNA. Since the c-myb protein is presumably the effector molecule that affects cellular functions, it is important to determine whether increased levels of this c-myb protein are produced. With the use of immunoprecipitation with an anti-v-myb reagent, we found high levels of c-myb protein in the lymph nodes of lpr mice. Detailed analysis showed that the c-myb protein is primarily expressed by an abnormal T lymphocyte population that does not express the mature T cell markers, L3T4 and Lyt-2. Analysis by two-dimensional gel electrophoresis showed that the c-myb proteins from normal thymocytes and from these L3T4-, Lyt-2-T cells are indistinguishable. DNA analysis with Southern hybridizations showed the lack of amplification, insertions, deletions, and rearrangements, which is in accord with results from the protein studies. Most interestingly, the c-myb gene in lpr L3T4-, Lyt-2- T cells is hypomethylated compared with normal controls. This suggests that a regulatory mechanism, rather than the structural alteration of the gene, is responsible for elevated expression of c-myb in these L3T4-, Lyt-2- cells.
Production of a mouse-human chimeric monoclonal antibody to CD20 with potent Fc-dependent biologic activity.
Authors: Liu A Y , Robinson R R , Murray E D , Ledbetter J A , Hellström I , Hellström K E
Published: December 2022

Mouse monoclonal antibody 2H7 recognizes the CD20 cell surface phosphoprotein that is expressed in normal as well as malignant B cells. CD20 may be a useful target for therapy of B cell lymphomas, since damaged normal B cells can be replaced by their antigen-negative precursors. Monoclonal antibody 2H7 is an IgG2b (kappa) immunoglobulin which cannot mediate antibody-dependent cellular cytotoxicity with human lymphocytes or complement-dependent cytotoxicity with human serum. We have now generated a chimeric 2H7 antibody by substituting the mouse constant domains of 2H7 with the human gamma 1 and kappa domains. This new antibody has the same binding specificities as 2H7 but is highly effective in mediating antibody-dependent cellular cytotoxicity with human effector cells and complement-dependent cytotoxicity with human complement.
Identification of three major target molecules of IgM antilymphocyte autoantibodies in systemic lupus erythematosus.
Authors: Minota S , Winfield J B
Published: December 2022

Three cell lymphocyte antigens of m.w. 55,000, 70,000, and 105,000 to 110,000 were identified by Western blotting as targets of IgM autoantibodies in serum from a group of 49 patients with systemic lupus erythematosus. The 55- and 70-kDa antigens were well expressed on unstimulated peripheral T cells, whereas the 105- to 110-kDa target was demonstrable only on mitogen-activated T cells and lymphoblastoid T cell lines. Localization of these molecules to the plasma membrane was established by cytoabsorption experiments in which IgM antibody staining of blotted antigens was specifically absorbed from systemic lupus erythematosus serum during 4 degrees C incubations with intact lymphocytes, and by their detection in purified lymphocyte plasma membranes. While the identity of these target antigens vis a vis known surface determinants was not defined, their expression on peripheral T cells from multiple donors and on cell lines of both undifferentiated (HSB-2) and phenotypically mature (Jurkat; HUT 78) types excluded alloantigens, major histocompatibility complex-encoded determinants, and most T cell differentiation antigens as candidates in this regard. Expression of the IgM autoantibody targets on HSB-2 cells argues against discrete T subset specificities as well. IgM reactivity with the 55-, 70-, and 105- to 110-kDa antigens by blotting was highly correlated with antilymphocyte antibody activity in complement-dependent cytotoxicity assays (Fisher's p less than 0.001), and paralleled flow microfluorimetric and microcytotoxicity quantitation of IgM antibody activity in serial observations of individual patients studied during different phases of disease activity. Taken together, these data suggest that IgM lymphocytotoxic antibodies in systemic lupus erythematosus are directed predominantly against a limited number of non-T cell subset-specific antigens.
Internalization of interleukin 2 (IL-2) by high affinity IL-2 receptors is required for the growth of IL-2-dependent T cell lines.
Authors: Kumar A , Moreau J L , Gibert M , Thèze J
Published: December 2022

During the growth of interleukin 2 (IL-2)-dependent T cells IL-2 binding is followed by internalization of the complex between IL-2 and the high affinity IL-2 receptor (HA-IL-2R). The respective role of IL-2 binding to HA-IL-2R and internalization of the complex has been examined. Monoclonal antibody 7D4 (IgM) blocks IL-2-dependent T cell growth although it does not affect IL-2 binding to HA-IL-2R. We show here that 7D4 inhibits T cell growth by blocking IL-2 internalization by HA-IL-2R. In contrast, Fab fragments prepared from 7D4 neither block IL-2 internalization nor inhibit T cell growth. Monoclonal 5A2, that recognizes an epitope related to the IL-2 binding site as well as its Fab fragment, inhibits T cell growth and IL-2 internalization. Monoclonal antibody 7D4, because of its pentameric structure, probably aggregates the IL-2R at the T cell surface and therefore prevents it internalization. The data presented in this paper suggest that simple occupancy of HA-IL-2R by IL-2 is not sufficient to transduce the T cell growth signal; this signal is transmitted only after internalization of the IL-2/HA-IL-2R complex.
A mouse T cell product that preferentially enhances IgA production. II. Physicochemical characterization.
Authors: Bond M W , Shrader B , Mosmann T R , Coffman R L
Published: December 2022

Certain subsets of helper T cells, following stimulation with concanavalin A, secrete factors that specifically enhance the production of IgG1, IgE, and IgA by lipopolysaccharide-stimulated B cells. In the previous report, we describe a factor from the helper T cell line MB2-1 which enhances IgA production. IgA-enhancing factor has been purified from serum-free supernatants of this cell line. The purified lymphokine is a family of microheterogeneous polypeptides presumably modified post-translationally. IgA-enhancing factor has a native m.w. of 45,000 to 60,000 with subunits of between 24,000 and 28,000 under reducing conditions. Upon Edman degradation, a single amino-terminal sequence is detected which is identical to that of the lymphokine interleukin 5. IgA-enhancing factor activity is thus mediated by the same polypeptide that has been characterized as type II B cell growth factor, T cell-replacing factor, and eosinophil-differentiation factor.
Lipid A-associated proteins provide an alternate "second signal" in the activation of recombinant interferon-gamma-primed, C3H/HeJ macrophages to a fully tumoricidal state.
Authors: Hogan M M , Vogel S N
Published: December 2022

Previous studies have shown that the activation of murine macrophages to a fully tumoricidal state requires that specific environmental signals be delivered to the macrophage in a stepwise manner: a "priming" signal first renders the macrophage responsive to a second or "trigger" signal. One potent "priming" signal has been identified as the T cell-derived lymphokine, interferon-gamma (IFN-gamma) and one often used "trigger" signal is lipopolysaccharide (LPS), the endotoxin derived from Gram-negative bacteria. In these studies, endotoxin-responsive C3H/OuJ (Lps(n)) and endotoxin-hyporesponsive C3H/HeJ (Lps(d)) macrophages were exposed in vitro to recombinant IFN-gamma (rIFN-gamma) and various preparations of endotoxin or purified lipid A-associated proteins (LAP). The resultant tumoricidal responses were evaluated to define the activation requirements of murine macrophages and to examine further the LPS defect exhibited by C3H/HeJ mice. The findings presented herein demonstrate that C3H/OuJ macrophages primed by rIFN-gamma respond to protein-free LPS (phenol-water extracted LPS), protein-rich LPS (butanol-extracted LPS), or purified LAP. In contrast, rIFN-gamma-primed C3H/HeJ macrophages failed to become cytolytic with phenol-water extracted LPS, but could be rendered fully tumoricidal if either butanol-extracted LPS or LAP were used as "second signals." These data indicate that C3H/HeJ macrophages are fully responsive to the priming effects of IFN-gamma, but remain restricted in their capacity to recognize protein-free LPS as a second signal. Alternate second signals, such as LAP, may provide a compensatory pathway by which these macrophages are rendered fully tumoricidal.
Carboxypeptidase A in mouse mast cells. Identification, characterization, and use as a differentiation marker.
Authors: Serafin W E , Dayton E T , Gravallese P M , Austen K F , Stevens R L
Published: December 2022

By using a conventional spectrophotometric assay with hippuryl-L-phenylalanine as the substrate, 10(6) BALB/c mouse serosal mast cells possessed 1.5 +/- 0.43 U (mean +/- SE, n = 5, range = 0.48 to 2.5) of carboxypeptidase A activity, while T cell factor-dependent, mouse bone marrow-derived mast cells (BMMC) had barely detectable levels of 0.01 +/- 0.001 U/10(6) cells (mean +/- SE, n = 3). In order to characterize the carboxypeptidase A present in the BMMC, a sensitive assay was developed that used angiotensin I as the substrate and reverse phase-high performance liquid chromatography to separate and quantify production of the cleavage product des-leu-angiotensin I. Using this assay, mouse BMMC carboxypeptidase A had a neutral to basic pH optimum and hydrolyzed angiotensin I with a Km of 0.78 mM. The antigen-induced net percent release of carboxypeptidase A from IgE-sensitized BMMC was proportional to that of the secretory granule component beta-hexosaminidase which indicates a secretory granule location for the exopeptidase. As defined by exclusion during Sepharose CL-2B chromatography, carboxypeptidase A was exocytosed as a greater than 1 X 10(7) m.w. complex bound to proteoglycans. Because BMMC cocultured with mouse skin-derived 3T3 fibroblasts are known to undergo an increase in histamine content and biosynthesis of 35S-labeled heparin proteoglycans, carboxypeptidase A activity was measured during BMMC/fibroblast coculture for 0 to 28 days. The carboxypeptidase A activity increased progressively during 28 days of co-culture from 0.004 +/- 0.002 U/10(6) starting BMMC (mean +/- SE, n = 3) to 0.36 +/- 0.10 U/10(6) co-cultured mast cells. These findings indicate that carboxypeptidase A, a neutral protease, is exocytosed from the secretory granules of mouse mast cells bound to proteoglycan and is increased during the in vitro differentiation of mouse BMMC from mucosal-like mast cells to serosal-like mast cells.
Differential expression of the amyloid SAA 3 gene in liver and peritoneal macrophages of mice undergoing dissimilar inflammatory episodes.
Authors: Rokita H , Shirahama T , Cohen A S , Meek R L , Benditt E P , Sipe J D
Published: December 2022

The three active serum amyloid A (SAA) genes of mice, SAA 1, SAA 2, and SAA 3, are coordinately expressed in liver during acute and chronic inflammatory stimulation and experimental amyloidosis. The genes, primarily SAA 3, are also expressed extrahepatically. The apoprotein SAA 2 is the precursor of the amyloid A (AA) fibril protein that is deposited as insoluble fibrils extracellularly in spleen and other organs when amyloidosis occurs secondarily to inflammation. The exact cause of AA fibril formation is unknown. Amyloid enhancing factor is a high m.w. glycoprotein extracted from amyloidotic organs. Administration of amyloid enhancing factor alters experimental inflammation to bring about accelerated deposition of amyloid A fibrils first in spleen and later in other organs. In this study, hepatic and extrahepatic expression of the SAA genes were compared during accelerated amyloidosis relative to inflammation uncomplicated by amyloidosis. Differences in kinetics and pattern of SAA gene expression by resident peritoneal macrophages and liver were detected during four dissimilar inflammatory episodes. Macrophages expressed the SAA 3 gene solely, and to a greater extent in chronic than in acute inflammation. In accelerated amyloid induction, macrophage SAA 3 expression increased as SAA 1 and SAA 2 expression in liver decreased. However, alpha-1-acid glycoprotein expression remained elevated throughout the course of amyloid induction. The greatly increased expression of the SAA 3 gene by macrophages and decreased expression of the SAA 1 and SAA 2 genes in liver during amyloidosis, suggests that altered SAA gene expression may play a pathogenetic role in experimental amyloid deposition.
Essential requirement of I-A region-identical host bone marrow or bone marrow-derived cells for tumor neutralization by primed L3T4+ T cells.
Authors: Ozawa H , Iwaguchi T , Kataoka T
Published: December 2022

The antitumor activity of Meth A-hyperimmunized BALB/c mouse spleen cells (Meth A-Im-SPL) was assayed by the Winn test in H-2 incompatible bone marrow chimeras in closed colony CD-1 (nu/nu), inbred DDD/1(nu/nu) (H-2s), or inbred BALB/c(nu/nu) (H-2d) mice as recipients. We found that Meth A-Im-SPL suppressed Meth A growth in the chimera nude mice which were reconstituted with bone marrow cells of the H-2d haplotype (i.e., BALB/c, DBA/2 and B10.D2), but not in the chimeras which were reconstituted with bone marrow cells of the H-2a, H-2b, or H-2k haplotype (i.e., B10.A, B10, and B10.BR). These results suggested that H-2 restriction occurred between Meth A-Im-SPL and bone marrow or bone marrow-derived cells in tumor neutralization. Furthermore, Meth A-Im-SPL did not suppress Meth 1 tumors (antigenically distinct from Meth A tumors) in the presence or absence of mitomycin C-treated Meth A in a Winn assay. These results suggested that there is tumor specificity in the "effector phase" as well as in the "induction phase". The phenotype of the effectors in the Meth A-Im-SPL was Thy-1.2+ and L3T4+, because Meth A-Im-SPL lost their antitumor activity with pretreatment with anti-Thy-1.2 monoclonal antibody (mAb) and complement or anti-L3T4 mAb and complement, but not with anti-Lyt-2.2 mAb and complement or complement alone. Positively purified L3T4+ T cells from Meth A-Im-SPL (Meth A-Im-L3T4), obtained by the panning method, suppressed the tumor growth in the chimera nude mice which were reconstituted with bone marrow cells of B10.KEA2 mice (that were I-A region-identical with Meth A-Im-L3T4 cells but not others in H-2) as well as B10.D2 cells (that were fully identical with Meth A-Im-L3T4 cells in H-2). We conclude that Meth A-Im-SPL (L3T4+) neutralized the tumors in collaboration with I-A region-identical host bone marrow or bone marrow-derived cells, and the neutralization was not accompanied by the "bystander effect."
Processing and presentation of insulin. I. Analysis of immunogenic peptides and processing requirements for insulin A loop-specific T cells.
Authors: Naquet P , Ellis J , Singh B , Hodges R S , Delovitch T L
Published: December 2022

The processing and presentation of insulin by B hybridoma cells to insulin A loop-specific T cell hybridomas was investigated. We found that the activation of these T cells requires insulin to be processed in a manner that permits unfolding of the molecule and prevents extensive proteolysis. An analysis of insulin peptides formed by either enzymatic digestion in vitro or solid phase synthesis revealed that a conformational determinant comprised of residues A1-A14 disulfide-linked to B7-B15 is most immunogenic to these T cells. Reduction and/or proteolysis of this peptide markedly decreases its immunogenicity. The pork insulin A1-A14/B7-B15 peptide differs only at residue A4 from its mouse insulin homolog. Thus, Glu A4 forms part of the antigenic site recognized by a pork insulin/I-Ad-specific mouse T cell. This insulin peptide can be induced to assume an alpha-helical configuration in a hydrophobic environment. In addition, virtually all of the residues of this peptide are identical with those predicted to be situated in amphipathic regions of the native insulin molecule. N-Ethylmaleimide and bacitracin, which inhibit the activity of two cytosolic enzymes that cleave insulin, enhance the antigen presentation of insulin. This suggests that these enzymes may participate in the nonlysosomal antigen processing of insulin by a B lymphocyte. A comparison of the relative avidity of several T cell hybridomas, which have the same apparent specificity for this insulin peptide, showed that an increase in their avidity was associated with a degeneracy in their fine specificity. Our data demonstrate that the efficiency of processing and presentation of a given antigenic determinant is related to the conformation of the determinant and the specificity and avidity of the T cell.
T-cell-derived IgE-binding factors. I. Cloned and transformed T cells producing IgE-binding factors.
Authors: Nutman T B , Delespesse G , Sarfati M , Volkman D J
Published: December 2022

In order to derive T cell clones that can produce factors that specifically regulate the IgE response, Fc epsilon receptor (Fc epsilon R)-positive T cells were isolated from two patients with hyper-IgE states (either the tropical pulmonary eosinophilia syndrome or the hyper-IgE syndrome) and transformed using the human T cell leukemia/lymphoma virus. After infection, these T4-positive, Fc epsilon R-positive T cells manifested a fourfold increase in the surface interleukin 2 receptor, a 10-fold increase in Fc epsilon R, and acquired viral markers not present in the uninfected cells. Cloning soon after transformation allowed for the isolation of 17 distinct T cell clones producing IgE-binding factors. The levels of these binding factors released in supernatant fluid from these clones ranged from 10 to 98 U (negative less than 1 U) using a radioimmunoassay. Furthermore, the presence and kinetics of production of these binding factors also could be demonstrated by metabolic labeling studies. These cells should prove potent immunologic tools for dissecting the various regulatory mechanisms involved in IgE production both in the normal state and in pathologic conditions.
In vitro generated human monoclonal trinitrophenyl-specific B cell lines. Evidence that human and murine anti-trinitrophenyl monoclonal antibodies cross-react with Escherichia coli beta-galactosidase.
Authors: Golding B , Inghirami G , Peters E , Hoffman T , Balow J E , Tsokos G C
Published: December 2022

Stable human antigen-specific monoclonal B cell lines were established without prior in vivo immunization. This was accomplished by expanding the anti-trinitrophenyl (TNP) B cells in vitro with the antigen TNP-Brucella abortus and then immortalizing them with Epstein-Barr virus. Five anti-TNP clones were selected by sequential limiting dilution. All five anti-TNP clones secreted IgM kappa antibodies. When tested against a panel of self and environmental antigens, all five anti-TNP clones exhibited cross-reactivity with an Escherichia coli-derived beta-galactosidase. To determine whether this was a more general phenomenon, a panel of murine monoclonals were tested and found to bind to beta-galactosidase. It is therefore possible that human and murine anti-TNP beta cell responses reflect reactivity against an environmental antigen, namely an epitope present on E. coli-derived beta-galactosidase. This approach of expanding human antigen-specific B cells by antigen stimulation in vitro, with a T-independent hapten-carrier conjugate before Epstein-Barr virus transformation, may prove useful in the development of human monoclonals for therapeutic purposes.
Lysis of cells infected with typhus group rickettsiae by a human cytotoxic T cell clone.
Authors: Carl M , Robbins F M , Hartzman R J , Dasch G A
Published: December 2022

Cytolytic human T cell clones generated in response to the intracellular bacterium Rickettsia typhi were characterized. Growing clones were tested for their ability to proliferate specifically in response to antigens derived from typhus group rickettsiae or to lyse targets infected with R. typhi or Rickettsia prowazekii. Two clones were able to lyse targets infected with typhus group rickettsiae. One of these clones was more fully characterized because of its rapid growth characteristics. This cytolytic clone was capable of lysing an autologous infected target as well as a target matched for class I and II histocompatibility leukocyte antigens (HLA). It was not capable, however, of lysing either a target mismatched for both class I and II HLA or a target partially matched for class I HLA. In addition, the clone exhibited specificity in that it was able to lyse an autologous target infected with typhus group rickettsiae, but did not lyse an autologous target infected with an antigenically distinct rickettsial species, Rickettsia tsutsugamushi. These results demonstrate, for the first time, that cells infected with intracellular bacteria can be lysed by human cytotoxic T lymphocytes.
The 230-kDa gamete surface protein of Plasmodium falciparum is also a target for transmission-blocking antibodies.
Authors: Quakyi I A , Carter R , Rener J , Kumar N , Good M F , Miller L H
Published: December 2022

Immunization with extracellular sexual stages of the malaria parasites can induce the production of antibodies which block the development of the parasites in the midgut of a mosquito after a blood meal. We have generated a number of monoclonal antibodies against gametes and zygotes of the human malaria Plasmodium falciparum. Two monoclonal antibodies (mAb) reacting with a 230-kDa gamete surface protein (mAb 1B3 and 2B4 both isotype IgG2a) were found to block transmission of P. falciparum to mosquitoes. Blocking was complement dependent and this was verified in vitro by the rapid lysis of newly formed gametes and zygotes in the presence of the mAb and active complement. Both mAb reacted by immunofluorescence with the surface of gametes and zygotes from isolates of P. falciparum from various geographical areas. Each mAb immunoprecipitated a 230-kDa protein from 125I-labeled surface proteins of newly formed gametes and zygotes and immunoblotted a protein doublet of about molecular mass 260 and 230 kDa from gametocytes and gametes of P. falciparum. Only the 230-kDa protein is expressed on the surface of newly formed macrogametes and zygotes. The 230-kDa gamete surface protein forms a molecular complex with two proteins of 48 and 45 kDa. The 48- and 45-kDa gamete surface proteins have previously been shown to be targets of mAb which block infectivity of P. falciparum to mosquitoes. The present study now demonstrates that antibodies against the 230-kDa gamete surface protein block transmission of P. falciparum to mosquitoes. The 230-kDa gamete protein is thus a potential candidate for a gamete vaccine.
The effect of dihydrofolate reductase-mediated gene amplification on the expression of transfected immunoglobulin genes.
Authors: Dorai H , Moore G P
Published: December 2022

Murine/human chimeric gamma 1 and K Ig genes were cloned adjacent to the gene coding for methotrexate-resistant dihydrofolate reductase. These constructs were introduced into myeloma cells, and lines containing stably integrated genes were selected. The integrated Ig genes were then amplified by selection of the cells in increasing concentrations of methotrexate. The extent of gene amplification, mRNA accumulation, and production of Ig was studied in transfectomas containing introduced light chain genes, heavy chain genes, or both. When the light chain gene was introduced alone, it was expressed at low levels, but after selection with methotrexate, light chain expression was increased as much as 63-fold. In contrast, the transfected heavy chain genes were highly expressed, but production of the corresponding protein was increased a maximum of only fourfold by methotrexate treatment. Cellular toxicity of unassembled heavy chain monomer was not observed, even at amounts equivalent to 2% of total cellular protein. Cointroduction of the heavy and light chain constructs with subsequent amplification resulted in as much as 25-fold increase in secretion of intact antibody relative to unamplified cells. The results demonstrate that amplification of Ig genes can induce transfectomas to secrete antibody at nearly the rate of hybridomas.