bluuebunny/crossref_metadata_2025 huggingface dataset:
Natural cytotoxicity against mouse hepatitis virus-infected cells. II. A cytotoxic effector cell with a B lymphocyte phenotype.
Authors: Welsh R M , Haspel M V , Parker D C , Holmes K V
Published: December 2022

The effector cell in mouse spleen which mediates natural cytotoxicity against mouse hepatitis virus (MHV)-infected target cells was characterized. The target cells were MHV-infected BALB/c 3T3, and the assay time was 3 hr. The effector cell, designated virus killer (VK) cell for the purpose of discussion, had the following phenotype: lymphocyte morphology, plastic-nonadherent, nylon wool-adherent, nonphagocytic, cyclophosphamide-sensitive; by antibody plus complement (C) depletion studies, it was asialo GM1-, NK 1.2 alloantigen-negative, Thy-1.2-, Lyt-5-, and macrophage antigen-negative; by rosetting techniques, it was Fc receptor-positive and surface Fab+; by flow cytometry (FACS) analysis, it was Lyt-2-, MAC-1-, Ia+, IgG (gamma)+, IgM (mu)+, IgD (delta)+, and B cell lineage antibody B-220+. NK cells, measured for cytotoxicity on YAC-1 cells, were similarly tested and were found to differ from the VK cell in the following properties: nylon wool-nonadherent, asialo GM1+, NK alloantigen-positive, Lyt-5+, surface Fab-, MAC-1+, Ia-, IgG-, IgM-, IgD-, and B-220-. The VK effector cell had a phenotype highly distinguishable from NK cells, effectors most commonly associated with antiviral natural cytotoxicity. The VK cell had a phenotype identical to that of a B lymphocyte and was identified as such. Although the effector cells displayed cell surface antibody, the antibody did not appear to be involved in lysis, because lysis could not be blocked by F(ab)'2 directed against Fab, mu, or delta. Cytotoxicity was more likely associated with recognition of the B lymphocyte surface by the MHV glycoprotein E2, as shown in the accompanying companion paper. This is the first demonstration that natural cytotoxicity can be mediated by B lymphocytes.
Higher frequency of Leishmania major-specific L3T4+ T cells in susceptible BALB/c as compared with resistant CBA mice.
Authors: Milon G , Titus R G , Cerottini J C , Marchal G , Louis J A
Published: December 2022

In previous studies, we reported that a) the adoptive transfer of parasite-specific L3T4+ T cells enhanced rather than inhibited the development of lesions induced by Leishmania major in normal BALB/c mice, and b) the depletion in vivo of L3T4+ T cells by administration of anti-L3T4 monoclonal antibody reversed the susceptibility of BALB/c mice to L. major. To further assess the role of specific L3T4+ T cells in the development of lesions induced by L. major in BALB/c mice, the frequency of parasite-specific T cells capable of mediating specific delayed-type hypersensitivity (DTH) reactivity was determined, by limiting dilution analysis, in the lymph nodes draining the lesions of susceptible (BALB/c) and resistant (CBA) mice. The numbers of L. major-specific DTH-mediating T cells was found to be substantially increased in the lymph nodes of infected BALB/c mice as compared with CBA mice. Moreover in CBA mice, analysis of the cell surface phenotype of the L. major-specific DTH-mediating T cells showed that these cells were equally represented in the L3T4+, Lyt-2-, and L3T4- Lyt-2+ subsets, whereas the majority of these cells in BALB/c mice expressed the L3T4+ Lyt-2- surface phenotype.
The mouse Thy-1.2 glycoprotein gene: complete sequence and identification of an unusual promoter.
Authors: Ingraham H A , Lawless G M , Evans G A
Published: December 2022

Recombinant bacteriophage and cosmid clones containing the gene for the mouse Thy-1.2 glycoprotein were isolated and characterized. The complete sequence of the gene was determined, including a previously unidentified exon located 2.1 kb upstream of the portion of the gene encoding the Thy-1.2 glycoprotein. The transcriptional initiation site was located by S1 nuclease protection mapping in both T lymphocytes and neural cells and was found to be located immediately upstream of this exon. S1 nuclease protection mapping was also used to define the 3' end of the Thy-1.2 transcription unit, and no evidence for alternate mRNA processing was found. Thus, the mouse Thy-1.2 gene is 5447 base pairs in length, including promoter sequences, rather than 2094 as previously described. The mouse and rat Thy-1 genes are highly homologous in both introns and exons. However, the mouse Thy-1 cDNA and rat Thy-1 cDNA differ significantly in sequence in the 5' untranslated region. This suggests that the transcriptional initiation site of the mouse and rat genes may be located at different positions within the genomic sequence and may be related to the differing tissue distribution of Thy-1 in the two species.
The antimetastatic function of concomitant antitumor immunity. I. Host Ly-1+2+ effector T cells prevent the enumeration of metastatic tumor cells in a biological assay.
Authors: Dye S E
Published: December 2022

A mouse survival assay was evaluated for its suitability to enumerate metastatic P815 tumor cells in the draining lymph node and spleen of a B6D2 F1 (H-2b X H-2d) host bearing a primary intradermal P815 tumor. The mouse survival assay is based on the linear relationship between the log10 number of P815 tumor cells (H-2d) injected i.p. into mice and their mean survival time. It was found that the assay is capable of quantifying as few as 10 tumor cells in lymph node and spleen, but only if cell suspensions of these organs are treated with anti-H-2b serum and complement, in order to selectively destroy H-2bd host cells. This was necessary because host cells from the lymph node and spleen of a tumor-bearing host possessed antitumor functions, in that they were capable of destroying the H-2d P815 tumor cells when admixed with the tumor cells and injected i.p. into 800-rad irradiated test recipients. The kinetics of acquisition and loss of host cells with antitumor function and the Ly phenotype of these host cells suggest that they are the same cells that give the tumor-bearing host the capacity to express concomitant immunity against a tumor implant.
IL 2 alone is mitogenic only for Tac-positive lymphocytes in human peripheral blood.
Authors: Taylor D S , Kern J A , Nowell P C
Published: December 2022

To investigate the ability of interleukin 2 (IL 2) alone to induce proliferation of resting human lymphocytes, we stimulated human peripheral blood mononuclear cells (PBMC) with an immunopurified preparation of IL 2 or with phytohemagglutinin (PHA). Proliferation and the percent of cells expressing IL 2 receptors were assessed over 6 days of culture. Regardless of the stimulus, the percent of cells bearing an IL 2 receptor paralleled the amount of proliferation, and proliferation was inhibited by an anti-IL 2 receptor monoclonal antibody (anti-Tac). When stimulated by IL 2 alone, less than 8% of PBMC expressed an IL 2 receptor after 24 hr of culture. Stimulation by IL 2 caused both proliferation and IL 2 receptor expression to increase over the entire culture period (routinely to 75,000 cpm and 50% respectively). When colchicine was added (to inhibit cell division), the percent of cells bearing an IL 2 receptor did not increase. IL 2 alone also induced proliferation of PBMC depleted of accessory cells, with the same kinetics but reduced peak response. Both accessory cells and supernatants that showed IL 1 but not IL 2 activity augmented this proliferation 50 to 100%. In contrast to the effect of IL 2, 25 to 50% of PBMC stimulated by PHA expressed an IL 2 receptor after 24 hr of culture. PHA-induced proliferation and IL 2 receptor expression peaked early in the culture period (routinely to 100,000 cpm and 50% respectively within 3 days), and colchicine did not inhibit the early induction of IL 2 receptors on PBMC. Our findings indicate that unlike PHA, IL 2 induces proliferation of PBMC (or PBMC depleted of accessory cells) by expanding the small percentage of cells in a resting population that already express IL 2 receptors. IL 2 does not appear to induce IL 2 receptors on cells previously lacking this molecule. We also find that IL 1 can enhance the response to IL 2 alone.
Establishment of an interleukin 2-dependent T cell line derived from a patient with severe aplastic anemia, which inhibits in vitro hematopoiesis.
Authors: Herrmann F , Griffin J D , Meuer S G , Meyer zum Büschenfelde K H
Published: December 2022

Circulating mononuclear cells from a patient developing severe aplastic anemia during the course of non-A, non-B hepatitis were found to be virtually entirely composed of in vivo activated suppressor T cells (Ia+T8+). These cells were used to establish a new permanent cell line, termed SMAA, by using phytohemagglutinin, Ebstein-Barr virus-transformed irradiated B cells, allogeneic irradiated peripheral blood mononuclear cells, and recombinant interleukin 2 to investigate the relationship of aplastic anemia-derived circulating T cells to bone marrow failure. SMAA cells, now in continuous culture for more than 9 mo, were shown to inhibit proliferation of purified myeloid progenitors and their differentiation into early and late appearing neutrophil and eosinophil colonies by 90%, whereas monocyte colonies were much less affected. Similarly, growth of erythroid colonies and bursts was almost completely inhibited, as was anti-mu-induced B cell proliferation and lectin-induced T cell proliferation. This inhibition of hematopoiesis was mediated by the release of a soluble factor that was sensitive to acid (pH 2), heat (56 degrees C), and trypsin. Monoclonal and polyclonal antibodies to interferon-gamma could abrogate the inhibitory effects of SMAA supernatant, but more than 10(4) neutralizing U/ml had to be added. The effects of SMAA could be duplicated by adding 10(4) U/ml of purified recombinant interferon-gamma to colony and proliferation assays. The concentration of interferon-gamma in SMAA supernatant was estimated to be greater than 3 X 10(3) National Institutes of Health reference U/ml by immunoradiometric assay. These results demonstrate that some patients with aplastic anemia have circulating T cells that are capable of prolonged in vitro secretion of interferon-gamma causing severe inhibition of in vitro hematopoiesis, and these cells can be expanded into permanent lines for studies on their regulatory properties.
Identification of Ki (Ku, p70/p80) autoantigens and analysis of anti-Ki autoantibody reactivity.
Authors: Francoeur A M , Peebles C L , Gompper P T , Tan E M
Published: December 2022

Anti-Ki (Ku, p70/p80) autoantibodies, named after the prototype patient Kikuta by Tojo et al., occur in approximately 10% of patients with SLE, often in association with anti-Sm autoantibodies. The immunofluorescent staining pattern characteristic of anti-Ki antibodies is diffuse speckled nuclear, although some substrates show nucleolar staining as well. Anti-Ki sera specifically immunoprecipitated two protein antigens, Ki86 (Mr 86,000) and Ki66 (Mr 66,000), from radiolabeled cell extracts. The Ki system was found to be immunologically identical to the Ku system described by Mimori et al. and the p70/p80 system described by Reeves. The Ki primary in vitro translation products were identified and proved similar in size to the cellular antigens. The Ki antigens were purified from human spleen by immunoaffinity chromatography followed by SDS-PAGE. The purified Ki antigens proved to be closely related by amino acid composition, and did not appear to be phosphorylated, glycosylated, or associated with RNA. The Ki antigens were found to bind to DNA, in agreement with the observations on the Ku and p70/p80 antigens. They were found to be widely conserved in mammals and were coordinately expressed in all tissues tested. Anti-Ki autoantibodies were purified by antigen-affinity chromatography and were tested by immunoblotting. The antibodies were classified as class I, II, or III, depending on their reactivity with the Ki antigens in immunoblots. Class I antibodies cross-reacted with both Ki antigens, class II antibodies reacted solely with Ki66, and class III antibodies reacted solely with Ki86. These results suggest that at least three different epitopes are present on the Ki autoantigens and that patients differ in their autoantibody response to each epitope.
Active suppression of host-vs-graft reaction in pregnant mice. VII. Spontaneous abortion of allogeneic CBA/J x DBA/2 fetuses in the uterus of CBA/J mice correlates with deficient non-T suppressor cell activity.
Authors: Clark D A , Chaput A , Tutton D
Published: December 2022

The mammalian fetus has been viewed as an unusually successful type of "allograft" and "unexplained" spontaneous abortion as a possible example of maternal rejection. Previous studies have shown the presence of small lymphocytic suppressor cells in the murine decidua which block the generation and reactivation of anti-paternal cytotoxic T lymphocytes (CTL) and lymphokine-activated killer cells (LAK) by elaborating a factor that inhibits the response to interleukin 2 (IL 2). A deficiency of these suppressor cells was associated with implants of xenogeneic Mus caroli embryos in the Mus musculus uterus which are infiltrated by maternal lymphoid cells and aborted. We have also shown a deficiency of such suppressor cells in the lymph nodes draining the uterus of CBA/J females in the process of aborting their semi-allogeneic CBA X DBA/2 F1 progeny. CBA/J females possess significantly lower levels of decidua-associated non-T suppressor cells on day 8.5 to 10.5 of allopregnancy than do mothers that will produce large litters of live babies. The F1 embryos are infiltrated by maternal lymphocytes prior to abortion, and the infiltration and abortion rate appears to be augmented by pre-immunization with paternal DBA/2 spleen cells. Susceptibility to spontaneous abortion is dependent upon maternal age and strain of male mate, and the high abortion rate of CBA/J mated to DBA/2 males can be reduced by immunization with BALB/c spleen cells. The CBA/J x DBA/2J mating combination provides a model of spontaneous abortion in which immunologic factors play an important role and demonstrates that the association between deficiency of decidua-associated suppressor cells and xenopregnancy failure also holds true for the failure of allopregnancies resulting from natural within-species mating.
Independent regulation of granulocyte-macrophage colony-stimulating factor and multi-lineage colony-stimulating factor production in T lymphocyte clones.
Authors: Kelso A , Metcalf D , Gough N M
Published: December 2022

When murine T lymphocyte clones were cultured with purified recombinant IL 2, a dose-dependent increase in the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) was observed. Whereas these clones produced both GM-CSF and multi-lineage CSF (multi-CSF) when cultured with concanavalin A, IL 2 induced the production of GM-CSF in the virtual absence of detectable multi-CSF. In addition, IL 2 synergistically enhanced the production of both GM-CSF and multi-CSF by some antigen- or Con-A-stimulated clones. Like Con-A-induced CSF production, GM-CSF production in the presence of IL 2 required protein synthesis but could occur in the absence of proliferation by the clone. Analysis of dose-response curves for stimulation of CSF production by Con A in the presence and absence of IL 2 suggested that Con A and IL 2 activated GM-CSF synthesis by different mechanisms. These results indicate that the coordinate production of two factors by a single T cell clone stimulated with Con A can be dissociated when the clone is stimulated with IL 2.
Membrane factors responsible for homologous species restriction of complement-mediated lysis: evidence for a factor other than DAF operating at the stage of C8 and C9.
Authors: Shin M L , Hänsch G , Hu V W , Nicholson-Weller A
Published: December 2022

Species-restricted lysis of complement refers to the relative inefficiency of complement to lyse cells from the homologous species. Restriction occurs at least at the steps involving C3/C5 convertase formation and the C9 insertion phase of the complement cascade, and is presumed to be mediated by inhibitory factors in the target cell membrane. In this study, we have examined whether decay accelerating factor (DAF), a membrane protein known to modulate C3/C5 convertase activities on cell surfaces, acts as a regulatory protein in species-restricted lysis of human erythrocyte (E). The role of DAF was assessed in homologous lysis by the classic pathway, in reactive lysis, and in lytic steps requiring C8 and C9. The results indicated that DAF participated in regulating C3/C5 deposition on the surface of homologous E, but had no effect on homologous restriction in reactive lysis and in the reaction of C8 and C9 with antibody-sensitized E C1-7. Treatment of E with pronase or with dithiothreitol (DTT) abolished the restricting effect of homologous C8/C9, indicating that species-restricted lysis by C5b-9 involves membrane factor(s) sensitive to pronase and DTT.
Molecular characterization of antibodies bearing Id-460. I. The structure of two highly homologous VH genes used to produce idiotype positive immunoglobulins.
Authors: Dzierzak E A , Janeway C A , Richard N , Bothwell A
Published: December 2022

The heavy chain immunoglobulin genes encoding a variety of antibodies specific for DNP or Pasteurella pneumotropica and bearing the dominant idiotype of MOPC 460, Id-460, were cloned and sequenced. The VH genes encoding the M460 and D35 (DNP binding) antibodies were found to be homologous but not identical to the VH gene encoding the LB8 (P. pneumotropica binding) monoclonal antibody. Two of at least eight genes in the VH460 cross-hybridizing gene family can encode Id-460 positive antibodies. The VH460 gene family overlaps with the gene family described by VH36-60 and more completely describes this germ-line VH gene family. We have previously demonstrated a genetic requirement for VK1 expression in order to observe the expression of Id-460 in anti-DNP antibody responses. Southern blot analysis of these monoclonal antibody-producing cells demonstrates, as with the cross-hybridizing VH, that two cross-hybridizing VK1 genes (VK1A and VK1C) can be used to encode Id-460-positive immunoglobulins. We demonstrate that immunoglobulin idiotype determinant expression can be the result of the expression of nonidentical but highly homologous genes in the VH460 cross-hybridizing VH gene family and also in the VK1 cross-hybridizing VL family.
Rabbit major histocompatibility complex. I. Isolation and characterization of three subregions of class II genes.
Authors: Sittisombut N , Knight K L
Published: December 2022

Approximately 300 kb of DNA from the rabbit major histocompatibility complex class II region has been cloned from two genomic libraries. Four alpha chain genes and ten beta chain genes were identified in the recombinant phage and cosmid clones by hybridization with human class II cDNA probes. These genes were classified into three subregions (R-DP, R-DQ, and R-DR) based on hybridization analyses with human DP, DQ, and DR subregion genes under stringent conditions. Two alpha genes and one beta gene were assigned to the R-DP subregion, one alpha gene and one beta gene to the R-DQ subregion, and one alpha gene and eight beta genes to the R-DR subregion. In each subregion, the alpha gene and at least one beta gene were closely linked and were oriented in a manner similar to those in the homologous subregions of human and mouse. A combination of cloning data and genomic blot analyses indicated that the rabbit genome contains a minimum of four alpha-chain genes. The results suggested that there has been evolutionary conservation of the subunit organization of the alpha- and beta-chain genes as well as the coding sequences of these genes and that the mammalian ancestor possessed three distinct MHC class II subregions before diversification of human, mouse, and rabbit.
Desferoxamine blocks IL 2 receptor expression on human T lymphocytes.
Authors: Carotenuto P , Pontesilli O , Cambier J C , Hayward A R
Published: December 2022

Thymidine uptake by PHA-stimulated human lymphocytes is reduced in the presence of 100 microM or greater concentrations of the iron-chelating agent desferoxamine (DF). We assessed expression of IL 2 receptor, 4F2 and Ia antigens, IL 2 production, and cell cycle progression by blood mononuclear cells (MNC) stimulated by PHA in the presence or absence of DF to determine whether the lack of T cell proliferation was a manifestation of inhibition of an earlier activation event. Tac antigen expression on PHA-stimulated MNC was inhibited by DF throughout 8 days of culture, and those cells which were positive had a low density of Tac antigen as compared with controls without DF. Expression of other activation antigens, 4F2 and Ia, was not impaired by DF. The supernatants of the DF-containing and control cultures contained equivalent IL 2 activity, as measured on the HT-2 cell line. Cell cycle analysis of these cultures shows that the addition of DF at the beginning of culture blocks most cells from undergoing G0 to G1 transition, whereas later addition of DF arrests the progression of the T cell blasts through the cell cycle. Separation of cells cultured with PHA and DF into Tac+ and Tac- subsets showed that progression from G0 to G1 was restricted to the former subset. These results suggest that interference with IL 2 receptor expression might contribute to the block in mitogen-induced proliferation caused by DF.
Evidence for direct renal injury as a consequence of glomerular complement activation.
Authors: Boyce N W , Holdsworth S R
Published: December 2022

An isolated perfused kidney (IPK) preparation was used to study the functional consequences of antibody-initiated glomerular complement activation in an environment devoid of circulating inflammatory cells. Control IPK, with antibody bound to the glomerular basement membrane (GBM) (mean +/- SEM, 165.0 +/- 5.7 micrograms globulin/g renal cortex), were perfused with a 5% albumin solution. Control urinary protein excretion was 0.306 +/- 0.112 mg/min, renal vascular resistance (RVR) was 4.72 +/- 0.69 mgHg/ml/min, and the glomerular filtration rate (GFR) was 0.41 +/- 0.01 ml/min/g. To produce glomerular complement activation, IPK with equal quantities of bound antibody (167.0 +/- 6.1 micrograms/g) were perfused with fresh plasma. Glomerular complement activation was associated with linear deposition of C3 on the GBM, a significant increase in protein excretion (3.317 +/- 1.077 mg/min; p less than 0.001) and RVR (10.15 +/- 1.85 mmHg/ml/min; p less than 0.001), and a decline in GFR (0.38 +/- 0.01 ml/min/g; p less than 0.05). Equivalent IPK perfused with decomplemented plasma demonstrated neither glomerular complement deposition nor augmented renal injury. By using both complement repletion and depletion techniques, this study demonstrates that antibody-initiated glomerular complement activation produces direct, neutrophil-independent renal injury. Thus, activated complement components may directly contribute to antibody-induced immune renal injury, in addition to their well established role in the recruitment of circulating inflammatory cells.
Immunotherapy for myasthenia gravis: a murine model.
Authors: Christadoss P , Dauphinee M J
Published: December 2022

In vivo therapy with monoclonal antibody (mAb) GK1.5, which recognizes a glycoprotein antigen designated L3T4 on murine helper T lymphocytes, either prevented or suppressed the development of murine lupus, autoimmune encephalomyelitis, and collagen arthritis. The L3T4 antigen in the mouse is analogous to the human Leu-3/T4 antigen expressed on helper T lymphocytes, because they both participate in the T cell response to class II major histocompatibility complex (MHC) antigens. Class II MHC genes and I-A antigens mediate murine experimental autoimmune myasthenia gravis (EAMG) induced by acetylcholine receptor (AChR) autoimmunity. We studied the efficacy of mAb GK1.5 as an immunotherapeutic agent for murine EAMG. Therapy with mAb GK1.5 not only suppressed established autoimmunity to AChR but also prevented loss of muscle AChR in mice with EAMG. Moreover, permanent remission of clinical muscle weakness was induced if mAb GK1.5 therapy was initiated after the onset of clinical disease. Because the function of the Leu-3/T4 determinant on human helper T lymphocytes is analogous to the murine L3T4 determinant, use of antibody to the Leu-3/T4 determinant as an immunotherapeutic agent may provide a way to control the progression of human MG.
Recognition of a hybrid HLA-DP-associated determinant by a human T lymphocyte clone.
Authors: Eckels D D , Hartzman R J , Johnson A H
Published: December 2022

Alloreactive, human T cell clones were derived from an HLA-DPw1-specific primed lymphocyte typing cell line by limiting dilution. The specificities of the clones were analyzed with allogeneic stimulator cells and in family segregation studies. One clone, TLC 56.94, recognized some, but not all, DPw1-positive stimulator cells and in two families, failed to proliferate in response to stimulatory cells from DPw1-homozygous individuals. The simplest explanation for these results is that TLC 56.94 recognizes a hybrid alloantigen produced by transcomplementation or transassociation between an element of DPw1 and some other gene product.
Induction and regulation by monokines of hepatic synthesis of the mouse serum amyloid P-component (SAP).
Authors: Le P T , Mortensen R F
Published: December 2022

The in vitro synthesis by mouse hepatocytes of the major acute-phase reactant, serum amyloid P-component (SAP), was induced either by inflammatory macrophages or by the addition of monokine(s), including IL 1. A single cell assay for enumerating SAP-secreting hepatocytes was developed. An increase in the frequency of SAP-synthesizing hepatocytes was found during the acute phase of inflammation. Macrophages elicited with a sterile inflammatory agent, when cultured with hepatocytes, both induced new SAP synthesis by the hepatocytes and increased the number of SAP-producing hepatocytes by sevenfold. Inflammatory macrophage culture supernatants induced new SAP synthesis in hepatocytes; however, the inducing activity did not correlate with the IL 1-dependent thymocyte-proliferating activity. Purified IL 1 alone increased SAP production without increasing the number of hepatocytes secreting SAP. A mixture of purified IL 1 with non-IL 1 monokines both increased the number of SAP synthesizing hepatocytes and the amount of SAP secreted per cell. Two non-IL 1 monokines of 70 to 80 Kd and 30 to 40 Kd were responsible for hepatocyte induction. The inducing activity was not neutralized by anti-mouse IL 1 antibody. IL 1 did contribute to the acute phase response by inducing more SAP synthesis per hepatocyte. The findings suggest that both the induction of nonsynthesizing hepatocytes into new SAP synthesis and the enhancement of the amount of SAP produced per hepatocyte are responsible for the increase in blood levels of SAP during the acute phase of inflammation.
A monoclonal anti-HEBFPP antibody with specificity for lymphocyte surface molecules mediating adhesion to Peyer's patch high endothelium of the rat.
Authors: Chin Y H , Rasmussen R A , Woodruff J J , Easton T G
Published: December 2022

Cell surface molecules involved in lymphocyte adhesion to high endothelial cell venules (HEV) of Peyer's patches (PP) have been studied in the rat by using a mouse monoclonal anti-HEBFPP (1B.2) antibody. We previously showed that rat thoracic duct lymph contains a high endothelial cell binding factor termed HEBFPP, which in vitro blocks lymphocyte binding sites of HEVPP but not HEVLN. Monoclonal 1B.2 antibody was produced by fusing P3U1 myeloma cells with spleen cells of a mouse immunized with this material. Immunoprecipitation studies with 125I surface-labeled rat thoracic duct lymphocytes (TDL) showed that the antibody recognized an 80-kilodalton protein. This antigen was present in the majority of TDL, spleen, LN, and PP cells but was found on few (5 to 10%) thymus and bone marrow cells (indirect immunofluorescence). Treatment of TDL with 1B.2 antibody blocked their ability to bind in vitro to HEVPP; antibody treatment did not interfere with TDL adhesion to HEVLN. Analysis of 1B.2 antigen isolated from lymph and detergent lysates of TDL by antibody-affinity chromatography showed that this material had the capacity to block lymphocyte binding sites of HEVPP but not HEVLN. In contrast, material with such blocking activity was not isolated from detergent lysates of thymocyte, a population deficient in HEV-binding cells. The results indicate that the 1B.2 antigen is a component of the lymphocyte surface recognition structure mediating adhesion to HEVPP and provide further evidence that distinct adhesion molecules of rat TDL mediate interaction with high endothelium of LN and PP.
The involvement of the major surface glycoprotein (gp63) of Leishmania promastigotes in attachment to macrophages.
Authors: Russell D G , Wilhelm H
Published: December 2022

The interaction between the macrophage and the parasite plays a central role in the continued success of Leishmania infection. The promastigote surface ligand, and its complementary macrophage membrane receptor, involved in attachment and phagocytosis are likely to exert considerable influence over the outcome of a new infection. In this study, we report experiments pertaining to one such parasite membrane protein. Initial examination of promastigote surface proteins by radiolabeling and two-dimensional-polyacrylamide gel electrophoresis revealed an abundant polypeptide with an apparent m.w. of 63,000. Lectin-binding studies indicated that it was a glycoprotein containing mannose, N-acetyl glucosamine, and N-acetyl galactosamine residues. Monospecific antiserum raised against this glycoprotein, gp63, decorated the entire promastigote plasmalemma. Univalent antibody fragments from this antiserum blocked the interaction between promastigotes and macrophages by inhibiting attachment. Anti-gp63-inhibition reduced parasite/macrophage binding to 30 to 35% of the control binding level. Additional evidence of the involvement of gp63 in attachment to macrophages was provided by studies that made use of gp63-containing proteoliposomes. These vesicles were avidly phagocytosed by macrophages. Uptake of the gp63-containing liposomes was suppressed by greater than 90% by both anti-gp63 F(ab) fragments and the oligosaccharide mannan, indicating that their phagocytosis was receptor dependent. These results demonstrate that the abundant glycoprotein gp63 plays an important role in attachment of promastigotes to macrophages, and attachment via this parasite ligand is sufficient to trigger phagocytosis.
Bacterial induction of human activated lymphocyte killing and its inhibition by lipopolysaccharide (LPS).
Authors: Tarkkanen J , Saxén H , Nurminen M , Mäkelä P H , Saksela E
Published: December 2022

Peripheral blood lymphocytes were incubated with glutaraldehyde-fixed Salmonella bacteria. This resulted in rapid activation of nonspecific cytotoxic potential of the lymphocytes. Both originally noncytotoxic, high-density Percoll-fractionated cells, and cytotoxic natural killer (NK) cell-enriched low-density cells were activated. The induction of originally noncytotoxic cells into activated killer (AK) cells was apparently independent of interferon (IFN), whereas the activation of the NK cell-enriched fractions also involved IFN production. Neither the AK nor NK activity were associated with significant bactericidal activity. The IFN-independent induction of AK activity was not dependent on the O-antigenic polysaccharide part of the lipopolysaccharide (LPS) on the bacterial cell surface, because both smooth (S) strains with differing O-antigenic structures (S-4,12 and S-6,7) and a rough (Re) strain without O-antigen were effective inducers. Isolated LPS, and especially alkali-hydrolyzed (O-deacylated, detoxified) LPS (ALPS) interfered with the induction of cytotoxicity. At concentrations of 10 to 30 micrograms/ml, ALPS totally inhibited the induction of AK activity without affecting the endogenous NK activity. Thus contact with bacteria can lead to the emergence of AK cells, and a bacterial product can effectively block this activation. These phenomena stress the complexity of interactions with host defenses that can take place during bacterial infection.
Identification of a unique receptor on a group A streptococcus for the Fc region of human IgG3.
Authors: Yarnall M , Boyle M D
Published: December 2022

Receptors that bind to the Fc region of all four human IgG subclasses have been described on a number of strains of group A streptococci. In this study, we have demonstrated that these immunoglobulin binding properties are mediated by two distinct Fc receptors. The first receptor, with a Mr of approximately 56,000, binds to human IgG1, IgG2, and IgG4, but not to IgG3. A second receptor, with a Mr of approximately 38,000, binds exclusively to human immunoglobulins of the IgG3 subclass.
T15 idiotype expression in the murine response to phosphorylcholine is actively regulated by genes linked to the Igh-C locus.
Authors: Stall A M , Quintáns J , Loken M R
Published: December 2022

In this report, we demonstrate the existence of a regulatory system that can determine the relative expression of individual clones (idiotypes) within the total immune response to PC. Studies of Igh-C-congenic mice and backcross analysis demonstrate that this regulation is controlled by genes linked to the immunoglobulin heavy chain locus. The regulation acts in a trans fashion. The regulatory genes of one parental allele can control the expression of idiotopes coded for by both parental alleles. This regulation does not appear to affect the quantity of the response, but could affect the quality of the response, depending on the potential repertoire and avidity of the individual clones available to respond to a given antigenic challenge.
Non-germ-line elements (NGE) are present in the T cell receptor beta-chain genes isolated from the mutant mouse, motheaten (me/me).
Authors: Hagiya M , Davis D D , Shultz L D , Sakano H
Published: December 2022

Extra nucleotides (which we call NGE, for non-germ-line elements) are inserted at the junctions of rearranged V, D, and J segments in the immunoglobulin heavy and T cell receptor beta-chain V region genes. NGE addition helps diversity HV3 regions of these genes. It is believed that NGE are added enzymatically and without template during the joining process. Terminal deoxynucleotidyl transferase (TdT) is thought to be the cause of NGE formation. TdT is normally detected in murine thymus and bone marrow cells, but its presence in the immunodeficient mutant mouse, Motheaten (me/me), is extremely reduced in these tissues. To determine whether this TdT deficiency could affect NGE formation during the V-D-J joining of antigen receptor genes, we cloned several rearranged T cell receptor beta-chain genes from thymocytes of me/me mice. Our sequence analysis revealed that Motheaten thymus beta-chain genes have approximately 4 base pair NGE, which are comparable in size to the NGE of wild-type genes. These results do not support the idea that TdT is the NGE-forming enzyme, although it is still possible that a low but residual level of TdT is capable of NGE formation in Motheaten. Alternatively, our data may suggest that the TdT activity in Motheaten T cells is normal; however, the number of TdT+ cells is greatly reduced in the Motheaten thymus, due to a severe defect in T cell development.
Analysis of T cell receptors in highly purified rat and human large granular lymphocytes (LGL): lack of functional 1.3 kb beta-chain mRNA.
Authors: Young H A , Ortaldo J R , Herberman R B , Reynolds C W
Published: December 2022

Recent reports have suggested that the T cell receptor for antigen is somehow involved in the cytotoxicity of natural killer (NK) cells. However, we now report that highly purified, freshly isolated large granular lymphocytes (LGL) from both the human and rat, as well as LGL cultured in the presence of recombinant IL 2, express only the 1.0 kb beta-chain mRNA. The lack of a 1.3 kb mRNA, indicative of a functional beta-chain rearrangement, strongly suggests that a functional T cell receptor beta-chain is absent in freshly isolated LGL, thus making it extremely unlikely that this molecule is involved in target cell recognition by NK cells. These results also suggest that LGL are derived from a lineage distinct from T cells or develop before a functional rearrangement of the T cell receptor beta-chain.
Enhanced transcription in a pre-B-like cell line transformed with cloned antibody genes after antigen-specific stimulation.
Authors: Ochi A , Hozumi N
Published: December 2022

The transducing vector, pSV2-neo, carrying the rearranged immunoglobulin (Ig) heavy (mu) and light (kappa) chain genes specific for the hapten 2,4,6-trinitrophenyl (TNP) was introduced into a pre-B cell line. The transformants expressed the TNP-specific IgM receptor on the surface. Furthermore, the addition of TNP-bovine serum albumin (hapten-carrier conjugate) to the culture media activated the expression of the transferred Ig genes and several endogenous genes such as v-abl and beta-tubulin. However, expression of the beta2-microglobulin gene was not affected. The results presented in this paper show that transfection of cloned Ig genes into B cells is a useful system for establishing monoclonal B cell lines expressing functional Ig receptor molecules with defined hapten specificity.
Inhibition of lymphoid cell growth by a lipid-like component of macrophage hybridoma cells.
Authors: Stallcup K C , Liu Y N , Dorf M E , Mescher M F
Published: December 2022

Previous studies have shown that plasma membranes of murine lymphocytes and lymphoid tumor cells can reversibly inhibit the growth of both normal and transformed lymphocytes. The inhibitor can be extracted with organic solvents and has properties consistent with it being a lipid or lipid-like component of the membrane. This report identifies a series of cloned macrophage hybridoma cell lines, obtained by fusion of splenic adherent cells and the P388D1 line, which have very high levels of lipid-like growth-inhibitory molecules. Furthermore, a survey of seven cloned lines indicated that the macrophages fell into two distinct groups with regard to their level of growth-inhibitory activity. Group 1 lines had little or no inhibitory activity when cells were examined for their effect on a B lymphocyte proliferative response. Organic extracts from these macrophages had inhibitory activity (on a per cell basis) comparable to that seen with extracts of the P388D1 parental cell line and lymphoid tumor cells. In contrast, relatively low numbers of Group 2 macrophages could profoundly inhibit B macrophage proliferation. The growth-inhibitory activity was quantitatively recovered in organic extracts of the macrophages. Although the precise nature of the lipid moiety remains undefined, the data argue against the involvement of oxidized cholesterol. These findings indicate that lipid-like inhibitors of cell growth are present and functional in these macrophage cell lines. In addition, the results demonstrate that the inhibitory activity found in plasma membranes and liposomes is present and active in the membranes of intact cells, which is in contrast to the possibility that the inhibitor is an artifact generated during subcellular fractionation. Thus, the inhibitor is likely to have a physiologic role in growth control and in macrophage-mediated immunoregulation, probably acting via a mechanism involving cell-cell contact.
Simultaneous cell cycle analysis and two-color surface immunofluorescence using 7-amino-actinomycin D and single laser excitation: applications to study of cell activation and the cell cycle of murine Ly-1 B cells.
Authors: Rabinovitch P S , Torres R M , Engel D
Published: December 2022

The DNA-binding, fluorescent dye 7-amino-actinomycin D (7AAD) is efficiently excited by the 488 nm laser line commonly used in flow cytometry, but yields fluorescence emission further into the red spectrum than alternative DNA-specific fluorochromes. In this report, we show that the spectral properties of 7AAD allow single-laser analysis of DNA content and cell cycle simultaneously with two cell surface markers labeled with fluorescein (green)-and phycoerythrin (orange)-conjugated antibodies. The use of 7AAD makes three-color analysis practical and feasible, using the most widely available flow cytometric instruments. The power of this technique was demonstrated in two systems. Staining of human peripheral blood lymphocytes (PBL) with 7AAD was demonstrated to be dependent on cell activation and chromatin conformation; PHA-stimulated cells which have become activated and express IL 2 receptors had greater 7AAD fluorescence than nonactivated, IL 2 receptor-negative cells. Cell cycle analysis of mouse splenocytes stained with fluorescent antibodies to IgM and to Ly-1 demonstrated that the proportion of S and G2 phase cells in native spleen varies strongly among the subsets of cells identified with these markers. Of particular interest was the striking finding that the Ly-1+/IgM+ subset (Ly-1 B cells) is greatly enriched for cells in the S phase fraction. This is important because Ly-1 B cells have been associated with the production of autoantibodies, and is consistent with reports that these cells have a lymphoblastoid or a plasmablast morphology. We hypothesize that Ly-1 B cells may belong to a subset of in vivo activated cells which are either rapidly proliferating or are arrested in S phase.
Modulation of antigen-induced T cell proliferation by alpha 2M-trypsin complexes.
Authors: Mannhalter J W , Borth W , Eibl M M
Published: December 2022

Proteinase-complexed alpha 2-macroglobulin (alpha 2M) could be shown to interfere with T cell proliferation in response to antigen presented by autologous antigen-pulsed monocytes (M phi) (antigen-induced M phi-T cell interaction, MTI). Addition of alpha 2M-trypsin (alpha 2M X T) complexes to cultures of T cells and antigen-pulsed M phi led to a dose-dependent decrease of T cell proliferation (up to 91% inhibition of the T cell response), whereas the same concentrations of free (native) alpha 2M had no effect on antigen-induced MTI. The observed interference with MTI could be attributed to residual enzymic activity of the alpha 2M X T complex. Addition of aprotinin, a low Mr protein proteinase inhibitor able to penetrate to the enzyme entrapped within the alpha 2M molecule and thus bind to and inactivate the enzyme's active site, resulted in a reversal of the alpha 2M X T-induced biological effect. Inactivation of the enzyme's active site within alpha 2M X T was monitored by a decrease in the hydrolytic activity of the complex. Kinetic studies (addition of alpha 2M X T 24 to 48 hr after culture onset was shown to be still inhibitory) indicated an effect at the level of the T cell or its mediators, but an overnight incubation of T cells with alpha 2M X T did not alter these cells' capacity to proliferate in response to an antigenic stimulus. An additional effect of alpha 2M X T on the antigen-presenting cell cannot be ruled out at present. However, alpha 2M X T did not alter the percentage of monocytes expressing HLA-DR, -DP, and -DQ or interfere with interleukin 1 release if added to M phi at concentrations that significantly inhibited MTI. Furthermore, incubation of M phi with alpha 2M X T for 1 hr before antigen pulsing had no effect on the M phi antigen presenting capacity.
Natural killer cell activity in the rat. V. The circulation patterns and tissue localization of peripheral blood large granular lymphocytes (LGL).
Authors: Rolstad B , Herberman R B , Reynolds C W
Published: December 2022

Large granular lymphocytes (LGL) and T cells were separated from blood by centrifugation on discontinuous gradients of Percoll, were labeled with [3H]uridine or [111In]oxine, and were injected i.v. into syngeneic euthymic or athymic nude rats. The tissue distribution of these labeled cells was monitored for up to 24 hr after transfer by scintillation counting of tissue homogenates and autoradiography of tissue sections. In normal euthymic rats, the main sites of LGL localization were the alveolar walls of the lungs and spleen red pulp; however, they were not detectable in the major traffic areas of T lymphocyte recirculation, the spleen white pulp, and lymph nodes. Furthermore, the density of labeled LGL was very low in the small intestine, thymus, kidney, and liver, although on a per-organ basis, about 10% of the injected radioactivity was found in the liver by 24 hr post-injection. When 111In-labeled LGL were injected i.v. into rats with an indwelling thoracic duct cannula, they completely failed to enter the thoracic duct lymphocyte (TDL) population over an observation period of 6 days. This finding was markedly different from the results obtained with T cells and was consistent with the lack of natural killer and antibody-dependent cellular cytotoxicity activity observed among TDL, even in rats pretreated with the biological response modifier, poly I:C. LGL in athymic nude rats also failed to recirculate between blood and lymph. However, in contrast to normal euthymic animals, a significant increase in the localization of radiolabeled LGL to lymph nodes was observed in nude rats between 30 min and 24 hr. Taken as a whole, these findings define the areas within the lungs and spleen in which blood LGL normally localize, and clearly demonstrate that LGL do not normally recirculate between blood and lymph.
The requirements for antigen multivalency in class I antigen recognition and triggering of primed precursor cytolytic T lymphocytes.
Authors: Herrmann S H , Mescher M F
Published: December 2022

In vitro generation of a secondary cytolytic T lymphocyte (CTL) response to Class I alloantigen requires two signals: recognition of the Class I antigen by precursor CTL (Signal 1), and subsequent interaction with lymphokine(s) (Signal 2). Previous work using subcellular antigen stimulation has demonstrated that the required lymphokine(s) is produced as a result of adherent cell uptake, processing, and Ia-restricted presentation of alloantigen to helper T cells. This pathway could be bypassed by addition to the cultures of supernatant from Con A-stimulated rat spleen cells. When an optimal level of lymphokine(s) is provided by addition of Con A supernatant, the magnitude of the CTL response obtained is dependent on the effectiveness of alloantigen recognition and triggering of the primed precursor CTL (pCTL). By using this approach, we examined the cellular and molecular requirements for generation of Signal 1. Previous results had indicated that pCTL were able to directly recognize subcellular antigen, and that cellular presentation of the antigen to pCTL was not required. Further evidence for this was provided by the finding that pulsing of the responder population for short times with liposomes containing purified H-2Kk resulted in effective stimulation of the response. Exposure of cells to antigen for 1 to 2 hr at 4 degrees C generated responses of comparable magnitude to those obtained when antigen was continuously present in the cultures. Experiments were also done to directly examine the ability of alloantigen-pulsed splenic adherent cells (SAC) to deliver Signal 1. Although the antigen-pulsed SAC were very effective in presenting to helper T cells to result in factor production, they were found to be very ineffective in providing Signal 1 to the pCTL. Having obtained strong evidence for triggering of pCTL occurring via direct recognition of the subcellular alloantigen, we then examined the role of antigen multivalency in recognition and triggering. Purified H-2Kk was prepared in a variety of forms of differing multivalency, ranging from monovalent papain cleavage product to large, highly multivalent liposomes and plasma membranes. The magnitude of the CTL responses obtained was found to be critically dependent on the multivalency of the antigen preparation. Examination of the antigen dose-response curves and maximal responses obtained suggests that valency of the antigen may be important both in determining the avidity of interaction between the pCTL and the antigen-bearing structure, and in determining the extent to which localized receptor cross-linking occurs on the cell surface to result in triggering.