Showing papers in "Immunology and Cell Biology in 1991"

Preparation and characterization of a chimeric CD19 monoclonal antibody.

Abstract: FMC63 is an IgG2a mouse monoclonal antibody belonging to the CD19 cluster. CD19 antibodies react with a 95kDa protein expressed by cells of the B lymphocyte lineage, from pre-B cells to mature B lymphocytes. CD19 antibodies have been suggested as candidates for immunological attack on leukaemic and lymphoma cells of the B lineage because the antigen is restricted to the B lineage. With the potential use of FMC63 in immunotherapy in mind, we have produced a mouse-human chimera in which the genes coding for the VDJ region of the heavy chain and the VJ region of the light chain derive from the FMC63 mouse hybridoma, while the C region genes code for human IgG1. The genes have been transfected back into a mouse myeloma line, which secretes low levels of immunoglobulin. (Ig). This Ig was purified and biotinylated in order to determine the specificity of the antibody. The chimeric antibody has a reaction profile concordant with the original FMC63 antibody, but has the properties of a human IgG1, including the ability to fix human complement. However, the antibody is not cytotoxic in vitro in the presence of complement or cells capable of mediating antibody-dependent cellular cytotoxicity. Possible reasons for this and ways of using the antibody are discussed.

Molecular cloning, expression and characterization of ovine TNFα

Abstract: Tumour necrosis factor alpha (TNF alpha) is a cytokine with a wide range of effects on both lymphoid and non-lymphoid cell types. By hybridization with a human TNF alpha cDNA probe the corresponding ovine cDNA was isolated from a lipopolysaccharide (LPS) stimulated alveolar macrophage cDNA library. The sequence of the cDNA clone showed that ovine TNF alpha encodes a polypeptide of 234 amino acids that, based on analysis of human TNF alpha, is processed to a protein of 157 amino acids. The nucleotide and amino acid sequences showed a high degree of homology to the equivalent human and mouse molecules. In a mammalian COS cell expression system the ovine cDNA was found to encode a protein which was able to lyse actinomycin-D treated WEHI-164 cells and induce COS cells to produce and secrete interleukin 6 (IL-6). Further experiments demonstrated the importance of sequences within the 3' untranslated region of the cDNA in determining the level of expression of ovine TNF alpha. Northern blot analysis was used to analyse the kinetics of induction of ovine TNF alpha mRNA in alveolar macrophages stimulated with a variety of mitogens. Addition of LPS increased mRNA encoding TNF alpha at 1 h and 5 h but not 24 h post stimulation. In contrast, addition of phorbol myristic acid (PMA) led to increased TNF alpha mRNA at 5 h while the combination of PMA and ionomycin increased the level of specific mRNA detected at 1 h, 5 h and 24 h. From genomic analysis ovine TNF alpha appears to exist as a single copy.

Human IgE-binding synthetic peptides of bovine beta-lactoglobulin and alpha-lactalbumin. In vitro cross-reactivity of the allergens.

Abstract: The allergenicity of cow's milk whey proteins, purified by high performance liquid chromatography (HPLC), was examined by the radio-allergosorbent test (RAST) against the sera of children immediately hypersensitive to milk beta-lactoglobulin and alpha-lactalbumin bound specific IgE in the sera of 63% and 75% of these patients respectively These allergens were tested for cross reactivity with each other by RAST inhibition Both inhibited the binding of IgE, in the sera of allergic patients, to the other protein Two possible determinant peptides, one from beta-lactoglobulin and one from alpha-lactalbumin, were selected by computer prediction of antigenic sites and synthesized by the fluorenylmethoxycarbonyl (FMOC)-polyamide method The peptides were adsorbed to nitrocellulose discs and used in further RAST studies with sera from the allergic children Both peptides bound specific IgE in the RAST assay

Modulation of the immune response by tachykinins.

Abstract: Neuro-immunology is becoming an increasingly important discipline of immunology. This review has examined the immunomodulatory function of one group of neuropeptides, the TK, particularly SP and NKA. These peptides are localized in primary afferent nerves which have been shown to innervate several immune organs. In addition, binding sites for the TK have been demonstrated in thymus, spleen and lymph node. Several immune cell types also express neurokinin receptors including human circulating lymphocytes with binding to the Th/i class predominating, murine T and B cells, a human T lymphoblastoid cell line, human monocytes, rabbit polymorphonuclear leucocytes and guinea-pig macrophages. The apposition of nerves with immune cells and receptors for neuropeptides thus produces an environment for interaction between the nervous and immune systems. Studies in vitro and, more recently, in vivo have examined how the TK regulate immune cell responses. The TK stimulate proliferation of T cells, enhance mitogen-induced release of cytokines including IFN-gamma, TNF-alpha, IL-1 and IL-6 from mononuclear cells and macrophages, enhance immunoglobulin secretion and affect cellular chemotaxis and phagocytosis. Studies in vivo have shown a role for TK in lymphocyte recirculation of sheep lymph nodes, reversal of stress-induced thymic involution and Ig production in both rat and mouse. Many of these effects appear to be mediated via NK-2 type receptors. To date, most of the work has involved studies in vitro, but the results from these are now being validated by studies in vivo where both the immune system and neuropeptides are able to interact at many anatomical sites. The complexities of the immune and the nervous systems mean that only a small number of potential interactions has been examined. Future studies can be expected to amplify these observations, especially with respect to the understanding of inflammatory and immune diseases in humans.

Monoclonal antibodies to Kunjin and Kokobera viruses

Abstract: Five monoclonal antibodies (MoAb) were produced to the envelope (E) protein of Kunjin (KUN) virus. Three of these were specific for the KUN/West Nile complex, and two reacted with epitopes that were common to the flavivirus family. These MoAb defined at least four distinct epitopes on the E protein of KUN virus. Eight MoAb were also produced to unidentified proteins of Kokobera (KOK) virus. Seven were specific for KOK, while the remaining antibody cross-reacted with Stratford virus and an unregistered flavivirus CS946. The application of these MoAb to arboviral surveillance is discussed.

Functional analysis of macrophages, B cells and splenic dendritic cells as antigen-presenting cells in West Nile virus-specific murine T lymphocyte proliferation.

Abstract: In this paper, the relative efficacy of macrophages, B cells and splenic dendritic cells (SDC) in presenting West Nile virus (WNV) antigens to WNV memory CD4+ T cells is examined. The results indicate that, under appropriate conditions, all these cell types can function as antigen-presenting cells (APC). Listeria-induced peritoneal macrophages induced higher proliferative responses than SDC or B cells derived from naive or 14 day WNV-primed mice. The ability of Listeria-induced macrophage populations to present antigen was specifically inhibited by anti-Class II major histocompatibility complex (MHC) antibodies. On a cell population basis, B cells obtained from mice primed with WNV 14 days previously evoked higher responses than resting B cells. B cells from mice receiving weekly injections of WNV over a period of 4 weeks elicited optimal responses with lower doses of antigen than naive or 14 day WNV-primed B cells. When macrophages were used as APC, addition of specific antibodies to WNV resulted in increased efficiency of presentation, probably due to increased uptake of antigen by opsonization. In contrast, addition of anti-WNV antibodies to hyperimmune B cells reduced their efficacy presumably by reducing uptake of antigen by B cell surface immunoglobulin. When SDC from C57BL/6 mice were used as APC, WNV-specific proliferative responses were directly related to the number of stimulator cells used, and the background proliferation with mock antigen was two- to five-fold lower than specific responses. Higher levels of background proliferation were stimulated by SDC from CBA/H mice so that the antigen-specific responses were always less than two-fold higher than background.

CD-13 ('gp150'; aminopeptidase-N): co-expression on endothelial and haemopoietic cells with conservation of functional activity.

Abstract: This report details experimental results which show the presence of enzymic aminopeptidase-N-like activity on endothelial cells, concomitant with cell surface expression previously detected by both ELISA and indirect immunofluorescence. This activity, detected using selected chromogenic substrates in 'functional' assays, is shown to be due (at least in part) to a molecule previously termed 'gp 150' and recognized by MoAb belonging to CD-13, since such antibodies can be shown to inhibit this activity. Activity, as detected on endothelial cells, is similar to that observed on various haemopoietic cells. These assays not only provide a functional basis to cell surface gp150 (CD-13) co-expression on haemopoietic and endothelial cells but have also been used to help define structural epitopes present on aminopeptidase-N/gp150, previously analysed using radiolabelled antibodies in competitive binding assays. Appraisal of these new data suggests that the number of distinct antibody binding sites on this molecule is greater than that previously demonstrated. This study is therefore an important first step in investigating the potential involvement of this selective peptidase molecule in the control of haemopoietic cell growth and differentiation and in haemostatic mechanisms.

Mucosal and systemic antibody formation in the rat after intranasal administration of three different antigens.

Abstract: Mucosal and systemic antibody formation in the rat after intranasal administration of three different antigens

Interleukin-2 and phytohaemagglutinin stimulate the proliferation of tunicate cells.

Abstract: Proliferative responses of cells in tunicate pharyngeal explants to human interleukins and mitogenic lectins were tested. Increased tritiated-thymidine [( 3H]-TdR) uptake was detected among pharyngeal cells incubated with recombinant human interleukin-2 (IL-2), and phytohaemagglutinin-P (PHA-P). Responses to IL-2 were dose-dependent and affected lymphocyte-like cells. Enhanced proliferation was stimulated by IL-2 in the absence of co-stimulants and was not synergized by co-incubation with human interleukin-1 (IL-1) or PHA-P. Anti-IL-2 polyclonal antibody inhibited the stimulatory activity of recombinant human interleukin-2 (rhIL-2). Of three lectins tested (concanavalin-A [Con-A], pokeweed mitogen [PWM] and PHA-P), only PHA-P proved to be mitogenic. Con-A and PWM did not significantly increase proliferative activity even though both lectins were capable of binding pharyngeal cells as revealed by flow cytometry and fluorescence microscopy. Similarly, human IL-1 had no effect on [3H]-TdR uptake either alone or in combination with IL-2 and PHA-P. These data suggest that the functions of some interleukin-like cytokines have been conserved during evolution.

Analysis of T cell receptor Vα and Vβ gene usage in synovia of patients with rheumatoid arthritis

Abstract: Analysis of T cell receptor V α and V β gene usage in synovia of patients with rheumatoid arthritis

Phenotype and activation of milk-derived and peripheral blood lymphocytes from normal and coeliac subjects.

Abstract: The phenotype of milk-derived and peripheral blood lymphocytes from normal and coeliac subjects was assessed for CD3, alpha beta-TcR (T cell receptor), gamma delta-TcR, CD4, CD8, HML-1 (human mucosal lymphocyte) determinants, and activation was measured by interleukin-2 receptor (IL-2R) expression. Milk cells from normal and coeliac subjects were analysed by manual immunofluorescence and milk and blood cells from normal subjects were analysed by flow cytometry. Milk cells from two coeliac subjects were tested for proliferation to gluten antigen. The CD4:CD8 ratio of milk lymphocytes from both normal and coeliac subjects was similar (0.78-1.1), but lower than that present in blood (1.5-2.1). The IL-2R expression of milk lymphocytes from both coeliac and normal subjects was increased by 3-6 times compared with peripheral blood cells. For example, IL-2R was present on 27.3% of milk CD3+ lymphocytes and on 8.0% of blood CD3+ lymphocytes from normal subjects. gamma delta- and HML-1+ T cells were increased 4.2-fold and 12-fold respectively compared with blood lymphocytes. Milk cells from coeliac subjects showed specific proliferation to gluten but not to soya bean antigen. We conclude that milk cells have a 'mucosal' phenotype, with increased gamma delta-TcR, CD8+ and HML-1+ T cells, and have an increased proportion of activated cells. Milk cells from coeliac subjects have no 'toxic' phenotype, but show functional reactivity by specific proliferation to gluten antigen.

Cloning and sequencing of the cDNA for ovine granulocyte-macrophage colony-stimulating factor (GM-CSF).

Abstract: Colony-stimulating factors (CSFs) are not only regulators of haemopoiesis but can also enhance the function of mature myeloid cells, and are therefore potential immune adjuvants. By use of the polymerase chain reaction (PCR) with primers based on the bovine granulocyte-macrophage CSF (GM-CSF) sequence, we have amplified the cDNA for ovine GM-CSF, produced from crude mRNA extracted from alveolar macrophages. The PCR product was cloned into pUC119, and electroporated into Escherichia coli. The complete nucleotide sequence of two clones, and the partial sequence of eight others, was determined. At the nucleotide and amino acid levels, the ovine and bovine GM-CSF sequences are 91% and 81% homologous, respectively.

Murine candidiasis: sex differences in the severity of tissue lesions are not associated with levels of serum C3 and C5.

Abstract: Mice deficient in the fifth component of complement are known to be extremely susceptible to lethal challenge with Candida albicans. However, male mice, that have significantly higher concentrations of serum C5 than females, were markedly more susceptible to infection. This difference was observed in both susceptible (CBA/H) and resistant (BALB/c) mice. Levels of serum C3 likewise showed no correlation with susceptibility.

VLA-2 is a collagen receptor on endothelial cells.

Abstract: In order to identify cell-substrate adhesion receptors on vascular endothelium, murine monoclonal antibodies (MoAb) were raised against human umbilical vein endothelial cells (HUVE). One anti-HUVE MoAb, RMAC11, identified the adhesion receptor VLA-2 as it immunoprecipitated a non-covalently linked heterodimer of 160 kD and 130 kD, which was identical to the heterodimer immunoprecipitated by the anti-VLA-2 MoAb, 12F1 and 5E8. Furthermore, proteolytic peptide maps of the VLA-2 alpha- and beta-chains were highly homologous with those of the RMAC11-recognized molecule. However, unlike other VLA-2 MoAb, RMAC11 also identified an 85 kD band which migrated to 90 kD under reducing conditions. This band was most likely a fragment of the 160 kD alpha-chain as a similar alpha-chain derived fragment has been demonstrated in the immunoprecipitates of some VLA-4 reactive monoclonals. However the possibility that this may be a novel molecule associated with VLA-2 has not been excluded. In vitro assays of HUVE adhesion to collagen types 1 and 4, laminin and fibronectin showed that RMAC11 blocked adhesion to collagen (types 1 and 4) and laminin, but had no effect on HUVE adhesion to fibronectin, confirming that VLA-2 is a collagen and laminin receptor for HUVE.

The induction of plasma leakage in skin by histamine, bradykinin, activated complement, platelet-activating factor and serotonin

Abstract: Vascular leakage of [125I]-human serum albumin into the skin of sheep was measured in response to intradermal injection of histamine, bradykinin, zymosan-activated plasma (ZAP), platelet-activating factor (PAF) and serotonin. Potency of the mediators was PAF greater than ZAP greater than bradykinin approximately histamine greater than serotonin. Maximal leakage occurred in the first 10 min following injection of bradykinin, histamine and PAF, and for histamine and bradykinin had effectively ceased by 40 min. In contrast, ZAP and serotonin induced relatively constant plasma leakage over the first 40 min following their intradermal injection. Prostaglandins E1 and E2 enhanced plasma leakage induced by histamine and bradykinin thus confirming the applicability of the two-mediator hypothesis of vascular leakage to permeability responses in skin of sheep.

Preferential binding of Chlamydia trachomatis to subsets of human lymphocytes and induction of interleukin-6 and interferon-gamma.

Abstract: The interactions between Chlamydia trachomatis and human blood mononuclear leukocytes were studied using flow cytometry, immunofluorescence, electron microscopy and cytokine assays. Under serum-free conditions, elementary bodies (EB) of C. trachomatis were found to bind to human T lymphocytes as well as to B cells and monocytes/macrophages (M phi). For all cell types the binding was saturable, rapid, temperature-independent and independent of the chlamydia-specific serological status of the donor. Similar proportions of T and B cells bound EB at similar levels. In the T cell population, proportionally less CD8+ cells bound EB. Whereas M phi phagocytosed and destroyed the bound micro-organisms for lymphocytes, the Chlamydia remained at the surface, adherent to morphologically featureless membrane areas and showed no evidence of uptake even after long periods at 37 degrees C. Host molecules modulated these basic binding patterns: a heat-stable serum factor inhibited EB binding to T cells and a heat-labile serum factor enhanced binding to B cells. Stimulation with C. trachomatis EB rapidly elicited cytokine production by lymphocytes including interleukin-6 from B cells and interferon-gamma (IFN-gamma) from T and/or nonT/nonB cells. The responses were irrespective of the serological status of the donor. The findings suggest that C. trachomatis-leucocyte interactions may differ from the interactions of other bacteria and human leucocytes. The possible relationship between leucocyte-binding, cytokine induction, and the pathognomonic development of lymphoid follicles during mucosal C. trachomatis infections is discussed.

The nature of immunoglobulin idiotypes and idiotype-anti-idiotype interactions in immunological networks.

Abstract: The nature of immunoglobulin idiotypes and idiotype-anti-idiotype interactions in immunological networks

The 'Burnet era' of immunology: origins and influence.

Abstract: An 'era' is a system of chronology starting from some particular point of time (1). Administratively, that point of time for the 'Burnet era' would be a memorable late afternoon in 1957. It was then that Burnet assembled his entire staff to announce that the virological work of the Walter and Eliza Hall Institute for Medical Research would cease so that he eould devote the remainder of his working career to making some significant contributions to immunology. The year 1957 also fits that 'point of time' since it was then that Burnet first published the clonal selection theory of acquired immunity. No other concept has had a greater influence on contemporary immunology. Scientifically, the beginning of the Burnet era is not so readily identifiable. The science of immunology developed in the 19th century, along with its sister science of bacteriology, now known as microbiology. Landsteiner noted that: 'after recovery from an infectious disease there remains an immunity for that particular disease, a fact which found its first practical application in Jenner's vaccination against small-pox. The search for the explanation of this remarkable phenomenon led to the discovery of a peculiar sort of substance in the blood stream, the so-called antibodies' (2). Burnet's thinking, as a microbiologist and a virologist, would have been influenced by immune responses to infections. Indeed, as noted by Fenner (3), he had employed serologieal techniques relating to typhoid fever from the very time of his entry into the laboratory in 1924; he was an early exponent of serology as a method of baeteriophage classification (1933) and his interest in immunity per se was heightened by his observations of the antibody response to staphyloeoceus toxoid (1932). These immunological interests led in 1941 to his first monograph eoncerning immunology. The Production of Antibodies (4). In that case. should 1940 be hailed as the beginning of the Burnet era? A justification for dating the Burnet era baek to 1940 is his own recollection of the origin of a dominant theme in his thinking and his writing. He dated his interest in self-discrimination in relation to biological processes back to 1937. Thus, in his historical book Walter and Eliza Hall Institute 1915-1965 Burnet recalled the origins of this idea (5). In his words:

Susceptibility of multipotent haemopoietic stem cell deficient W/Wv mice to Plasmodium berghei-infection

Abstract: The susceptibility of haemopoietic stem cell deficient W/Wv mice to infection with Plasmodium berghei was examined. The mean survival time of W/Wv mice after the infection was shorter than that of the +/+ mice. Splenomegaly, a characteristic pathological change of the host after infection with malaria parasites was not observed in W/Wv mice. When haemopoietic activity of the infected mice was examined, a substantial increase in number of multipotent haemopoietic stem cells (CFU-S) and the committed stem cells for granulocytes and macrophages (CFU-GM) or for erythrocytes (CFU-E) was observed in the bone marrow and spleen of +/+ but not of W/Wv mice. CFU-S were not detected in W/Wv mice before or after infection. The number of CFU-GM and CFU-E in bone marrow and spleen of W/Wv mice decreased after infection. Bone marrow grafting from +/+ to W/Wv mice 8 weeks before infection prolonged the mean survival time of the mice and effectively restored the number of CFU-S in the spleen of W/Wv mice. These results indicate that multi-potent haemopoietic stem cells play an important role in the host's defence mechanisms against P. berghei-infection.

Deoxyspergualin inhibits cytotoxic T lymphocytes but not NK or LAK cells.

Abstract: Deoxyspergualin (DOSP) is a new immunosuppressive agent which probably inhibits various functions of monocytes, B cells and T cells. We examined the effects of deoxyspergualin on cellular cytotoxicity, including cytotoxic T lymphocyte (CTL) mediated killer, natural killer (NK) cell and lymphokine activated killer (LAK) cell killing. Deoxyspergualin inhibited cellular cytotoxicity generated by 7 days allo-antigenic challenge; it also inhibited cell killing if added on day 6 of this 7 day culture period. The drug did not significantly inhibit NK or LAK cell killing. The inhibitory effects of deoxyspergualin, however, were dependent on the serum used in the culture medium. Normal human serum (NHS) was associated with less inhibition than fetal calf serum (FCS). Finally, interleukin 2 (IL-2) was able to prevent the inhibitory effects of deoxyspergualin on antigen-specific cytotoxicity.

Effects of Freund's adjuvants on local, draining and circulating lymphocyte populations in sheep.

Abstract: Lymphatic cannulation and serial biopsy were used to examine changes in cell traffic and lymphocyte populations after injection of adjuvant in order to explain the mechanisms of the subversive effect that Freund's adjuvants (FA) have on protective immunity against nematode parasites in sheep. Within 4 days of intradermal injection of complete or incomplete FA into sheep there was a selective depletion of the T19+ TCY gamma delta+ T lymphocyte subpopulation from efferent lymph draining the local lymph node. Transient depletion of CD8+, T19+ and CD5+ cells from jugular blood was evident at day 2. The adjuvant-induced granuloma was a connective tissue/macrophage/T lymphocyte phenomenon, with no B cell follicles, and the T cell content continued to increase for the 21 days. The early granuloma (days 2-4) contained a disproportionate number of T19+ gamma delta + lymphocytes. It appeared that the adjuvants induced selective sequestration of this T cell subset during the initial period of granuloma formation.

Active immunization with EPF suppresses the formation of immune ascites in BALB/c mice.

Abstract: The production of early pregnancy factor (EPF) is not confined to pregnancy--EPF has been detected as a product of tumour and transformed cells in vivo and in vitro. In this study, EPF (or an EPF-like substance) was detected also in the serum of BALB/c mice, 7 days after intraperitoneal (i.p.) administration of the mineral oil pristane. Furthermore, EPF was present in serum from mineral oil-induced plasmacytoma-susceptible mice throughout the latent period potentially leading to tumour development, peaking around the time neoplastic cells were identified in ascites. Since mineral oil-induced granuloma is essential to development of immune ascites, involvement of EPF in this process was investigated. Active immunization of BALB/c mice with EPF was shown to suppress the production of immune ascites, induced by multiple i.p. injections of antigen in complete Freund's adjuvant. Of the mice immunized with EPF (n = 19), only 47% produced ascites, compared with 94% of mice receiving saline or human chorionic gonadotrophin and 100% of mice receiving keyhole limpet haemocyanin. Further investigations revealed that ascites was only produced in mice that maintained free-circulating EPF. These mice displayed the classic mineral oil-induced granuloma covering the tissues of the peritoneum. In contrast, the serum of mice that did not produce ascites tested negative for EPF and the peritonea of these mice were devoid of the oil granuloma. These studies suggest that EPF is involved in the initiation and maintenance of the inflammatory response of the peritoneum to mineral oil.

The effect of intrathymic injection of donor blood on the graft versus host reaction and cardiac allograft survival in the rat.

Abstract: Our previous paper reported that it was difficult to induce tolerance using donor specific transfusion (DST) in Lewis rats, while in DA and PVG rats it was possible to induce tolerance using DST In this paper we investigated the effects of DST in a local graft versus host reaction (GVHR) model using these rat strains The following results were obtained: (i) DST suppressed the GVHR in the DA rat but not in the Lewis rat; (ii) DST did not suppress GVHR in PVG rat strain, but lymphocytes, prepared from the PVG rats that were tolerant of Lewis heart grafts induced by DST, could suppress the the GVHR; and (iii) in Lewis rats, intrathymic injection of whole blood suppressed the GVHR, suggesting this route of antigen delivery might be effective in prolonging allograft survival Cardiac allograft survival in this strain was prolonged in some cases after pretreatment with intrathymic donor whole blood These results suggest the value of the GVHR in the assessment of new protocols to induce allograft tolerance in rats

Effect of high ligand concentration on West Nile virus-specific T cell proliferation.

Abstract: In this paper the phenomenon of suppression of proliferation in vitro of 14 day primed, West Nile Virus (WNV)-specific, murine CD4+ T cells by large numbers of antigen-presenting macrophages and B cells has been investigated. Suppression was apparently not mediated by prostaglandins, as the use of indomethacin in cultures at four times the usual concentration did not reverse suppression. Experiments were designed to evaluate the contribution of major histocompatibility complex (MHC) Class II and nominal WNV antigens in causing suppression of T cell proliferation. Listeria- or thioglycollate-induced macrophages from CBA/H (H-2k) mice, when treated with heat-killed Listeria in vitro for 1 h to maintain or increase, respectively, MHC Class II levels before the addition of alloreactive Iak-specific T cells caused inverse dose-responses; the highest T cell proliferation occurred at a stimulator to responder (S : R) ratio of 0.25 and profound suppression at a S : R ratio of 1 or 2. In contrast, untreated thioglycollate-induced macrophages, which express low MHC Class II levels, gave a direct dose-response with increasing T cell proliferation as antigen-presenting cell (APC) numbers increased. Addition of anti-Ia antibodies (or their Fab fragments) to cultures caused a significant reversal of suppression of anti-WNV T cells imposed by high numbers of Listeria-induced macrophages or 14 day WNV-primed B cell APC. Suppression was also reversed by reducing the concentration of WNV antigen. These observations support the notion that the suppression of T cell proliferation observed at high S : R ratios was due to high concentrations of ligand (WNV-derived peptide complexed with Class II MHC) on APC.

Intercellular adhesion molecule (ICAM-1) inhibition can induce tolerance in vivo

Abstract: Antigen specific unresponsiveness can be induced in vivo by administration of antibody against CD4, major histocompatibility complex (MHC) Class II or LFA-1 at the time of antigen exposure. Since target cell depletion is not necessary for this effect it was hypothesized that interference with intercellular interaction was responsible. To test this hypothesis, antibody against the ligand for LFA-1, ICAM-1, was administered during primary immunization with human gamma-globulin (HGG) and long-term secondary antibody responses monitored. It was found that HGG specific unresponsiveness resulted from this treatment (less than 10% of normal secondary antibody response). Delayed-type hypersensitivity responses to HGG were also suppressed suggesting unresponsiveness in some T cell subsets. These findings suggest a role for ICAM-1/LFA-1 interaction in determining the long-term outcome of contact with antigen.

Low affinity binding of mouse immunoglobulin to human CD5+ B cells.

Abstract: Polyreactive immunoglobulin (Ig) secreted by CD5-bearing B cells has the capacity to bind a broad range of self and foreign antigens. Flow cytometric analysis was used to detect low-affinity binding of mouse Ig molecules to the surface of CD5-bearing B cells from patients with chronic lymphocytic leukaemia (CLL). Mouse Ig isotypes G, A, and M, and IgG subclasses G1, G2a, and G3 bound to the B cell surface via a mechanism not involving the antigen-binding site of the mouse Ig molecule. Fab and F(ab')2 fragments of mouse Ig associated with the CLL cells in a similar manner to intact Ig, indicating that Fc receptor interactions were not involved. Dissociation of the mouse Ig from the B cell surface by three washing steps distinguished this lower-affinity binding from high-affinity binding which occurs through the antigen-binding site of a mouse monoclonal antibody (MoAb) when it recognizes a specific cell surface epitope. Blocking studies suggest that the low-affinity binding occurred via surface IgM and surface IgD (sIgM/sIgD) on the CD5-bearing B cells. The results are consistent with the expression of polyreactive Ig on the surface of CD5-bearing B cells in CLL.

In vitro activation of rGM-CSF derived bone marrow macrophages by cisplatin and lipopolysaccharide

Abstract: Treatment of fresh non-adherent bone marrow cells (NABMC) with cisplatin or lipopolysaccharide (LPS) did not render them tumoricidal. NABMC incubated in medium alone or medium containing recombinant granulocyte macrophage colony stimulating factor (rGM-CSF) for 4 days matured to macrophages that were positive for non-specific esterase staining. Bone marrow-derived macrophages cultured with medium alone did not respond to cisplatin or LPS by the induction of tumoricidal activity, whereas rGM-CSF derived bone macrophages showed significantly enhanced cytotoxicity after treatment with cisplatin or LPS. Culturing of NABMC with rGM-CSF enhanced cell survival compared to the cells incubated in medium alone. These results suggest that bone marrow cells not only mature in rGM-CSF, but are also primed by rGM-CSF for induction of tumoricidal activity.

Temporal variations in the fine specificity of IgM anti-fluorescyl antibodies.

Abstract: This study compares the fine specificities of the primary and secondary fluorescein (FITC)-specific immunoglobulin M (IgM) repertoires in BALB/c mouse serum and monoclonal antibodies (MoAb) and has found reproducible, immunization-dependent differences. FITC and four of its homologues; iodoacetamido fluorescein (IAF), dichlorotriazinyl aminofluorescein (DTAF), substituted rhodamine isothiocyanate (XRITC) and tetramethyl rhodamine isothiocyanate (TRITC), each conjugated to bovine serum albumin (BSA), were used to determine reactivity patterns of serum IgM from mice immunized once or twice with FITC-haemocyanin (FITC-Hy). Reactivity patterns were also obtained for 20 IgM MoAb, eight of which were produced by fusions of SP2/0 myeloma cells with splenocytes from mice immunized once (primary) and 12 from mice immunized twice (secondary) with FITC-Hy. Each MoAb exhibited a unique fine specificity pattern, evidence of extensive heterogeneity in the FITC-specific repertoire. Reactivities of IgM MoAb with certain homologues were found to be more characteristic of either the primary or secondary response. Polyclonal serum IgM also showed reproducible immunization-dependent variations in fine specificity. Such a pattern could result from idiotypic suppression of primary antibodies, from the expansion of subsets of IgM memory cells utilizing novel genes and/or from somatic mutation absent in primary IgM antibodies.

T lymphocyte participation in acute serum sickness glomerulonephritis in rabbits.

Abstract: The extent and timing of glomerular T lymphocyte infiltration was studied in acute serum sickness (AcSS) glomerulonephritis (GN) in rabbits. AcSS was initiated by a single intravenous injection of bovine serum albumin (BSA). Rabbits developed circulating BSA anti-BSA immune complexes, and rapid immune elimination of the circulating antigen was associated with the deposition of immune complexes in the kidney and the onset of a diffuse endocapillary proliferative GN. On the day of immune elimination (defined as when less than 1% of the injected antigen remained in the circulation), rabbits developed significant proteinuria (98 +/- 36 mg/24 h; normal 14 +/- 1 mg/24 h, P less than 0.01), glomerular macrophage accumulation (44.3 +/- 21.1 macrophages per glomerulus [mac/glom]; normal 0.28 +/- 0.18 mac/glom, P less than 0.01), and a significant glomerular T lymphocyte influx (3.0 +/- 0.9 cells/glomerular cross-section [c/gcs]; normal 0.47 +/- 0.13 c/gcs; P less than 0.005). On the day prior to immune elimination, increased T cells numbers were observed in some rabbits (2.4 +/- 2.1 c/gcs) together with a minor macrophage presence (7.6 +/- 3.6 mac/glom) and minimal proteinuria (17.6 +/- 3 mg/24 h). These studies demonstrate the influx of T lymphocytes together with macrophages at the onset of proteinuria in serum sickness nephritis and are consistent with a role for cell-mediated immunity in the pathogenesis of this lesion.