Specific murine B-cell activation by synthetic single-and double-stranded polynucleotides.

The synthetic single- and double-stranded polynucleotides, poly I, poly C, and poly I.C, were shown to induce thymidine incorporation in six inbred strains of murine spleen cells. This stimulation was shown to be secondary to B-cell activation and not due to contamination of the polynucleotides with bacterial lipopolysaccharide (LPS). The ability of poly I.C to act as a B-cell mitogen, in addition to its behavior as a thymic-independent antigen, suggested that these two phenomena may be related. The similarity of the molecular structure of poly I.C to LPS, a material which also acts as a thymic-independent antigen and a B-cell mitogen, supports the hypothesis that the polyvalent nature of these materials accounts for their functional interaction with murine B cells.

B-lymphocyte heterogeneity: development and characterization of an alloantiserum which distinguishes B-lymphocyte differentiation alloantigens.

CBA/N mice and F1 male mice, which are hemizygous for the CBA/N X chromosome, have an immune defect which is associated with the absence (deficiency) of a subpopulation of mature or late developing B lymphocytes. This characteristic was utilized to develop an antiserum that was specific for this subclass of B cells. C57BL/L mice were immunized with DBA/2 spleen calls, and the resulting antisera was absorbed with lymphoid cells derived from immunologically abnormal (CBA/N female X DBA/2 male)F1 male mice. The absorbed antisera was cytotoxic for a subpopulation of lymphocytes that was present in the spleens of adult DBA/2 and (CBA/N female X DBA/2 male)F1 female mice. The cells killed by the absorbed antisera were Ig-bearing, complement receptor-bearing B lymphocytes, which had a low-to-intermediate density of total surface Ig. Moreover, the cells remaining after treatment of adult (CBA/N female X DBA/2 male)F1 female spleen cells with the absorbed antisera and C had a high ratio of surface IgM to IgD. The development of this cytotoxic alloantisera, which is specific for a late developing (mature) subpopulation of B lymphocytes, will allow the functional characterization of subclasses of B lymphocytes.

Lymphocyte activation in subacute sclerosing panencephalitis virus and cytomegalovirus infections. In vitro stimulation in response to viral-infected cell lines.

Efforts to stimulate lymphocytes from measles seropositive and two patients with subacute sclerosing panencephalitis (SSPE) with either commercially available measles virus or virus isolated from a known case of SSPE failed to show any significant data using a microculture assay. Similar results were obtained using lymphocytes from two patients with active cytomegalovirus (CMV) infections and CMV seropositive individuals using CMV suspensions. On the other hand, lymphocytes from the patients with subacute sclerosing panencephalitis exhibited in vitro blastogenesis in culture with SSPE virus-infected HeLa cells. Similarly, lymphocytes from the CMV-infected patients demonstrated blastogenesis when cocultivated with CMV-infected WI-38 cells. This affords a new method for determining the cell-mediated immune capacity of patients with "slow" virus diseases.

Demonstration of a blocking factor in the plasma and spinal fluid of patients with subacute sclerosing panencephalitis. I. Partial characterization.

Conflicting reports on the immune responsiveness of patients with subacute sclerosing panencephalitis (SSPE) have been reported. This report shows that the leucocytes from four SSPE patients exhibited strong sensitivity to both measles and SSPE virus preparations as measured by the macrophage migration inhibition test, mixed lymphocyte virus infected cell culture test, and the lymphotoxin assay. Earlier suggestions that a factor may be operating to suppress cellular reactivity are confirmed by the demonstration that the response of lymphocytes from SSPE patients could be blocked by the addition of SSPE spinal fluid or plasma. It was determined that the blocking factor was stable at -20 degrees C, heat labile at 56 degrees C for 30 minutes, trypsin and neuraminadase sensitive, and had a mol wt greater than 150,000 as determined by Sephadex G-200 gel chromatography. The blocking factor appeared to be specific for SSPE virus and did not block the response of lymphocytes to nonspecific mitogenic agents and other viral and bacterial agents.

Mixed leukocyte stimulation of normal peripheral leukocytes by autologous lymphoblastoid cells.

Lymphocyte culture lines can be established by using buffy coat cells from normal or diseased individuals. Established lymphocytoblasts from a normal donor were used in the one-way mixed leukocyte culture test and were found to stimulate the peripheral lymphocytes of that donor. These lymphocytoblasts were also capable of stimulating allogeneic lymphocyte populations.

Theta-sensitive cell and erythropoiesis: identification of a defect in W/Wv anemic mice.

Nonirradiated mice of the W/WV genotype were injected with normal (+/+) bone marrow cells that had been treated with antiserum to Thy 1.2 and complement (C'). Such bone marrow cells had no effect on the number of macroscopic colonies formed in the spleens of these mice, but did not cure the anemia. The addition of +/+ thymocytes to these bone marrow cells restored their ability to cure the anemia in W/WV mice. These data suggest that a theat-sensitive cell is required in the promotion of differentiation of murine hematopoietic stem cells into erythrocytes, and that there is a deficiency of such a cell in the W/WV mouse.

Human T-cell heterogeneity as delineated with a specific human thymus lymphocyte antiserum. In vitro effects on mitogen response mixed leukocyte culture, cell-mediated lymphocytotoxicity, and lymphokine production.

Human peripheral blood lymphocytes (PBL) were evaluated by their responses to phytohemmagglutinin (PHA-P), concanavallin A (con-A), and pokeweed mitogen (PWM), both before and after treatment with an antiserum against human thymic lymphocyte antigens (HTLA) that had been made T-cell-specific by multiple absorptions with immunoglobulin EAC-positive lymphoblast cell lines (B cells). Cells treated with HTLA were examined for their ability to react in a mixed lymphocyte culture (MLC) and to form killer cells in a cell-mediated lymphocytotoxicity (CML) system. Sensitized cells were also examined for their ability to respond to purified protein derivative (PPD) by blastogenesis, migration inhibitory factor release (MIP), and lymphotoxin (LT) production, both before and after treatment with HTLA and complement. The HTLA was in itself highly stimulatory to PBL. However, with the addition of complement and subsequent cell destruction, a marked decrease in its stimulatory response was noted. PBL treated with HTLA and complement exhibited marked inhibition of responsiveness to con-A with little decrease in PHA-P -OR PWM stimulation except at very high concentration of HTLA. MLC reaction was inhibited only when responder cells were treated with HTLA + C'. Treatment of stimulator cells with HTLA + C' did not significantly alter the MLC response. The HTLA + C'-treated cells failed to form killer cells in the CML reaction and inhibited PPD-induced blasto-genesis from PPD-sensitized individuals; however, treatment of sensitized cells with HTLA + C' had little effects on the release of MIF and LT. It is suggested that subpopulations of T-cells carry surface antigens that bind with this specific antisera, and that the con-A-responsive cells, the responder cells in the MLC, and killer T-cells comprise a separate subset from cells responding to PHA-P or PWM, OR THE MIF-and LT-producing cells.

Distribution of Lyb-4.1 and other membrane antigens on murine lymphoid tumors.

The distribution of membrane antigens on 6 DBA/2-derived tumors (L1210, L5178Y, P815, ABLS 11, ABLS 12, and ABLS 13) was studied by direct cytotoxicity and quantitative absorption assays. Lyb-4.1 antigen was found solely on the L1210 tumor. Iad antigens were absent from all tumors, and H-2Kd and H-2Dd antigens were present on all tumors. Immunoglobulin was adsorbed to the ascites tumors and lost after 3 days or more in tissue culture. These studies were performed to characterize the distribution of DBA/2 membrane antigens on DBA/2-derived tumors as a base line for functional and chemical studies with these tumors and with their solubilized proteins.

Protein transfer of glycosyl-phosphatidylinositol-B7-1 into tumor cell membranes: a novel approach to tumor immunotherapy.

Modification of tumor cells with one or more costimulatory adhesion molecules has been proposed as a means to develop therapeutic cancer vaccines for use in human immunotherapy. Expression of B7-1 (CD80) in tumors by gene transfer creates an immunogenic tumor cell that induces antitumor immunity and protects mice from further challenge with wild-type tumor cells. In this report, we demonstrate that protein transfer of glycosyl-phosphatidylinositol (GPI)-anchored costimulatory molecules into tumor cell membranes could be used as an alternative to gene transfer for tumor immunotherapy. Incubation of isolated tumor membranes with purified GPI-anchored B7-1 results in stable incorporation of B7-1 on tumor cell membranes within a few hours. Immunization of C57BL/6 mice with EG7 tumor membranes modified to express GPI-B7-1 by protein transfer induces tumor-specific T-cell proliferation and CTLs. In addition, immunization with these EG7 membranes protects mice from parental tumor challenge. The protein transfer approach used here does not require foreign vectors or live tumor cells and is completed within a matter of hours. Irradiated cells or membrane preparations from fresh or frozen tumor tissue can be used. Therefore, protein transfer of glycolipid-anchored molecules provides an efficient and novel approach to modify tumor membranes for human immunotherapy. This approach is not limited to costimulatory molecules because any cell surface protein can be converted to a GPI-anchored form by recombinant techniques.

Induction of major histocompatibility complex antigens within the myocardium of patients with active myocarditis: a nonhistologic marker of myocarditis.

The histologic diagnosis of active myocarditis is frequently difficult to establish. A nonhistologic marker of immune activation would be clinically useful in identifying cases of immune-mediated myocarditis. A viral etiology with subsequent autoimmunity to cardiac antigens has been implicated in human myocarditis. Because autoimmunity and viral disease are commonly associated with increased expression of major histocompatibility complex (MHC) antigens on targeted tissue, we examined endomyocardial biopsy samples from patients with active myocarditis for abnormal levels of MHC antigen expression. Thirteen patients with active myocarditis and eight control patients with other well-defined cardiac diagnoses (coronary disease, amyloidosis or neoplasm) were studied. A sensitive radioimmunoassay was developed that utilized monoclonal antibodies to human MHC class I and class II antigens in order to quantitate the expression of both of these antigens within each biopsy. Abnormal MHC class I and class II antigen expression was present in 11 of 13 myocarditis specimens and 1 of 8 control samples (specificity 88%, sensitivity 84.6%). Active myocarditis samples had approximately a 10-fold increase in MHC class I and class II expression. Immunoperoxidase staining localized abnormal MHC expression primarily within microvascular endothelium and along myocyte surfaces (11 of 13). This study is the first to demonstrate a marked increase in major histocompatibility complex antigen expression within the myocardium of patients with active myocarditis. The identification of abnormal histocompatibility antigen expression within an endomyocardial biopsy may prove a useful adjunct to the histologic diagnosis of myocarditis.

Tritiated thymidine incorporation and cell-mediated lympholysis as correlates of acute graft-versus-host reaction.

The graft-versus-host (GVH) reaction remains a serious consequence of administration of allogeneic immunocompetent cells to an immunosuppressed host even if donors and recipients are matched for major histocompatibility loci. This report describes a murine model for acute GVH reactions. Spleen cells from C3H/He (H-2k) mice, after intravenous injection of BALB/c (H-2d) spleen cells, were specifically cytotoxic for C3H target cells in vitro 4 days after irradiation and reconstitution. The cells in the recipients apparently are of donor genotype. The spleen cells exhibited rapid proliferation in vitro as measured by the uptake of 3H-TdR. The in vitro proliferation was distinguished from erythropoiesis by an assay of 59Fe incorporation. The kinetics of the in vitro incorporation of 3H-TdR and the in vivo uptake of 59Fe are reported.

CSF-1 deficiency in the op/op mouse has differential effects on macrophage populations and differentiation stages.

Osteopetrosis and the absence of colony-stimulating factor 1 (CSF-1) in op/op mice are associated with decreased cellularity of the bone marrow (to one tenth of the normal), a very significant reduction in the number of cells recovered from peritoneal, pleural, and alveolar lavages, moderate leukopenia, and a slight decrease in the number of cells per spleen and thymus. Furthermore, op/op mice possess deficiencies in the number of macrophages in various organs. These cells are apparently absent in the bone marrow, severely reduced (5%-15% of the normal number) in peritoneal and pleural cavities and in the lungs. In addition, a marked decrease in the frequency and total number of circulating monocytes is present (5% of the normal). The deficiency of macrophages is less severe in the liver, spleen, and thymus of op/op mice (approximately 30% of those seen in normal). There is a concomitant redistribution of macrophage progenitor cells (granulocyte-macrophage colony-forming units, CFU-GM) in op/op mice from the marrow to the spleen and liver, associated with an increased sensitivity to interleukin 3 (IL-3). Their total number is decreased at least threefold compared to control mice. Moreover, op/op mice have at least a fivefold reduction in the total number of day-11 spleen colony-forming units (CFU-S) associated with their redistribution to the spleen and liver. These data suggest that the macrophage system in op/op mice is reduced at all levels tested, that is, at the level of mature macrophages, the level of progenitors, and the level of stem cells, whereas the redistribution of progenitor and stem cells could be viewed as a secondary consequence of osteopetrosis. Furthermore, these data suggest that macrophage dependency in vivo on CSF-1 is limited and different in various organs. Particularly in the liver, spleen, and thymus, other growth factors may significantly compensate for CSF-1 deficiency. Based on the relative decrease in the number of CFU-GM in the op/op mice, it appears that the population size of these progenitors is less dependent on CSF-1 than the hematopoietic stem cell population size as evidenced by the day-11 CFU-S assay. The day-11 CFU-S population is severely reduced in op/op mice, suggesting a physiological involvement of CSF-1 in expanding its size. These data provide evidence that CSF-1, besides acting on the final and intermediate stages of macrophage maturation, may also play a role in early stages of hematopoiesis.

Engraftment of bone marrow transplants in W anemic mice measured by electronic determination of the red blood cell size profile.

Defective stem cells of WBB6F1-W/Wv mice produce macrocytic red blood cells (RBCs); stem cells of WBB6F1-+/+ mice produce normocytic RBCs. Utilization of the Coulter counter channelyzer permitted good dissociation between the size distribution of populations of +/+ and W/Wv RBCs. Peaks (mean cell volumes) for +/+ and W/Wv RBCs have been determined to be between the 30th and 40th channel and 50th and 60th channel, respectively. Variability of profiles for individual mice of both genotypes did not exceed the variability of separate determinations of the same cell suspension from a single mouse. Admixture (approximately 15%) of either type of erythrocytes could be quantitatively detected by this method. One week after transplant of 10(7) +/+ marrow cells into W/Wv recipients, 25% of donor type erythrocytes were detected. Eighteen days post-graft, concentration of +/- normocytes exceeded the concentration of macrocytes in the W/Wv recipients' circulation. Approximately 45 days post-transplant, the proportion of macrocytes decreased below the 10% detectable level. Calculation of the daily RBC production rate during repopulation and estimation of the number of RBCs produced by a single hematopoietic colony were determined. The RBC size profile was found to be a convenient method for studying the effect of implantation of W/Wv marrow into lethally irradiated +/+ mice. This method proved suitable for repetitive determination of the size population in individual transplanted mice.

Correction by CSF-1 of defects in the osteopetrotic op/op mouse suggests local, developmental, and humoral requirements for this growth factor.

Mice that are mutant at the op locus have a severe deficiency of mononuclear phagocytes due to an inactivating mutation in the CSF-1 (macrophage colony-stimulating factor, M-CSF) gene. op/op mice are toothless, possessing skeletal abnormalities, a low body weight, and compromised fertility; they are osteopetrotic due to a deficiency of osteoclasts. The congenital osteopetrosis, toothless phenotype, osteoclast deficit, and the defects in splenic and femoral macrophages were corrected by routes of administration of human recombinant CSF-1 that maintained normal circulating CSF-1 concentrations. Early restoration of circulating CSF-1 was required for rescue of the toothless phenotype, but only partially restored body weight. In contrast, the deficiencies of pleural and peritoneal cavity macrophages and the reduced female fertility were not corrected by restoration of circulating CSF-1. These results suggest that although circulating CSF-1 is required for osteoclast and macrophage production, local synthesis and action of the growth factor are important for certain target cell populations.

Requirements for simian immunodeficiency virus antigen-specific in vitro proliferation of T cells from infected rhesus macaques and sooty mangabeys.

The measurement of cell-mediated immunity against the etiologic agent of human AIDS (HIV) in the non-human primate model of AIDS (simian immunodeficiency virus, SIV) has been difficult. In general, culture of peripheral blood mononuclear cells from HIV-1- and SIV-infected humans and monkeys, respectively, with purified inactivated HIV and SIV virus preparations has given inconsistent or negative proliferative responses. However, we describe herein an assay which consists of coculturing monocytes that have been pulsed with inactivated SIVsmm with nylon-wool-purified autologous T cells, leading to antigen-specific T-cell proliferation. The proliferative response, which predominantly occurs in CD4+ T cells, is major histocompatibility complex (MHC) class II-restricted and requires antigen processing. This assay will greatly facilitate the identification of the immunodominant epitopes recognized by T cells in sooty mangabeys, which are naturally infected but remain clinically asymptomatic, and in rhesus macaques, in which experimental infection leads to clinical symptomatology similar to human AIDS, eventually resulting in death.

Evidence for simian immunodeficiency virus-specific IgM and IgG response in peripheral blood mononuclear cells of serum enzyme-linked immunosorbent assay-negative nonhuman primates.

In vitro polyclonal activation of peripheral blood mononuclear cells (PBMCs) from 70% of the simian immunodeficiency virus (SIV) serum enzyme-linked, immunosorbent assay (ELISA)-negative sooty mangabeys leads to synthesis and release of low but significant and reproducible levels of SIV-reactive antibodies, as determined by ELISA and Western blot analysis. The predominant isotype of SIV-reactive antibodies in the pokeweed mitogen (PWM) supernatant fluids from serum ELISA-negative mangabeys is IgM, whereas the predominant isotype of SIV-reactive antibodies in seropositive mangabeys is IgG. Depletion of CD8+ cells led to a marked increase in the levels of SIV-reactive antibodies detected in supernatant fluids from PWM-induced cultures from the serum ELISA-negative mangabeys. No evidence for such SIV-reactive antibodies has been found, to date, in similar unfractionated or CD8+ T-cell-depleted PWM-induced PBMC cultures from uninfected macaques. Supernatant fluids from PWM cultures of PBMCs from a select group of serum ELISA-negative mangabeys, when concentrated five times, were shown to give a Western blot profile against SIV, similar to the profile seen with plasma from seropositive infected macaques and mangabeys. Evidence is presented to show that these serum ELISA-negative mangabeys are most likely latently infected with SIV. This evidence, which was obtained in samples from such ELISA-negative mangabeys, includes the detection of reverse transcriptase activity and the presence of SIV p27 in supernatant fluids of phytohemagglutinin-stimulated PBMCs in vitro. In addition, the data show the presence of CD8+ T cells that regulate SIV-specific Ig synthesis and show the detection of gag sequences by the polymerase chain reaction. Thus, the PWM assay described herein may provide a valuable additional tool for detection of lentivirus infection before or in the absence of seroconversion.

Characteristics of the 'active rosette test'. I. Technical considerations of the test and comments.

Using normal subjects, it was found that the 'Active Rosette Test' is both reproducible and consistent if performed exactly as described by Wybran et al. (1972). Variations in incubation time, centrifugal force while sedimenting rosettes, resuspension time after centrifugation, SRBC lymphocyte ratio, and increasing concentrations of fetal calf serum all produced marked deviations in test results. Rosette inhibition studies using a specific anti-T cell antisera showed inhibition of 'active' and total rosettes at the same level, suggesting that there are no marked variations in surface T antigens among respective T cells. Unless differences in functional activities can be demonstrated between the 'active' rosette-forming cells and the other T cells, the test will be of an empiric nature.

Detection of helper and suppressor T cell lines to soluble antigens using a modified ELISA system.

Helper and suppressor activity of T cell lines were investigated using an assay system which is based on measurement by the enzyme-linked immunosorbent assay (ELISA) of the antibody produced in vitro. T cell lines were established from spleens of BALB/c mice immunized with ovalbumin and human serum albumin. The T cells were expanded in culture with irradiated spleen cells and antigen or concanavalin A supernatant. The culture system used for assay of T cell activities contained antigen (Ag)-primed unfractionated spleen cells or Ag-primed B cells. Ag-primed cells and cultured T cells were incubated together with Ag (0.05 ng/ml-100 micrograms/ml) for various times, washed at varying intervals, and supernatants assayed for specific antibody activity by an ELISA adapted for this purpose. A total incubation time of 9 days was required for significant antibody production. Complete reconstitution of the antibody response was observed with Ag-primed B cells and Ag-primed T cells were combined. In one experiment, a helper cell line was shown to restore specific antibody production to approximately 50% of the normal response while a suppressor cell line suppressed antibody production by 90%. A linear response of between 0.2 and 1.4 OD units was observed in the ELISA allowing easy detection of help or suppression. As little as 80 ng of specific antibody could be detected. This technique allows quantitative determination of antibody production for a large number of individual microcultures.

Purification and optimization of functional reconstitution on the surface of leukemic cell lines of GPI-anchored Fc gamma receptor III.

Purified glycosyl phosphatidyl inositol (GPI)-anchored cell surface proteins can be reincorporated spontaneously into the cell membrane by incubating the cells with these proteins. This unique property provides a novel way of introducing cell surface receptors on live cell membranes without the use of gene transfection. Since any classical transmembrane cell surface protein can be converted to a GPI anchored protein by recombinant techniques, this method provides a means of studying ectodomain associated receptor functions on various cell types. Moreover, in some circumstances, it can be used to correct deficient cellular functions resulting from lack of cell surface protein expression. Using GPI-anchored Fc gamma receptor III (CD16B), a low affinity Fc gamma receptor, we have systematically studied the optimal conditions for reconstitution of a functional receptor on nucleated cells. CD16B is purified to homogeneity from neutrophil lysates by single step immunoaffinity chromatography. The purified CD16B is functionally active as evidenced by its ability to bind IgG opsonized erythrocytes. CD16B incorporation on nucleated cells is temperature dependent with an optimum of 37 degrees C. The level of expression of incorporated CD16B is also depend on the concentration of CD16B available and the duration of incubation. The incorporated CD16B retains its ability to bind ligand and also mediates endocytosis of the bound ligand. In summary, our results demonstrate that purified, functionally active GPI-anchored receptors can be expressed on desired cells in a controlled manner and retain some functional properties.

Flow-cytometric detection of circulating immune complexes.

In an effort to develop an assay which would rapidly detect and analyze circulating immune complexes, we have adapted the Raji cell radioimmunoassay (RIA) to a flow cytometric (FCM) analysis. The advantages of immune complex analysis by FCM are many. Foremost is the effectiveness and efficiency of the FCM method relative to the radioimmunoassay (RIA) method. The data demonstrated that FCM detection is three times more sensitive than RIA detection. Only populations of viable Raji cells bearing immune complexes are analyzed because parameters of the FCM analysis permitted the elimination (gating out) of dead cells. The determinations are rapid and the data are immediately available for several additional analyses. Because of the availability of many fluorescent monoclonal antibodies to complement components, viral antigens, light chains and immunoglobulin isotypes, it is possible to detect many components that might be present in the Raji cell bound complexes. Finally, the Raji cells can be characterized in different stages of their cell cycle to generate information about the state of the cells and the density of the receptors involved in binding the complexes.