Characterization of an antiserum to guinea pig antithrombin III (AT III). I. Reactivity with guinea pig T lymphocytes

In the course of studies investigating the effects of antisera prepared against a variety of guinea pig proteins on lymphocyte function, a goat antiserum prepared against a guinea pig gamma-globulin preparation was found to react with guinea pig T lymphocytes. This antiserum, serum 592, contained a significant titer of antibodies that were cytotoxic for a subpopulation of lymph node cells and thymocytes, and mitogenic for lymph node T cells. Immunoelectrophoretic analysis and selective absorptions of the antiserum demonstrated that the antigen recognized on thymocytes was also present on an alpha 2 globulin in guinea pig plasma, which, on the basis of physiochemical characteristics and heparin-binding affinity, appeared to be guinea pig antithrombin III (AT III). Although the antiserum was shown to contain antibodies to both protein and carbohydrate determinants on the AT III molecule, studies comparing the effects of 7 M guanidine and periodate oxidation on the antigenicity of the AT III determinant also recognized on the thymocytes indicated that this shared antigenic determinant was carbohydrate in nature. The thymocyte membrane molecule bearing this determinant was also isolated and was found to be a 210,000-dalton macromolecule that was very sensitive to proteolytic and/or autolytic degradation. These data raise the interesting possibility that guinea pig lymphoid cells may have a membrane-associated protease inhibitor related to plasma AT III.     

T and B lymphocytes and serum alpha 1-antitrypsin activity in children with bronchial asthma treated with broncho-vaxom

The aim of this study was to evaluate an effect of Broncho-Vaxom treatment on T and B lymphocytes and serum alpha 1AT in children treated at "Zuch" sanatorium in Szczawno-spa. The trial involved 46 school aged children suffering from infectious or infectious-atopic asthma. The post-Broncho-Vaxom treatment values for T lymphocytes were significantly higher in infectious, and significantly lower for B lymphocytes in infectious-atopic asthma. Serum alpha 1AT activity in children suffering from infectious asthma decreased significantly after the treatment. A correlation between the efficacy of the treatment and the lymphocyte percentage was observed. In children with very effective clinical results of Broncho-Vaxom treatment, the significant increase in T lymphocyte, and decrease in B lymphocyte populations was observed. Changes in T and B lymphocyte percentage were analysed in respect to alpha 1AT pre-treatment activity. In children with high alpha 1AT value, T lymphocytes after the treatment increased significantly in infectious, and B lymphocytes decreased significantly in infectious-atopic group.     

Specific triggering by concanavalin A of a secondary T killer cell-mediated anti-tumor immune response.

Spleen cells from C57 B1/6 mice which rejected a Moloney sarcoma virus (MSV)-induced tumor elicited a strong cytolytic reaction against MSV-associated antigens when incubated for 7 days with a mitogenic dose (5 μg/ml) of concanavalin A (Con A). The cytolytic activity evaluated in a chromium release test was shown to be mediated by T lymphocytes, by anti-Thy 1–2 serum treatment, and independent of remaining Con A, by treatment with d-[α-methyl]mannopyranoside. A similar reactivity was obtained with phytohemagglutinin (PHA) at mitogenic doses but not with the B-cell mitogen lipopolysaccharide (LPS). This “secondary-like” response was regenerated up to 50 days post-MSV inoculation but decreased regularly. The cytolytic activity had an antigenic pattern indistinguishable from that of the relevant tumor cell reactivation and was directed by the same H-2 restriction.     

Immunoregulatory human T lymphocytes triggered as a consequence of viral infection: clonal analysis of helper, suppressor inducer and suppressor effector cell populations

The regulatory functions of a series of human T cell clones specific for an autologous Epstein-Barr virus transformed B lymphoblastoid cell line were examined. Two T4+ T cell clones, termed AT4II and AT4IV, and one T8+ clone, AT8III, were maintained in culture for greater than or equal to 9 months and were characterized in detail. Both T4+ clones provided helper function for autologous B cell immunoglobulin production when added to unstimulated peripheral blood mononuclear cells. In addition, these same clones produced soluble inducer factors after specific antigenic stimulation. However, when AT4II, AT4IV and their subclones were tested on pokeweed mitogen stimulated peripheral blood mononuclear cells, it was found that AT4IV provided help for immunoglobulin production whereas AT4II cells were strongly suppressive. This suppression by AT4II was indirect and required the presence of fresh, autologous, unirradiated T8+ cells. In contrast, the T8+ AT8III clone markedly inhibited Ig production by autologous B cells in the absence of any additional T8+ cells from peripheral blood and produced a soluble suppressor factor upon specific antigenic triggering. Thus, after stimulation with autologous Epstein-Barr virus transformed cells, at least three discrete regulatory human T cell populations can be defined at the clonal level: helper, inducer of suppression and suppressor effector clones.     

HLA-DR antigens and mitogenic factors released by PWM-stimulated T lymphocytes share the framework determinant recognized by the monoclonal antibody Q5/13

Human T lymphocytes release factors which enhance the mitogenic response of B lymphocytes to PWM. These mitogenic factors share with HLA-DR antigens the framework determinant recognized by the monoclonal antibody Q5/13 (MoAb Q5/13) since adsorption of T-cell medium with an excess of insolubilized MoAb Q5/13 significantly reduces the enhancing activity of the T-cell supernatant on the proliferative response of B cells to PWM. On the other hand, incubation of the T-cell supernatant with an excess of insolubilized anti-HLA-A,B MoAb Q1/28 did not effect the activity of the T-cell supernatant in the proliferative assay.     

Recognition of hepatitis B surface antigen by human T lymphocytes. Proliferative and cytotoxic responses to a major antigenic determinant defined by synthetic peptides

The antigenic sites for human T lymphocytes on hepatitis B surface Ag (HBsAg) were studied by using synthetic oligopeptides. T cell lines of the helper/inducer class, which were isolated from hepatitis B vaccine recipients, were found to react strongly and in an Ag-specific way with peptides corresponding to a sequence of 10 to 30 amino acids near the amino terminus of the HBsAg molecule. Cells with surface expression of the antigenic determinant contained in these synthetic peptides induced both proliferative and cytotoxic responses in the hepatitis B-specific T cells. The results indicate that amino acid residues 24-27 of HBsAg could be directly involved in this T cell determinant. Inhibition studies with mAb to MHC class II Ag and target cells from various HLA-typed individuals suggest that some T cell responses to this determinant of HBsAg might be restricted by the DPw4 molecule. However, the possibility exists that more than one of the MHC class II molecules could be involved as restricting elements of T cell responses to this synthetic peptide. In vivo experiments with synthetic peptides such as those described here are needed to demonstrate the possibility of enhancing HBsAg immune responses in some individuals.     

Description of the effect of polyclonal mitogens on a population of splenic lymphocytes in the presence of serum against isologous aggregated mouse immunoglobulins

Antiserum against isologous aggregated mouse immunoglobulins was shown to be mitogenic for spleen cells during the first 48 hours of culture. The mitogenic effect of the serum factor is mediated through T lymphocytes and may be macrophage-dependent. Whe incubated with spleen cells the serum was demonstrated to inhibit the DNA synthesis in B cells in response to sulfate dextran and lipopolysaccharide. Experiments with B cells and the thymidine "suicide" test suggest that the target cells for the serum factor may reside in the population of radiosensitive and highly proliferating B lymphocytes. The degree of suppressing by the antiserum factor depends on the differentiation stage and the presence of antigen-binding receptors on the membrane of B cells.     

Murine natural killer cells express the Ly24 (Pgp-1) marker on their surface

The Ly24 (Pgp-1) marker is expressed on some, but not all, mature T lymphocytes. It has recently become apparent that the development of Ly24- T lymphocytes is dependent on the presence of an intact thymus and that virgin Ly24- T cells rapidly acquire this marker upon antigenic or mitogenic stimulation. Although natural killer (NK) cells can develop and function in the absence of an intact thymus, some NK cell subsets express certain markers normally associated with T lymphocytes. The experiments in this report were undertaken to determine if NK cells express Ly24 and whether such an expression could be used to delineate distinct NK cell subsets. We found that mature functional NK cells expressed the Ly24 marker as defined by the monoclonal antibody 9F3. Double-color fluorescence analysis using C57BL/6 splenocytes (whose NK cells express the NK1.1 marker) showed all the NK1.1+ cells to be Ly24+ as well. For C3H/HeN (an NK1.1- strain), double-color fluorescence analysis utilizing asialo GM1 and Ly24 revealed a distinct subset positive for both markers and containing most of the functional NK cell activity. Whereas the Ly24 marker did not illuminate an NK cell subset, these findings demonstrate that this determinant can be useful for the further characterization and isolation of NK cells.     

Antigenicity of licensed whole virion and subvirion influenza vaccines in "high risk" persons.

The frequency and magnitude of serum antibody response to type A and B influenza virus induced by whole virion and subvirion vaccines were essentially comparable. Immunization was followed in vaccinated individuals by an antihemagglutinin antibody response to the common antigenic determinant shared by the type A H3N2 viruses. Relatively few individuals developed antibody to the type-specific determinant.     

The decline in murine splenic PHA and LPS responsiveness with age is primarily due to an intrinsic mechanism

Changes in splenic B and T lymphocyte number and mitogenic activity with age were quantitated in (A X C57BL/6)F1 (AB6F1) hybrid mice. Although both the B and T lymphocyte proliferative reactivity to their respective mitogens, lipopolysaccharide (LPS) and phytohemagglutinin (PHA), declined significantly with age, an earlier and more marked reduction was recorded for the T cell response. The decline in B and T lymphocyte mitogenic activity with age could not be correlated with a corresponding reduction in the percentage of splenic B or T lymphocytes. The main focus of this study was to determine if the reduction in T and B lymphocyte mitogenic activity with age results primarily from a mechanism intrinsic to the lymphoid lineage itself or from adverse extracellular factors that increase with age. Bone marrow cells (BMC) derived from individual young and old donor AB6F1 mice were transplanted into the neutral environment of young, lethally irradiated syngeneic recipients. Number and mitogenic activity of splenic T and B lymphocytes were recorded for the original BMC donors as well as for the recipients of the young and old BMC lines 9 mo after the BMC transplants. A predominance of the donor (male) rather than recipient (female) karyotype within the mitogen-responding populations of recipient mice confirmed a donor BMC take. The PHA and LPS response levels exhibited by the old donors were 30% and 70% of those of the young donors, respectively. These differences in PHA and LPS reactivity recorded between young and old donors were maintained between recipients of young and old donor BMC lines. Thus, even under the influence of a young recipient environment, old BMC were incapable of giving rise to mitogen responding cells with a functional competence equivalent to that of their younger counterparts. This finding would lend further support to the theory that an intrinsic mechanism is responsible for the decline in murine mitogenic activity with age.     

Production of interleukin 1 activity by normal human peripheral blood B lymphocytes

Interleukin 1 (IL 1) production by normal human B lymphocytes was investigated. Normal human peripheral blood B lymphocytes were purified by sequential separation with the use of Ficoll-Hypaque gradient centrifugation, sheep red blood cell rosette formation, Percoll gradients, and treatment with monoclonal antibodies (anti-Leu-M1, B73.1, and T101) and complement. Both purified large B lymphocytes (BL) and small B lymphocytes (BS) produced IL 1-like (thymocyte co-mitogenic and fibroblast mitogenic) activities in response to lipopolysaccharide. Maximal production of IL 1 activity by both BL and BS occurred at 48 hr. The m.w. of IL 1 activities from both BL and BS were about 20,000 with high pressure liquid chromatography, and the major isoelectric point of BL- and BS-derived IL 1 activity was 7.0. A rabbit anti-human monocyte IL 1 antiserum inhibited the activity of B cell-derived IL 1, suggesting antigenic similarities of monocyte- and B lymphocyte-derived IL 1 moieties. These data suggest that normal B lymphocyte-derived IL 1 activity is biochemically and immunologically similar to monocyte-derived IL 1.     

Brugia malayi antigens associated with lymphocyte activation in filariasis

Filarial parasites induce immune response in humans which are still poorly characterized. To define the antigens responsible for inducing lymphocyte responses, fast protein liquid chromatography was used to fractionate the antigens of Brugia malayi adult worms which were then tested on lymphocytes from patients with filariasis and normal controls. From an anion exchange column (Mono Q), three peaks of lymphocyte-stimulating activity were eluted which were further fractionated by gel filtration (Superose-12). Peak I induced both a proliferative response as well as the production of filaria-specific antibody in patient lymphocytes. Peak II, capable of inducing only a proliferative response (without antibody production) in patient lymphocytes, was a glycoprotein with phosphocholine as one of the antigenic determinants. Peak III induced proliferative responses in both patient and normal lymphocytes and thus appears to be mitogenic. Two-dimensional gel electrophoresis was then used to identify changes in the major cellular proteins associated with the activation of patient lymphocytes by these partially purified antigens. Stimulation of patient lymphocytes with peak I resulted in increased synthesis of immunoglobulin heavy, light, and J chains. Further, these were the only major secreted proteins found in the culture supernatants. Peak II resulted in quantitative changes in proteins associated with T and not B lymphocyte stimulation. Further analysis of these antigens should help to elucidate the mechanism of host-parasite interaction at both the cellular and molecular levels.     

The mitogenic activity of type III bacterial Ig binding proteins (protein G) for human peripheral blood lymphocytes is not related to their ability to react with human serum albumin or IgG

The mitogenic potential of bacterial IgG Fc binding proteins for human PBL is controversial. Wild type and recombinant type III IgG Fc binding proteins induce a wide spectrum of proliferative responses ranging from non-mitogenic to potent responses. To understand the reason for these differences, three recombinant forms of a type III IgG Fc binding protein derived from a single human group C streptococcal strain, 26RP66, were generated. Form I bound human IgG and human serum albumin, form II bound IgG alone and form III bound human serum albumin alone. These functionally distinct forms were compared with the corresponding wild type preparation from the same strain for mitogenic potential. A mitogenic response was induced only with the form I recombinant or the native wild type protein. These proteins shared the functional characteristics of binding human serum albumin and IgG. Mixtures of the IgG binding (form II) and human serum albumin binding fragments (form III) failed to reconstitute the mitogenic potential of the full length proteins. These results demonstrate that the type III IgG Fc binding protein has mitogenic potential for human PBL that is not related to its ability to react with human serum albumin or IgG.     

Mitogenic and antigenic activity of Plasmodium falciparum in primate and rodent lymphocytes

Goldenser J., Marva E., Spira D. T., Gabrielsen A. A. and Jensen J. B. 1985. Mitogenic and antigenic activity of Plasmodium falciparum in primate and rodent lymphocytes. International Journal for Parasitology15: 435–440. Considerable reaction of human leucocytes to a wide range of concentrations of plasmodial preparations derived from in vitro cultures of Plasmodium falciparum was observed. Highest responses were recorded after 6 days in culture. This differed from the response to PHA or CON-A which peak with a narrow range of concentrations after 3 days in culture. Parasitized erythrocytes (PE) or parasites released from PE as well as soluble antigens obtained from the particulate preparations had a pronounced mitogenic activity which was unaffected by heating to 56°C for 1 h. Peripheral lymphocytes from man and monkey but not from rats reacted to P. falciparum preparations. Spleen cells obtained from normal rats did not react towards any P. falciparum preparation. Spleen cells of rats immune to P. berghei, responded to normal human erythrocytes but the response against P. falciparum antigens was much higher, indicating cross-reactivity with genus specific antigens. The combination of experimental procedures using human peripheral and rat spleen lymphocytes is suggested for differentiation between mitogenic and antigenic activity. Heat inactivation of some proteases present in the plasmodial preparations, while retaining mitogenic activity, may enable further purification of the mitogenic factors.     

Characterization of a serum inhibitor of MLC reactions. III. Specificity: HL-A-related antibodies.

A mixed leukocyte culture (MLC)-inhibitory serum from a healthy multipara, JH, has been characterized with regard to the specificity of its inhibitory antibody. When added directly to MLC, JH serum will inhibit most combinations. However, when lymphocytes intended as responder or stimulator cells in MLC were preincubated with this serum, the specificity narrowed considerably. Four groups of lymphocyte donors were recognized, depending upon whether their lymphocytes were inhibited as responders, as stimulators, as both, or as neither. Absorptions of inhibitory activity, followed by assay of the absorbed sera in pretested MLC combinations, yielded reliable data for determining which donors' cells shared pertinent antigens. An association of MLC inhibition by JH serum with the HL-A types of the involved lymphocytes was observed and these relationships are summarized in Table 4. The three HL-A specificities identified, W19, W29, and 12, correspond with the HL-A typing of the husband of the serum donor. Various cell types absorbed relevant inhibitory activity (against responder and stimulator functions) in the following order of efficiency: LCL cells, B lymphocytes, T lymphocytes, fibroblasts, and erythrocytes. When the above three HL-A specificities were removed by absorption, the serum was no longer inhibitory in any combinations. Whether the inhibitory activity of JH serum is directly related to anti-HL-A antibodies or to antibodies against closely related MLR determinants will depend to a large extent upon the degree of linkage disequilibria found between W19, W29, and 12 antigens and the MLR locus.     

Antigen-specific helper factor production by immortalized clone of OVA-specific T cells. I. In vitro activity

Immortalized clones of virally transformed OVA-specific T cells produce antigen-specific helper factor upon stimulation in vitro. The helper factor activate DNP-primed B cells to multiply and synthesize IgG anti-DNP antibodies. The trigger of the helper clone is antigen specific and the B cell-stimulating hapten must be coupled to the specific T cell carrier in order to transfer the help signal from the activated T clone to the B lymphocytes. Activation of the helper clone is performed by antigen-pulsed macrophages and cannot be achieved by the free soluble antigen. However, cell-free supernatant of the antigen-pulsed macrophages can stimulate the helper cells. Thus the antigenic determinant must be presented to the helper cell in the form of macrophage-processed antigen. These requirements for antigenic stimulation and the activity of the secreted helper factor demonstrate that the immortalized helper clone preserved the cellular components which control the antigen-specific immune function of the normal T lymphocyte.     

Preliminary characterization of accessory cells in the nonadherent fraction of human blood mononuclear cells

We investigated why peripheral blood mononuclear cells rigorously depleted of adherent cells by sequential incubation on plastic and nylon wool remained fully responsive to both antigenic and mitogenic signals. Nylon wool nonadherent cells (NWNA) depleted of cells expressing HLA-DR by monoclonal antibody and complement lysis did not respond to tetanus toxoid (TT) or suboptimal concentrations of phytohemagglutinin (PHA). Addition of adherent accessory cells to these NWNA HLA-DR- cells reconstituted the response to stimuli. NWNA, fractionated by discontinuous density gradient centrifugation, contained high density cells which were unresponsive alone to optimal concentrations of both TT and PHA. All the lower density fractions contained cells which were accessory for higher density cell responses to stimuli. The lowest density fraction was approximately 30% monocytes (esterase and peroxidase positive) and less than or equal to 3% B lymphocytes (surface IgG bearing). The other low density fractions contained large granular lymphocytes but rarely monocytes and no B lymphocytes. Depletion of OKT3+, OKM1+, and Leu-11+ cells from lower density cells by monoclonal antibody and complement lysis did not abolish their accessory activity, but depletion of HLA-DR+ cells or gamma irradiation of these cells decreased their accessory activity for PHA and eradicated accessory activity for TT. Thus, the responsiveness of NWNA to soluble antigenic and mitogenic signals is due, in part, to the presence of low density cells which are radiosensitive and phenotypically HLA-DR+ OKT3-OKM1-Leu-11-. Accessory activity in NWNA seems to reside, therefore, in a cell which is not a typical monocyte, natural killer cell, nor B or T lymphocyte.     

Stimulation of human peripheral blood lymphocytes by periodate, galactose oxidase, soybean agglutinin, and peanut agglutinin: differential effects of adherent cells.

Blastogenic responses of normal human peripheral lymphocytes to three distinct groups of mitogens were studied: Group I--phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM); Group II--soybean agglutinin (SBA) and peanut agglutinin (PNA); and Group III--galactose oxidase (GO) and sodium periodate (IO4-). SBA was mitogenic for human cells, and this effect was enhanced by treating the cells with neuraminidase (NA). PNA was mitogenic only after cells had been treated with NA. GO was effective before and activity was increased after lymphocytes were treated with NA. Responses to Group II and III mitogens were more variable than were those to Group I mitogens. Studies with purified T and B cells indicated that SBA and PNA were T cell mitogens, whereas IO4- and GO failed to stimulate either T or B cells. Adding macrophages back to this system indicated that they were both T cell mitogens with strict macrophage requirements. T cell responses to SBA and PNA were enhanced over responses to unfractionated cells to a degree that could not be explained simply by enrichment of the cultures with T cells. Removal of adherent cells from unfractionated cell suspensions again revealed a marked enhancement of responses to SBA and PNA, a consistent decrease in responses to IO4-, and a variable decrease in responses to GO. Similar results were found with 14C-leucine and 3H-uridine incorporation, as well as 3H-thymidine for the assessment of bastogenic response. Mechanisms responsible for these differential effects of macrophage depletion on lymphocyte responses to different groups of mitogens are yet to be determined. Either different mitogens require different lymphocyte to macrophage ratios for optimal stimulation, or some mitogens (i.e., SBA and PNA) form inhibitory complexees in the lymphocyte-macrophage mixture. In any case, variability in response to mitogenic agents in normal as well as pathologic states may be dependent on adherent cell populations, rather than on the lymphocytes themselves.     

Heterogeneity of mouse Thy 1.2 antigen expression revealed by monoclonal antibodies

A different sensitivity of T cells from C57B1/6 and DBA/2 mice to treatment with the monoclonal anti-Thy 1.2 F7D5 serum as compared with a conventional alloantiserum is reported. Depletion of T helper cells, Con A-, PHA-, MLC-, and GVH-reactive cells from a DBA/2 or C57B1/6 spleen cell population was readily achieved with the conventional alloserum. In contrast, the F7D5 antiserum abolished all T functions studied in C57B1/6 spleen cells whereas it was totally or partially ineffective on DBA/2 spleen cells when T helper, MLC, or GVH reactivity were assayed. It did however eliminate the capacity of DBA/2 spleen cells to respond to stimulation with Con A or PHA. Analysis in an Ortho-Cytofluorograf of thymocytes and sIg^? lymphocytes labeled with either GAMB-F or F7D5 + RAM Ig-F showed no difference at the level of the thymocytes: Thy 1.2 antigen as revealed by either GAMB or F7D5 is similarly expressed in the two mouse strains. The fluorescence profiles of splenic T lymphocytes indicated a reduced representation per unit cell basis of the Thy 1.2 antigenic determinant recognized by F7D5 in DBA/2 mice. Moreover, this same determinant is expressed in only 70% of all Thy 1.2-positive cells detected in DBA/2 sIg^? population. This implies that, in DBA/2 mice, maturation of T cells is accompanied by a complete or partial loss of the F7D5 Thy 1.2 determinant and that T helper functions and MLC and GVH reactivity are mediated by T cells which express little or none of this F7D5 Thy 1.2 determinant.     

Target level blocking of T-cell cytotoxicity for human malignant melanoma by monoclonal antibodies

Cytotoxic T lymphocytes (CTL) for autologous malignant melanoma in culture of a patient AV were induced by restimulation of PBL (peripheral blood leukocytes) with AV melanoma cells in vitro and subcultured in interleukin 2 (IL-2) conditioned media. Monoclonal antibodies detecting six antigenic systems on melanoma cell surfaces were tested for blocking activity on the effector function of subcultured cytolytic T lymphocytes for autologous melanoma cells. The monoclonal antibodies R₂₄ (γ3), specific for the G_D3 disialoganglioside on melanoma cell surfaces and I₂₄ (γM), detecting a similar antigenic determinant, blocked autologous T lymphocytotoxicity for malignant melanoma cells on the target level. The effector function of alloantigen activated cytolytic T lymphocytes generated by coculture of allogeneic PBL with Epstein-Barr virus (EBV) transformed AV B lymphocytes, was blocked by monoclonal antibody R₂₄ when tested against AV melanoma targets, but not when tested against AV B lymphocyte targets. It is concluded that blocking by mAb R₂₄ occurs in this system as a nonspecific effect, unrelated to the specific target antigen recognition by cytotoxic T lymphocytes. Steric hindrance or antibody induced membrane changes may account for the blocking effect of monoclonal antibody R₂₄.