Tolerogen-mediated suppression of the immune response. Suppressor cells as passive transfer agents.

The anti-dinitro-phenyl (Dnp)IgM antibody response in mice was inhibited by administration of either a non-immunogenic form of Dnp-polyacrylamide (Dnp-Pa) or an excess amount of an immunogenic form of Dnp-Pa. Spleen cells, alive or heat-killed, from mice tolerized in vivo by either method, inhibited the anti-Dnp response of naive spleen cells co-cultured in vitro with antigen. Conversely, donor cells tolerized in vivo by a high dose of immunogenic Dnp-Pa, when titrated into a naive cell culture which contained no antigen, produced a stimulatory dose-response curve. Both the dose-dependent inhibition and stimulation correlated strongly with the amount of 125I-labelled Dnp-Pa carried over by tolerized spleen cells into the naive cell culture system. Because the doses and experimental procedures used were comparable to those commonly used for suppressor cell generation and assay, it is suggested that antigen-specific suppressor cells may produce their effects through passive transfer of antigen and/or tolerogen.

[Antibodies reacting with thymus and skin epithelium and antibodies to cell nuclei in immunization with streptococcal group A polysaccharide conjugated with synthetic polyelectrolytes]. / Antitela, reagiruiushchie s épiteliem timusa i kozhi, i antitela k iadram kletok pri immunizatsii polisakharidom streptokokka gruppy A, kon''iugirovannymi s sinteticheskimi poliélektrolitami.

The antibodies to streptococcal group A polysaccharide (A-PS) have been obtained upon immunization of BALB/c mice with A-PS conjugated with synthetic polyelectrolytes (PEL). Prolonged immunization in the majority of cases revealed antibodies to cross-reactive determinant of A-PS reacting with human and mouse epithelium of the thymus and basal skin layer. These antibodies belong to autoantibodies. Later on, after the beginning of immunization some animals produced antibodies reacting with cellular nuclei. The formation of autoantibodies to nuclei is not related to crossreactions with A-PS, because A-PS do not inhibit these reactions. No antibodies reacting with the epithelial cells or with cellular nuclei have been observed upon immunization with A-PS in Freund adjuvant or with PEL alone. The production of autoantibodies to cellular nuclei is probably a result of immunoregulatory disorders associated with the damage of thymus epithelium by autoantibodies during immunization with A-PS conjugated with PEL.

Xid mouse lymphocytes respond to TI-2 antigens when co-stimulated by TI-1 antigens or lymphokines.

Spleen cells from male (CBA/N x DBA/2) F1 hybrid mice do not significantly respond to in vitro stimulation by trinitrophenyl-conjugated polyacrylamide beads (TNP-PAA), whereas the same antigen elicits high PFC responses in female F1 hybrid cells. Therefore, this antigen could be classified as a T-independent type 2 (TI-2) antigen. When male spleen cells were co-stimulated by TNP-PAA and TI type 1 antigen, either LPS or Brucella abortus, they produced vigorous anti-TNP responses. A similar increase of the in vitro responsiveness of male F1 hybrid spleen cells to TNP-PAA antigen was provoked by the addition of supernatants from P 388-D1 cells stimulated by muramyl-dipeptide (MDP) mainly containing interleukin-1 (IL-1) or supernatants from phorbol 12-myristate 13-acetate (PMA)-stimulated EL-4 cells that contained T-cell factors. The PFC response to another TI-2 antigen, TNP-Ficoll, was also significantly enhanced after co-stimulation by P 388-D1 supernatants. The response to TI-2 antigens being macrophage dependent, the influence of supernatants of peritoneal macrophages from male and female F1 hybrids incubated with TNP-Ficoll on the PFC response of normal DBA/2 mouse spleen cells to sheep erythrocytes was assessed. It was found that macrophage supernatants from female hybrids regularly increased by more than two times this anti-SRBC PFC response, whereas macrophage supernatants from male F1 hybrids did not. Moreover, in a specific proliferation test measuring IL-1 activity, when macrophage supernatants from female F1 produced a 13-fold increase of thymidine incorporation, supernatants from male F1 only produced a three-fold increase. It is concluded that, in addition to the known defects of B cells from Xid mice, their macrophages are also defective.

Polyacrylonitrile complement activation attenuated by simultaneous ultrafiltration and adsorption.

The highly permeable synthetic polyacrylonitrile (PAN) membrane (Filtral; Hospal, Basle) is regarded as biocompatible for its slight complement activation and leucocyte sequestration. The low C3a and C5a concentrations during PAN dialysis may be due to a lack of complement activation potential of this polymer, but also to elimination of activated complement components by the dialyser through adsorption and/or ultrafiltration (mol wt of C3a and C4a 9000 daltons; of C5a 11,000 daltons). Comparing arteriovenous differences throughout the study, higher concentrations of C3a (+23%; n.s.) and C5a (+80%; P less than 0.05) were measured in the efferent blood lines, suggesting a slight complement activation by the alternate pathway. A significantly lower C4a concentration in the efferent blood lines (-30%, P less than 0.05) indicates an elimination within the dialyser. Significantly higher arteriovenous concentration differences at the beginning of dialysis and increasing sieving coefficients suggest elimination by adsorption, mainly in the first 20 min of haemodialysis. The continuous decrease of C3a and C4a plasma concentrations in the afferent blood line suggests transport across the membrane and removal by the dialysate. Accordingly, measurable amounts of these complement components were detected in ultrafiltrate.

Functional heterogeneity of nonresting B cells in human blood.

Among human peripheral blood B cells we localized the precursors of two interleukin-dependent B cell activation processes: the specific response to a particulate antigen, trinitrophenylated polyacrylamide beads (TNP-PAA) and the polyclonally induced response to pokeweed mitogen. In both cases the precursors belong to the OKB7+, sIgD-, mouse red blood cell- subpopulation. However, they differ when cell density, reflecting the stage of activation reached by B cells in peripheral blood, is considered. Only B cells of intermediate density respond to TNP-PAA, whereas the optimal response to pokeweed mitogen is obtained with the cells displaying the lower density. The lack of response of the more dense (resting) B cells to TNP-PAA suggests that the T dependency of this antigen is not based on linked recognition, and fits with our demonstration that this particulate antigen can trigger B cells in the presence of T cell factor. More importantly, our results show that nonresting B cells are functionally heterogeneous according to their degree of preactivation: the responsiveness to specific signals provided by a nonmitogenic hapten-carrier conjugate would be acquired before that to polyclonal activators.

Production of an antibody-like factor in the sea star Asterias rubens: involvement of at least three cellular populations.

Cells from the axial organ of sea stars stimulated in vivo with TNP coupled to polyacrylamide beads and subsequently cultured in vitro were able to produce an antibody-like factor which induced the lysis of mammalian red cells sensitized with TNP. The axial organ cells were fractionated in two populations, adherent and non-adherent to nylon wool. The release of the antibody-like factor required the contact of both populations. When the adherent cells were disrupted by sonication the factor was not produced, but the non-adherent cells could be substituted by their membranes. Destruction by silica of the phagocytic cells present in the adherent population inhibited the production of the factor. The addition of mercaptoethanol to the cultures was essential and did not neutralize the effect of silica. It is concluded that at least three types of cells are involved in the production of the antibody-like factor adherent and non-adherent cells, and phagocytes.

[Comparative study of the antineoplastic and immunoadjuvant activity of acrylic acid (co)polymers]. / Sravnitel'noe izuchenie protivoopukholevoi i immunoad"iuvantnoi aktivnosti (so)polimerov akrilovoi kisloty.

The paper deals with the carcinostatic and immunomodifying action of synthetic polyelectrolytes containing acid and basic groups as well as of polyampholytes and nonionogenic polymers. Polyacrylic acid was found to possess the highest antitumour and adjuvant activity. The antitumour effect of polyacrylic acid increases with its molecular mass. Introduction of other polymers into a polymer chain of acrylic acid results in a decrease in its antitumour and immunoadjuvant action.

[Activation of human B lymphocytes by a particulate antigen]. / Activation des lymphocytes B humains par un antigène particulaire.

A specific IgM antibody response toward the trinitrophenol (TNP) hapten can be induced in mononuclear blood cell suspensions upon culture with a particulate antigen: polyacrylamide beads conjugated with the TNP hapten (TNP-PAA). The response, and its specificity, are demonstrated by an increase in the number of TNP binding B lymphocytes (specific rosette forming cells), by the appearance of cells producing anti-TNP antibody at a high rate (haemolytic plaques), (ELISA test). The anti-TNP response requires monocytes, the role of which is to produce interleukin-1 (IL-1) and T lymphocytes (belonging to the T4 helper subset) the role of which is to produce interleukins (the characterization of which is under study). We propose a model or B cell activation based on the following signals: an early specific signal, provided by the particulate antigen; several non specific signals, provided by T derived interleukins. The anti-TNP response is negatively regulated by monocytes, the functional states of which can be modified in certain situations (autoimmunity, aging) or influenced by glucocorticoids. Suppressor T lymphocytes of this response (not exclusively of the T8 phenotype) can be induced and this can allow the evaluation of T suppressor cell function. This was used in adult idiopathic thrombocytopenic purpura treated with high doses of intra-venous gammaglobulins.

Influenza virus antigens conjugated with a synthetic polyelectrolyte: a novel model of vaccines.

Haemagglutinin (HA), a mixture of haemagglutinin and neuraminidase (HA + NA), and matrix (M) protein were isolated from the influenza A virus and covalently coupled to a synthetic polyelectrolyte (P). A single injection into mice of the resultant conjugates (virogates) brought about efficient stimulation of the primary immune response specific to the corresponding viral antigens. Mice immunized with virogates HA.P or (HA + NA).P were largely protected against a lethal challenge infection with homologous virus. Immunization of mice with M.P virogate containing M protein originated from a 1934 influenza strain resulted in pronounced protection against a lethal challenge infection with a 1980 strain. Virogates are discussed as a novel model of artificial vaccines.

[Relation of the immunogenicity of artificial antigens to the number of polyelectrolyte-bound protein molecules]. / Zavisimost' immunogennosti iskusstvennykh antigenov ot kolichestva sviazannykh poliélektrolitom belkovykh molekul.

Complexes and covalent conjugates of protein antigens with polyelectrolytes of different molecular mass have been synthesised. The structure and composition of the resulting water-soluble complex particles were determined. Artificial antigen immunogenicity was shown to depend on the amount of protein molecules complexed with polyelectrolytes. Direct correlation between immunostimulating activity of the polymer-carrier, immunogenicity of complex antigens and size-dependent capacity of the polymer molecule to aggregate protein globules has been established.

[Secondary immune response to flagellin conjugates with polyacrylic acid]. / Vtorichnyi immunnyi otvet na kon''iugaty flagellina s poliakrilovoi kislotoi.

The conditions of the activation of immunological memory by means of flagellin and its conjugate with polyacrylic acid (PAA) have been studied. The flagellin-PAA conjugate has proved to be capable of inducing the appearance of memory cells when introduced in doses, considerably lower (0.01 and 0.001 micrograms) than those of native protein (0.1, 1 and 10 micrograms). At the same time no manifest differences in the reactivation of flagellin-induced and conjugate-induced memory cells by the antigen have been established. Immunization with protein, as well as with its conjugate, has been found to induce the formation of mainly anti-Hi : 1,2 IgG in secondary immune response.

Antigen valence determines the binding of nominal antigen to cytolytic T cell clones.

We have shown that cytotoxic T cell clones specific for the nominal antigen FL will bind high molecular weight (600,000 to 2,000,000) polyacrylamide and Ficoll polymers conjugated with 200-600 FL groups per molecule. Low molecular weight polymers (40,000) with the same epitope density did not give stable binding. A high molecular weight polymer with a lower epitope density also failed to bind. Taken together, these results suggest that a substantial degree of multivalence is a necessary factor in the stable binding of nominal antigen to T cell clones.

B cell activation: role of dendritic and T cell factors in the response to thymic-independent and -dependent antigens.

We described a cloned dendritic cell, clone Den-1, that is a potent accessory cell for some B cell responses. Clone Den-1 produces a novel lymphokine that is distinct from previously described factors produced by T cells. In the present study, we compare the role of nonspecific helper factors produced by Den-1 (Den-1 SN) or the T cell thymoma EL4 (EL4-SN) in promotion of B cell plaque-forming cell (PFC) responses to a variety of antigens. We find that the antigen in culture determines the B cell requirement for dendritic and/or T cell factors. B cell PFC responses to TNP-Brucella abortus (BA) and TNP-lipopolysaccharide (LPS) are greatly increased by EL4-SN but show little, if any, enhancement with Den-1 SN. Responses to TNP-polyacrylamide are reconstituted by either Den-1 SN or EL4-SN, whereas responses to TNP-Ficoll, TNP-dextran and TNP-levan are reconstituted by Den-1 SN and are much less sensitive to factors present in EL4-SN. Responses to SRBC require the presence of both Den-1 SN and EL4-SN. We also show that the time at which Den-1 SN must be provided to the B cell is dependent on the antigen in culture. Our findings are discussed in terms of present classification of antigens based on their ability to stimulate various B cell subpopulations.

Bone marrow function. I. Peripheral T cells are responsible for the increased auto-antiidiotype response of older mice.

After immunization with trinitrophenyl (TNP)-Ficoll, mice produced both anti-TNP antibodies and auto-anti-idiotype (auto-anti-Id) antibodies specific for the anti-TNP antibody. Older animals produced more auto-anti-Id than did young animals. When mice were exposed to a normally lethal dose of irradiation while their bone marrow (BM) was partially shielded, they survived and slowly (6 wk) regained immune function, as indicated by the number of nucleated cells in their spleen and the in vitro primary plaque-forming cell (PFC) response of their spleen cells to TNP-treated aminoethylated polyacrylamide beads. Recovery is presumably the result of repopulation of the peripheral lymphoid system by cells originating in the BM. By enzyme-linked immunosorbent assay (ELISA), and by hapten-augmentable PFC assay, we show that, after recovery from irradiation with their BM shielded, old animals produce low auto-anti-Id responses, like those of young animals. The transfer of splenic T cells into mice irradiated with their BM shielded provided evidence that the magnitude of the auto-anti-Id response is controlled by the peripheral T cells. Thus, mice that received splenic T cells from aged donors produced high levels of auto-anti-Id while those that received splenic T cells from young donors produce low levels of auto-anti-Id.

[Enhancement of the immune response to surface antigens of the influenza B virus as a result of their covalent binding with a synthetic polymer carrier]. / Usilenie immunnogo otveta k poverkhnostnym antigenam virusa grippa v rezul'tate ikh kovalentnogo sviazyvaniia s sinteticheskim polimerom-nosi-telem.

The primary immune response (the number of antibody-forming cells, AFC) and the delayed type hypersensitivity (DTHS) were studied in mice immunized either with isolated glycoproteins of influenza virus (hemagglutinin, HA and HA plus neuraminidase, HA plus NA) or with their conjugates with an acrylic acid copolymer (CP) and N-vinylpyrrolidone of equimolar composition. Immunization of mice with conjugates containing virus proteins (virogates-HA-CP or HA plus NA-CP--entailed a 50-100 increment of the number of IgM- and IgG-AFC, anti-HA as compared with analogous parameters during immunization of animals with isolated virus proteins. Immunization of mice with the virogate HA-CP gives rise to the development of a more pronounced DTHS to HA. The authors discuss the possibility of the use of this basically new approach to the design of highly immunogenous vaccine preparations, effective in the control of influenza and other virus diseases.

Complement activation during hemodialysis: clinical observations, proposed mechanisms, and theoretical implications.

Human C3a radioimmunoassay techniques were employed to define both the temporal profile and the amount of complement activation taking place in the extracorporeal circuit during maintenance hemodialysis. Prospective studies demonstrated that C3a formation, like hemodialysis-associated leukopenia, was a transient phenomenon that occurred predominantly during the first 30 min of dialysis. Quantitative comparisons revealed that new Cuprophan hemodialyzers displayed somewhat greater complement-activating potential than cellulose acetate dialyzers. By contrast to new Cuprophan membranes, both reused Cuprophan and polyacrylonitrile dialyzers exhibited only a modest ability to activate human complement. These findings are compatible with the known mechanisms of complement activation and suggest that certain chemical and biochemical methods might be exploited to enhance the biocompatibility of cellulose dialysis membranes.

Interleukin 1 can replace monocytes for the specific human B-cell response to a particulate antigen.

It is shown that the anti-trinitrophenyl (TNP) response of human B cells to trinitrophenyl polyacrylamide beads (TNP-PAA) is monocyte dependent. This response is abolished by extensive adherent cell depletion and restored by the addition of monocytes. The optimal response is obtained with 3% monocytes, higher numbers being suppressive. Supernatants from muramyl dipeptide (MDP)-activated monocytes can restore the response of monocyte-depleted preparations even when cells are cultured at suboptimal concentration. A partially purified preparation of interleukin (IL-1) has a comparable restorative ability. The following arguments suggest that monocytes do not function as antigen-presenting cells for this particulate antigen: (i) antigen-pulsed monocytes induce neither an anti-TNP response nor a specific T-cell proliferative response; (ii) allogeneic monocytes function as well as autologous monocytes to restore the response of nonadherent cells; (iii) HLA-DR-negative cells from the human leukemia cell line K562 can replace monocytes for this response. Monocyte supernatants do not replace T cells for the response of B-enriched lymphocytes, showing that T cells are directly involved in B-cell activation.

Helper T cell activation for the human B cell response to trinitrophenylated polyacrylamide beads: involvement of the T4 antigen.

The monoclonal antibody (mAb) OKT4A (but not OKT4) inhibits the in vitro antibody response of human peripheral blood mononuclear cells. The target of OKT4A mAb is the helper T cell, as the helper cells for the antibody response to trinitrophenylated polyacrylamide beads (TNP-PAA) are exclusively in the T4 subset. The OKT4A mAb is still suppressive when the anti-TNP response of cultures of monocyte-depleted cells is supported by purified interleukin 1. Both the OKT4A mAb and anti-DR mAb suppress the non-specific T cell proliferation in the cultures leading to the in vitro mAb response. A parallel inhibition is observed for the autologous mixed lymphocyte reaction, the nonspecific B cell response, but the T cell response to mitogens is not affected. These results suggest that the recognition of self major histocompatibility complex class II determinants by the T4 molecule plays a major role in the activation of T helper cells for antibody production to this particulate antigen.