Antigen-specific suppression of human antibody responses by allogeneic T cells. II. Cell interactions involved in the generation of suppression

Specific antibody responses to influenza virus were obtained in vitro from human blood mononuclear cells (PBMC). Antibody production in these cultures was profoundly suppressed by the addition of allogeneic T cells with the surface phenotype Leu2a+ (CD8+), Leu8-. Suppression by allogeneic T suppressor (Ts) cells required interactions only between T-depleted B (E-) cells and allogeneic Leu2a+. No evidence was obtained for T-T cell interactions, or for Ts inducer cells similar to those described for nonspecific antibody responses to pokeweed mitogen. Moreover, allogeneic E+, or allogeneic Leu2a+ cells were able to suppress specific antibody responses by E- cells when help was provided by T cell-replacing factor showing that the target of suppression was the responding E- cells, and not T helper cells. In contrast to allogeneic T cells, allogeneic E- cells did not suppress antibody production when added to cultures of unfractionated PBMC (E- + E+). That is, Ts cells activated to allogeneic E- were unable to suppress antibody production by the syngeneic E- cells present in the same culture tube. This result shows that alloactivated Ts cells were specific for the allogeneic E- target cells, and that suppression was not mediated by nonspecific allogeneic effects. Allogeneic Ts cells therefore differ from Ts cells in pokeweed mitogen responses by their specificity, and by their activation in the absence of Ts inducer cells.     

The allogeneic effect: bystander effect in the primary immune response in vitro

There is a strong stimulation of the primary immune response when spleen cells of the CBA/H and CBA/J strains of mice are cocultured in the Mishell-Dutton system and stimulated with foreign erythrocytes or hapten-erythrocyte conjugates. This stimulation occurs regardless of carrier (sheep red blood cells) priming of one donor of the spleen cells. The enhancement of the primary immune response is most evident early in the course of the immune response. During mixed spleen cell culture, cells of the CBA/H strain can recognize and proliferate in response to CBA/J spleen cells, but not vice versa. When CBA/H spleen cells are cultured with irradiated CBA/J spleen cells, there is a strong stimulation of the immune response of the B cells of the CBA/H strain even though they are bystanders to the ensuing allogeneic response. Supernatants of 20-hour cultures of mixtures of CBA/H and CBA/J spleen cells restored the immune responsiveness of spleen cells from nu/nu mice. The generation of this B cell stimulatory activity is dependent on the presence of Θ-bearing cells of CBA/H origin during the 20-hour culture. These findings are consistent with the release of a B cell stimulatory activity from mixtures of spleen cells of mice which differ at a locus associated with a mixed lymphocyte response which is not linked to the H-2 gene complex (M locus).     

Cell adhesion-related proteins as specific markers of sponge cell types involved in allogeneic recognition

Sponge immunocyte identification is of interest to comparative immunologists since characterizing these cells will allow investigations into the mechanisms of non-self recognition in the oldest animal phylum. Here, we report that polyclonal antibodies raised against the core protein of a proteoglycan involved in cell adhesion in the marine sponge Microciona prolifera are specific markers for archaeocytes, the totipotent sponge cells. Archaeocytes are mobilized upon allogeneic contact and they accumulate in the contact zone. A second type of cell, the gray cells, are specifically recognized by monoclonal antibodies raised against CD44, a hyaluronan receptor. Gray cells do also accumulate in the contact area. Specific staining of a third sponge cell type, the rhabdiferous cells, shows that these do not accumulate upon allografting. These specific cell markers allow tracking of archaeocytes and gray cells, and show that they play an active role in sponge allogeneic reactions.     

Quantity of antibody synthesized in a single cell during the primary immune response.

Following immunization of mice with SRBC the kinetics of antibody synthesis by individual immunocompetent cells during the primary immune response were investigated. The diameter of the average hemolytic plaque using a modified Jerne plaquing technique, was taken to be an estimate of the antibody production by a single cell. We found an increase, followed by a decrease, of the average plaque diameter during the primary immune response. The increase of average plaque diameters is probably due to the raised rate of synthesis of antibody molecules. This increase can be accelerated by adjuvants. The decrease late in the primary immune response could be explained by an internally regulated switch from IgM to IgG antibody synthesis. The results are consistent with the mathematical approach of HELLWIG (1,2).     

The allogeneic affect in vitro: activation of antibody synthesis in aggressor and target lymphocyte populations by allogeneic interaction

Normal F₁ hybrid spleen cells, interacted in tissue culture with cortisone-resistant thymocytes (CRT) of parental origin, were activated to increased antibody synthesis against SRC (sheep red cells) as compared to F₁ hybrid lymphocytes mixed with syngeneic CRT. However, an equal or greater stimulation occurred when parental spleen cells were mixed with F₁ hybrid CRT as compared to syngeneic mixtures. When the experiments were performed in cultures containing fetal calf serum, large doses of T cells suppressed the immune response to SRC in both directions. Supernatants from a mixture of parental CRT and normal F₁ spleen cells stimulated the immune response of both parental and F₁ spleen cells to SRC. Analogous results were obtained when the secondary response was studied. Thus, spleen cells from mice hyperimmunized to SRC were activated to increased synthesis of indirect plaque-forming cells by allogeneic confrontation. However, hyperimmune parental spleen cells were activated by confrontation with nonimmune F₁ lymphocytes to the same extent or more than hyperimmune F₁ spleen cells mixed with normal parental cells. It is concluded that the stimulating effect on the immune response caused by allogeneic interaction does not exhibit specificity for the genotype of the target B lymphocytes. Possible mechanisms by which specificity can be detected by in vivo experiments are discussed.     

Effect of carrier priming on antibody avidity in the in vivo and in vitro immune response.

The effect of carrier priming on antibody avidity was investigated under several experimental conditions. Basically, mice were carrier primed with HRBC (horse red blood cells) prior to immunization with TNP (2,4,6-trinitrophenyl) conjugated to HRBC. Immunization was performed either in vivo or in spleen cell culture, and avidity of anti-TNP antibodies was estimated from inhibition of direct PFC (plaque-forming cells) by free TNP-BSA (-bovine serum albumin).The data indicate the appropriate conditions under which carrier priming can enhance antibody avidity. The carrier effect is maximized by priming the animals with 10⁴-10⁵ HRBC 3-7 days before immunization with a low dose of TNP-HRBC. Hyper-immunization by repeated injections of a high dose of the conjugate does not modify the carrier effect on avidity but it delays the fall of avidity in both carrier primed and unprimed animals. These results are interpreted in terms of T- and B-cell co-operation within the framework of the maturation theory of antibody affinity.Carrier priming was also found to increase the number of direct PFC of the IgM and, mostly, of the non-IgM classes, a finding in agreement with the notion that T cells can help IgM production and the shift to IgG.     

Effect of exogenous lipids and lipoproteins on the primary immune response in vitro

Cholesterol and certain lipoproteins have regulatory effects on the primary immune responses of murine spleen cells in vitro. The plaque-forming cell (PFC) responses to sheep red blood cells of trinitrophenylated Brucella abortus were studied in complete, lipid-depleted or lipoprotein-reconstituted media. The requirement for exogenous low density lipoprotein (LDL) and its cholesterol moiety was established by comparison of the yield of PFC in cell cultures deprived of lipoproteins with that in cultures to which specific classes of lipoproteins were added. The spleen cells in complete medium yielded about 10-fold greater PFC responses than cells in lipoprotein-deficient medium. In lipoprotein-deficient media, human LDL completely reversed the decreased immune response, LDL lipids and free cholesterol partially reversed the deficit, the human high density lipoproteins and an apo B phospholipid complex were ineffective. In complete media, cholesterol at higher concentrations (100--200 microgram/ml) and LDL lipids partially inhibited the primary immune response. Exogenous cholesterol was required for the in vitro response to both thymus-dependent and thymus-independent antigens.     

Cell interactions in the immune response in vitro. I. Metabolic activities of T cells in a collaborative antibody response

Cell collaboration between thymus-derived (T) and non-thymus-derived (B) lymphocytes in induction of the response to SRC was investigated in an in vitro test system. Different populations of T cells were found to vary in helper activity depending on their degree of differentiation following exposure to antigen. Activated thymus cells obtained from the spleen of irradiated mice injected with syngeneic thymocytes and SRC proved the most effective, followed by primed T cells and then unprimed T cells. The interaction between T cells and B cells as measured here was specific for both classes of lymphocytes. The mechanism of T cell action in the collaborative response was analyzed by treating these populations of T cells with agents which became firmly cell bound and could selectively inhibit different aspects of cell metabolism. Mitomycin C was chosen as an inhibitor of DNA synthesis and cell division, actzinomycin D to interfere with RNA synthesis, and antimycin A to block protein synthesis. To exclude significant leakage of inhibitor from T cells to B cells, cultures were stimulated with DNP-POL, which immunizes B cells directly, as well as with SRC. The development of a normal anti-DNP response in all cases was confirmation of the integrity of B cell function. Mitomycin treatment (40 μg/ml) of normal T cells or T cells from mice primed to SRC at least four weeks previously markedly reduced helper activity. In contrast, activated T cells which had recently divided in response to antigen and were enriched for specific antigen reactive cells were resistant to mitomycin; thus effective collaboration with B cells could take place in the absence of further differentiation and division by T cells. All T cell populations, whether normal, primed or activated, were sensitive to treatment with actinomycin D at a concentration of 0.1 μg/ml, or 10^?5 M antimycin A. Taken together, these experiments suggest that T cells divide and differentiate over a period of approximately 48 h in vitro. During this time antibody production by B cells is initiated by a process requiring active RNA and protein synthesis in T cells.     

Effect of hydrocortisone on the in vitro human antibody response: interaction with monocytes and prostaglandins

The specific plaque-forming cell response induced by trinitrophenyl polyacrylamide beads in cultures of nonadherent human peripheral blood mononuclear cells (PBM) is resistant to the inhibitory effect of hydrocortisone (HC). Previous results showed that low concentrations (up to 10(-7 M) of HC inhibit the same response in cultures of unfractionated human PBM. It is shown that the addition of 5-10% monocytes to nonadherent PBM renders their response HC-sensitive. One mechanism of the interaction between HC and monocytes is the potentiation of prostaglandin E2-mediated suppression by HC, which can be demonstrated in vitro and after in vivo administration of HC. However, interaction with a non-prostaglandin-mediated suppression by monocytes should be involved, particularly with high (10(-6) M) concentrations of HC.     

Effect of azathioprine on in vitro antibody response. Differential effect on B cells involved in thymus-dependent and independent responses.

The effect of azathioprine (Az) on the primary in vitro antibody response of mouse spleen cell cultures has been studied. The response towards T cell-dependent antigens is suppressed by low Az concentrations (50% inhibition by 10(-2) mug/ml and 100% suppression by 10(-1) mug/ml). The same pattern is observed when Az addition is delayed until day 2, but the suppression is absent or partial when Az is added on day 3. In the early period (day 0 to day 1) the effect of Az is reversible upon addition of an excess of purine nucleosides. In contrast, the response to a T cell-independent antigen (TNP-T4) is relatively insensitive to Az, since 100-fold higher drug concentrations are required to obtain an inhibition. With the assumption that T helper cells are likely to be highly sensitive to Az, the effect of low Az concentrations on the other two cell populations involved in T cell-dependent responses has been evaluated. Adherent cells appear unaffected. In contrast, the B-cell response is markedly sensitive to Az, as shown by the effect of Az on the response of nude mouse cells to a T cell-dependent antigen in the presence of T-cell products, either specific or non-specific. On the other hand, the B-cell response to mitogens is resistant to az. Thus, Az has a differential effect on B-cell response according to the thymus dependency of the antigen. This may suggest the existence of two pathways for B-cell activation or two different B-cell subpopulations.     

Antibody-mediated suppression of the immune response in vitro: III. Low zone tolerance in vitro

Immunological tolerance was induced by the exposure of mouse spleen cells to mixtures of polymerized flagellin and antibody in vitro for 6 hours, at 37°, prior to thorough washing and in vitro challenge with optimally immunogenic doses of polymerized flagellin. The induction of antibody-mediated tolerance depended critically on the ratio of antigen and antibody to which the cells were initially exposed. Low zone tolerance could be induced with subimmunogenic concentrations of monomeric flagellin, in amounts as low as 2 pg/ml (about 10^-14 molar) and the appropriate concentration of antibody (a titre of 10^-3). In contrast tolerance could not be induced with subimmunogenic concentrations of polymeric flagellin in the presence of a wide range of antibody doses.

There was a remarkable similarity between the tolerant states obtained in vitro with tolerogenic doses of polymeric flagellin, and with immunogenic and subimmunogenic concentrations of flagellin in the presence of antibody, suggesting that the cellular basis of the tolerant state remains the same. An antigen-focussing hypothesis has been proposed to integrate the mechanisms of high zone and antibody-mediated tolerance at the cellular level.

     

Depressed primary in vitro antibody response in rheumatoid arthritis.

We have studied the primary in vitro antibody response toward a hapten in cultures of peripheral blood lymphocytes from twenty-two patients suffering from regular rheumatoid arthritis (RA). These patients were not receiving immunosuppressive drugs or corticosteroids and had not taken Aspirin or non-steroidal anti-inflammatory agents for at least 72 hr. The control groups included thirty-two healthy subjects and twenty-seven control patients. The mean anti-TNP response of the RA patients was significantly lower than that of both control groups. No pre-existing anti-TNP or IgG response could be detected. A search for suppressor cells in co-cultures of RA and normal lymphocytes was negative. On the contrary, the extent of allogeneic enhancement in such co-cultures was comparable to that observed when control lymphocytes were co-cultured. RA serum added to normal lymphocytes cultures showed a dramatic inhibitory effect in only two out of nine cases. A follow-up study has strongly suggested that RA lymphocytes could increase their in vitro antibody response upon treatment.     

Regulation of cellular antibody synthesis: Selective suppression by antibody of the immune response induced by a high antigen dose

Mice immunized with a high dose of sheep red blood cells and 24–48 hours later given specific antiserum produced on the average only 1 per cent of the direct and indirect number of plaque-forming cells (PFC) found in the non-antibody treated controls, whereas animals given a low antigen dose followed by antiserum produced 30 per cent of the number of PFC found in the controls. It is suggested that the low antigen dose preferentially stimulated antigen-sensitive cells having a receptor for the antigen of high affinity, whereas the high antigen dose in addition stimulated cells having lower affinity receptors and that, therefore, the passively transferred antibody would compete more efficiently for the antigen with the antigen-sensitive cells triggered by a high antigen dose than with the higher affinity cells stimulated by a low antigen dose.

When antigen and antibody were introduced simultaneously the selective suppression of the immune response to a high antigen dose did not occur. Possible mechanisms for this finding are discussed.

     

Measurement of parasite-specific immune responses in vitro: evidence for suppression of the antibody response to Trypanosoma cruzi

Using the Mishell and Dutton culture system, we have developed an assay for eliciting and quantifying parasite-specific immune responses in vitro. The ability of spleen cells from noninfected and Trypanosoma cruzi-infected mice to respond to parasite-associated antigens was assessed by examining the primary plaque-forming cell response to trinitrophenylated T. cruzi (TNP-TC). The response to TNP-TC of both normal, noninfected, unprimed mice and mice infected with T. cruzi is T cell dependent and appeared to involve recognition of parasite antigens by T. cruzi-specific T cells. In most experiments, spleen cells from infected mice respond to TNP-TC at levels equal to, or below, that of spleen cells from normal mice. This near "normal" response is in apparent contrast to the suppressed response of spleen cells from infected mice to another antigen (sheep red blood cells) or TNP on a different carrier (TNP-chicken erythrocytes). Demonstration that the response of spleen cells from infected mice to TNP-TC can be potentiated by addition of interleukin 2-containing supernatants or by depletion of plastic and Sephadex G-10-adherent cells suggests that the mechanisms which control the response of infected mice to nonparasite antigens may also limit parasite-specific immune responses.     

Effect of various antisera on intensity of the immune response during induction of antibody formation by allogeneic macrophages

Data on the effect of various antisera on the induction of antibody formation by immune allogeneic macrophages are described. A considerable decrease in the intensity of the immune response was observed after injection both of allogeneic antiserum and of antimacrophagal serum during the first 2 days after transplantation of the allogeneic macrophages. Injection of these sera on the following days had no significant effect on the intensity of the immune response. Antierythrocytic serum prevented the accumulation of antibody-forming cells if injected at various times after transplantation of the allogeneic macrophages.Institute of Medical Genetics, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR P. D. Gorizontov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 9, pp. 1094–1096, September, 1976.     

Effect of antibody on the rickettsia-host cell interaction

A recent study demonstrated that polyclonal antibodies to Rickettsia conorii and monoclonal antibodies to outer membrane proteins A (OmpA) and B (OmpB) provided effective, Fc-dependent, passive immunity, even in severe combined immunodeficient mice with an established infection. In order to determine the mechanism of protection, mouse endothelial and macrophage-like cell lines were infected with R. conorii that had been exposed to polyclonal antibodies, monoclonal antibodies to OmpA or OmpB, Fab fragments of the polyclonal antibodies, or normal serum or that were left untreated. At 0 h, Fc-dependent antibody enhancement of R. conorii adherence to endothelial and macrophage-like cell lines was inhibited by the presence of normal serum, suggesting Fc receptor-mediated adherence of opsonized rickettsiae. At 3 h, the opsonized rickettsiae had been internalized. After 72 h, inhibited survival of rickettsiae exposed to polyclonal antibodies or monoclonal antibodies to OmpA or OmpB was evident compared with growth of untreated and normal serum-treated and polyclonal Fab antibody-treated R. conorii. Polyclonal antibodies and an anti-OmpB monoclonal antibody inhibited the escape of R. conorii from the phagosome, resulting in intraphagolysosomal rickettsial death. At 48 h of infection, rickettsicidal activity of macrophages by opsonized rickettsiae was inhibited by NG-monomethyl-L-arginine, superoxide dismutase, mannitol, or supplemental L-tryptophan, and endothelial rickettsicidal activity against opsonized rickettsiae was inhibited by NG-monomethyl-L-arginine, superoxide dismutase, catalase, or supplemental L-tryptophan. Thus, Fc-dependent antibodies protected against R. conorii infection of endothelium and macrophages by opsonization that inhibited phagosomal escape and resulted in phagolysosomal killing mediated by nitric oxide, reactive oxygen intermediates, and L-tryptophan starvation.     

The effect of cyclosporin A on the primary immune response to allogeneic red cells in rabbits.

Cyclosporin A (CSA) has been used in an attempt to suppress the primary immune response of HgA(A)-negative rabbits to A-positive red cells. The immune response was assessed by measuring the survival of a small intravenous (i.v.) dose of 51Cr-labelled A-positive cells and by testing the serum of the immunized rabbits for anti-A. In one experiment, eight A-negative rabbits were given a first i.v. injection of A-positive red cells, and CSA (25 mg/kg/day) in olive oil was given by mouth for 17-34 days. There was no evidence of impaired alloimmunization compared with the responses in control animals treated with olive oil alone. In a second experiment, eight A-negative rabbits were given a first injection of A-positive muscularly (i.m.), and CSA (25 mg/kg/day) in miglyol was given by im.m. injection for 10 days. Six of these rabbits were rendered unresponsive, and the remaining two, who showed impaired survival of the monitoring red cells, produced only low anit-A titres. Seven out of eight controls given i.m. miglyol without CSA responded with good anti-A production. Rabbits that were unresponsive to A-positive red cells responded normally to sheep red blood cells 15 weeks after CSA treatment. Higher serum levels of CSA were found following i.m. administration of the drug but treatment by this route as associated with severe toxicity in some rabbits.     

Effect of thiosulfate on the immune response in mice, in vitro.

It was found that intravenous application of thiosulfate increased non-specifically immunoglobulin concentration in mice sera. Thiosulfate stimulated the immune response to T-dependent antigens.     

Neutralizing antibody immune response in children with primary and secondary rotavirus infections.

We have characterized the neutralizing antibody immune response to six human rotavirus serotypes (G1 to G4, G8, and G9) in Brazilian children with primary and secondary rotavirus infections and correlated the response with the G serotype of the infecting rotavirus strain. Twenty-five children were studied: 17 had a single rotavirus infection, 4 were reinfected once, and 4 experienced three infections. Two of the reinfections were by non-group A rotaviruses. Among the 25 primary infections, we observed homotypic as well as heterotypic responses; the serotype G1 viruses, which accounted for 13 of these infections, induced mostly a homotypic response, while infections by serotype G2 and G4 viruses induced, in addition to the homotypic, a heterotypic response directed primarily to serotype G1. Two of the primary infections induced heterotypic antibodies to 69M, a serotype G8 virus that by RNA electrophoresis analysis was found not to circulate in the population during the time of the study. The specificity of the neutralizing antibody immune response induced by a virus of a given serotype was the same in primary as well as secondary infections. These results indicate that the heterotypic immune response induced in a primary rotavirus infection is an intrinsic property of the virus strain, and although there seem to be general patterns of serotype-specific seroconversion, these may vary from serotype to serotype and from strain to strain within a serotype.