Tolerance induction in resting memory B cells specific for a protein antigen.

Resting memory B lymphocytes specific for the model protein Ag cytochrome c have been shown to be susceptible to tolerance induction in in vitro splenic fragment cultures. This induction of nonresponsiveness is dependent upon the strength of the interaction between surface Ig and specific Ag, where concentration, valency, affinity, and time of exposure all appear to be important factors, as is the case for tolerance induction in immature or primary B cells. The induction of nonresponsivenes in greater than 80% of Ag-specific memory B cells was achieved by incubation with 1 microM cytochrome polymer for 24 h in the absence of T cell help. Not only were memory B cells unresponsive to specific Ag, they were also unable to become activated through nonspecific uptake and presentation of an Ag to which T cells have been primed, demonstrating that the induction of nonresponsiveness involves more than a modulation or blockade of surface Ig receptors. Although soluble factors collected from activated T cells failed to prevent memory B cells from becoming nonresponsive after surface Ig cross-linking, the direct activation of T cells within splenic fragment cultures did partially inhibit tolerance induction in splenic fragment memory B cells. In addition, the induction of tolerance was partially blocked by protein tyrosine kinase inhibitors, suggesting a physiologic change within the B cells associated with the state of nonresponsiveness and resulting from tyrosine-specific phosphorylation.     

Tolerance and immunity in antigen-specific B-lymphocyte lines: early receptor binding of either antigen or tolerogen initiates an immune response

Studies of the effect of tolerance-inducing compounds on B lymphocytes have been complicated by the fact that it is technically difficult to completely isolate the antigen-specific B cell from the effects of T cells or T-cell factors. We have used our cell lines of nonmalignant dinitrophenyl (DNP)-specific B lymphocytes derived from normal mice, which have no contaminating T cells, to study the effect of DNP-murine IgG2a (DNP-MGG), a tolerogen which is not normally immunogenic, on antigen-specific B lymphocytes. Preincubation with DNP-MGG for 48 hr, both in the presence and absence of T-cell factors from EL-4 supernatant prior to adding the antigen DNP-Ficoll, can induce tolerance in cell line B lymphocytes. The suppression is antigen-specific since preincubation with fluorescein-MGG or unconjugated MGG does not suppress the anti-DNP response. At least a 36-hr incubation is required for tolerance induction in B lymphocytes, but a 6-hr preincubation with DNP-MGG augments the immune response to DNP-Ficoll. Lymphocytes incubated for 6 or 24 hr with DNP-MGG prior to adding EL-4 supernatant and filler cells without DNP-Ficoll exhibited an immune response equal to that elicited by DNP-Ficoll and T-cell factors. A 6-hr pulse with a DNP-conjugated polymer of D-glutamic acid and D-lysine (DNP-dGL), a B-cell tolerogen which does not bind to Fc receptors, elicited the same immune response as seen with a 6-hr pulse of DNP-MGG but a 48-hr preincubation with DNP-dGL induced tolerance. Thus, it is likely that the initial binding of the tolerogen to the immunoglobulin receptor on the mature B cell elicits an activation signal similar to that seen with the antigen. The suppressive effect of the tolerogen itself appears to occur at a later stage of the process of the B-cell activation, proliferation, and differentiation.     

Lipopolysaccharide-induced suppression of the primary immune response to a thymus-dependent antigen.

The immune response to a thymus-dependent antigen was depressed in vivo and in vitro in spleen cells from mice injected with LPS i.p. a few days before challenge with the antigen. Spleen cells from LPS-injected mice could, however, respond with increase DNA synthesis after activation with polyclonal B and T cell activators in vitro. The LPS-activated spleen cells could actively suppress normal cells in their response to the antigen sheep red blood cells. The suppressor cells contained in the LPS-activated spleens were most likely B lymphocytes, and the possible mechanism for their inhibitory function is discussed.     

Macrophage handling of a tolerogen and the role of IL 1 in tolerance induction in a helper T cell clone in vitro

The human gamma-globulin (HGG)-specific helper T cell clone AB.7.D7 can reconstitute the plaque-forming cell response of HGG-primed B cells. Tolerance induction at the level of T cell help results from exposure of the AB.7.D7 cells to 10 micrograms monomeric HGG. The monokine IL 1 was found to interfere with tolerance induction in AB.7.D7 cells in a dose-dependent manner. Furthermore, interference with tolerance induction was dependent upon the T cells being presented with IL 1 at the same time as monomeric HGG, the tolerogen. IL 1 and monomeric HGG could not be demonstrated to interact to make nontolerogenic soluble aggregates, however. It was found that monomeric HGG was unable to stimulate the production of either membrane or secreted IL 1 by splenic macrophages and in addition was not degraded by peritoneal exudate cells. Heat-aggregated HGG, which is highly antigenic and nontolerogenic, is a good stimulus for IL 1 production and is processed by macrophages into peptides of varying sizes. These data are consistent with the suggestion that a tolerogenic signal results from T cell recognition of a nondegraded antigen in the absence of a signal from IL 1. It is possible, however, that small amounts of processed antigen, undetectable by us, are involved.     

In vitro and in vivo induction of effector T cells mediating DTH responses to a protein and a synthetic polypeptide antigen.

We have developed an in vitro system for the activation of T cells in order to get a better insight into the genetic and molecular mechanisms involved in the generation of delayed-type hypersensitivity (DTH) effector T cells. Low doses of fowl γ-globulin (FγG) as well as the synthetic polypeptide (T,G)-A-L were bound to splenic adherent cells and served as immunogens for the in vitro sensitization of lymphocytes. In parallel, (T,G)-A-L-specific T cells were activated in vivo in irradiated recipient mice. The ability of the in vitro- and in vivo-activated cells to mediate DTH responses was determined in naive recipient mice by the radioisotopic ear assay. Twenty to thirty × 10⁶ “educated” cells were sufficient to elicit significant DTH responses. Irradiation of the spleen cells prior to their transfer resulted in higher responses. The DTH reactivity was transferable by nylon wool-enriched T cells but not by a Thy 1.2-depleted population indicating the T-cell dependency of the response. The in vitro and in vivo antigen-activated T-cell population exhibited also helper-cell activity as determined by their cooperation with B cells in adoptive transfer experiments.     

Tolerance to hepatitis B antigen: a hypothesis for its termination with 'immune-RNA'

Immunologic tolerance to hepatitis B antigen, evidenced by a lack of specific cellular and humoral immune response to HB_sAg, produces a chronic carrier state which serves as an epidemiologic reservoir for the transmission of viral hepatitis type B. It is proposed that cell-mediated immunity transferred with immune-RNA may serve to terminate immunologic tolerance to hepatitis B antigen and abolish the carrier state. The efficacy and safety of ‘immune-RNA’ administration to chronic carriers can be validated by leukocyte migration inhibition techniques $\text{in vitro}$ and in HB_sAg positive chimpanzees $\text{in vivo}$.     

Requirement for BSF-1 in the induction of antigen-specific B cell proliferation by a thymus-dependent antigen and carrier-reactive T cell line

We report here a role of B cell stimulatory factor 1 (BSF-1) in the induction of antigen-specific proliferation of affinity-purified small B lymphocytes by a thymus-dependent antigen and a carrier-reactive T cell line. By using an ovalbumin-reactive T cell line (designated Hen-1), which does not produce BSF-1 following activation, it was possible to demonstrate that the antigen-specific proliferative response of trinitrophenyl (TNP)-binding B cells to TNP-ovalbumin required exogenous BSF-1 in addition to direct interaction with irradiated Hen-1 T cells. The activation obtained under these conditions was highly efficient, being sensitive to antigen doses as low as 0.001 microgram/ml. The addition of saturating amounts of BSF-1 did not alter the antigen-specificity or the requirements for hapten-carrier linkage or major histocompatibility complex-restricted T-B interaction in this system. The involvement of BSF-1 was confirmed by the ability of 11B11 anti-BSF-1 antibody to specifically suppress the response of TNP-binding B cells to TNP-ovalbumin, BSF-1, and irradiated Hen-1 T cells. Finally, this response was augmented by addition of the monokine interleukin 1. These data indicate that the proliferative response of small B cells to the thymus-dependent antigen and carrier-reactive T cell line used in our experiments can be regulated by the same factors that govern B cell proliferation induced by thymus-independent type 2 antigens or anti-IgM antibodies.     

Force microscopy of nonadherent cells: a comparison of leukemia cell deformability

Atomic force microscopy (AFM) has become an important tool for quantifying mechanical properties of biological materials ranging from single molecules to cells and tissues. Current AFM techniques for measuring elastic and viscoelastic properties of whole cells are based on indentation of cells firmly adhered to a substrate, but these techniques are not appropriate for probing nonadherent cells, such as passive human leukocytes, due to a lateral instability of the cells under load. Here we present a method for characterizing nonadherent cells with AFM by mechanically immobilizing them in microfabricated wells. We apply this technique to compare the deformability of human myeloid and lymphoid leukemia cells and neutrophils at low deformation rates, and we find that the cells are well described by an elastic model based on Hertzian mechanics. Myeloid (HL60) cells were measured to be a factor of 18 times stiffer than lymphoid (Jurkat) cells and six times stiffer than human neutrophils on average (E(infinity) = 855 +/- 670 Pa for HL60 cells, E(infinity) = 48 +/- 35 Pa for Jurkat cells, E(infinity) = 156 +/- 87 for neutrophils, mean +/- SD). This work demonstrates a simple method for extending AFM mechanical property measurements to nonadherent cells and characterizes properties of human leukemia cells that may contribute to leukostasis, a complication associated with acute leukemia.     

Conversion of type III pneumococcal polysaccharide low responders to high responders by immunization with a thymus-dependent form of antigen

C57BL/Ks mice immunized with 0.6 μg Type III pneumococcal polysaccharide (S3) or with 10⁹ S3 conjugated sheep erythrocytes (S3-SRBC) produced 5–7 times fewer S3-specific plaque-forming cells than similarly immunized BALB/c mice. However, when mice were primed with the SRBC carrier prior to challenge with S3-SRBC the low responder C57BL/Ks mice responded as well as the high-responder BALBc strain. The cell activated by the carrier priming was shown to be a thymus-derived (T) cell and the antibody produced by primed mice was mercaptoethanol sensitive (presumably IgM). Nonspecific T cell activation by unrelated antigens did not enhance C57BL/Ks responses to the same degree as specific carrier priming. These findings are discussed in relation to the possible cellular basis for genetic control of the S3 immune response.     

B cells as antigen-presenting cells: antibody production in vitro against a T-dependent antigen

It was examined whether B cells can serve as antigen-presenting cells (APC) in the antibody response to a T-dependent antigen, trinitrophenyl-ovalbumin (TNP-OVA). B cells purified from mice primed with TNP (TNP-B cells) responded to TNP-OVA in the presence of purified T cells sensitized with OVA (OVA-T cells). OVA-T cells required the addition of APC to proliferate in response to TNP-OVA. APC activity of TNP-B cells in the T-cell proliferation was abolished by 4000 R irradiation. Our experiments also revealed that an antibody response requires more adherent cells than the T-cell proliferation. These results indicate that adherent cells possibly accompanying the T- and B-cell preparations were at a less than functional level. There was genetic restriction between T and B cells for the antibody response. B cells in the pellet fraction of 70% Percoll density sedimentation behaved similarly to the unfractionated TNP-B cells in the antibody response. A T-cell clone specific for human gamma-globulin (HGG) also induced an anti-TNP antibody response in B cells from unprimed mice in the presence of TNP-HGG. These results suggest that B cells are able to elicit an antibody response to a T-dependent antigen in the presence of carrier-primed T cells without the participation of macrophages.     

Plaque-forming cells fater in vitro stimulation with theta-AKR antigen

Spleen cells of inbred mice strains carrying θ-C3H allele have been cultured in the presence of AKR thymus cells and their in vitro primary PFC response against thymic alloantigen θ-AKR was studied.The responses of a magnitude which was comparable with that obtained in previous in vivo experiments were obtained 4 days after stimulation. The strain-dependent variability of the magitude of anti-θ-AKR responses was observed in vitro. RR and C58/J spleen cells produced much more PFC than C57BL/6J and DBA/2J spleen cells. This was in agreement with previous in vivo studies on the genetic control of the anti-θ AKR responses.In the absence of AKR thymus cells, spleen cells of high responders, RR, developed in vitro PFC which released antibodies lytic to AKR thymus cells. Their number was ten-times lower than in stimulated cultures. Spleen cells of all strains tested produced also small numbers of PFC secreting antibodies against θ-identical allogenic thymus cells and even to syngenic thymus cells.     

Expression of IgG memory response in vitro to thymus-dependent and thymus-independent antigens

A model is described in which expression of IgG secondary antihapten responses of large magnitude can be initiated in vitro without resorting to in vivo boosting prior to culture. The number of IgG plaque-forming cells (PFC) is frequently as much as 100-fold greater than that of IgM PFC. Spleen cells from mice primed with trinitrophenylated keyhole limpet hemocyanin (TNP-KLH) several months earlier are stimulated in vitro to produce an anti-TNP plaque-forming cell response 7–10 days later. The in vitro IgG response can be elicited with either a thymus-dependent antigen (TNP-KLH) or thymus-independent antigens (TNP-T₄ bacteriophage or DNP-dextran). The kinetics of the responses to these two forms of antigen differ in that the thymus-independent response peaks two days earlier. The IgG response to both forms of antigen requires the presence of 2-mercaptoethanol (2-ME) even though macrophages are not depleted prior to culture. In the absence of the reducing agent both thymus-dependent and thymus-independent IgG responses were diminished ≥90%. The magnitude of the response to thymus-independent antigens emphasizes the ability of these materials to elicit IgG expression in memory B cells provided optimal conditions for memory development and in vitro expression exist.     

Tolerance rendered by neonatal treatment with anti-idiotypic antibodies: induction and maintenance in athymic mice

Congenitally athymic (nude) mice of BALB/c background and their littermates were rendered unresponsive to the phosphorylcholine (PC) determinant by neonatal injection of anti-idiotypic antibodies. The kinetics of recovery from unresponsiveness were found to be similar for both groups when measured over a 20-week period. Spleen cells from suppressed, athymic mice did not respond to PC and were able to inhibit the response of normal cells to PC when tested in vitro. These results indicated that a population of specific suppressor cells, which may be responsible for induction and/or maintenance of unresponsiveness, can be generated in the absence of a thymic environment.     

Effect of antimicrobial surface-active agents on the immune response to thymus-dependent antigen in mice

The study showed that miramystin, a cationi surfactant, was an immunostimulant inducing an increase in humoral and cellular immunity in response to sheep red blood cells. The observed dose-dependent stimulating effect of miramystin on antibody production and development of DTH recommends its use as an immunologic adjuvant in the laboratory practice. It also indicated to the prospects in investigation of immunologic adjuvant properties of other preparations belonging to surface-active substances.     

Tolerance and contact sensitivity to DNFB in mice. VII. Functional demonstration of cell-associated tolerogen in lymph node cell populations containing specific suppressor cells.

Adoptive tolerance to contact sensitivity to DNFB is mediated by suppressor T cells. These cells are induced by iv injection of the hapten DNB-SO₃. Experiments were carried out to investigate the question of simultaneous transfer of tolerogen (DNB-SO₃ or its conjugation product DNP) with the suppressor cells. The results showed that tolerant lymph node cells pretreated in vitro with anti-TNP serum before transfer were unable to induce unresponsiveness to DNFB. Tolerant cells treated with either anti-TNP serum which had been passed over a TNP-affinity column or with polyvalent anti-immunoglobul in serum were not inhibited. These results functionally demonstrate that LN cell populations containing DNFB suppressor cells have accessible hapten (e.g., DNP) associated with their membrane, which is necessary for induction of adoptive tolerance. The hapten (tolerogen) appears to be bound directly to the cell surface rather than as an immune complex.