B cell tolerance induced by polymeric antigens. VI. Kinetics and reversibility of the inhibition of antibody-forming cells by antigen.

Injection of mice already making antibodies to 2,4-dinitrophenylated (DNP) Ficoll with tolerizing doses of DNP-pneumococcal polysaccharide (DNP-lys-S3) markedly inhibits the secretion of anti-DNP antibodies by IgM antibody-forming cells. The present study shows that the degree of inhibition depends not only on the dose of DNP-lys-S3 but also on the duration of exposure to antigen. DNP-lys-S3 was detectable on the surface of antibody-forming cells at a time when their rate of secretion was unimpaired, thus suggesting that the inhibition involves intracellular events subsequent to the binding of antigen to the cell membrane. The inhibition was reversible if antibody-forming cells were exposed to antigen for 24 h, and then cultured for 18 h in its absence, but became irreversible if the treatment period was extended to 48 h. The relevance of this model of inhibition of lymphocyte function by antigen to possible mechanisms of B cell tolerance is discussed.     

B cell tolerance induced by polymeric antigens. V. Different avidities of primed and virgin precursor cells for paucivalent antigen.

We have compared the avidity of virgin and primed hapten-specific precursor cells for DNP-lys-S3 (dinitrophenylated type 3 pneumococcal polysaccharide) by measuring its capacity to prevent them from binding highly radioactive DNP-hemocyanin, and thereby protect them from committing hapten-specific "suicide". Moderately substituted conjugates (e.g. DNP-lys2.7S3) protected virgin and memory B cells impartially. In contrast, DNP-lys0.6S3 protected memory cells efficiently, but protected virgin cells only at considerably higher concentrations. These results are consistent with previous evidence - from this and other systems - that there is a higher density of receptors on primed than on unprimed B cells - the significance of which is discussed.     

B cell tolerance induced by polymeric antigens. II. Effects of tolerance on hapten-binding lymphocyte levels in primary and secondaryantibody responses

Tolerogenic doses of hapten [2,4-dinitrophenyl (DNP)]-coupled type 3 pneumococcal polysaccharide (DNP-lys_2.5?S3) totally abolished the anti-DNP rosetteforming cell (RFC) response to primary immunization with DNP-hemocyanin in mice, while lightly substituted antigen (DNP-lys_0.6?S3) had little effect. Both antigens suppressed secondary anti-DNP RFC responses to DNP-KLH. Limiting doses of DNP-lys-S3 preferentially suppressed antibody-secreting cell levels, and had less effect on RFC. DNP-lys_2.5?S3 was 500—1000-fold more potent in “blockading” primary RFC in vitro than DNP-lys_0.6?S3, whereas both antigens were equally effective in blocking secondary RFC. These results suggest that the sensitivity of primed B lymphocytes to inactivation by DNP-lys-S3 is related to their high avidity for antigen. Furthermore, this appears to be largely due to a high density of immunoglobulin receptors on primed cells, since the affinitiesof primary and secondary RFC for monovalent hapten were indistinguishable. Treatment of primarily immunized mice with DNP-lys_2.5?S3 2 h before assay abolished 90 % of RFC. Therefore, the reduction in RFC levels in tolerant mice may be due to cellular blockade by persisting tolerogen. However, it seems unlikely that simple blockade of antigen-reactive cells is the sole mechanism operative in this system.     

B cell tolerance induced by polymeric antigens. III. Dissociation of antibody formation and memory generation in tolerant mice

Hapten (2,4-dinitrophenyl (DNP))-substituted type 3 pneumococcal polysaccharide (DNP-lys-S3) effectively suppresses both primary and secondary anti-DNP antibody responses to DNP-proteins. The present experiments show that tolerizing doses of DNP-lys-S3 have much less effect on B cell memory (re)generation in three distinct situations: (i) in serial transfers of DNP-protein-primed cells, (ii) in virgin mice tolerized before priming with DNP-hemocyanin, and (iii) in tolerized, hemocyanin-primed mice boosted with DNP-hemocyanin. Under some conditions tolerized mice developed 10-50% of normal memory in the absence of significant antibody formation. Isoelectric focusing analyses revealed that many B cell clones proliferate in tolerant mice, but produce very little antibody until transferred to further hosts. Clones that escape suppression in partially tolerant mice do not appear to be resistant to DNP-lys-S3, when retested. Since there is independent evidence that the generation of B memory cells is less T cell-dependent than the development of antibody-forming cells, these data are best explained by assuming that DNP-lys-S3 blocks lymphocyte cooperation. This would be expected to preferentially suppress antibody formation, and to spare memory generation.     

B cell tolerance induced by polymeric antigens. II. Effects of tolerance on hapten-binding lymphocyte levels in primary and secondary antibody responses.

Tolerogenic doses of hapten [2,4-dinitrophenyl (DNP)]-coupled type 3 pneumococcal polysaccharide (DNP-lys2.5-S3) totally abolished the anti-DNP rosette-forming cell (RFC) response to primary immunization with DNP-hemocyanin in mice, while lightly substituted antigen (DNP-lys0.6-S3) had little effect. Both antigens suppressed secondary anti-DNP RFC responses to DNP-KLH. Limiting doses of DNP-lys-S3 preferentially suppressed antibody-secreting cell levels, and had less effect on RFC. DNP-lys2.5-S3 was 500--1000-fold more potent in "blockading" primary RFC in vitro than DNP-lys0.6-S3, whereas both antigens were equally effective in blocking secondary RFC. These results suggest that the sensitivity of primed B lymphocytes to inactivation by DNP-lys-S3 is related to their high avidity for antigen. Furthermore, this appears to be largely due to a high density of immunoglobulin receptors on primed cells since the affinities of primary and secondary RFC for monovalent hapten were indistinguishable. Treatment of primarily immunized mice with DNP-lys2.5-S3 2 h before assay abolished 90% of RFC. Therefore, the reduction in RFC levels in tolerant mice may be due to cellular blockade by persisting tolerogen. However, it seems unlikely that simple blockade of antigen-reactive cells is the sole mechanism operative in this system.     

B cell tolerance induced by polymeric antigens. I. Comparison of thedose and epitope density requirement for inactivation of primed and unprimed B cells in vivo

Hapten [2,4-dinitrophenyl (DNP)]-specific tolerance was induced in nonimmun or DNP-hemocyanin (DNP-KLH) primed mice by administering hapten-conjugated type 3 pneumococcal polysaccharide (DNP-lys-S3). The dose of DNP-lys_2.5?S3 required to suppress the primary anti-DNP antibody responses was appaprox-imately ten times higher than that required to suppress the secondary response Large doses of lightly substituted antigen (DNP-lys_0.6?S3) had no effect on prim-ary antibody responses, while small doses of this conjugate suppressed 90—95 % of the secondary response. The conclusion from this (presumably B cell) tolerance model is that B lymphocytes “mature” in their susceptibility to tolerization following primary contact with immunogen, since primed cells are inactivated by lower doses of tolerogen, and by tolerogen with lower epitope density, than no immune B cells. These and other data suggest that the tolerance threshold of B lymphocytes is related to their state of differentiation, and especially to their antigen-binding characteristics.     

B cell tolerance induced by polymeric antigens. I. Comparison of the dose and epitope density requirements for inactivation of primed and unprimed B cells in vivo.

Hapten [2,4-dinitrophenyl (DNP)]-specific tolerance was induced in nonimmune or DNP-hemocyanin (DNP-KLH) primed mice by administering hapten-conjugated type 3 pneumococcal polysaccharide (DNP-lys-S3). The dose of DNP-lys2.5-S3 required to suppress the primary anti-DNP antibody responses was approximately ten times higher than that required to suppress the secondary response. Large doses of lightly substituted antigen (DNP-lys0.6-S3) had no effect on primary antibody responses, while small doses of this conjugate suppressed 90-95% of the secondary response. The conclusion from this (presumably B cell) tolerance model is that B lymphocytes "mature" in their susceptibility to tolerization following primary contact with immunogen, since primed cells are inactivated by lower doses of tolerogen, and by tolerogen with lower epitope density, than nonimmune B cells. These and other data suggest that the tolerance threshold of B lymphocytes is related to their state of differentiation, and especially to their antigen-binding characteristics.     

Lymphoma models for B cell activation and tolerance. IV. Growth inhibition by anti-Ig of CH31 and CH33 B lymphoma cells

CH31 and CH33 are B cell lymphomas whose growth in vitro is inhibited by anti-Ig reagents, including both polyclonal and monoclonal anti-mu antibodies, and an anti-idiotype antiserum. Antibodies against class I or class II major histocompatibility complex antigens do not affect the growth of these cells. Inhibition is dependent on surface Ig cross-linking and does not require ligand binding to Fc receptors. Interestingly, the inhibition of growth by anti-mu is reversed in CH31 (but not CH33) by E. coli lipopolysaccharide. These lymphomas should provide excellent models to study the mechanisms of growth inhibition mediated by surface Ig cross-linking and the pathways of its reversal.     

Ontogeny of the antibody-forming cell line in mice. I. Kinetics of appearance of mature B cells

The development of functionally mature B cells in the liver and spleen of fetal and neonatal CBA mice has been followed. A mature B cell is defined as one giving rise to antibody-producing progeny within 2 days in culture. Very few such B cells are found during fetal life. After birth there is a rapid rise in the numbers reaching 1/3 of adult levels in the 7-day-old spleen.     

Depletion by monolayer binding of specific precursors of antibody-forming cells directed against cellular antigens

Eight cases of the recently reported ''primary mediastinal clear cell lymphoma of B-cell type'' (Möller et al., 1986) were examined immunohistologically for the expression of cytoplasmic and/or surface antigens of MHC class I and II with mAbs directed against framework determinants of HLA-A,B,C (W6/32; B9.12.1), HLA-DP,DR,DQ (2.06), -DQ (Leu 10; Tü22), -DR (Tü34) gene products, and with mAbs specific for beta 2-microglobulin (BBM-1) and the HLA-D associated invariant chain (Vic-Y1). Besides the reported Ig-deficiency, the neoplastic B-cells of 7/8 tumours have variable defects in MHC antigen expression. Three lack both class I and class II antigens, one tumour lacks class I antigens but expresses HLA-DQ and -DR on the majority of neoplastic cells, three others contain varying proportions of MHC-antigen deficient tumour cells. The expression of Ii is closely correlated with HLA-D(R) expression and its antigenic sites are strictly located in the cytoplasm. Against the background of current knowledge, the variable and occasionally severe defects in MHC antigen expression within the herein presented series of B-cell lymphomas suggest that this unusual feature might be another characteristic of a novel lymphoma type.     

Ontogeny of the antibody-forming cell line in mice. I. Kinetics of appearance of mature B cells.

The development of functionally mature B cells in the liver and spleen of fetal and neonatal CBA mice has been followed. A mature B cell is defined as one giving rise to antibody-producing progeny within 2 days in culture. Very few such B cells are found during fetal life. After birth there is a rapid rise in the numbers reaching 1/3 of adult levels in the 7-day-old spleen.     

Pathway of programmed cell death in HeLa cells induced by polymeric anti-cancer drugs

Synthesis of anticancer polymeric materials plus their biological applications is one of the most charming and active research areas in biological functional materials. However, the predominant mechanisms for controlling cancer cell viability are not yet clear. In this work, cell culture polymeric materials co-immobilized with death signal proteins interferon-γ (IFN-γ)/tumor necrosis factor-α (TNF-α) on the surface were prepared by photochemical method to develop an anticancer polymeric drug model. Various characterizations on the microstructures and compositions, including the Fourier transform infrared spectroscopy, UV absorption spectroscopy, fluorescence measurement, atomic force microscopy, and electron spectroscopy for chemical analysis, were performed. For addressing the biological applications, we investigated systematically the death pathways of HeLa cells attached onto the drug model by means of a series of cell-biology techniques. It was demonstrated that the IFN-γ plus TNF-α co-immobilized on the polymeric material surface exhibited more notable inhibitive effects than the free IFN-γ plus TNF-α, and the induced HeLa cells were mainly along apoptosis-like PCD with the translocation of EndoG from the cytoplasm to the nucleus. These findings indicate that the polymeric drugs with the co-immobilized IFN-γ plus TNF-α may offer significant potentials for therapeutic manipulation of human cervical cancer.     

Lymphoid cell apoptosis induced by trophoblastic cells: a model of active foeto-placental tolerance.

To test the hypothesis that CD95-L (Fas-L) present on trophoblastic cells plays a part in establishing foeto-placental tolerance by inducing apoptosis of immune defence cells, we cocultured trophoblasts with lymphoid cells and scored the frequency of cell death in these cultures. We prepared human trophoblastic cells from term placentas removed by C-section and placed them in culture for 48 h before introducing the lymphoid cells. We added Jurkat cells, a CD3 + lymphoid cell line, or purified T cells from human blood to the cultured trophoblasts and monitored apoptosis by electron microscopy and flow cytometry after TUNEL or annexin V labelling. The frequency of cell death in the CD3 + cell population was higher when the lymphoid cells were cocultured with trophoblastic cells than when they were cultured alone. This frequency increased with time but was reduced when anti-CD95-L antibodies were added to the culture medium. Cell death was less frequent in the lymphoid cell population when trophoblasts were replaced with human fibroblasts not expressing CD95-L.     

B cell positive selection by self antigens and counter-selection of dual B cell receptor cells in the peripheral B cell pools

The presence of B cells expressing two B cell receptors (BCR), described in BCR-transgenic, gene-targeted and normal mice, may represent an autoimmune hazard. We generated RAG-2-deficient mice bearing two complete rearranged immunoglobulin transgenes. In these mice most mature resting B cells express chains from the two transgenes. We studied selection of these dual receptor B cells in the presence of self antigens. In spite of the reduced surface density of the anti-self receptor, self-reactive B cells are deleted in the presence of membrane-bound self antigens. In contrast, the presence of soluble self antigen positively selects single receptor B cells expressing the self-reactive receptor. At the periphery these positively selected B cells down-regulate surface IgM expression and become unresponsive. A few dual receptor cells, however, escape tolerance induction. We examined the peripheral fate of the dual receptor B cells and showed that they are poorly selected into the activated B cell compartment and show a poor competitive capacity when in presence of populations of single receptor B cells. These results indicate that peripheral selection contributes to the very low frequencies of dual receptor B cells in normal mice and that multiple safeguard mechanisms operate to minimize the autoimmune hazard that allelically included B cells could represent.     

Normal tolerance characteristics of the antibody-forming cell precursors of the NZB mouse

Splenic PFC dose-response curves were measured in normal mice and in mice of the autoimmune NZB strain for the thymus-independent antigens pneumococcal polysaccharide type 3 (SIII) and bacterial levan. There were differences between the strains in the maximal response achieved to each antigen, but the level at which high dose tolerance occurred was the same in all the strains.     

Deregulation of mouse antibody-forming cells in vivo in cell culture by streptococcal pyrogenic exotoxin.

An unregulated, elevated rebound of antibody levels in rabbits was shown to follow late (10 to 15 days) after steptococcal pyrogenic exotoxin (SPE)-induced immunosuppression. Because of that result we have suggested that SPE acts by preferentially inhibiting a regulatory cell which normally limits the extent of full expression of antibody formation by B-cells. We are currently testing this hypothesis in mice. NIH (Swiss Webster) mice (+/+) or NIH (Swiss Webster) mice heterozygous (+/nu) for the mutant athymic nude gene and phenotypically normal showed an elevated plaque-forming cell (PFC) response to sheep erythrocytes (SE) late (10 to 15 days) after immunosuppressive SPE treatment similar to that described in rabbits. Homozygous nude mice (nu/nu) that are phenotypically athymic normally show a reduced early (4 day) PFC response to SE (a T-cell-dependent antigen) as compared with +/nu littermates or +/+ parent strain mice. This cryptic early 4-day response was improved by injection of purified endotoxin (a B-cell mitogen), but these relatively elevated nude PFC responses had decreased to normal control (SE only)nude PFC levels before 10 days. In similar SE-injected nude mice treated instead with SPE, no elevation at 4 days was observed and, more pertinently, the late (10 to 15 day) elevated rebound of PFC levels observed in normal response controls (+/nu or +/+) was not observed. Similar experiments were subsequently conducted in Marbrook-type spleen PFC cultures during periods of 12 days. The results of these experiments paralleled the in vivo results above, and in addition showed that SPE induced a large proliferation of either +/+ or +/nu cells (T-and B-cells) in culture but had no such effect on nu/nu cells (B-cells) in culture. Purified endotoxin, the Bcell mitogen, had a better sparing effect on nu/nu cells in this respect. These results are consistent with our premise that SPE inhibits preferentially the function of a regulator of the antibody response. The regulator appears to be a T-cell and is likely a suppressor T-cell.     

Generation of antibody dieversity. IV. Variation within single clones of antibody-forming cells developing in vivo

Previous experimental work demonstrated that clonal variation occurs in vitro. The present experiments were designed to test for clonal variation in vivo. B cells were transferred at limiting dilution, with antigen, into irradiated recipients. Seven days later spleens were assayed for plaque-forming cell (PFC) colonies. Control experiments showed that these PFC colonies were clones, that is, they were derived from a single B cell precursor. When the clones were analyzed for heterogeneity of the PFC population, using cross-reactivity on various mixtures of red blood cells as a method of detecting differences in antibody specificity, form 23–83 % of the clones contained variants. By adjusting the amounts of helper activity and antigen available to a developing clone, we have been able to influence this variation; high levels of help and/or antigen favour pure clones, while low levels of either produce mainly mixed clones.     

Receptor editing is the main mechanism of B cell tolerance toward membrane antigens

Self-reactive B cells specific for ubiquitous membrane-bound autoantigens are eliminated in the bone marrow by two mechanisms of tolerance: receptor editing and clonal deletion. However, the relative contributions of clonal deletion and receptor editing to B cell tolerance in a polyclonal B cell population have not been established. Here we show that tolerance toward a membrane antigen-reactive B cell clone acts by receptor editing with very minimal cell loss. The capacity of receptor editing to rescue almost all autoreactive B cells from deletion relies on the availability of multiple joining light chain gene segments as substrate for secondary immunoglobulin light chain gene rearrangement and is independent of the affinity of the autoantigen and the presence of non-autoreactive B cells. Our data further suggest that clonal deletion is a default pathway that functions only when receptor editing has been exhausted.     

Tolerance to class I major histocompatibility complex antigens in chicken B cell chimeras. Effect of B cell depletion on transferability of tolerance

B cells from bursa of Fabricius of newly hatched chickens are able to reconstitute the B cell compartment of chemically bursectomized chickens. The resulting B cell chimerism can be detected with monoclonal antibodies against donor B cell alloantigen. Chimeric chickens accept donor-type skin grafts and are unresponsive to donor major histocompatibility complex (MHC) antigens in graft-vs.-host splenomegaly assay and mixed lymphocyte reaction. To study the capability of B cells to induce tolerance to selected MHC antigens, we transplanted class I or total MHC-incompatible bursa cells into cyclophosphamide-treated recipients. The recipients of class I or total MHC-incompatible bursa cells were equally tolerant of donor-MHC antigens. To further analyze the mechanisms of tolerance to class I antigens vs. total MHC, spleen cells from tolerant chickens were transferred to irradiated, histocompatible secondary hosts. The secondary recipients were also unresponsive to bursa cell donor-strain MHC antigens. However, if the chimeric B cells were depleted before the spleen cell transfer, the transfer of tolerance to total MHC was severely inhibited. Instead, most recipients of B cell-depleted spleen cells tolerant of class I antigens were still tolerant of bursa cell donor MHC. Our results indicate differences in the transferability of tolerance to class I antigens vs. entire MHC, although in primary recipients of bursa cells the tolerance is similar. These data suggest that a mechanism that is not dependent on the presence of donor cell chimerism contributes to the maintenance of tolerance to donor class I antigens. The transfer of tolerance to total MHC disparity requires the presence of chimeric cells indicating that donor alloantigen expression is needed for induction of tolerance in the secondary hosts.