Ontogeny of B-lymphocyte function with respect to the heterogeneity of antibody affinity-1,2.

Neonatal liver or adult spleen was used as a source of B-lymphocytes in reconstituting lethally irradiated, syngeneic mice. Recipients were all given excess adult, syngeneic thymus cells and were immunized with dinitrophenylated bovine gamma globulin. The distribution of avidities of plaque-forming cells produced by immunized recipients of neonatal liver was highly restricted in comparison with animals reconstituted with adult spleen indicating a restriction of B-lymphocyte heterogeneity in the neonatal mouse.     

Ontogeny of B-lymphocyte function. VII. Effect of cyclic AMP on the heterogeneity of the antibody produced by B lymphocytes from immature donors.

Liver cells from neonatal mice were incubated for 1 hr with or without cyclic AMP before they were used as the source of B lymphocytes to reconstitute lethally irradiated mice. All recipients also received 1 × 10⁸ adult thymus cells and were immunized with DNP-BGG. Mice reconstituted with neonatal B cells which had been incubated with cyclic AMP produced an anti-DNP response which was of greater magnitude, higher affinity and greater heterogeneity of affinity than that produced by mice reconstituted with neonatal B cells incubated without cyclic AMP. The possibility that cyclic AMP plays a role in the differentiation of B-cell function is suggested.     

T cell control of the antibody response to the T-independent antigen, DAGG-Ficoll

C57BL/6J nu/nu mice respond to the type 2 TI antigen DAGG-Ficoll, but not to the TD antigen SRC. A comparable difference can also be seen in vitro, but only at high spleen cell density and in the presence of selected batches of FBS. At low spleen cell density and in the absence of FBS, the DAGG-Ficoll-induced B cell response is strictly dependent on soluble helper factors or cloned specific helper T cells. The B cell response so induced requires that the T cell-depleted spleen cells be compatible in the I-A subregion of the H-2 complex. These helper factors, induced by antigen in an I-A-restricted T cell-macrophage interaction, provide helper for T cell-depleted spleen cells irrespective of their H-2 haplotype. Under conventional culture conditions, the stringent requirement for helper factors in the in vitro response to DAGG-Ficoll is obscured by FBS. In vitro culture of low numbers of spleen cells, in serum-free medium instead of FBS, provides a sensitive assay for helper factors. We have compared the helper activity for a B cell response to SRC or DAGG-Ficoll as provided by antigen-induced supernatants of various individual EA-specific T cell clones. There was a remarkable and consistent heterogeneity among individual T cell clones: their helper activity in the response to TI and TD antigens did not correlate, nor was there any correlation between helper activity and antigen-induced TCGF (interleukin 2) activity.     

Ontogeny of B-lymphocyte function: XII. Evidence that the ability to generate memory cells precedes the ability to produce antibody-secreting cells

Using a cell transfer system it was shown that lymphoid cells of B.C-9 mice mature to be able to generate a primary response of high magnitude and high affinity to dinitrophenylated γ-globulin at about 4 weeks of age. Irradiated mice reconstituted with lymphoid cells from B.C-9 or C57BL/6 donors younger than 4 weeks of age fail to produce high-affinity plaque-forming cells and their serum antibody responses were of low magnitude. Nevertheless such recipients give adult-like, high-affinity, high-magnitude secondary responses when assayed at both the plaque-forming cell and serum antibody levels. The antibody produced by the reconstituted mice, in both the primary and secondary responses, was shown to be of donor cell origin in transfers between allotype congenic pairs. Thus, naive, immature lymphoid cells, although not capable of giving rise to high-affinity antibody-secreting cells in a primary response, are capable of giving rise to high-affinity memory B cells which in turn can give an adult-like, high-affinity secondary response following boosting.     

X-ray snapshots of the maturation of an antibody response to a protein antigen

The process whereby the immune system generates antibodies of higher affinities during a response to antigen (affinity maturation) is a prototypical example of molecular evolution. Earlier studies have been confined to antibodies specific for small molecules (haptens) rather than for proteins. We compare the structures of four antibodies bound to the same site on hen egg white lysozyme (HEL) at different stages of affinity maturation. These X-ray snapshots reveal that binding is enhanced, not through the formation of additional hydrogen bonds or van der Waals contacts or by an increase in total buried surface, but by burial of increasing amounts of apolar surface at the expense of polar surface, accompanied by improved shape complementarity. The increase in hydrophobic interactions results from highly correlated rearrangements in antibody residues at the interface periphery, adjacent to the central energetic hot spot. This first visualization of the maturation of antibodies to protein provides insights into the evolution of high affinity in other protein-protein interfaces.     

Ontogeny of B-lymphocyte function. XIV. Maturation of the capacity of B cells to re-express surface immunoglobulin after treatment with anti-immunoglobulin

The maturation of the ability of the B-cell population to re-express surface immunoglobulin (sIg) after its removal by treatment with rabbit anti-mouse immunoglobulin (RAMIg) was studied in LAF1, C57BL/6, and C57L mice. As demonstrated by previous workers, the B-cell population from immature mice failed to re-express sIg after treatment with RAMIg. We have shown that the age at which the B-cell population acquires the capacity to re-express sIg is different in different strains and that the order in which the B-cell population of the different strains acquires the capacity to re-express sIg is different from the order in which their B-cell populations acquire the capacity to produce high-affinity antibodies. This suggests that these represent distinct differentiation events in the development of the B-cell population. In all of the strains studied the maturation of the capacity to re-express sIg occurred in two steps. After the first maturation step the B-cell population was able to re-express sIg after treatment with RAMIg for 1 hr but did not re-express sIg after treatment with RAMIg for 24 hr. After the second maturation step the B-cell population could re-express sIg even after 24 hr treatment with RAMIg. It has been suggested by previous workers that the inability of the immature B-cell population to re-express sIg could represent one of the mechanisms responsible for the development of B-cell self-tolerance. It is suggested here that the existence of a period during which cells become tolerant only upon prolonged exposure to antigen could protect the developing B cells from becoming unresponsive to transiently experienced foreign antigens but still permit them to become tolerant to self antigens which are continuously present.     

Antigen presentation by hapten-specific B lymphocytes. IV. Comparative ability of B cells to present specific antigen and anti-immunoglobulin antibody

The ability of trinitrophenyl (TNP)-binding murine B lymphocytes to present native rabbit IgG (RGG), TNP-modified RGG, and rabbit anti-mouse Ig (RAMG) to an Ia-restricted, RGG-specific helper/inducer T cell clone was compared. By three independent assays (lymphokine secretion, T cell proliferation, and B cell differentiation), TNP-RGG was presented at 10(2)- to 10(3)-fold lower concentrations than RGG, and RAMG at 10(2)- to 10(3)-fold lower concentrations than TNP-RGG. The available data suggest that the efficiency of antigen presentation is dependent primarily on the avidity of binding of a ligand to B cell surface Ig and/or the extent of subsequent endocytosis (modulation). Despite the observed quantitative differences between anti-Ig (RAMG) and specific antigen (TNP-RGG), these results demonstrate that qualitatively both are essentially similar in their ability to mediate specific T-B interactions. Thus, anti-Ig antibodies are valid models for analyzing cognate interactions between antigen-specific B and helper T lymphocytes.     

Measurement and comparison of the proliferative and antibody response of neonatal, immature and adult murine spleen cells to T-dependent and T-independent antigens.

Mouse spleen cell antigenic responses to the thymic-dependent antigen sheep red blood cells (SRBC), and the thymic-independent antigens, E. Coli lipopolysaccharide (LPS) and pneumococcal polysaccharides Type I and II (SI, SII) were studied as as a function of age, employing both in vitro spleen cell stimulation and plaque-forming cell (PFC) assay systems. Primary spleen cell proliferative and PFC responses to SRBC, were either absent or meager in comparison to adult (8–12 weeks) values for the first 3 weeks of life. Thereafter responses rose achieving adult values between 4 and 8 weeks of age. The inability of young mice to respond to SRBC was not because of a different immunizing dose requirement for SRBC, since immunization with SRBC over a 200-fold range did not enhance their capability to respond. Also, addition of adherent cells or macrophages from adult mice did not enhance the immune responses of young mice. Furthermore, immunization of 2–4 week old mice with SRBC inhibited the secondary response to SRBC. In contrast, young murine spleen cell proliferative and PFC responses to SI, SII, and LPS were approximately the same as the adult by 7–14 days of life. These data suggest that B-cell immunologic activity, as measured by immunologic assays utilized in this study, develops much earlier than does T-cell responsiveness.     

IL-10 augments antibody production in in vitro immunized lymphocytes by inducing a Th2-type response and B cell maturation

An in vitro immunization (IVI) protocol enables antigen specific antibody production from L-Leucyl-L-Leucine methyl ester (LLME)-treated human peripheral blood lymphocytes (PBL) upon antigen stimulation in the presence of IL-2, IL-4, and muramyl dipeptide. In the course of our studies, we have found that IL-10 added at the antigen sensitization significantly augmented antibody production level from the LLME-treated PBL. In the present study, we tried to demonstrate the role of IL-10 in the augmentation of antibody production in an IVI protocol by clarifying the cytokine expression profiles in CD4(+) and CD8(+) T cells. The results showed that IL-10 skewed the Th1/Th2 balance to Th2-type responses by suppressing Th1-type cytokine production and augmenting Th2-type cytokine production in CD4(+) and CD8(+) T cells, as well as in CD19(+) B cells. Furthermore, IL-10 augmented the expression of CD38, an antigen marker of plasma cells, on B cells, which clearly indicates that IL-10 promoted differentiation and maturation of B cells in an IVI protocol. These results indicate that IL-10 plays an important role in setting the cellular milieu to produce antibodies in an IVI protocol.     

LPS regulation of the immune response: suppression of immune responses to orally administered T-independent antigen

The regulation of immune responses to gastrically administered TI antigens has been investigated, and the characterization of a regulatory cell population has been performed. Intragastric administration of TNP-haptenated homologous erythrocytes (TNP-MRBC) induced splenic IgM anti-TNP PFC responses in LPS nonresponsive C3H/HeJ mice that were higher than those in LPS-responsive C3H/HeN mice and similar to those noted in athymic (nu/nu) C3H/HeN animals. The simultaneous intragastric administration of LPS with TNP-MRBC augmented immune responses in a manner similar to that previously reported for parenterally administered LPS and antigen. Further, LPS-induced augmentation of TNP-MRBC responses was greater in athymic mice. These findings were substantiated using in vitro spleen cultures. Intragastric challenge with a 2nd TI antigen, TNP-LPS, induced approximately 8-fold higher splenic anti-TNP PFC responses in athymic C3H/HeN mice compared with those in euthymic littermates. By admixture of B and T cell populations, it was demonstrated that the host responsiveness to TNP-LPS was negatively regulated by suppressor cells. Suppressive activity resided in a Thy 1.2-bearing, irradiation-resistant, nylon wool-nonadherent cell population. These cells could be demonstrated in spleen and Peyer's patches from young or old LPS-responsive C3H/HeN mice, but not in tissues from LPS nonresponsive C3H/HeJ mice. The specificity of the regulator cells was not limited to TNP-LPS responses, since immune responsiveness to another TI antigen, TNP-dextran, was also under the control of this cell population. These studies confirm the TI nature of TNP-MRBC and indicate that immune responses to gastrically administered antigens such as TNP-LPS, TNP-dextran, and possibly TNP-MRBC are negatively regulated by a suppressor T cell population. A role for endogenous LPS in the generation of regulator cells and the effect of these cells on host responses to gut-derived antigens is discussed.     

Depletion of mouse alpha beta T cell antigen receptor bearing lymphocytes by neonatal monoclonal antibody treatment.

Neonatal treatment with a monoclonal antibody specific for the alpha beta TCR results in mice with a long term, severe depletion in the number of alpha beta T cells in the periphery. Significant numbers of T cells reappear in the periphery about age 65 days, but these cells tend to lack expression of CD4 or CD8. Splenocytes of antibody-treated mice are less sensitive to mitogen stimulation or stimulation with MHC allogeneic cells. The level of serum IgG but not IgM was decreased by the treatment. Anti-alpha beta TCR antibody treatment decreased single-positive T lymphocytes that express high levels of the CD3/alpha beta TCR complex from the thymus, suggesting that the treatment could act in part by affecting negative selection of alpha beta TCR+ thymocytes. This treatment does not, however, detectably affect either the homing or the numbers of gamma delta T cells which are abundant in the intestinal epithelium, but which remain a minor population in the spleen and lymph nodes. This supports the hypothesis that gamma delta T cells are developmentally autonomous from alpha beta T cells. These mice provide an excellent model system for assessing the developmental and functional role of gamma delta T lymphocytes in vivo.     

Modulation of the B cell response to T-independent antigens by prostaglandins: evidence for effector system cross-talk.

The extracellular environment is a major factor in determining the responsiveness of a cell to particular stimuli. For example, E series prostaglandins suppress B cell responses to T-independent antigens, mitogen stimulation of DNA synthesis and proliferation, and the primary immune response. We investigated the effects of prostaglandins on the intracellular signals generated by receptor-coupled effector systems in B lymphocytes. Pretreating splenocytes from athymic nude mice with forskolin, PGE1, or PGE2 decreased the magnitude of anti-IgM-induced changes in cytosolic free [Ca2+]. Addition of 8-Br-cAMP, forskolin, PGE1, or PGE2 following stimulation with anti-IgM resulted in a decrease in the intracellular calcium signal measured by fluorescence-activated cell sorting using Indo-1 as a Ca2+ indicator. This decrease was not a result of an inhibition of influx across the plasma membrane. Thus activation of adenylate cyclase by prostaglandins modifies the generation of signals by phosphoinositidase C. This effector system cross-talk between adenylate cyclase and phosphoinositidase C is consistent with and may account for the inhibitory effects of prostaglandins in B cell responses.     

Suppression of anti-hapten antibody responses by hapten-specific cytolytic T lymphocytes: role of I-A-associated antigen on target B lymphocytes

Cytolytic T lymphocytes (CTL) specific for 2,4,6-trinitrophenyl (TNP) determinants suppress the effector phase of a secondary anti-TNP antibody responses of murine syngeneic spleen cells in vitro. The cells mediating this suppression are hapten-specific, H-2-restricted, and possess properties typical of CTL. Moreover, the targets of the suppression appear to be antigen-primed B lymphocytes that are recognized by CTL via soluble antigen bound noncovalently to their Ig receptors. The effect of the CTL can be blocked by the addition of monoclonal antibodies directed against I-A molecules but not I-E or H-EK-encoded molecules on the target B cells, even in strain combinations in which the CTL-B cell interaction is restricted only by the H-2K and I regions of the MHC. This result suggests that B lymphocyte-bound antigen tends to associate preferentially with I-A rather than H-2K/D-encoded determinants, and that the suppressive effect of the CTL population is attributable to the minor subset that recognizes hapten-modified Ia antigens. These findings are also discussed in terms of the possible immunoregulatory function of Ia-restricted CTL.     

The proliferative response of human lymphocytes to antigen is suppressed preferentially by lymphocytes precultured with the same antigen.

Human blood lymphocytes activated in vitro with antigen to which the donor is reactive are capable of suppressing the secondary proliferative response of autochthonous fresh cells to antigen. Both antigen-specific and antigen-nonspecific suppression can be detected in each experiment. These suppressor cells act by decreasing the number of lymphocytes entering the proliferative response rather than by slowing or otherwise inhibiting ongoing proliferation. The suppressor cells must be added soon after fresh cells are stimulated with antigen to be effective, but the suppressor cells themselves need not proliferate to exert their effect. Suppressor cells are optimally effective when added in numbers equal to those of the responding population, but still exert a significant effect at one-eighth that number.     

B cell maturation factor (BMF): a lymphokine or family of lymphokines promoting the maturation of B lymphocytes

Two in vitro B cell tumor lines have been used to characterize and partially purify a lymphokine, or family of lymphokines, from monoclonal helper T cell immune response supernatants. These lymphokines induce the pre-B-like 70Z/3 tumor cell to synthesize Ig L chains and express complete Ig molecules on its cell surface, and cause the mature B cell-like WEHI-279 tumor cell to increase its ratio of secretory to membrane mu production, begin high rate Ig secretion, and then die. Most of the activity responsible for these changes co-purifies during five different separation procedures, implying the existence of a discrete molecule or closely related class of molecules able to mediate all of these effects. The molecules active in these systems appear distinct from the other lymphokines IL 1, IL 2, G/M-CSA, TRF, IFN, BCGF, and the activity variously termed IL 3/BPA/PSF/HCGF/MCGF, etc. We call these B cell-differentiating molecules BMF, or B cell maturation factor(s). The BMF molecules are mildly acidic (pI 5 to 6 in various conditions), extremely hydrophobic, probably heterogeneously glycosylated glycoproteins, with an apparent m.w. of 50,000 to 55,000 by gel permeation chromatography and 16,000 by SDS-PAGE. BMF has been purified approximately 3000-fold by three sequential chromatographic steps, with the use of the B tumor line assay systems. BMF molecules thus purified also cause normal resting splenic B cells to mature to the state of active Ig secretion.     

Role of self carriers in the immune response and tolerance. IV. Active T cell suppression in the maintenance of B cell tolerance to a "T-independent" antigen.

Previous studies indicated that T cells are required for tolerance induction by hapten-modified syngeneic spleen cells (TNP-SC) in vivo. The role of T cells in the maintenance of this unresponsive state has been examined herein. By three criteria--limiting dilution precursor analysis, removal of T cells by anti-Thy-1 + C, and direct mixing experiments--we show that T cells are required for the continued suppression of the B cell response to the T-independent antigen, TNP-POL. Suppressor cells can also be induced by TNP-teratoma cells, which lack detectable H-2 antigens. Both anti-Ly-1 + C and anti-Ly-2 + C treatment reversed suppression induced by TNP-SC. These results demonstrate that normal B cell reactivity is present in the spleens of mice rendered tolerant by haptenated self, but that Ly-1,2,3 or Ly-1 + Ly-2,3 suppressor T cells prevent their responsiveness.