Bondada Subbarao

Properties of anti-Lyb-2-mediated B-cell activation and the relationship between Lyb-2 molecules and receptors for B-cell stimulatory factor-1 on murine B lymphocytes

The murine B-cell differentiation antigen Lyb-2 has been shown to be involved in B-lymphocyte activation and has been postulated by some to be related to a receptor for B-cell stimulatory factor I (BSF-1) (H. Yakura et al., J. Immunol. 137, 1475, 1986). Here we have demonstrated that monoclonal antibody (mAB) to Lyb-2 resembles BSF-1 in its ability to activate small resting B cells and enhancement of surface Ia. Anti-Lyb-2 antibodies bound B cells with very high avidity and were able to induce mobilization of cytosolic-free calcium. Anti-Lyb-2 mAB differs from BSF-1 in that BSF-1 but not anti-Lyb-2 is able to synergize with anti-mu in induction of B-cell proliferation. The relation between Lyb-2 molecules and BSF-1 receptors was tested in assays that measure binding of anti-Lyb-2 or BSF-1 in B cells and were found not to compete with each other. It appears that the two B-cell agonists anti-Lyb-2 and BSF-1 may exert their effects on B cells through different cell surface moieties as well as different intracellular pathways.

Early events in B-cell activation: Anti-Lyb2, but not BSF-1, induces a phosphatidylinositol response in murine B cells

Previous studies have indicated that the murine surface antigen Lyb2 is involved in an activation pathway that apparently does not involve the surface immunoglobulin receptor. As sIg has been shown to transduce its activation signal through the breakdown of phosphatidylinositol (PI), and since activation via Lyb2 does not involve sIg, it was of interest to determine if binding to Lyb2 generates a PI response. We have demonstrated that an allele-specific monoclonal antibody to Lyb2 (anti-Lyb2 mab), which has previously been shown to drive B cells into S, also activated PI metabolism in these cells. This activation occurred in a dose-dependent and allele-specific manner. Antibodies to other B-cell surface molecules such as Ia did not induce a PI response. The effect of anti-Lyb2 mab was always less in magnitude than that induced by anti-IgM, but the effects of the two antibody preparations were most comparable in larger, presumptively preactivated cells. To explore the issue that Lyb2 may represent a receptor for a growth factor, possibly the early-acting B-cell growth factor BSF-1, we studied the PI response to BSF-1 and the effect of BSF-1 on Lyb2-induced PI turnover. BSF-1 neither induced a PI response nor inhibited competitively the response induced by anti-Lyb2 mab.

Isolation and immunological characterization of a group of new B lymphomas from CBA mice

We have isolated and characterized a new series of B lymphoma which occurred spontaneously in a group of CBA/N mice that were transferred with spleen or lymph node cells from 24-month-old CBA/Ca mice. Tumor cell lines from six CBA/N mice that received spleen cells were rescued and designated as BKS-2, BKS-3, BKS-4, BKS-5, BKS-6, and BKS-7. Also, tumor cells from a recipient of lymph node cells were rescued and the resulting cell line was designated BKL. These tumor cells expressed membrane immunoglobulin (mu, kappa), major histocompatibility complex Class I and Class II molecules, B220, Lyb8, Fc receptors, J11d, interleukin 2 receptors, and Ly1. All of the tumors did not express the T cell specific markers Thy 1.2, L3T4, and Lyt2.1. They appeared to be clonal in origin, since they exhibited common rearrangements at both heavy and light chain immunoglobulin loci. Phenotypically, these lymphomas appeared to be analogous to immature B cells. Also, these lymphomas displayed different functional reactivities when treated with various B cell mitogens and growth factors in vitro. Anti-mu antibodies which normally induce B cell growth inhibited the proliferation of these lymphoma cells in vitro, whereas they responded to lipopolysaccharide, T cell-derived growth factors, and interleukin 5 by enhanced proliferation. These tumor cells expressed constitutively high levels of c-myc mRNA.

A specific defect in the proliferative capacity of B cells from old mice stimulated with autoreactive T cells

B lymphocytes from aged mice were found to be defective in their ability to proliferate in response to stimulation with an autoreactive T cell clone D1.4. The differentiative response leading to antibody secretion was also impaired in the auto D1.4 T cell-stimulated B cells from old mice in comparison to similarly stimulated B cells from young mice. The B cells from old mice were competent in activating the autoreactive T cells such that the T cells were induced to proliferate. The B cell defect appears to be restricted to a certain phase of B cell activation, since old mouse B cells responded to the auto D1.4 T cells by increasing cell surface Ia as well as size, but failed to incorporate tritiated thymidine. The responsiveness to interleukin-4 was found to be similar between B cells from young and old mice. It appeared that the B cells from old mice are specifically defective in progressing from the G0 phase of cell cycle into the S phase when stimulated with the auto D1.4 T cells.

Effects of cigarette smoke on the antibody responses to thymic independent antigens from different lymphoid tissues of mice

The influence of cigarette smoke on the humoral immune response of mice was investigated in lymphocytes derived from the spleen, bone marrow (BM) and mesenteric lymph nodes (MLN). Mice of the DBA/2J or C57BL/6 strain were exposed to cigarette smoke of a standard research cigarette, 2R1, twice a day, ten puffs each in morning and afternoon for 20, 40 or 60 weeks. At the end of the smoking period, animals were immunized intraperitoneally with the thymic independent antigens polyvinyl pyrrolidone (PVP) or trinitrophenyl (TNP)-Ficoll. The antibody reponses were analyzed using sheep red blood cells coated with PVP or TNP, in a plaque forming cell (PFC) assay. The results indicate a statistically significant inhibition of the antibody response induced by PVP but not by TNP-Ficoll in splenic B cells of smoke exposed mice compared to sham controls. When tested in other lymphoid organs, there was higher anti-TNP PFC response from the BM and MLN cells of smoke exposed animals compared to sham controls.

Phenotypic and functional properties of B lymphocytes from aged mice

Phenotypic and functional properties of B lymphocytes from individual young and old mice of different inbred strains were studied. B lymphocyte subpopulations defined by the ratios of the densities of cell surface IgM and IgD were found to be altered with age. However, such alterations in B cell subsets were found only in 30-40% of the old mice. B cell mitogenic responses to anti-mu and anti-Lyb2 antibodies were decreased in a majority of DBA/2 mice. Proliferative responses to LPS and anti-mu were reduced only in a minority of CBA/Ca mice but there was a very good correlation in the responsiveness of the old mice to LPS and anti-mu. The anomalous properties of the individual old mice of these inbred strains may be due to a heterogeneity in the effects of aging or due to environmental influences.

Macrophages but not B cells from aged mice are defective in stimulating autoreactive T cells in vitro

In the present study the effect of aging on the capacity of Ia+ cells to stimulate autoreactive T cells in the syngeneic mixed lymphocyte reaction (SMLR) was investigated. Using young CD4+ T cells as responders, it was observed that unseparated whole spleen cells from aged mice had normal stimulatory activity comparable to that of young spleen cells. Interestingly, however, when purified splenic adherent cells (SAC) enriched for macrophages or splenic B cells were used as stimulators, aged SAC but not aged B cells were found to be defective in stimulating autoreactive T cells. This defect in aged SAC was not due to decreased expression of Ia antigens since the percentage of Ia+ SAC and density of Ia antigen expression was similar in both young and old mice. Also, the B cells from aged mice expressed normal levels of Ia antigens. Aged SAC, when mixed with young SAC could also actively suppress the normal SMLR. However, this suppression was not due to increased prostaglandin production but was found to be associated with interleukin-1 (IL-1) regulation, inasmuch as addition of exogenous IL-1 could completely reconstitute the defective stimulatory activity of aged SAC and also abolished the suppressor activity of the SAC. Aged mice also demonstrated an intrinsic defect in the CD4+ T cells responding in the SMLR. Together, our studies on the SMLR demonstrate an age-related defect in responder autoreactive T cells and in stimulator splenic macrophages but not in the stimulatory activity of B cells.

Priming of peripheral lymph node B cells with TNP-ficoll : role of lymphokines in B cell differentiation

We have previously shown that peripheral lymph node (PLN) B lymphocytes of adult DBA/2J mice failed to make an antibody response to type 2 antigen TNP-Ficoll, but exhibited a good antibody response to type 1 antigen TNP-Brucella abortus. In the present study we wanted to find out whether the unresponsiveness of PLN B cells to TNP-Ficoll is due to defects in the early activation and proliferation stage or in the final differentiation stage of B cells. Therefore, we have used a two-step protocol of in vivo immunization of mice with TNP-Ficoll and the subsequent in vitro challenge with TNP-Brucella abortus and studied the anti-TNP plaque-forming cell (PFC) responses. The results indicate a three- to sixfold increase of PFC responses in PLN cell cultures derived from TNP-Ficoll-primed animals compared to saline control mice. This increased antibody response was TNP-specific as 93% of the PFCs were inhibited by TNP-lysine. Limiting dilution experiments confirm that the increase in anti-TNP PFC response from the TNP-Ficoll-primed animals was indeed due to an increase in TNP-specific precursor B cells. Further, the addition of rIL-5 or rIL-6 induced anti-TNP PFC in the TNP-Ficoll-primed and in control PLN cell cultures in the presence of antigen. However, in primed PLN cells lymphokines alone were sufficient to restore anti-TNP PFC response. In conclusion, our results show that in PLN, the TNP-Ficoll can induce proliferation of hapten-specific B cells but not final differentiation. These primed PLN B cells mature into antibody-secreting cells upon stimulation with TNP-BA or lymphokines.

Impaired autoreactive T cell-induced T cell-T cell interaction in aged mice

Self-Ia-reactive (autoreactive) L3T4+ T cell clones have been shown earlier to stimulate the proliferation of syngeneic naive L3T4+ T cells and initiate a T cell-T cell (T-T) interaction leading to the generation of immunoregulatory circuits. Since aging has been shown to be associated with a decline of the immune responsiveness, age-related alterations in the T-T interaction was investigated in the present study. Using several I-Ed-specific autoreactive T cell clones isolated from 2- to 3-month-old (young) DBA/2 mice as stimulators, it was observed that L3T4+ T cells from 22- to 24-month-old (aged) DBA/2 mice, failed to demonstrate a significant response to the autoreactive T cells. In contrast, L3T4+ T cells from young mice responded strongly to the autoreactive T cell clones. The deficient T-T cell interaction in aged mice correlated with an impaired syngeneic mixed lymphocyte reaction in these mice, thereby suggesting that aging induces a defect both in the autoreactive T cells and in T cells which react with the autoreactive T cells. When exogenous recombinant interleukin 2 (rIL-2), recombinant interleukin 4 (rIL-4), or a combination of these was added to the interaction, it was observed that rIL-4 but not rIL-2 enhanced the T-T interaction in young mice. However, rIL-4 or a combination of rIL-2 and rIL-4 failed to correct the defective T-T interaction in aged mice. Since the T cell network is believed to play an important role in the maintenance of normal immune system homeostasis, the present study suggests that age-related alterations in T and B cell functions and increased susceptibility to autoimmune diseases with age may result from a defect in the T cell network regulation.

A Role for Ia Antigens in B Cell Activation: Studies with Normal B Cells and A B Cell Lymphoma

Monoclonal anti-Ia antibodies enhanced anti-μ induced proliferation of murine B lymphocytes. Anti-Ia induced augmentation of B cell proliferation was allele specific and did not require participation by T cells and adherent cells. The effects of anti-Ia antibodies on B cell activation are neither due to induction of interleukin-1 secretion in the cultures nor due to interaction with the Fc receptors and can be demonstrated under serum-free culture conditions. Monoclonal antibodies against both IA and IE region encoded Ia molecules were able to synergize with anti-μ in inducing B cell proliferation. Antibodies to both IA and IE coded antigens induced IgM secretion from the B lymphoma cell lines BKS3 and BKL. The anti-Ia antibodies did not affect the proliferation of the B lymphoma cells. These results suggest that the interaction of B cell surface Ia with the anti-Ia antibodies results in an activation signal and that the B cell surface Ia antigens may function as signal transducer molecules.