Polyclonal B cell activation by B cell differentiation factor B151-TRF2. I. Involvement of self-Ia recognition process mediated by B cells

The present study examined the functional role of Ia antigens on B cells in polyclonal B cell activation induced by a B cell differentiation factor, B151-TRF2. The polyclonal IgM PFC responses by B151-TRF2 were inhibited by monoclonal antibodies specific for class II MHC antigens (Ia antigens) but not class I MHC antigens. Such inhibition by anti-Ia antibodies was haplotype-specific and was observed in the absence of both T cells and accessory cells. Moreover, the anti-Ia antibody-induced inhibition of the B151-TRF2 responses was not due to the blocking of binding of B151-TRF2 to the corresponding B cell receptor. A series of kinetic studies revealed that some Ia-mediated cellular activation process occurs before the resting B cells become responsive to B151-TRF2. Thus, the B151-TRF2-mediated B cell responses consist of at least two distinct phases. The early phase is an Ia-dependent but B151-TRF2-independent process, whereas the late phase is an Ia-independent but B151-TRF2-dependent process. To further characterize the functional role of Ia antigens on B cells, an additional experiment was carried out by using F1 B cells which co-dominantly express both parental Ia antigens on the surface. Interestingly, it was observed that the degree of inhibition of the B151-TRF2-mediated responses of F1 B cells by anti-parental Ia antibody was, at best, one-half that of the parental B cells, suggesting that F1 B cells may be separated into two subpopulations with the restriction specificity for the respective parental Ia antigens. To examine this possibility, (B10 X B10.BR)F1 B cells were separated into adherent and nonadherent cell populations by their ability to bind to either one of the parental B cell monolayers, and the specificity of inhibition of their responses to B151-TRF2 by anti-Ia antibodies was assessed. It was found that the responses of (B10 X B10.BR)F1 B cells adherent to the B10 B cell monolayer or the B10.BR B cell monolayer were almost completely inhibited by anti-I-Ab and anti-I-Ak antibodies, whereas those of nonadherent cells were now selectively inhibited by anti-I-Ak and anti-I-Ab antibodies, respectively. These findings are interpreted as indicating that the B151-TRF2-responsive F1 B cells consist of at least two subpopulations with the restriction specificity for either one of the parental Ia antigens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Polyclonal activation of primed rat B cells

In recent years, murine and human virgin B lymphocytes have been used to examine the steps necessary for polyclonal activation. In these models mitogens are used in conjunction with lymphokines to determine which signals are responsible for regulating B-cell triggering, proliferation, and differentiation. While progress has been made in understanding these events as they occur in virgin B cells, very little evidence exists to suggest whether these models of activation also apply to the memory B-cell population. In this report we have described an antigen-specific, secondary in vitro immune response using cells isolated from lymph nodes draining the site of antigen injection. Unfractionated cells, B cells, and size-fractionated cells from dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH)-primed rats were challenged in vitro with DNP-KLH, lipopolysaccharide plus dextran sulfate (LPS/DxS), and T-cell factors. We have consistently found, under all these conditions, that antigen challenge of primed cells results in the production of DNP-specific IgG antibody while stimulation with LPS/DxS plus T-cell factors results only in the polyclonal activation of virgin B cells; no antigen-specific IgG secretion is seen. This suggests that acquisition of memory status is associated with a loss in responsiveness to LPS/DxS-induced differentiation.     

Polyclonal activation of human lymphocytes and induction of cytotoxic lymphocytes by streptococcal preparations

Polyclonal activation of human peripheral blood lymphocytes (PBLs)in vitro by preparations ofStreptococcus pyogenes Su strain (OK-432) and other heat-killed strains was investigated. The streptococcal preparations tested induce a proliferative response of PBLs via interleukin-2 (IL-2)-independent pathways. The proliferative response is accompanied by the generation of lymphoblastic cells (LBCs), which consist of heterologous lymphocyte populations: CD4⁺ helper type of T cells, and CD4^–CD8^– double-negative (DN) lymphocytes, including both CD3⁺ TcR ⁺ T cells and CD2⁺CD3^– immature type of T or non-T cell type of lymphocytes. Almost all the LBCs express Leu19, TfR (transferrin receptor), LFA-1 and CD38 (OKT10) antigens, which are expressed on activated T cells, NK cells and some other lymphocytes. The proliferative response of human PBLs is also accompanied by the generation of potent cytotoxic activity against NK-sensitive and -resistant targets. C-dependent cytolysis and cell sorting experiments of OK-432-activated LBCs revealed that both CD3⁺ and CD3^– types of CD4^–CD8^– DN lymphocytes, but not CD4⁺ helper T cells, may be major populations responsible for the cytotoxicity induced. On the other hand, CD4^–CD8 T cells may be required for the proliferation of PBLs and generation of cytotoxic effector cells. These results suggest that the OK-432 and other streptococcal preparations stimulate the human PBLsin vitro to induce the proliferation/activation of CD4⁺ T cells, mediating the following generation of DN cytotoxic effector lymphocytes.     

Polyclonal activation of xid B cells by auto-Ia-reactive T cell clones

The mechanism of T cell-dependent activation of xid B cells into Ig-producing cells was studied by employing H-2-restricted, antigen-specific T cell clones. Helper factors (B cell stimulatory factors, BSF) released from KLH-specific T cell lines could induce polyclonal Ig production in B cells from (CBA/N X BALB/c)F1 (NBF1) female mice but not from CBA/N or NBF1 male mice. Direct addition of helper T cell lines induced Ig production in xid B cells from CBA/N or NBF1 male mice. A T cell clone, MK6, which was derived from NBF1 male mice and specific against Iad determinant, could activate NBF1 male but not CBA/N B cells. Another clone, CK4, derived from CBA/N mice and having specificity against KLH plus I-Ak determinant could activate both CBA/N and NBF1 male B cells into IgM- and IgG-producing cells in the absence of KLH, and monoclonal anti-I-Ak antibody specifically blocked such activation. These results suggest that xid B cells are able to be activated by the signal provided by the recognition of Ia molecules on B cells by auto-Ia-reactive T cells. Xid B cells from CBA/N mice that had been co-cultured with a T cell line specific against I-Ak determinant for 24 hr became reactive to BSF and capable of differentiating into Ig-producing cells in the presence of BSF. The results showed that even xid B cells could be responsive to BSF if they were in a certain activation stage.     

T cell activation of antigen-specific antibody responses by large B cells is MHC restricted

The activation of small, resting B cells for antibody synthesis by helper T cells has been proposed to require an MHC-restricted interaction between the T and B cells. Large, activated B lymphocytes were, in contrast, thought to be activated by an unrestricted pathway. We re-examined this issue and found that both large and small size fractionated murine B lymphocytes required an MHC-restricted interaction with helper T cells to be activated for specific antibody synthesis. Polyspecific antibody synthesis in the same cultures was not dependent upon an MHC-restricted T-B interaction for any size category of B cell. These results are interpreted as reflecting the ability of antigen-specific B cells to focus and present antigen to T cells, in contrast to B cells of random specificity, which have no effective focusing mechanism for a given experimental antigen. We found that the polyspecific response required much higher antigen concentrations than the antigen-specific response, a result consistent with the antigen-focusing hypothesis.     

Regulation of lymphocyte responses by human gangliosides. I. Characteristics of inhibitory effects and the induction of impaired activation

Gangliosides obtained from normal human brain were found to inhibit the in vitro activation of human lymphocytes by nonspecific mitogens and allogeneic cells at concentrations between 3 to 50 microgram/1.5 to 1.7 X 10(5) lymphocytes/0.2 ml culture. Ganglioside inhibition did not represent cytotoxic effects or altered lectin binding and was independent of the mitogen concentration. In addition to concentration, the degree of inhibition was dependent on the mode of presentation to lymphocytes, since gangliosides incorporated within liposomal membranes displayed a synergistic inhibitory effect greater than predicted from the cultures receiving either gangliosides or liposomes alone. In binding experiments, radiolabeled ganglioside GM1 became associated with human lymphocytes within 10 min. However, approximately 72 hr pre-exposure of human lymphocytes to gangliosides was required to induce impaired lymphocyte responses to mitogens and allogeneic cells. Thus, concentrations of human gangliosides equivalent to the levels occurring in the sera of patients with certain malignancies are capable of actively inhibiting lymphocyte stimulation in addition to inducing impaired lymphocyte responses.     

Regulation of antibody secretion by hybridoma cells. I. Suppression of antibody secretion by coculture of hybridoma cells with idiotype-induced suppressor cells

In this study, we have demonstrated that antibody secretion by hybridoma cell lines can be down-regulated by idiotype-specific immune spleen cells or by nylon wool nonadherent spleen cells. This suppression of antibody secretion can be abolished by treating the idiotype-specific immune spleen cells with anti-Thy 1.2 plus complement. The hybridoma we used for most of our experiments secretes IgM specific for the cross-reacting haptens 2,4,6-trinitrophenyl (TNP) and 2,4-dinitrophenyl (DNP). Suppression was achieved by direct coculture of hybridoma cells with immune cells from animals which were injected with affinity-purified hybridoma antibody-coupled syngeneic spleen cells. The suppressed and control cultures contained similar numbers of viable hybridoma cells, suggesting that a simple cytotoxic effect is not responsible. Idiotype specificity was established in experiments showing that two idiotype immune animals immunized with antibody from two different IgM anti-TNP hybridomas could suppress the hybridoma to which they were immunized but could not affect the other hybridoma. Immune spleen cells required 3-4 days of coculture with hybridoma cells before maximum suppression was achieved. The kinetics of the response suggest that the final effector suppressor cell is generated during the coculture period and that a second signal, perhaps a product of the hybridoma cells, may be required.     

Analysis of the mechanisms of T cell-dependent polyclonal activation of human B cells. Induction of human B cell responses by fixed activated T cells.

Coculture of resting human B cells with T cells stimulated with immobilized mAb to the CD3 molecular complex induces polyclonal activation and the production of Ig of all isotypes. The current experiments were carried out to determine the nature of the signals provided to B cells by the anti-CD3-activated T cells. For these experiments, fresh T cells or T cell clones were activated with immobilized mAb to CD3 and then fixed with 1% paraformaldehyde. Upon coculture, the fixed activated T cells or T cell clones induced B cell RNA synthesis and IL-2R expression, but only minimal DNA synthesis and no Ig production. Induction of B cell RNA synthesis by fixed activated T cells was not inhibited by mAb to the alpha-chain of the IL-2R, and was not enhanced by supplementing cultures with IL-2, IL-4, IL-6, or supernatants of mitogen-activated T cells. Upon the addition of IL-2, but not IL-4 or IL-6, to cultures of B cells and fixed activated T cells, sustained proliferation was noted along with the production of Ig. Control fixed T cells or T cell clones did not induce any of these responses. The presence of cycloheximide or cyclosporin A during the activation with anti-CD3 prevented T cells from developing the capacity to provide help for B cells. The use of mAb to a variety of cell surface molecules indicated that several T cell surface molecules including CD11a/CD18, CD44, CD54, and class I MHC molecules are involved in the induction of B cell responses. Among the mAb that inhibited B cell DNA synthesis and/or Ig production, only mAb to CD11a, CD18, or CD54 inhibited initial B cell activation as assessed by RNA synthesis. Even though mAB to CD11a/CD18 inhibited the capacity of fixed activated T cells to induce B cell responses, the finding that fixed activated CD18 deficit clones provided help for B cells indicated that expression of the beta 2 family of integrins by T cells was not necessary. These results indicate that activated T cells acquire the capacity to stimulate B cells polyclonally and induce cytokine responsiveness, proliferation, and Ig production by utilization of a variety of surface molecules. Moreover, these results indicate that the initial activation of the B cell is independent of the metabolic activity of the T cell and the production of cytokines.     

Tumor necrosis factor selectively inhibits activation of human B cells by Epstein-Barr virus

We have investigated the effect of recombinant human tumor necrosis factor-alpha (rTNF-alpha) on human B cell activation and differentiation. Among several T cell-dependent and independent B cell stimulation systems tested (anti-mu, pokeweed mitogen, Epstein-Barr virus), only the activation by Epstein-Barr virus was inhibited by rTNF-alpha. rTNF-alpha inhibited in a dose-dependent manner both the proliferation and differentiation (Ig secretion) of Epstein-Barr virus-stimulated B cells when added at the beginning or within 48 hr of a 6 to 8-day culture period. Maximal suppression (80 to 95%) was found at rTNF-alpha concentrations of 10 to 50 ng/ml. Inhibition of B cell activation required the presence of significant numbers (25%) of plastic adherent macrophages within the B cell population. Suppression was not due to lysis of Epstein-Barr virus-infected B cells by rTNF-alpha-treated macrophages. As shown by double chamber experiments where macrophages and B cells were separated by a 0.45-micron membrane, macrophages elaborated factors in response to rTNF-alpha, which, alone or synergistically with rTNF-alpha, inhibited B cell activation. These factors were different from prostaglandin E2, interferon-alpha, and interleukin 1. We conclude that rTNF-alpha can dramatically modulate certain normal immune responses in vitro.     

Polyclonal activation of murine B lymphocytes by immune complexes

Murine splenic B lymphocytes are stimulated by homologous immune complexes to proliferate and secrete polyclonal antibody. The use of antibody from whole serum or monoclonal antibodies to form complexes resulted in the stimulation of mouse B lymphocytes. The ratio of antibody to antigen appears to be critical for the generation of the polyclonal antibody response. Because antigen and antibody are added independently at culture initiation, the exact nature of the complex is unknown, but optimal polyclonal antibody formation occurs in slight antigen excess. Immune complex-induced polyclonal antibody production requires the presence of both macrophages and T cells, whereas B cell proliferation requires only macrophages. The role of the macrophage appears to be to cleave a low m.w. (17,000) fragment from the complex, which is responsible for lymphocyte activation.     

Polyclonal activation of murine B lymphocytes by Fc fragments. I. The requirement for two signals in the generation of the polyclonal antibody response induced by Fc fragments

Fc fragments derived from human IgG1 induce murine splenic B lymphocytes to undergo proliferation and differentiation to antibody-secreting cells. The polyclonal antibody response was found to require both the presence of macrophages and T cells. Spleen cell cultures from nude mice or T cell-depleted normal mice proliferate to the level of untreated control mice but do not produce polyclonal antibody unless T cells are added. Regulation of the Fc fragment induced B cell differentiation to antibody synthesis apparently occurs through two distinct signals. One signal is provided by Fc fragments for proliferation and the other by T cells for differentiation. This suggestion is supported by the observation that spleen cell preparations, devoid of T cells, are capable of proliferation to the level of normal spleen cell cultures in response to Fc fragments, but are incapable of making a polyclonal antibody response. The cell population that responds to the differentiation signal also responds to the proliferative signal. "Hot pulse" experiments demonstrated that proliferation precedes polyclonal activation.     

Specificity of initiation factor preparations from poliovirus-infected cells.

Crude initiation factor preparations from poliovirus-infected cells stimulated the translation of poliovirus RNA in vitro, but were inactive for the translation of host cell or vesicular stomatitis virus mRNA's. In contrast, similar preparations from either uninfected or vesicular stomatitis virus-infected cells supported the initiation of translation of host cell mRNA's and both viral mRNA's. These results reflect a specific alteration of some components(s) of the initiation factor preparation from poliovirus-infected cells which is consistent with the ability of the virus to inhibit the translation of host cell and vesicular stomatitis virus-directed protein synthesis.     

Polyclonal activation of human lymphocytes in vitro. I. Characterization of the lymphocyte response to a T cell-independent B cell mitogen

The staphylococcal cell wall component protein A (SpA) and formalinized, Cowan I strain Staphylococcal organisms (STA) were compared with the lectins phytohemagglutinin, concanavalin A, and pokeweed mitogen for their ability to trigger proliferation of normal human lymphocytes, lymphocyte subpopulations, and cells from patients with primary immune deficiency diseases. SpA was found to be a potent T cell mitogen, very similar to the other lectins tested. It failed to stimulate purified non-T cells and peripheral blood lymphocytes from patients with different forms of severe combined immunodeficiency disease (SCID). STA, treated to prevent the leakage of soluble SpA during culture, exclusively stimulated non-T cells: the responding cell population was characterized to be E-rosette negative but positive for C3 receptors, surface Ia, a receptor for STA itself, and likely carried surface immunoglobulin. Normal responses to STA were found in patients with the adenosine deaminase-positive form of SCID. In 18 patients with humoral immune deficiency syndromes, the presence of STA responses was correlated with the presence of circulating, surface immunoglobulin-bearing cells. A commercial STA preparation was rendered B cell specific after reformalinization, a procedure that eliminated the shedding of soluble SpA under culture conditions.     

Regulation of human cytotoxic T-lymphocyte responses by a soluble suppressor factor

We describe some aspects of the biology of a suppressor factor (SF) secreted by actively metabolizing and dividing alloantigen-primed T cells which functions by regulating human cytotoxic T-lymphocyte (CTL) activation. The SF functions most effectively during the first 24 hr of CTL activation, while it does not function at the CTL effector stage. Both T cells and adherent cells are capable of absorbing out the biological activity from suppressor factor supernatants. Experiments demonstrated that either fresh adherent cells or the addition of interleukin 2 (IL-2) into the test system could reverse the effects of the SF on CTL activation. These data suggest that the SF could be acting by either indirectly restricting IL-2 availability to proliferating CTLs by limiting adherent cell interleukin 1 (IL-1) secretion or, alternatively, SF acting directly on the IL-2-producing T cells.     

Polyclonal activation of the murine immune system by an antibody to IgD. X. Evidence that the precursors of IgG1-secreting cells are newly generated membrane IgD+B cells rather than the B cells that are initially activated by anti-IgD antibody

Injection of BALB/c mice with an affinity-purified goat antibody to mouse IgD (GaM delta) stimulates T cell-independent B cell activation as well as later T cell activation. Activated T cells then induce polyclonal differentiation of B cells into IgG1-secreting cells, which results in an approximately 100-fold increase in serum IgG1 level. It is not known whether the same B cells that are initially activated by GaM delta are the progenitors of the IgG1-secreting cells. To investigate this issue a system was developed in which CB20 mice, which are congenic to BALB/c mice but express Ig of the beta allotype rather than the BALB/c alpha allotype, were injected with GaM delta and simultaneously or subsequently also received BALB/c B cells. The IgG1 response generated by the donor BALB/c B cells was quantitated by an assay specific for IgG1 of the alpha allotype. Our experiments with this system indicate that: 1) BALB/c B cells transferred 2 days after CB20 mice were injected with GaM delta generate a much larger IgG1 response than do BALB/c B cells transferred simultaneously with GaM delta antibody; 2) B cells that express membrane IgD generate the great majority of this response; 3) differences in the magnitudes of the responses of BALB/c B cells transferred at different times after CB20 mice were injected with GaM delta antibody cannot be explained by differences in homing of the donor B cells to the host spleen or by short survival of donor BALB/c B cells after their transfer; and 4) the response made by donor BALB/c B cells transferred 2 days after CB20 mice were injected with GaM delta is proportionate to donor cell representation in the host spleen 1 day after their transfer, whereas the response made by donor cells transferred simultaneously with GaM delta is disproportionately small. These observations suggest that most of the IgG1 antibody made by GaM delta-injected mice is generated by newly produced, mIgD+ B cells that appear approximately 2 days after GaM delta injection, rather than by those B cells that are present in the spleen at the time of GaM delta injection, and support the view that signals that induce B cell secretion of Ig require an interaction with at least partially activated Th cells.     

Suppression of antibody responses in allogeneic mice by products of lymphoid tissue. I. Allogeneic suppressive factor (ASF) from spleens repopulated with thymus cells.

A cellfree extract prepared from the spleen cells of C3H mice is capable of suppressing antibody responses to SRBC when extract material is exposed to alloantigens. The observed immunosuppression was attributed to a soluble factor in the extract. This allogeneic suppressive factor (ASF) was detected in extracts prepared from the spleen cells of unirradiated mice as well as those of irradiated mice repopulated with thymocytes, provided that mice were previously immunized with SRBC. Donors of actively suppressive ASF preparations did not need to be previously exposed to alloantigens. Extracts from thymus and marrow cells of unirradiated mice and the spleen cells of irradiated mice repopulated with marrow cells (or no cells) did not contain ASF. C3H thymocytes stimulated with SRBC generated more ASF activity in spleens of C3BF1 hosts than in those of C3H hosts, indicating that alloantigenic stimulation enhances the production or activity of ASF. Once produced, C3H ASF was able to suppress antibody responses in cell transfer experiments only if exposed to C3BF alloantigens of either donor lymphoid cells or irradiated hosts. Once exposed to alloantigens, ASF appears to be capable of suppressing antibody responses of syngeneic C3H or semi-allogeneic C3BF cells. When both donor lymphoid cells and hosts were syngeneic with the donor of the ASF, there was enhancement of antibody formation in cell transfer experiments. C3H ASF did not interfere with education of C3BF thymocytes to SRBC or with the generation of precursors of anti-SRBC antibody-forming cells by C3BF1 marrow cells. ASF may interfere with cellular cooperative events necessary for humoral immune responses or with terminal differentiation of B cells. Production of ASF could partially account for the suppression of antibody responses observed during graft-vs-host reactions.