Interleukin 6 is essential for antibody secretion by human in vivo antigen-induced lymphoblastoid B cells

In vivo immunization of normal subjects with a variety of antigens generates circulating lymphoblastoid (LB) B cells, which in vitro spontaneously secrete significant levels of specific antibody. Since activation and initial differentiation of these cells occurs in vivo, they provide a useful model for the study of the later stages of B cell maturation. In the present study, we investigated the requirement of interleukin 6 (IL-6) for the "spontaneous" in vitro production of IgG-Tet by LB B cells. Addition of IL-6 to cultures of LB B cells in medium supplemented with 10% fetal calf serum failed to increase the levels of IgG-Tet produced in vitro. However, addition of anti-IL-6 antibodies decreased IgG-Tet production as much as 70%, and this inhibition could be reversed by the addition of IL-6. LB B cells cultured in serum-free medium in order to restrict endogenous IL-6 production secreted only low levels of antibody, unless exogenous IL-6 was added. Addition of 2.5 units/ml of IL-6 to serum-free cultures induced an increase in IgG-Tet secretion nearly comparable to that seen in cultures supplied with serum. The magnitude of the increase in IgG-Tet secretion in response to exogenous IL-6 was inversely related to the number of cells in culture, which was due in part to increased endogenous IL-6 production in cultures with higher cell concentrations. Experiments including hydroxyurea in serum-free cultures indicated that IL-6-dependent enhancement of LB B cells' IgG-Tet secretion was not primarily mediated by cell growth. These observations suggest that in vivo generated LB B cells are not totally committed to antibody secretion, and that IL-6 is essential for in vivo antigen-induced LB B cells to reach the antibody-secreting stage.     

Optimization of an ovine antibody-secreting cell assay for detection of antigen-specific immunoglobulin production in peripheral blood leukocytes

Enumeration of antibody-secreting cells in peripheral blood by enzyme-linked immunospot (ELISPOT) has been used in human studies to detect antigen-specific antibody production at mucosal tissue sites. An alternative assay for detecting and quantitating antigen-specific antibody responses involves culturing circulating peripheral blood antibody-secreting cells and quantitating specific antibody production in culture supernatant by ELISA. In the present study, antigen-specific peripheral blood lymphocytes were isolated from subcutaneously immunized sheep and the parameters for maximizing in vitro antibody production by in vivo-induced antibody-secreting cells optimized for this species. Maximum antibody-secreting cell responses were observed in peripheral blood collected four days after antigen challenge. The addition of lipopolysaccharide and antisheep immunoglobulin had no effect on in vitro antibody secretion by blood antibody-secreting cells, while the effects of pokeweed mitogen were highly variable. However, the combination of anti-Ig and recombinant ovine interleukin-6 to peripheral blood lymphocyte cultures was found to markedly and consistently enhance specific antibody production. In unstimulated cultures, the optimal peripheral blood lymphocyte concentration for generating the greatest antibody responses was 5.0 x 107 cells per mL, but in cultures stimulated with recombinant ovine interleukin-6/antisheep immunoglobulin, the optimal cell concentration was lowered to approximately 1.0 x 107 cells per mL. In vitro, peak immunoglobulin production was usually achieved by day one in unstimulated cultures. In recombinant ovine interleukin-6/antisheep immunoglobulin-stimulated cultures, antibody levels were similar to unstimulated cultures by day one, however, the levels continued to rise during incubation to reach a maximum between days four and five of incubation. This optimized antibody-secreting cell culture assay is amenable for increasing the sensitivity and reducing the cell numbers required for quantitating antigen-specific antibody induction in large-scale immunization trials in sheep and other large animal species.     

Recombinant human interleukin 6 (rhIL-6) promotes the terminal differentiation of in vivo-activated human B cells into antibody-secreting cells

Antigen-activated peripheral blood B cells were induced by parenteral immunization of healthy individuals with a polyvalent pneumococcal vaccine, or diphtheria toxoid. Seven to nine days after immunization, high frequencies of antigen-specific antibody-secreting cells, representing in vivo activated lymphoblastoid B cells, were detectable in peripheral blood or spleen. The B cell-enriched fractions were stimulated for 7 days with different concentrations of rhIL-6. Both the frequency of antibody-secreting cells and the secreted amount of antibody to the immunizing antigen were increased by rhIL-6 in a dose-dependent fashion. Stimulation with rhIL-6 did not alter the isotype distribution of antibody-secreting cells. A polyclonal anti-IL-6 serum completely abrogated the stimulatory effect of rhIL-6 on the in vitro antibody secretion. Fluorescence-activated cell sorter analysis revealed that 25-29% of cells in the large B cell fraction which presumably contained the in vivo activated cells bore the IL-6 receptor. Thus, rhIL-6 enhances the terminal differentiation of in vivo activated B cells into antibody-secreting plasma cells.     

NK and T cell subsets regulate antibody production by human in vivo antigen-induced lymphoblastoid B cells

This study demonstrates the existence of two different suppressive systems for the regulation of antitetanus toxoid antibody production by human lymphoblastoid (LB) B cells. These B cells appear in the circulation 5 to 7 days after in vivo immunization and spontaneously secrete antibody during a 3-day in vitro culture. One suppressive system was mediated by large granular lymphocytes that exhibited high natural killer activity. This suppressive cell subset spontaneously inhibited the antibody production by autologous LB cells, and this effect could be enhanced by the addition of interferon. This inhibition of antibody synthesis could be readily reversed by the addition of as few as 10(2) K-562 cells to the culture. Additionally, the activity of this suppressive cell population could be reduced by complement (C)-mediated lysis with Leu-7 antibody. These results strongly suggest that this autologous suppression was mediated by NK cells. The other suppressor system was contained in the fraction of high density T cells depleted of Fc receptor-bearing cells, which was low in NK activity. This subset inhibited LB function in the presence of pokeweed mitogen but not interferon, and even the addition of up to 10(6) K-562 NK target cells only minimally reversed this inhibition. These results indicate that two distinct subsets of cells share regulatory functions on the in vivo induced B lymphoblastoid cells. The observation that NK cells can inhibit these highly differentiated B cells expands our view of the spectrum of natural targets recognized by NK cells.     

Inhibition of human antigen-induced lymphoblastoid B-cell function by an in vivo-induced suppressor T cell

Lymphoblastoid (LB) B cells which spontaneously produce antitetanus toxoid IgG antibodies (Tet-IgG) in short-term cultures (3 days) appear in the circulation 5-7 days after immunization with tetanus toxoid. Addition of pokeweed mitogen (PWM), normally a stimulator of antibody production, caused instead a reduction in the in vitro synthesis of Tet-IgG by the LB cells. In order for this inhibition of antibody production to occur, T cells had to be present, and the inhibition was proportional to the number of T cells added to the culture, demonstrating the existence of PWM-inducible suppressor cells. The cells mediating the suppression had the OKT8 phenotype and also exhibited the following characteristics: (1) a PWM pretreatment period as little as 14 hr was enough to complete activation; (2) conventional inhibitors of suppressor T cells as hydrocortisone and cyclosporin A only partially reversed its effect; and (3) DNA synthesis was not required. The T-suppressor activity was detectable in the circulation before immunization, increased two- to fourfold by 5-12 days after boosting, and waned after 3 weeks. The mechanism of action of this suppression does not appear to involve conventional cytotoxic T cells as (1) the suppression was mediated across allogeneic barriers and (2) the suppression could not be reversed by inclusion of anti-Leu-2a antibodies in the culture. These results suggest that this suppressor T-cell subset may be important in the normal regulation of activated stages of human B lymphocytes.     

Human peripheral blood lymphocytes from recently vaccinated individuals produce both type-specific and intertypic cross-reacting neutralizing antibody on in vitro stimulation with one type of poliovirus

An in vitro system of poliovirus-specific antibody production by peripheral blood B cells on stimulation by the virus has been developed. Virus-neutralizing antibodies in culture supernatant fluids, or virus-specific antibody-secreting cells (ASC) were detected by microneutralization assay and ELISA-SPOT test, respectively. After booster immunization with polio vaccine, anti-poliovirus-neutralizing ASC were present in circulation. This response was measurable between 5 and 12 days after booster vaccination. At between 12 and 90 days, another subset of B cells was found in peripheral blood that only produced poliovirus-specific neutralizing antibody after in vitro antigenic stimulation. The in vitro virus-induced response required B cells, monocytes, and T4+ (T helper) cells, and was shown to result from de novo protein synthesis. The anti-poliovirus-neutralizing response in vitro could be dissected in a type-specific and intertypic cross-reactive response by using various antigen concentrations for in vitro stimulation. Evidence was obtained by absorption studies for the existence of intertypic cross-reactive neutralization-inducing epitopes.     

Primary anti-trinitrophenyl memory cells investigated by plaque-forming cells and ELISA.

Primary anti-trinitrophenyl antibody production was investigated from spleen cells of mice immunized with trinitrophenylated-keyhole limpet hemocyanin, using the plaque-forming cell method and ELISA. Cells taken 5 days after antigen injection do not produce IgE, but do produce IgM and IgG1 anti-trinitrophenyl antibodies as demonstrated by plaque-forming cells. Substantial increase of IgM, IgG1, and IgE antibody production was seen from cells taken 7 days after immunization, followed by a rapid decline. By ELISA it was seen that cells taken 3 days after immunization already produce small amounts of anti-trinitrophenyl antibodies. Presence of antigen from the start of the cultures did not increase antibody production from cells taken 3 days after immunization, but potentiated antibody secretions from cells taken 5 days or later after immunization. This potentiation was interpreted as recruitment of antibody-forming cells from early memory B cells. The presence of IL-4 from the start of the cultures had no appreciable effect. Cell sorting with specific antibody-coated magnetic beads showed that plaque-forming cells from nonsorted cells, membrane IgE+ or membrane IgE- cells secreted similar amounts of anti-trinitrophenyl IgG1 and IgE antibodies. No difference in anti-trinitrophenyl IgM, IgG1, or IgE production was found in controls; cells sorted negatively or positively for CD23. The data show that memory B cells can be demonstrated already on Day 5 after immunization, and their antigen-induced antibody secretion is IL-4 dependent.     

In vivo obtained antigen presented by germinal center B cells to T cells in vitro

Shortly after secondary immunization germinal center (GC) B cells obtain antigen from follicular dendritic cells (FDC) in the form of immune complexes. This antigen appears to be degraded by the GC B cells and may be processed for presentation to T cells. The present study was undertaken to determine whether GC B cells can process and present antigen obtained from FDC in vivo to appropriate T cells in vitro. GC B cells were isolated from immune mice with the use of Percoll density separation followed by a panning procedure which utilizes the ability of the plant lectin, peanut agglutinin (PNA), to selectively bind to GC B cells. The enriched GC B cells were approximately 80% highly positive for PNA, 97% positive for Ia and surface IgM, but less than 0.01% positive for Thy-1.2 or esterase. In some experiments, this population was further purified to near 100% highly PNA-positive cells with the use of fluoresceinated PNA and a fluorescence-activated cell sorter. Cell sorting analysis indicated that the antigen (125I-labeled ovalbumin (OVA)) was restricted to the highly PNA-positive cell fraction. The capacity of these highly PNA-positive B cells to present antigen was assessed by monitoring interleukin 2 (IL-2) production by the OVA-specific T cell hybridoma, 3DO-54.8. GC B cells obtained from mice 3 wk or more after secondary immunization did not elicit IL-2 production in the absence of added OVA. However, GC B cells isolated as early as 1 day and for over 1 wk after a challenge with OVA, were able to stimulate high levels of IL-2 production, in the absence of adding OVA to the cell cultures. This response was maximal on day 5 and corresponded precisely with the kinetics of the ultrastructural studies which document the uptake of antigen by GC B cells in vivo. The FDC-derived antigen was remarkably immunogenic when compared with exogenous antigen. These findings demonstrated that antigen obtained in vivo by GC B cells could be processed and presented to T cells. In vivo, GC B cells may induce the T cell help needed for the germinal center reaction, generate B memory cells, and help induce the high titers of antibody associated with the secondary antibody response.     

Comparison of the influenza virus-specific effector and memory B-cell responses to immunization of children and adults with live attenuated or inactivated influenza virus vaccines

Cellular immune responses to influenza virus infection and influenza virus vaccination have not been rigorously characterized. We quantified the effector and memory B-cell responses in children and adults after administration of either live attenuated (LAIV) or inactivated (TIV) influenza virus vaccines and compared these to antibody responses. Peripheral blood mononuclear cells were collected at days 0, 7 to 12, and 27 to 42 after immunization of younger children (6 months to 4 years old), older children (5 to 9 years old), and adults. Influenza virus-specific effector immunoglobulin A (IgA) and IgG circulating antibody-secreting cells (ASC) and stimulated memory B cells were detected using an enzyme-linked immunospot assay. Circulating influenza virus-specific IgG and IgA ASC were detected 7 to 12 days after TIV and after LAIV immunization. Seventy-nine percent or more of adults and older children had demonstrable IgG ASC responses, while IgA ASC responses were detected in 29 to 53% of the subjects. The IgG ASC response rate to LAIV immunization in adults was significantly higher than the response rate measured by standard serum antibody assays (26.3% and 15.8% by neutralization and hemagglutination inhibition assays, respectively). IgG ASC and serum antibody responses were relatively low in the younger children compared to older children and adults. TIV, but not LAIV, significantly increased the percentage of circulating influenza virus-specific memory B cells detected at 27 to 42 days after immunization in children and adults. In conclusion, although both influenza vaccines are effective, we found significant differences in the B-cell and antibody responses elicited after LAIV or TIV immunization in adults and older children and between young children and older age groups.     

In vitro induction of a Haemophilus influenzae type b polysaccharide-specific antibody response in human peripheral blood lymphocytes of individuals recently vaccinated with an oligosaccharide-protein conjugate.

In this paper an in vitro culture system for the induction of an antibody response to the Haemophilus influenzae type b polysaccharide (PRP) is described. Anti-PRP IgM and IgG antibody-secreting cells (ASC) and anti-diphtheria toxoid (DT) IgG ASC were detected in cultures of blood B and T cells derived from donors 4 to 6 wk after immunization with Haemophilus influenzae type b oligosaccharide-mutant diphtheria toxin (CRM197) conjugate (HbOC) and required in vitro restimulation with HbOC. When lymphocytes from HbOC-vaccinated donors were stimulated with PRP, anti-PRP IgM and IgG ASC could be detected in 50% offGe cases. Lymphocytes from PRP-vaccinated donors or non-vaccinated donors consistently failed to generate anti-PRP antibodies after in vitro stimulation with HbOC. Optimal in vitro responses were observed at concentrations of 0.06 to 0.6 micrograms/ml of Ag. At higher doses of Ag (6 micrograms/ml) anti-PRP and anti-DT antibody responses were suppressed. The in vitro generation of anti-PRP and anti-DT ASC, as detected by a spot-forming cell assay was shown to be T cell dependent, Ag dependent, and Ag specific. This culture system provides a model for the study of human B cell activation and immunoregulation by polysaccharide-protein conjugates and polysaccharides.     

Heterologous antibody responses in mice with chronic T. cruzi infection: depressed T helper function restored with supernatants containing interleukin 2

Antibody production to sheep erythrocytes (SRBC) or hapten-conjugated SRBC (TNP-SRBC) was studied in mice with chronic Trypanosoma cruzi infections. Studies in vivo demonstrated that both IgM and IgG anti-SRBC responses were suppressed during chronic infection. Secondary IgG responses were suppressed regardless of whether the primary immunization was given before or after infection. The ability of cells from infected mice to provide help for antibody production was examined in vitro. Anti-SRBC responses were restored to cultures of whole spleen cells from infected mice by the addition of interleukin 2 (IL 2)-rich supernatants, indicating that these cells were capable of antibody production when sufficient help was provided. T cells from SRBC-primed infected mice were unable to provide significant help to normal B cell/M phi cultures for in vitro anti-TNP or anti-SRBC responses. The percentages of Thy-1+, Lyt-1+, and Lyt-2+ spleen cells were not significantly different between normal and infected mice. Anti-TNP and anti-SRBC responses were restored to cultures that contained T cells from infected mice and normal B cell/M phi by the addition of IL 2-rich spleen cell supernatants. The suppression of in vitro antibody responses in mice with chronic T. cruzi infections was associated with a lack of T cell help, which was provided by exogenous spleen cell supernatant.     

Inhibition of experimental autoimmune thyroiditis in mice by anti-I-A antibodies

Experimental autoimmune thyroiditis is induced in mice by immunization with thyroglobulin emulsified in Freund's complete adjuvant. The disease is characterized both by thyroid infiltration with mononuclear cells and by circulating thyroglobulin antibodies. The magnitude of the thyroid infiltration and the titer of thyroglobulin antibodies are controlled by genes in the I-A subregion of the major histocompatibility complex (H-2). We investigated the in vivo effect of monoclonal anti-Ia antibodies on experimental autoimmune thyroiditis in susceptible mice. Antibodies were given around the time of immunization, later after immunization, and to mice with established disease. Monoclonal antibody produced by the hybridoma line 10-3.6 (anti-I-Ak, s, u, v, z, f) completely prevented both production of thyroglobulin antibodies and thyroid infiltrates, when given shortly before or at the time of antigen administration. This effect was dose-dependent and this monoclonal antibody decreased the severity of the disease when given after the antigen challenge but did not fully suppress established thyroiditis. The same antibody markedly decreased the number of B lymphocytes in the spleen and decreased the thyroglobulin-induced spleen cell proliferation when either given in vivo or added in vitro to cell cultures. Antibodies produced by the hybridoma line 11.2.12 (anti-I-Ak) did not show an inhibitory effect on the disease. These experiments suggest that in this model of murine thyroiditis anti-Ia antibodies act on antigen-presenting cells. Furthermore, only one monoclonal antibody, anti-Ia, suppressed the immune response to thyroglobulin, suggesting a possible role for the isotype and specificity of anti-Ia antibody.     

Human B lymphocytes capable of spontaneous Ig production in short-term cultures: characterization in the circulation and lymphoid tissues

In the present work we have studied the occurrence and the characteristics of human B lymphocytes which are capable of spontaneous immunoglobulin (Ig) production in short-term cultures. It was found that this type of B cells secreted predominantly IgG and were present in the circulation as well as in the lymphoid tissue (tonsil and lymph node) of normal subjects. Tissular and circulating cell subsets exhibited many similarities: These cells produced Ig during a 3-day culture period without apparent need for T cells or monocytes. Protein and DNA synthesis were required for Ig secretion to occur. In Percoll fractionation experiments these cells showed low density, as they were mainly collected in the 42.5-45% Percoll fractions. These subsets consisted of cells considerably larger in size than the majority of B lymphocytes, as determined by Ig sedimentation. They were commonly defined as SmIg- Ia+ B cells by panning fractionation techniques. All these common characteristics suggest that these B cells have reached an advanced stage of maturation in vivo in both circulation and lymphoid tissue. Further surface marker analysis demonstrated that tissular but not circulating B-cell subset reacted with peanut agglutinin and the BA-2 monoclonal antibody which probably indicates their germinal center origin.     

Asialo-GM1+ natural killer cells directly suppress antibody-producing B cells

Natural killer (NK) cells were tested for their ability to suppress antigen-induced antibody responses in vitro. Asialo-GM1+ (ASGM1+) cells were prepared from nylon-wool-nonadherent spleen cells obtained from normal mice. After depletion of Ig+, L3T4+ and Lyt-2+ cells, the ASGM1+-enriched cell population had high NK activity which was abrogated by treatment with anti-ASGM1 and C'. This NK-enriched ASGM1+ cell fraction significantly suppressed the generation of antibody-producing cells when added to in vitro immunization cultures of primed spleen cells. Treatment of the NK-enriched cell population with anti-ASGM1 and C' abrogated the ability of these cells to suppress antibody responses. In vitro antibody production by purified B cells was also suppressed in the presence of the NK-enriched cell population, although the kinetics of the suppression differed from that observed with unfractionated spleen cells. In addition, the NK-enriched cell population suppressed the proliferation of the B cell line WEHI-279.1. Suppression of WEHI-279.1 cells was abrogated when the NK-enriched cell population was treated with anti-ASGM1 and C'. These results suggest that normal NK cells suppress the generation of antibody-producing B cells and that this occurs, at least in part, through a direct regulation of the B cell.     

In vitro Induction of primary antibody responses to particulate and soluble protein antigens in T cell—replaced murine spleen cell cultures

Specific antibody responses could be induced in serumfree condition.Specific anti-SRBC or anti-SRBC ghost antibody were induced from anti-Thy treated (T-depleted) murine spleen cells in serum-free culture in the presence of Con A conditioned medium.This induction system may facilitate the study of lymphokine functions on antigen triggered B cells. In T cell-replaced cultures,the antibody responses of B cells could be successfully induced when soluble SRBC membrane proteins were used as antigens.It thus indicates that antigen together with lymphokines are sufficient to drive B cells to become antibody secreting cells in the absence of T cells.The T cell-replaced system provides a more stable way for in vitro immunization and may be applied to monoclonal antibody production when in vivo immunization is difficult to be carried out.     

Generation of nondividing high rate Ig-secreting plasma cells in cultures of human B cells stimulated with anti-CD3-activated T cells.

The capacity of human B cells to differentiate into high rate nondividing antibody-secreting plasma cells was investigated. Highly purified human peripheral blood B cells were stimulated with polyclonal B cell activators in the presence of a variety of recombinant cytokines (IL-2, IL-4, IL-6). Maximal production of Ig of all isotypes was observed when B cells were stimulated with intact T cells that were activated with mAb to the CD3 molecular complex. In these cultures, Ig production continued for more than 16 days. Moreover, differentiation to nondividing high rate Ig-producing cells was induced, as evidenced by a ninefold increase in the amount of Ig produced per Ig-secreting cell and the acquisition of resistance of ongoing Ig secretion to the inhibitor of DNA synthesis, hydroxyurea. To determine whether intact T cells were required for the entire culture period to achieve maximal Ig production, B cells were cultured with activated T cells for various lengths of time, reisolated and cultured with fresh activated T cells or various cytokines, then analyzed for Ig secretion. B cells preactivated for 6 days with anti-CD3-stimulated T cells required contact with intact T cells for continued Ig secretion. However, after 9 days of preactivation, dividing B cells responded maximally to anti-CD3-stimulated T cells, whereas cytokines were able to drive continued IgG secretion by nondividing B cells in the absence of intact T cells. IL-6 alone, or in combination with either IL-2 or IL-4, was the major cytokine driving ongoing Ig secreting by nondividing preactivated B cells. These results suggest that continued clonal expansion of Ig-secreting B cell blasts requires intact anti-CD3-activated T cells, whereas terminal differentiation of B cells into plasma cells after extensive clonal expansion is driven by cytokines, most notably IL-6.     

Activation, proliferation, and differentiation of circulating B cells in autoimmune thyroid disease

Several studies of thyroid autoantibody production in vitro have been reported with the use of pokeweed mitogen, but the conclusions that have resulted regarding the immunoregulation of B cell function in thyroid disease are difficult to interpret due to the relatively nonphysiologic nature of pokeweed mitogen stimulation. We have therefore examined the responses of circulating B cells in Graves' disease and Hashimoto's thyroiditis by using a combination of lymphokines and other stimuli that act at various stages of the B cell cycle. In patients with autoimmune thyroid disease, nonspecific B cell proliferation and differentiation into IgG-secreting cells were both normal. However, a previously unsuspected heterogeneity among patients was found in their ability to produce autoantibodies in vitro. B cells of certain patients produced maximal autoantibody in response to pokeweed mitogen, some in response to Staphylococcus aureus Cowan strain I, and some in response to the lymphokines contained in the supernatants of stimulated T cell cultures. There was no correlation between serum autoantibody levels and those achieved in vitro. Attempts to stimulate antibody production by autoantigen (thyroglobulin) were unsuccessful, even when B cells were cultured with purified autologous OKT4+ T cells to avoid potential suppressor effects in the OKT8+ population. However, OKT4+ T cells enhanced pokeweed mitogen-driven autoantibody production. Our results show that several different functional stages of B cells exist in the circulation of patients with autoimmune thyroid disease, and that circulating B cells from such patients do not manifest a uniform response to B cell stimulators. This is presumably the result of differences in migration of circulating B cells and in their level of activation at the major sites of autoantibody production, such as the thyroid gland itself. In the light of these findings, caution is required in interpreting the results obtained from studies of circulating B cells as a means of elucidating the pathophysiology of autoimmune thyroid disease.     

Requirements for the adoptive transfer of antibody responses to a priming antigen in man

Antibody responses to recall vaccines can be adoptively transferred after marrow transplantation in man. Transfer of responses to priming Ag has not been successful, although this would broaden the range of organisms to which recipients could be protected. To investigate the importance of T cells and Ag in such transfer we primed marrow donors with keyhole limpet hemocyanin (KLH) 1 or 3 wk before marrow harvesting. B cells secreting IgM and IgG anti-KLH antibody were present in donor marrow at both 1 and 3 wk after immunization. After T cell depletion, donor marrow was infused into chemo-irradiated recipients, half of whom were immunized pretransplant with KLH. We found no evidence for the transfer of the IgM component of the response. Clonal expansion of the transferred IgG antibody-secreting cells with a corresponding rise in recipient serum IgG antibody levels was seen only when donors were primed 3 wk before marrow harvest and when the recipients were also immunized. IEF and immunoblotting demonstrated that successful transfer coincided with maturation of the IgG primary response from a polyclonal to an oligoclonal pattern and confirmed that donor oligoclonal bands appeared in the recipient serum. We conclude that the immunization protocols required for the transfer of antibody responses to priming Ag reflect the initial dependence of unprimed B cells on T cell help and on prolonged Ag stimulation. Ag-stimulated primary B cells in T cell-depleted marrow respond only to the noncognate growth and differentiation signals available in the chemo-irradiated recipient after an initial period of clonal selection and expansion in the donor which is both T cell and Ag dependent. Even after this initial selection, continued expansion of antibody-secreting clones in recipients retains an absolute dependence on Ag stimulation. Immunization techniques to protect transplant recipients against organisms such as Pseudomonas and CMV may need to be modified accordingly.     

Selective activation of antigen-specific human B cells in recently immunized individuals by nonspecific factors in the absence of antigen

The in vitro effect of nonspecific factors (derived from mixed lymphocyte culture [MLC] supernatants) on human B cell responses was studied in individuals recently immunized in vivo to keyhole limpet hemocyanin, tetanus toxoid, and/or diphtheria toxin. In T cell-depleted fractions of peripheral blood mononuclear cells, nonspecific factors alone, without antigen, selectively induced a specific antibody response to the antigen to which the individual had been recently immunized, at dilutions that did not generate a significant polyclonal response in the remainder of the B cell repertoire. The source of these factors, with respect to MLC donors, did not affect the antibody response. Supernatants of MLC from nonimmunized individuals induced a specific antibody response as effectively as supernatants of MLC from immunized individuals, when added to B cells plus monocytes from recently immunized individuals. Studies in which the same individuals were followed over time showed that these factor-sensitive B cells are seen in the peripheral blood of recently immunized individuals for only a finite period of time. Thus, in vivo immunization with a specific antigen results in the transient appearance in the peripheral blood of B cells that are specific for the antigen in question. These B cells are probably preactivated in that nonspecific factors selectively induce in vitro their further differentiation into antibody-secreting cells, in the absence of added antigen or mitogen. These studies may add further insight into our understanding of the sequential steps involved in the activation and differentiation of human B lymphocytes and provide a model for the combined in vivo and in vitro study of human B cell physiology.     

Rapid changes in the regulatory potential of autologous anti-idiotopic T cells during an antigen-driven primary response

The antibody response of C57BL/6 strain mice to Streptococcus pneumoniae R36a (Pn) is dominated by the T15 idiotype, but the responding cells appear to be idiotypically heterogeneous, in that individual antibody plaque-forming cells (PFC) may express some but not all idiotopes (Id) of the T15 complex. The presence of these distinct Id on the PFC was detected by a plaque-inhibition assay with three different monoclonal anti-Id antibodies, designated AB1-2, MaId5-4, and B36-82. A periodic change in the expression of AB1-2 and MaId5-4 Id was observed during primary (IgM) antibody response to Pn in the spleen. Those two Id were poorly expressed in the log phase of the response between day 2 and day 4 after immunization (few PFC in the spleen bore the Id), but they became detectable on the majority of PFC at the peak of the response, day 5 to day 7. The proportion of the Id-(AB1-2 or MaId5-4) positive PFC declined, again at day 10 after immunization. In contrast, the B36-82 Id was expressed on greater than or equal to 80% PFC throughout the entire primary response. The possibility that the apparent changes in the Pn-reactive cell populations are regulated by autologous anti-Id T cells was tested in vitro. Normal, unimmunized B cells were cultured with Pn, either alone or in the presence of syngeneic T cells isolated from the spleen of mice at the appropriate intervals after immunization: day 2 (T2), day 5 (T5), and days 10 to 14 (T10 to T14); T cells from unimmunized donors (T0) served as a control. The specific response after 4 days in culture was determined in regard to the total PFC as well as the proportion of PFC expressing the Id. Pn-stimulated B cells, alone or with the control T0 cells, produced moderate, variable levels of AB1-2+ and MaId5-4+ PFC. The expression of these two Id in the assay cultures was suppressed by addition of either T2 cells or T10-14 cells, but it was enhanced if T5 cells were added. However, these various T cell populations did not differ in their effect on the total PFC response. Also, the proportion of PFC bearing the third Id, B36-82 was high, and it was not consistently influenced by the added T cells.(ABSTRACT TRUNCATED AT 400 WORDS)