The nature of the suppressive effect of interferon and interferon inducers on the in vitro immune response

Viral-induced interferon inhibition of the primary in vitro plaque-formong cell (PFC) response in the mouse (C57B1/6) involves a dynamic relationship between the nature of the antigen, the concentration of interferon added to antigen-stimulated cultures, and the time of addition of interferon relative to antigen addition. The PFC response to a thymus-dependent antigen (sheep red blood cells) was more easily suppressed by viral-induced interferon than was that to a thymus-independent antigen (E. coli 0127 LPS), both in terms of inhibitory concentrations of interferon and the time at which the interferon could be added to cultures after antigen and still inhibit the PFC response. These differential effects of interferon could be related to the difference in cellular requirements (B and T lymphocytes) of the two antigens. Interferon was effective in inhibiting the in vitro PFC response of antigen-primed spleen cells, indicating that it can block the response of memory lymphocytes. By using interferon inducers as inhibitors of the in vitro PFC response, it was possible to show that at least two antigenically distinct interferons may be involved in suppressing the immune response. It is known that one type of interferon is induced by virus and synthetic double-stranded polyribonucleotides. The other type is stimulated by antigen and T cell mitogens. A model is proposed to explain the nature of these interferon inhibitory effects in terms of mediation of immune suppressor cell effects.     

Synergistic Induction of Interferon α through TLR-3 and TLR-9 Agonists Identifies CD21 as Interferon α Receptor for the B Cell Response

Maternal antibodies inhibit seroconversion and the generation of measles virus (MeV)-specific antibodies (both neutralizing and non-neutralizing antibodies) after vaccination whereas T cell responses are usually unaffected. The lack of seroconversion leaves individuals susceptible to vaccine-preventable infections. Inhibition of antibody secretion is due to the inhibition of B cells through a cross-link of the B cell receptor with the inhibitory FcγIIB receptor (CD32) by maternal antibody/vaccine complexes. Here, we demonstrate that a combination of TLR-3 and TLR-9 agonists induces synergistically higher levels of type I interferon in vitro and in vivo than either agonist alone. The synergistic action of TLR-3 and TLR-9 agonists is based on a feedback loop through the interferon receptor. Finally, we have identified CD21 as a potential receptor for interferon α on B cells which contributes to interferon α-mediated activation of B cells in the presence of maternal antibodies. The combination leads to complete restoration of B cell and antibody responses after immunization in the presence of inhibitory MeV-specific IgG. The strong stimulatory action of type I interferon is due to the fact that type I interferon uses not only the interferon receptor but also CD21 as a functional receptor for B cell activation.     

Size-dependent B lymphocyte subpopulations: relationship of cell volume to surface phenotype, cell cycle, proliferative response, and requirements for antibody production to TNP-Ficoll and TNP-BA

Murine splenic B lymphocytes were separated into size-dependent subpopulations by using counterflow centrifugation. Spleen cells were rigorously depleted of T lymphocytes to yield a population of cells that were greater than 90% surface immunoglobulin (Ig)-positive and that had a mean cell volume of 136.6 +/- 3.3 microns. From this population, five fractions of cells were obtained with mean cell volumes that ranged from 115.8 +/- 3.7 microns in fraction 1 to 168.0 +/- 6 microns in fraction 5. The cells in these five subpopulations were characterized by analysis on a fluorescence-activated cell sorter after staining with acridine orange to evaluate RNA and DNA content, and with fluorescein-conjugated anti-mu, anti-delta, and anti-Ia antibodies to evaluate their surface membrane phenotypes. DNA analysis revealed that virtually all of the cells in fractions 1 to 4 had 2 N DNA. Between 7 and 21% of fraction 5 cells were either in S-phase or contained 4 N DNA. In contrast, RNA content increased through the fractions, suggesting a transition from G0 to G1 in the subpopulations with increasing B cell size. As another measure of cell activation seen with increasing cell size, we observed a progressive increase in the expression of surface Ia and a decrease in the expression of surface IgD. In the absence of in vitro stimulation, the larger cells showed significantly higher levels of thymidine incorporation. When polyclonal B cell activators such as LPS or anti-Ig antibody were added, peak proliferative responses were similar in all of the fractions, but the time necessary to achieve a maximal response was shorter for the larger-sized cell subpopulations than it was for the smaller-sized cell subpopulations. Unprimed, size-dependent B lymphocyte subpopulations exhibited spontaneous or "background" antibody formation that occurred primarily in the subpopulations containing the largest cells. T cell factors present in EL4 supernatant enhanced the efficiency of in vitro differentiation of these same subpopulations. When cultured in the absence of T cell help, the thymus-independent type 1 (TI-1) antigen TNP-Brucella abortus (TNP-BA) or the thymus-independent type 2 (TI-2) antigen TNP-Ficoll induced the largest anti-TNP plaque-forming cell (PFC) responses in the fractions containing intermediate-sized cells, suggesting that in vitro, antigen-specific responses came primarily from B cells that have been influenced in vivo to leave their small resting state. The subpopulations containing the smallest size B cells required the presence of both a TI antigen and EL4 supernatant for efficient differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunological and biochemical characterization of an epitope of the transferrin receptor involved in the production of interferon-gamma and B-cell growth factor

We have investigated the effect of a newly developed monoclonal antibody, MoAb NDA9, on human lymphocyte function. This MoAb inhibits the capacity of peripheral blood lymphocytes to display blastogenic responses and to produce immunoglobulins when stimulated in vitro with PWM or with soluble antigens. The inhibitory effect seems to result from the decreased ability of T lymphocytes to produce B cell growth factors (BCGF) in the presence of MoAb NDA9. This antibody also blocks the capacity of polyclonal or monoclonal populations of activated human T cells to produce immune interferon (gamma) but has no direct effect on B cell activation and growth in T-cell-independent systems. Immunochemical studies of the antigen recognized by MoAb NDA9 showed that it is an epitope of the transferrin receptor molecule which is distinct from that recognized by the MoAb OKT9.     

Suppressor cells in mice infected with Trypanosoma brucei.

Within 2 to 3 days of infection with Trypanosoma brucei strain S42, the ability of spleen cells from infected CBA mice to mount a primary in vitro antibody response to sheep red blood cells (SRBC) is profoundly reduced, and suppressor cells are generated as detected by cell mixture experiments. Suppressor cell activity lies in the T and adherent cell compartments of spleens from infected mice, but not in the B cell compartment, although antibody responses to a thymus-independent antigen, DNP-Ficoll, are significantly reduced. Suppression of antibody responses of normal spleen cells depends on viable cells from infected mice. The trypanosome, itself, plays no direct role in suppression, and we have ruled out the possibility of antigenic competition as a mechanism of suppression. Our data is consistent with the model of suppressor T cells induced by concanavalin A mitogenesis. We hypothesize that trypanosome antigens may directly stimulate T cells with the concomitant release of factors with affinity for macrophage surfaces thus becoming suppressive for T and B cell responses.     

In vitro studies of the genetically determined unresponsiveness to thymus-independent antigens in CBA/N mice.

The X-chromosome-linked B lymphocyte defect of CBA/N mice has been studied in vitro by comparing the ability of (CBA/N X DBA/2)F1 (X-/X- X X+/Y) male (X-/Y) and female (X-/X+) spleen cells to respond to the thymus-independent antigen DNP (or TNP)-AECM-Ficoll. (CBA/N X DBA/2)F1 male spleen cells failed to generate significant in vitro anti-TNP antibody responses to DNP- or TNP-AECM-Ficoll, in contrast to spleen cells from F1 female (X-/X+) mice which responded normally to these T-independent antigens. Spleen cells from male F1 mice responded almost as well as F1 female cells to the thymus-dependent antigen, TNP-sheep red blood cells (TNP-SRBC) in vitro. Adding F1 male cells to F1 female cells failed to reduce the response of the latter to DNP-AECM-Ficoll, suggesting that the inability of F1 male cells to respond was not due to active suppression. The response of F1 male spleen cells to TNP-SRBC was not impaired by adding high concentrations of TNP-AECM-Ficoll indicating that the mechanism of unresponsiveness was not tolerance induction in all TNP-specific precursors. Lymphocytes from F1 male mice were capable of forming anti-TNP antibody after stimulation with lipopolysaccharide (LPS) in high concentrations; DNP-AECM-Ficoll had no effect on this polyclonal response. B lymphocytes from mice bearing only the X-chromosome of the CBA/N strain thus display a profound defect in B cell activation. This functional defect may represent either an inability of the defective B cells to be activated by thymus-independent antigens or the absence of a sub-class of B cells which respond to thymus-independent antigens.     

Mechanisms of the adjuvant effect of nystatin on in vitro antibody response of mouse spleen cells: indication of nystatin as a B-cell mitogen and as a stimulant for polyclonal antibody synthesis in B cells.

Adjuvanticity of nystatin, one of the polyenic antifungal antibiotics having as its primary target the membrane sterol of eukaryotic cells, was investigated by examining its effect on several functions of mouse spleen cells relevant to immunological phenomena in vitro. Nystatin was found to stimulate significantly DNA synthesis in thymus-independent (B) cells but not in thymus-dependent (T) cells. Like the other B-cell mitogens such as bacterial lipopolysaccharide (LPS), nystatin elicited nonspecifically polyclonal antibody synthesis in mouse spleen cell cultures, and also restored antibody response of T cell-deficient spleen cells of congenitally athymic nude mice to heterologous erythrocytes (RBC; thymus-dependent antigen). Thus, nystatin and LPS appeared to cause similar changes in the functions of spleen cells relevant to immunological events. However, antagonism but no additive effect in the adjuvanticity was revealed between the two adjuvants. As an interesting finding, the polyclonal generation of anti-RBC antibody-forming cells (AFC) in the spleen cell cultures by stimulation with B-cell mitogen, i.e., either nystatin or LPS, was not inhibited at all by inclusion of any anti-RBC antiserum, whereas, as is well known, the generation of AFC by stimulation with the antigen was specifically suppressed by the corresponding antiserum, indicating a difference in the genesis between the mitogen-induced AFC and the antigen-induced AFC.     

Detection and characterization of idiotype-specific enhancing cells generated in mice immunized with idiotype

Cellular interaction between MOPC-104E (M104E) cross-reactive idiotypic (CRI) antibody-producing B lymphocytes and lymphocytes generated by immunization with the relevant idiotype, M104E, was investigated. Adoptive transfer of M104E idiotype-primed and normal spleen cells into 600R x-irradiated syngeneic recipient mice resulted in striking enhancement of the M104E-CRI positive antibody response upon simultaneous immunization of recipients with dextran B1355S. The enhancement was not attributable to a simple additive effect but was due to synergistic cooperation between the two lymphocyte populations. This synergistic enhancement of the anti-idiotype immune cells producing CRI antibody was specific for MOPC-104E CRI, and was reproducible in an in vitro culture system. Because of the cellular characteristics of the enhancing cells, they were assumed to be B lymphocytes specific for the corresponding idiotype, since the activity was not abrogated by treatment with anti-Thy-1, anti-Lyt-1, anti-Lyt-2, or anti-brain-associated theta antisera plus complement, but was eliminated by means of a planning method using a rabbit-anti-mouse immunoglobulin-coated or idiotype-coated dish. The mechanisms of interaction between the CRI-positive B cells and anti-idiotypic B cells in response to the thymus-independent antigen dextran B1355S are discussed.     

Thymus-independent anti-DNP antibody responses to DNP-casein and DNP-gelatin.

Although studies on the molecular nature of thymus-independent antigens suggested that the polymeric structure with repeated antigenic determinants and slow metabolism are responsible for thymus-independence, we found that anti-DNP antibody responses to DNP-casein and DNP-gelatin were thymus-independent as well as macrophage-independent. These antibody responses were not affected by in vivo treatment with carrageenan or anti-thymocyte serum. In addition, responses of athymic nude mice to both antigens did not show any significant differences when compared with heterologous nu/+ mice. The findings were confirmed by in vitro experiments; non-adherent spleen cells or T cell-depleted spleen cells responded well to both antigens to the same extent as normal spleen cells. Since both casein and gelatin are polyclonal B cell activators and are not presumed to be high polymer or slow-metabolizing substances, we suggest that thymus-independence in many kinds of antibody response should be reconsidered.     

Idiotope antigens (Ab2 alpha and Ab2 beta) can induce in vitro B cell proliferation and antibody production

We previously showed that immunization of various strains of mice with three types of antigen--PC-Hy (nominal antigen), F6-Hy (Ab2 alpha-Hy, and 4C11-Hy (Ab2 beta-Hy)--induces a differential PC-specific, T15-Id+ antibody response. In this report, the in vitro phosphorylcholine (PC)-specific B cell responses induced by these three antigens were studied. A hemocyanin-specific long-term T helper cell line was used to provide help for primary and secondary in vitro T cell-dependent B cell responses. At low doses (0.005 to 0.5 micrograms/ml) of antigen, a significant increase in the proliferation of PC-OVA-primed BALB/c B cells was observed with Ab2-Hy or PC-Hy conjugate, but not unconjugate, antigens. Similar low doses of antigen could stimulate naive B cells to secrete IgM and stimulate PC-OVA- or 4C11-Hy-primed B cells to secret IgM and IgG1 anti-PC antibodies. The percentage of T15-Id of the PC-specific antibodies produced in the in vitro T-B culture was found to be less dominant than that produced by in vivo immunization, suggesting that certain regulatory mechanisms occur in the in vivo environment that may help to maintain the T15-Id dominance. Taken together, our in vivo and in vitro results indicate that idiotope antigens can function like nominal antigens to induce antigen-specific B cell responses. The mechanisms of thymic-dependent B cell activation induced by idiotope and nominal antigen are similar in that the T-B interaction is MHC-restricted and requires cognate recognition.     

B lymphocyte regulation of the immune system. I. In vivo biologic activity of the novel lymphokine, B cell-derived enhancing factor (BEF)

B cell-derived enhancing factor (BEF) is a lymphokine of B cell origin which was originally identified and characterized by its ability to enhance in vitro antibody responses, an effect shown to be due to the ability of BEF to reduce the activation of suppressor T cells. The present study was undertaken to determine whether BEF could also be active in modulating antibody responses in vivo. The data presented here demonstrate that BEF is biologically active in vivo, as manifested by significantly enhanced primary IgM and IgG antibody responses in mice that were either injected with BEF prepared exogenously or implanted with growing BEF-secreting cells of a B cell line. Moreover, BEF was shown to enhance subsequent development of immunologic memory in mice pretreated with BEF at the time of primary immunization; these mice then displayed enhanced secondary responses when challenged with the same antigen some weeks later. The mechanism by which BEF exerts biologic activities to positively modulate in vivo antibody responses and immunologic memory reflects the ability of BEF to modulate one or more T cell functions, as evidenced by the following findings. 1) Transient in vitro exposure to BEF of T cells, but not of B cells, endowed such cells with the capacity to adoptively transfer enhanced primary antibody responses to irradiated recipients. 2) Utilizing adoptive in vivo antibody responses, in which fractionated B cell or T cell populations were obtained from BEF-pretreated mice, revealed that one effect of BEF which results in enhanced immunologic memory is related to its activity on T cells during the priming phase of the immune response. Finally, the existence of this B cell-derived lymphokine and the demonstration of its in vivo regulatory effects on the immune system provide yet another example of the emerging biologic importance of B lymphocytes in the overall regulation of the immune system.     

Regulation of the anti-alpha(1----3) dextran IgG antibody response of BALB/c mice by idiotype-specific T suppressor lymphocytes.

The immune response of BALB/c mice against the so-called thymus-independent bacterial Ag alpha(1----3) dextran (Dex) is restricted to the expression of few major idiotypes (Id). It is furthermore under the control of T lymphocytes which regulate the isotype expression in such a way that they prevent anti-Dex IgG antibody production upon immunization. At the same time these T cells are part of a regulatory system for Dex-specific B cell memory formation. The underlying Ts cell activity has previously been analyzed by using euthymic and athymic congenic animals. Now we have isolated CD4-positive Id-specific T cell lines and clones which by several criteria are representatives of the above Ts cells. They inhibit in vitro proliferation and antibody secretion of Dex-specific hybridoma B cells. They prevent Id-restricted in vivo IgG anti-Dex antibody formation in T cell-reconstituted BALB/c nu/nu mice. At the same time they enforce, again Id-specific, accumulation of Dex-specific B memory cells. As has been shown previously under the influence of splenic Ts cells, these B memory cells are arrested in the original host but can be expanded and activated for anti-Dex IgG antibody formation upon adoptive transfer into X-irradiated allotype congenic nonresponder BALB.Ighb mice. The data show that the regulatory influence of T cells on the anti-Dex response is Id specific. It can now be studied by means of cloned Ts cells.     

Distinct proliferative T cell clonotypes are generated in response to a murine retrovirus-induced syngeneic T cell leukemia: viral gp70 antigen-specific MT4+ clones and Lyt-2+ cytolytic clones which recognize a tumor-specific cell surface antigen

After immunization of B6 mice with the syngeneic retrovirus-induced T cell leukemia/lymphoma FBL-3, two major tumor-specific proliferative T cell clonotypes were derived. T cell clones derived from long-term lines propagated by in vitro culture with irradiated tumor cells and syngeneic spleen cells were exclusively of the Lyt-2+ phenotype. Such clones were cytolytic, retained their proliferative phenotype indefinitely when expanded by repeated cycles of reactivation and rest, and recognized a tumor-specific cell surface antigen in association with class I MHC molecules. This tumor cell antigen was not present on nontransformed virus-infected cells. Class II MHC-restricted MT4+ clones specific for the viral antigen gp70 were derived from lymph node T cells of FBL-3 tumor-immune mice only by in vitro culture with purified Friend virus in the presence of syngeneic splenic APC. Once derived, however, such clones could be stimulated in the presence of FBL-3 tumor cells and syngeneic spleen cells, demonstrating the reprocessing of tumor-derived gp70 antigen by APC in the spleen cell population. In contrast, no reprocessing of the tumor cell surface antigen by splenic APC for presentation to the class I MHC-restricted T cell clones could be demonstrated. Evidence is presented that FBL-3 T leukemia/lymphoma cells function as APC for Lyt-2+ class I MHC-restricted clones, and that no concomitant recognition of Ia molecules is required to activate these clones. Both Lyt-2+ and MT4+ clones were induced to proliferate in the presence of exogenous IL2 alone, but this stimulus failed to result in significant release of immune interferon. In contrast, antigen stimulation of both clones resulted in proliferation as well as significant immune interferon release. Immune interferon production is not required for the generation of MHC-restricted cell-mediated cytolytic function.     

Anti-Ia inhibits the activity of B cells but not a T cell-derived helper mediator

The effects of addition of anti-Ia sera to cultures of B cells responding to a number of different stimuli were studied. Anti-Ia sera inhibited the responses of B cells to thymus-dependent and thymus-independent antigens. The effects of antisera directed at different subregions within theI region were examined, using the same anti-Ia serum and mouse strains congenic atI. There was some indication that antisera directed at theI-C region might be more efficient at inhibiting responses to a thymus-independent antigen than to a thymus-dependent antigen. An antiserum to another B-cell surface component controlled by theH-2 complex, the D glycoprotein, had no effect on the response of B cells to a thymus-dependent antigen. By contrast, anti-Ia serum added to cultures had no effect on the activity of a T cell-derived, nonspecific, B-cell helper mediator (NSM). We concluded that the binding of anti-Ia sera to B-cell surfaces inhibited B-cell responses to antigen, either by competing directly with the binding of signal molecules, or by delivering an inhibitory signal to the B cell, such that it was subsequently refractory to stimulatory signals.     

In vivo priming of helper T cells in the absence of B cell activation

This paper describes an adjuvant-free immunization regimen that results in the priming of T cells but not B cells. B10.A mice were primed s.c. with syngeneic spleen cells that had been pulsed with the peptide 81-104 derived from pigeon cytochrome c. The T cell response was measured by using a sensitive limiting dilution assay that measures lymphokine production. The precursor frequency of Ag-specific cells found in these mice was indistinguishable from the frequency found in mice primed in the footpads with 81-104 in CFA. A striking difference in antibody induction was found, however, when these two immunization regimens were compared. Mice primed with 81-104 in CFA developed significant serum antibody responses against the peptide, whereas mice primed with Ag-pulsed spleen cells produced no detectable anti-peptide antibodies. This lack of antibody did not result from detectable differences in the T cells that were primed: no differences were seen in IL-2 and IL-4 production or in the ability to provide help to B cells in vitro. In vitro stimulation with LPS suggested that the B cells were not primed by the Ag-pulsed spleen cells. The B cells were not tolerized, however, because boosting the mice with Ag in CFA resulted in the induction of an antibody response. The failure to induce an antibody response by priming with Ag-pulsed spleen cells was not caused by the site of immunization or the total amount of Ag used for priming. The critical variable may be the introduction of the Ag on the surface of an APC; in this form, B cell Ag recognition was apparently inefficient, whereas T cell Ag recognition was optimal.     

Adjuvant properties of thermal component of hyperthermia enhanced transdermal immunization: effect on dendritic cells

Hyperthermia enhanced transdermal (HET) immunization is a novel needle free immunization strategy employing application of antigen along with mild local hyperthermia (42°C) to intact skin resulting in detectable antigen specific Ig in serum. In the present study, we investigated the adjuvant effect of thermal component of HET immunization in terms of maturation of dendritic cells and its implication on the quality of the immune outcome in terms of antibody production upon HET immunization with tetanus toxoid (TT). We have shown that in vitro hyperthermia exposure at 42°C for 30 minutes up regulates the surface expression of maturation markers on bone marrow derived DCs. This observation correlated in vivo with an increased and accelerated expression of maturation markers on DCs in the draining lymph node upon HET immunization in mice. This effect was found to be independent of the antigen delivered and depends only on the thermal component of HET immunization. In vitro hyperthermia also led to enhanced capacity to stimulate CD4+ T cells in allo MLR and promotes the secretion of IL-10 by BMDCs, suggesting a potential for Th2 skewing of T cell response. HET immunization also induced a systemic T cell response to TT, as suggested by proliferation of splenocytes from immunized animal upon in vitro stimulation by TT. Exposure to heat during primary immunization led to generation of mainly IgG class of antibodies upon boosting, similar to the use of conventional alum adjuvant, thus highlighting the adjuvant potential of heat during HET immunization. Lastly, we have shown that mice immunized by tetanus toxoid using HET route exhibited protection against challenge with a lethal dose of tetanus toxin. Thus, in addition to being a painless, needle free delivery system it also has an immune modulatory potential.     

Development of B cell subsets: effect of priming on in vitro TNP-LPS responsiveness.

Mice were injected with DNP-derivatives of thymus dependent (DNP-OA) and thymus-independent (DNP-Ficoll and TNP-LPS) antigens, and the response to TNP-LPS in vitro was studied after several priming periods. DNP-OA priming decreases the amount of cells responding to TNP-LPS in vitro. In the case of DNP-Ficoll and TNP-LPS-primed cells, there is an initial burst of responsiveness to TNP-LPS, which progressively decreases until an abolishment of TNP-LPS responsiveness is found at day 40 after immunization. The sensitivity to TNP-LPS reappears as new precursor cells differentiate into mature cells. We suggest that B cells progressively gain the capacity to respond to thymus-independent and thymus-dependent antigens (B1-B2 differentiation) and that challenge with a particle antigen increases the ratio of maturation throughout the pathway.     

Regulation of antibody production by helper T cell clones in experimental autoimmune myasthenia gravis

Ten acetylcholine receptor (AChR)-specific T cell clones from Lewis rats were studied. These clones had various AChR subunit and peptide specificities, and proliferated in response to antigen on appropriate APC. All the T cell clones were CD4+CD8- and OX22-, helped anti-AChR antibody production by AChR-primed lymph node B cells, and could secrete IL-2. However, several lines of evidence suggested that IL-2 was not the lymphokine that mediated T cell help. B cells primed with native AChR and then exposed in culture to very low concentrations of native AChR effectively presented the Ag to the T cell lines, presumably due to uptake via Ag receptors, but primed B cells were no more effective than were non-specific APC at presenting a synthetic AChR peptide which is recognized by AChR-specific T cells but not by AChR-specific B cells. Increasing AChR doses produced an antibody production response that was bell shaped and low doses stimulated, whereas higher AChR concentrations suppressed the antibody production response. Evidence suggested that AChR exerted its inhibitory effect through the T cells, but not via IL-2.     

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.     

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.