The role of interleukin 2 in inducing Ig production in a pokeweed mitogen-stimulated mononuclear cell system

Cyclosporin A (CsA) has been found previously to block mitogen-stimulated T cell proliferation and production of discrete T cell-derived lymphokines such as interleukin 2 (IL 2) and interferon (IFN)-gamma. In addition, CsA blocks pokeweed mitogen (PWM)-driven T cell-dependent differentiation of B cells into immunoglobulin (Ig)-secreting cells. Recently, we reported that CsA (1 microgram/ml) inhibited PWM-induced T cell production of IL 2 and IFN-gamma, but supernatants retained B cell differentiation factor (BCDF)-like activity. The present study demonstrates the ability of CsA to suppress T cell functions in PWM-driven Ig production in mononuclear cells (MNC), and the capacity of exogenous T cell lymphokines to reverse CsA-induced suppression. CsA profoundly suppressed PWM-driven PFC formation (greater than 95%). However, Ig production was substantially reconstituted by the addition of IL 2 at concentrations of 10 to 50 U/ml. In contrast, no effects were observed by the addition of IFN-gamma or BCGF. The kinetics of CsA inhibition of Ig production and IL 2 secretion were found to be closely related. In addition, to obtain effective reconstitution in the CsA-treated PWM-MNC system it was necessary to add IL 2 at the initiation of culture. T cells themselves were also required for B cell differentiation in this system. However, surface Ig+ cells obtained by cell sorting after 3 days of culture could differentiate in the absence of T cells but only in response to IL 2, not in response to IFN-gamma or BCDF. Thus, in PWM-driven B cell differentiation T cells are necessary early in culture, whereas IL 2 is essential from the initial stage of B cell activation through the final stage of B cell differentiation.     

Lymphokines regulate immunoglobulin isotype expression in an antigen-specific immune response

The control of immunoglobulin class switching appears to involve T cell-derived lymphokines. Such lymphokines have been shown to affect isotype expression in polyclonally activated B cells. We show in this paper that the same lymphokines similarly control isotype expression in an antigen-specific response acting in concert with a "T cell independent" antigen. In this situation, B cell growth factor II (BCGF II) enhances the production of antigen-specific IgM antibodies, whereas the production of antigen-specific IgG1 antibodies is only observed in the presence of B cell differentiation factor gamma (BCDF gamma). These results suggest that these lymphokines (and perhaps additional ones) are involved in the control of isotype expression in antigen-specific responses.     

Two independent pathways of helper activity provided by a single T cell clone

Data presented in this paper demonstrate the existence of two separate pathways by which a single T cell clone can induce B cell differentiation. With the use of high doses of antigen, a T cell clone can induce a primary antibody response in unprimed B cells. With the use of low doses of antigen, the same T cell clone can induce an immunoglobulin (Ig)G response in primed B cells. The primary response is accompanied by T cell proliferation and lymphokine production (interleukin 2, B cell growth factor, B cell differentiation factor for immunoglobulin M, and B cell differentiation factor for immunoglobulin G). The secondary response does not require proliferation and occurs independently of detectable lymphokine production. Variants of the wild type T cell helper clone have been isolated. One variant can provide help to unprimed B cells when high doses of antigen are used. This variant cannot provide help to primed B cells when low doses of antigen are used, nor can it provide help to CBA/N "xid" B cells at any antigen concentration tested. Additional variants have been isolated that proliferate on antigen-pulsed-presenting cells, but fail to secrete detectable lymphokines and do not induce B cell differentiation. These results suggest that a single T cell helper clone has multiple functional activities that can be independently expressed.     

Regulation of the anti-alpha (1,3) dextran response: two populations of dextran-reactive B cells that differ in their T cell requirements for induction to antibody synthesis

The in vitro antibody response to dextran B1355S, a thymus-independent Type 2 antigen, requires T cell-derived lymphokines but is not thought to require an activation signal from an antigen-specific T helper cell. The present study demonstrates that there are two dextran-reactive B cell populations in BALB/c mice with respect to the T cell requirements for the generation of antibody-forming cells. One population found among dextran-reactive spleen B cells from 12- to 14-mo-old BALB/c mice generated anti-dextran PFC in the presence of B cell growth factor (BCGF II) and IL 2 or the combination of BCGF II, IL 2, and IFN-gamma. A second population of dextran-reactive B cells found in spleen and Peyer's patches of 2-mo-old unprimed mice did not respond to these same lymphokines, but did generate anti-dextran plaque-forming cells in the presence of Thy-1.2+, L3T4+ T cells from Peyer's patches. However, splenic B cells obtained from 2-mo-old mice that had been primed with dextran 2 to 3 days after birth were shown to be responsive to the same lymphokines as dextran-reactive B cells from 12- to 14-mo-old mice. These results suggest that previous priming with dextran B1355S induces a dextran-specific B cell population that can be activated to antibody-forming cells in the presence of antigen and T cell-derived lymphokines, whereas a second, unprimed population requires an additional activation signal from L3T4+ T cells.     

Interleukin 1 (IL 1)-dependent lymphokine production by human leukemic T cell line HSB.2 subclones

Cloning of a human T cell leukemic cell line, HSB.2, was performed by a limiting dilution method to obtain clones with high levels of IL 2 production. None of the subclones that were obtained produced IL 2 constitutively, and only a low level of IL 2 was produced by the stimulation of these subclones with phytohemagglutinin (PHA) alone. High levels of IL 2 production (greater than 300 U/ml) were observed in several clones when stimulated with a cocktail of PHA and IL 1. Among them, HSB.2-A7-D2, A7-D9, or C5-B2 subclones, which were selected after cloning twice, were most effective in IL 1-dependent IL 2 production. HSB.2 subclones exhibited IL 1-dependent production of a variety of lymphokines other than IL 2, e.g., interferon-gamma (IFN-gamma), B cell growth factor (BCGF), and colony-stimulating factor (CSF). We observed that subclones with high IL 2-producing capacity tended to produce high levels of IFN-gamma or BCGF as well, while the capacity of CSF production was not parallel to these properties. Although several subclones were found to produce IFN-gamma and BCGF simultaneously with minimal IL 2 activity, no subclones with an exclusive BCGF production were obtained. Furthermore, when supernatants from the stimulated A7-D9 subclone were applied to an Ultro-gel AcA54 gel chromatography, it was revealed that IL 2 activity (m.w. 17K to 18K) and IFN-gamma (40K to 45K) were clearly separated, whereas two peaks of BCGF activity coincided with each peak of IL 2 and IFN-gamma, respectively. On the other hand, CSF activity was eluted at a different peak (30K to 35K). These data indicate that IL 2, IFN-gamma, and CSF activities are based on distinct molecules, whereas BCGF activities are indistinguishable from IL 2 and IFN-gamma. The HSB.2 subclones thus selected will provide a useful model for delineating the mechanism of IL 1-dependent lymphokine(s) production, and are a promising candidate for better lymphokine(s) producers.     

The relationship between immune interferon production and proliferation in antigen-specific, MHC-restricted T cell lines and clones

The release of immune or gamma interferon (IFN-gamma) by major histocompatibility complex (MHC)-restricted pigeon cytochrome c-specific Lyt 1+2-, interleukin 2 (IL 2)-producing proliferative T cell clones when cultured with antigen and antigen-presenting cells (APC) is a sensitive measure of the state of activation of the cell. In general, the fine specificity of T cell activation was similar when activation was measured either by IFN-gamma production or by proliferation. In response to antigen and the correct Ia molecule, the T cell clones produced both high titered IFN-gamma and a strong proliferative response. However, IFN-gamma production and the degree of proliferation of the T cell clones differed at high antigen concentrations. As antigen concentration increased, the magnitude of proliferation became submaximal whereas the IFN-gamma response became maximal suggesting that IFN-gamma produced by the cells might act as an autoregulatory molecule inhibiting the proliferative response. Stimulating the T cell to divide via its IL 2 receptor by adding exogenous IL 2 produced high levels of proliferation but only low titers of IFN-gamma activity. In addition, irradiation of the clone eliminated the IFN-gamma release induced by IL 2 but did not affect the IFN-gamma release induced by antigen and Ia. Thus proliferation is not essential for IFN-gamma production and unlike antigen and Ia, IL 2 functions predominantly as a proliferative signal and not as a signal for factor release. Two T cell clones showed a dissociation of IFN-gamma production and proliferation. In one case, a clone that proliferated in response to both allogeneic and antigenic stimuli released IFN-gamma in response to antigen but failed to produce IFN-gamma in response to the allogeneic stimulus. A second clone that showed a strong proliferative response to pigeon cytochrome c but no proliferative response to a species variant of cytochrome c, tobacco hornworm moth (THWM) cytochrome c, produced IFN-gamma when stimulated with either of these antigens. Thus, the sensitivity of detecting activation of T cell clones as measured by the release of an individual lymphokine varies from one clone to another.     

Clonal analysis of B cell growth and differentiation activities induced from T lymphocytes upon triggering of T3-Ti and T11 pathways

The cellular origin of B cell growth factors (BCGF) and differentiation factors (BCDF) was investigated in the present study. For this purpose, T4+ and T8+ T cell clones were obtained from human peripheral blood, activated via stimulation of either the antigen/MHC receptor (T3-Ti molecular complex) or the antigen-independent alternative pathway (T11 molecule), and subsequently examined for their capacity to induce B cell proliferation and immunoglobulin production. The results showed that 1) BCGF is produced by both T4+ and T8+ T cells at the population level as well as at the clonal level; 2) BCDF activity, in contrast, is largely but not exclusively restricted to the T4+ subset; and 3) both the T3-Ti and T11 pathways activate individual clonal T cell populations to promote B cell growth and differentiation.     

Immunologic and molecular characterizations of T cell-derived T cell activating factor

Culture supernatants from several subclones of a human T hybrid line (24A) stimulated with PMA showed co-stimulatory activity in the proliferation of Con A-stimulated murine thymocytes, but did not show any IL 2 activity. Some subclones did not show co-stimulatory activity even when stimulated with PMA, excluding the possibility of a carry-over effect. The factor found in the culture supernatants increased IL 2 production in normal T cells stimulated with a suboptimal concentration of PHA. The factor also induced IL 2 production in a T hybrid clone, T-394.1, when the latter was stimulated with a suboptimal concentration of mitogens, indicating a direct effect by this T cell-derived factor on mitogen-stimulated T cells inducing IL 2 production. This factor also induced the generation of other lymphokines such as BCDF and IFN-gamma. Northern blot analysis showed that the factor induced an increase in mRNA for IL 2 as well as IL 2 receptor. These results indicated that T cells could secrete a factor with IL 1-like activity. However, Northern blot analysis showed that mRNA from a T hybrid clone does not cross-react with cDNA for IL 1 (beta) derived from human monocytes.     

Characterization of a T4+/Leu-8+ T cell clone that directly helps B cell Ig production by secreting B cell differentiation factor

A human T4+/Leu-8+ T cell clone (YA2) was established by phytohemagglutinin activation and interleukin 2 (IL 2) propagation. Functional characterization of this clone demonstrated that it provided potent help towards Ig production by pokeweed mitogen-stimulated B cells in the presence of small numbers of autologous T cells or by Staphylococcus aureus Cowan I (SAC)-activated B cells in the presence of B cell growth factor (BCGF). YA2 provided no help to resting B cells and minimal help to either unactivated B cells cultured with BCGF or SAC-activated B cells. Supernatant generated from clone YA2 by IL 2 stimulation had significant B cell differentiation activity but no BCGF or IL 2 activity. Thus, YA2 is a T4+/Leu-8+ potent direct helper only to B cells that are activated and proliferating due to its selective secretion of a differentiation factor, and not an activation and growth factor. The availability of phenotypically defined cloned populations of T cells with restricted functional helper activity related to the secretion of selected B cell tropic factors should prove useful in the dissection of the role of individual T cell subsets in the regulation of the human B cell cycle.     

The direct effects of interleukin 1, interleukin 2, interferon-alpha, interferon-gamma, B-cell growth factor, and a B-cell differentiation factor on resting and activated human B cells

A wide variety of cytokines have been demonstrated to affect B-cell function. However, it is unclear which of these mediators actually exert direct effects on the B cells themselves. In the present study, the direct role of interleukin (IL) 1, IL-2, Interferon-gamma, or Interferon-alpha in human B-cell activation, proliferation, or differentiation was examined and compared with the effects of a B-cell growth factor (BCGF) or a B-cell differentiation factor (BCDF). Highly purified human B lymphocytes were separated according to size into two nonoverlapping populations. The fraction of small B cells was incubated with IL-1, IL-2, Interferon-gamma, Interferon-alpha, BCGF, or BCDF, and cell size changes, RNA synthesis, DNA synthesis, or supernatant immunoglobulin (Ig) production were measured. Neither IL-1, IL-2, Interferon-alpha, Interferon-gamma, nor the BCGF induced substantial cell size changes, RNA synthesis, DNA synthesis, or Ig production by the small fraction of B lymphocytes; however, the BCDF could directly activate a proportion of resting B lymphocytes to secrete Ig. The fraction of large B cells was also incubated with these cytokines. While neither IL-1, Interferon-alpha, nor Interferon-gamma enhanced DNA synthesis or Ig production by the fraction of large B lymphocytes, DNA synthesis was augmented 23-fold by BCGF and IgG production was increased 7-fold by BCDF. Additionally, IL-2 slightly enhanced both proliferation and differentiation of large B cells but substantially less so than BCGF and BCDF; DNA synthesis was increased 4-fold, while Ig production in the presence of IL-2 was increased by approximately 50%. Thus, the most important lymphokines modulating the function of these two fractions of tonsillar lymphocytes were a BCGF and a BCDF.     

Signal requirements for growth and differentiation of activated murine B lymphocytes

Mouse B lymphocytes were stimulated at high cell concentrations with goat anti-IgM antibodies, which leads to the induction of B cell proliferation without the addition of any growth factors. After 48 hr, blast cells were purified and cultured at low cell concentrations. Proliferation and differentiation of purified B lymphocyte blasts is then dependent on the addition of either mitogens (e.g., LPS) or certain lymphokines derived from activated T cells or macrophages. One such lymphokine was isolated from supernatants of various activated T cells and characterized by gel filtration as a material with an apparent m.w. of 40,000 to 50,000, similar to BCGF II. It supports the proliferation of the B cell blasts and induces their differentiation into plaque-forming cells. Lymphokines such as BCGF I, interleukin 2, and BCDF gamma could neither maintain growth nor induce differentiation of B lymphocytes preactivated by goat anti-IgM.     

Activation of Lyt-1+ and Lyt-2+ T cell cloned lines: stimulation of proliferation, lymphokine production, and self-destruction

Antigen-induced activation of a chicken gamma-globulin (CGG)-specific Lyt-1+ T cell clone measured both as a function of proliferation and immune interferon (IFN-gamma) production is restricted by a class II determinant of the major histocompatibility complex (MHC) mapped to the I-A subregion, as determined by studies with both recombinant inbred lines and monoclonal antibodies. Activation of Lyt-2+ picryl chloride (PC1)-specific cloned T cell lines by trinitrophenyl (TNP)-coupled spleen cells results in proliferation and the production of at least two lymphokines: lymphotoxin (LT) and IFN-gamma. This antigen-specific activation is restricted to a class I determinant of the MHC complex encoded in the K region. Thus, the common intracellular pathway leading to production of IFN-gamma by Lyt-1+ and Lyt-2+ T cells is mediated and restricted through different surface recognition units. The LT that is produced by antigen-specific activation of T cells not only kills fibroblasts, but it inhibits interleukin 2 (IL 2)-maintained T cells as well. Activation of T cells by concanavalin A (Con A) results in suicidal inhibition of proliferation and cell death by those clones that make LT, but not by those that produce only IFN-gamma under such induction conditions. These results indicate that it is neither Con A nor IFN-gamma that kills T cells, but LT. These results strongly suggest a self-regulatory role of LT in limiting continuing unrestricted T cell response to antigen activation.     

Distinct signals are required for proliferation and lymphokine gene expression in murine T cell clones

Experiments were performed to assess the capacity of lectin (Con A), ionomycin, phorbol ester (PMA), and recombinant IL 2 to mediate proliferation as well as the expression of cell surface IL 2 receptors, two lymphokine genes, IL 2 and IFN-gamma, and the c-myc proto-oncogene in cloned T cell populations. Stimulation of T cell clones with recombinant IL 2 resulted in proliferation and sustained expression of the c-myc cellular proto-oncogene, but did not induce the expression of mRNA for the lymphokines IFN-gamma and IL 2. In contrast, stimulation of cloned T cells with lectin alone induced significant IFN-gamma and IL 2 mRNA expression, up-regulation of the number of cell surface IL 2 receptors, and transient c-myc expression. Ionomycin alone was not a sufficient signal for lymphokine mRNA induction. The phorbol ester PMA alone induced neither proliferation nor lymphokine gene expression but potentiated lectin and ionomycin-mediated signals. We also performed experiments to examine whether the T cell response to extracellular stimuli was a function of the activation state of the cell. Reexposure of 48-hr antigen-activated cloned cells to identical stimuli revealed several differences. Low but significant levels of IFN-gamma mRNA were now also reinduced in activated clones cells in response to IL 2 or PMA alone. Activated cells were refractory to reinduction of IL 2 mRNA by any stimulus, which may reflect a physiologic mechanism to limit clonal expansion after antigenic stimulation. This could be partially reversed by restimulation with lectin in the presence of cycloheximide, suggesting a role for a labile protein repressor in the down-regulation of IL 2 mRNA expression. PMA alone induced an IL 2-independent proliferative response. We demonstrate that distinct signals are required for lymphokine gene expression vs cellular proliferation in cloned T lymphocyte populations, and that the capacity of extracellular stimuli to reinduce expression of lymphokine genes or to mediate cell proliferation is altered by prior activation.     

Immortalization of BGDF (BCGF II)- and BCDF-producing T cells by human T cell leukemia virus (HTLV) and characterization of human BGDF (BCGF II)

Human peripheral T cells were transformed by human T cell leukemia virus (HTLV), and T cell lines producing BGDF (BCGF II) and BCDF were established. Among these cell lines, a cell line, TCL-Na1, secreted the highest level of both BGDF and BCDF, and the amount of BCDF secreted by TCL-Na1 cells was 900-fold more than that produced by PHA-stimulated T cells. Within the limits of our examination, none of the HTLV-transformed T cell lines produced IL 2 or BSF-p1 (BCGF I). BCDF produced by TCL-Na1 cells had a m.w. of 35,000 and a pI value of 5.5, being separated from BGDF, which was eluted in the fractions corresponding to m.w. of more than 60,000 and pI values of 5 to 6. BGDF induced both proliferation and IgM secretion in a mouse leukemic B cell line, BCL1, and these activities were not separated by either isoelectric focusing or gel filtration in the presence or absence of 0.1% Triton X-100, suggesting that the molecule designated BGDF exerted both growth and differentiation activities. BGDF acted on normal mouse B cells to induce proliferation as well as IgM secretion. The target cells of BGDF were in vivo activated B blast cells. BGDF acted on DXS-activated murine B cells to induce both proliferation and IgM secretion but not anti-Ig-activated B cells, indicating that BGDF and BSF-p1 were different molecules.     

Role of BCGFII in the differentiation to antibody secretion normal and tumor B cells

This report describes the effects of B cell growth factor (BCGFII) and other lymphokines in the differentiation of normal and tumor B cells. We compared BCL1 tumor B cells, normal B cells giving rise to a polyclonal response without the intentional addition of antigen, and an antigen-driven, SRBC-specific response. BCL1 tumor B cells gave maximum PFC responses when partially purified BCGFII was added or when suboptimal doses of BCGFII were mixed with one of several putative terminal differentiation factors we call B cell differentiation factors BCDF. IFN-gamma was not active as any of these factors. Maximum polyclonal responses of B cells were seen when either IL 2 or BCGFII were mixed with BCDF. In contrast, SRBC-specific responses showed a strict requirement for IL 2, and BCGFII and BCDF synergized with IL 2 to give a maximum response. The involvement of BCGFII in all of these responses suggests that BCGFII acts as a growth factor for a population of B cells that has differentiated much of the way towards Ig secretion, and that many B cells become responsive to this growth factor. In addition, the fact that different lymphokine requirements were seen in the different experimental systems raises the possibility that there are multiple pathways to Ig secretion, and suggests that different subpopulations of B cells defined either by different lineages or by different stages of development within a single lineage have requirements for distinct lymphokines that regulate their growth and differentiation.     

Different antigen-presenting cells differ in their capacity to induce lymphokine production and proliferation of an apo-cytochrome c-specific T cell clone

The activation of an apo-cytochrome c-specific T cell clone was found to differ, depending on the antigen-presenting cell population. Whereas total syngeneic spleen cells and bone marrow macrophages could be shown to trigger proliferation, IL 2, and MAF production by the T cell clone, a B cell lymphoma only induced MAF secretion. Further studies demonstrated that this effect was not due to a different antigen processing by the B lymphoma or to limiting amounts of Ia and antigen molecules on the B lymphoma cell surface. The dissociation of induction of MAF production from IL-2 production/proliferation found with the different antigen-presenting cells indicates strongly that molecules other than Ia and antigen may be required for the complete functional activation of antigen-specific T cell clones.     

Release of lymphokines after Epstein Barr virus infection in vitro. I. Sources of and kinetics of production of interferons and interleukins in normal humans

Infection of human lymphocytes with Epstein Barr virus (EBV) activates the release of lymphokines. Previous experiments have emphasized the ability of interferon-gamma (IFN-gamma) to prevent EBV-induced B cell transformation. However, the factors that regulate IFN-gamma synthesis and release during in vitro EBV infection are controversial. In the present investigation we have systematically evaluated the kinetics of production, cellular origins, and accessory cell requirements for IFN-alpha and IFN-gamma and for IL 1 and IL 2, after EBV infection. Our data indicate that IFN-alpha is released entirely by natural killer (NK) cells and B cells, in the absence of accessory cells, independently of the other lymphokines and within 24 hr of infection. In contradistinction, IFN-gamma secretion is exclusively of T cell origin, is absolutely dependent on the prior elaboration of IL 1 and IL 2, and is maximal 8 days after EBV infection. IL 2 secretion by T cells peaks on day 5 and requires the earlier release of IL 1. Both NK cells and monocytes are a source of IL 1. Secretion of IL 2 and IFN-gamma occurs in the presence of either one of these cell types but not in the absence of both. Antibody against IL 1 blocks EBV-induced IL 2 and IFN-gamma generation, and antibody against IL 2 decreases production of IFN-gamma. Thus, the production of IFN-gamma, the lymphokine that prevents EBV-induced B cell transformation, is the final outcome of a cascade of lymphokine-mediated events that involve interactions between virus-infected B lymphocytes that serve as antigen-presenting cells, NK cells and monocytes as sources of IL 1, and T lymphoblasts. Dysfunctions of any or all of these cell types would be expected to impair the regulation of EBV transformation.     

Characterization of lymphokines mediating B cell growth and differentiation from monoclonal anti-CD3 antibody-stimulated T cells

Addition of anti-CD3 mAb 147 (IgG1), 446 (IgG1), or 454 (IgG2a) to cultures of T plus non-T cells can result in both B cell growth and differentiation. To determine whether lymphokines mediating these activities were similar to those described from conventional mitogen-induced T cell activation, normal peripheral blood T cells were stimulated with anti-CD3 mAb for 48 h. The supernatants were assayed for factors inducing B cell growth or differentiation (BCDF). A marked increase in Ig secretion was observed when either EBV-transformed B cell lines or normal B cells, pre-activated with Staphylococcus aureus Cowan I strain, were cultured in the presence of mAb 446 (anti-CD3) stimulated T cell supernatant whereas no significant increase in Ig secretion was noted with either mAb 454- or 147-induced T cell supernatant despite equivalent T cell proliferative responses to these antibodies. In contrast, IL-2 secretion was detectable in T cell supernatants from T cells stimulated with either mAb 454 or 147 but not 446. Factors promoting B cell proliferation were detected in all antibody-stimulated T cell supernatants but, contrary to BCDF, appear to act only on non-activated B cells. To determine whether these effector activities were due to distinct lymphokines, supernatants were pooled and concentrated by ammonium sulfate precipitation. Superose 12 permeation chromatography revealed BCDF activity with an apparent Mr of approximately 30,000 Da. The growth factor activity eluted over a wider and higher molecular weight range which overlapped the differentiation factor activity. Fractions containing BCDF activity were pooled, dialyzed, applied to a Mono Q anion-exchange column, and eluted with a linear NaCl gradient. The growth factor activity came off in a single-peak while BCDF was found divided into two major areas. The growth factor eluted at an ionic strength between the two BCDF activities. BCDF has an apparent isoelectric point (pI) of 6, in contrast to the reported pI 5 for IL-6 and more acidic than the documented basic pI of IFN-gamma. Lastly, peaks with BCDF activity were not active in assays for either IL-2 or IL-4. In addition, a rabbit anti-IL-6 heteroantiserum failed to inhibit the pI 6 BCDF, suggesting non-identity between IL-6 and anti-CD3 induced BCDF. Thus, anti-CD3 activated T cells generate both growth factor activity and BCDF as separate molecular entities distinct from IFN-gamma, IL-2, IL-4, and conventional IL-6.     

Cognate interactions between helper T cells and B cells. II. Dissection of cognate help by using a class II-restricted, antigen-specific, IL-2-dependent helper T cell clone

In order to determine the involvement of T-B cell contact vs lymphokine production in mediating B cell cycle entry and progression, Th cell clones "defective" in lymphokine production were cloned. Th-3.1 is one such clone that required IL-2 to produce significant levels of IL-4 and IFN-gamma. Unlike conventional Th clones, Th-3.1 induced B cell proliferation only in the presence of Ag and IL-2. In contrast to the absolute requirement of IL-2 for Th-3.1-induced B cell proliferation, IL-2 was not required for the formation of stable Th-3.1-B cell conjugates or Th-3.1-induced B cell entry into the G1 phase of the cell cycle. In the absence of IL-2 and under conditions that promoted Th-B cell interactions, Th-3.1 induced 10 to 20% of resting B cells to enter G1. B cell entry into the cell cycle was not inhibited by anti-lymphokine mAb or promoted by exogenous lymphokines, suggesting that endogenous lymphokine activity was not required for Th-3.1-induced G0 to G1 transition. The data suggested that the IL-2-independent induction of B cells into G1 by Th-3.1 was a cell contact-dependent event. Direct proof that Th-3.1-B cell contact was necessary for B cell cycle entry was provided by comparative in situ analysis of the RNA synthetic activity and the RNA content of B cells that were in physical contact with Th-3.1 or not in contact with Th-3.1. In situ autoradiography of RNA synthesis illustrated that a high frequency of B cells in contact with Th-3.1 expressed heightened RNA synthetic activity, whereas "bystander" B cells were less frequently induced into cycle. In situ laser cytometry of B cell size and total RNA content showed that B cells in physical contact with Th-3.1 had a higher RNA content and were larger than "bystander" B cells present in the same microcultures. This model system has allowed the dissection of T cell help into IL-2-dependent and IL-2-independent phases. Early cell contact-dependent events and B cell cycle progression into G1 were IL-2 independent, whereas the production of lymphokines (IL-4, IFN-gamma) by Th-3.1 and Th-3.1-induced B cell proliferation was IL-2 dependent.     

Antigen-specific B cells efficiently present low doses of antigen for induction of T cell proliferation

Previous experiments suggested a role for specific B cells in the induction of antigen (SRBC)-specific T cell proliferation. Two models were proposed: in the first, B cells directly presented antigen to T cells; alternatively, B cells secreted antibody, which opsonized antigen for presentation by macrophages. Experiments to distinguish between these possibilities are presented here. Three lines of evidence support the conclusion that antigen is presented directly by specific B cells. First, nonimmune splenic adherent cells (SAC), which efficiently induced proliferation of appropriately primed T cells to antigens such as OVA and GAT, were unable to induce SRBC-specific proliferation. Secondly, a slope analysis of the logarithmic plot of T cell proliferation vs the number of irradiated B cells suggested that two cells were limiting within the presenting population. The addition of IL 1 or SAC reduced the slope to 1 (although in serum-free conditions, the addition of IL 1, but not SAC, reduced the slope of the line). Specificity of the B cells for the antigen continued to be required in the presence of exogenous IL 1 or SAC. These results suggested that presentation by specific B cells and the amount of IL 1 were the limiting requirements for the induction of SRBC-specific T cell proliferation. The third line of evidence was the demonstration of a restricted interaction between T cells and B cells. The addition of irradiated, allogeneic SRBC-specific B cells to T cell lines and syngeneic SAC failed to support proliferative responses. We further show that a GAT-specific T cell clone was triggered to proliferate by either SAC or B cells, but that antigen-specific B cells were necessary at low doses of antigen. This finding is important in two respects. First, the T cell clone previously has been shown to act as a helper; secondly, when low doses of antigen are used, the requirement for priming of the B cells to the specific antigen is true for a soluble, as well as a particulate, antigen. We propose that at low (physiologic) doses of antigen, presentation to secondary T cells takes place mainly at the surface of antigen-specific B cells. At high doses of antigen,h presentation can also be accomplished by nonspecific cells such as other B cells, macrophages, or dendritic cells.