Identification of a novel thymocyte growth-promoting factor derived from B cell lymphomas

We found a unique thymocyte growth-promoting activity in supernatants (SN) from subclones of the B cell lymphoma CH12.LX. We have tentatively named this activity B-TCGF (for B cell-derived T cell growth factor) and characterized the activity produced by the CH12.LX.4866 subclone. This SN did not induce thymocyte proliferation alone, however, it enhanced both adult and fetal (Day 15 of gestation) murine thymocyte proliferation in the presence of IL-2, IL-4, or IL-7. Other known cytokines were screened for a B-TCGF-like activity using both adult and fetal thymocytes. IL-6 was found to be active only on adult thymocytes, while TNF alpha and GM-CSF were found to be active only on fetal thymocytes. However, neutralizing antibodies against these cytokines did not block the B-TCGF activity present in CH12.LX.4866 using either adult or fetal thymocytes. These observations suggest that the B-TCGF activity is mediated by a novel factor(s). The apparent molecular weight of this novel molecule(s) was 27-50 kDa determined by sizing HPLC.     

B cell growth-promoting activity of recombinant human interleukin 4

Human interleukin 4 (IL-4), also known as B cell stimulatory factor 1, is a T cell-derived glycoprotein consisting of 129 amino acids for which a cDNA has been recently isolated. IL-4 displays little or no B cell growth factor (BCGF) activity in the standard anti-IgM costimulatory assay using suboptimal concentrations of soluble anti-IgM antibody whereas the low m.w. BCGF is very active. When insolubilized anti-IgM was used as the costimulating agent, both IL-4 and the low m.w. BCGF were found to promote B cell proliferation. Human IL-4 is able to induce the proliferation of B lymphocytes preactivated for either 1 day with insolubilized anti-IgM antibody or for 3 days with Staphylococcus aureus strain Cowan I. However, IL-4 is poorly mitogenic for B cells preactivated for 1 day with the Staphylococcus strain whereas the low m.w. BCGF strongly enhances the proliferation of these B cells. These two findings demonstrate that the preactivation signal necessary to induce human B cells to proliferate in response to IL-4 is critical. The increased tritiated thymidine ([3H]dThd) uptake in preactivated B cell cultures with IL-4 reflects cel proliferation because cell cycle analysis demonstrates that IL-4 induces activated B cells to enter the S and G2/M phases of the cell cycle and the addition of IL-4 to preactivated B cell cultures permits the recovery of three- to fourfold more B cells after 4 days of culture. IL-4 and the low m.w. BCGF act in concert to induce the proliferation of anti-IgM-preactivated B cells as demonstrated by [3H]dThd uptake and cell cycle analysis. In striking contrast to the demonstrated antagonistic effect of interferon-gamma on the IL-4-induced expression of the low affinity receptor for IgE (Fc epsilon RL/CD23), on B cells, it was found that interferon-gamma enhanced the IL-4-induced proliferation of anti-IgM-preactivated B cells. Finally, it was found that IL-4 had to be present continuously during the culture period to exert an optimal growth-promoting effect on B cell blasts. As a conclusion, IL-4 is able to induce the proliferation of an appropriately activated subpopulation of human B cells.     

B-cell-derived interleukin 1 (IL-1)-like factor. I. Relationship of production of IL-1-like factor to accessory cell function of Epstein-Barr virus-transformed human B-lymphoblast lines

The relationship of production of interleukin 1 (IL-1)-like factor to accessory function of Epstein-Barr virus (EBV)-transformed B lymphocytes was examined. Six of eight human EBV-B cell lines spontaneously produced and released detectable levels of thymocyte comitogenic factor in vitro, but no interleukin 2 (IL-2) activity. Eight of eight produced fibroblast proliferation activity. Culture supernatants from the two apparent nonproducers of thymocyte comitogenic activity induced the proliferation of the IL-1-dependent murine helper-T-cell clone D10G4.1 in the presence of concanavalin A (Con A). One of the EBV-B cell lines produced a potent inhibitory factor in addition to IL-1-like thymocyte comitogenic and fibroblast proliferation factors. The inhibitory factor inhibited mouse thymocyte proliferative response to Con A, and the proliferation of the IL-2-dependent CT6 cell line, but not human fibroblast growth. All but one of the eight EBV-B cell lines tested, the exception being the line that produced an inhibitory factor, were able to serve as antigen-presenting cells that enabled purified human T lymphocytes to proliferate in one-way mixed lymphocyte reactions (MLR) and in response to Con A. The supernatants of 14 of 16 clones derived from two of the EBV-B cell line cells contained thymocyte comitogenic activity and all 16 stimulated fibroblast proliferation. The phenotypic characteristics of the EBV-B cell lines were heterogeneous, but there was no clear-cut relationship between the cell surface phenotypes of either the cloned or uncloned EBV-B cells and their ability to produce these factors. These studies show that all of the EBV-B cell lines that can function as accessory cells have the capacity to produce an IL-1-like factor.     

Disparate functional properties of two interleukin 1-responsive Ly-1+2- T cell clones: distinction of T cell growth factor and T cell-replacing factor activities

We describe the properties of two Ly-1+2- T cell clones (Ly-1.14 and Ly-1.21), which are maintained in long-term culture in the absence of other cell types. The clones require media containing a source of interleukin 1 as well as interleukin 2. They retain physiologic responses to interleukin 1, which is required for optimal production of T cell lymphokines by these clones in response to concanavalin A (Con A). The two Ly-1+2- T cell clones differ in their production of lymphokines after stimulation by Con A. The supernatant of clone Ly-1.21 promotes the proliferation of T cells maintained in long-term culture, induces antibody synthesis in cultures of B cells and antigen, and induces the differentiation of cytolytic cells in cultures of thymocytes and antigen; these assays define the properties of T cell growth factor (TCGF), T cell-replacing factor for B cells (TRF-B), and T cell-replacing factor for cytolytic cells (TRF-C), respectively. In contrast, the supernatant of clone Ly-1.14 contains only TCGF activity and does not promote antibody synthesis by B cells or differentiation of cytolytic cells from thymocytes. The results indicates that TCGF and TRF activities reside on independent, although perhaps related, molecules.     

Recombinant interleukin 4 promotes the growth of human T cells

Recently, we reported the isolation of a cDNA clone that encodes a polypeptide which has B cell and T cell growth factor activities. The amino acid sequence of this polypeptide deduced from the nucleotide sequence of the cDNA clone showed significant homology with mouse B cell stimulating factor-1. Because of its multiple biologic activities, it was designated interleukin 4 (IL-4). Here we describe the effects of supernatants of Cos-7 mouse cells transfected with the IL-4 coding cDNA clone in a mammalian expression vector, on human thymocyte T cells and T cell clones. The T cell growth-promoting effect of IL-4 on preactivated T cells was not inhibited by monoclonal antibodies against IL-2 or the IL-2 receptor, indicating that the IL-4 activity is independent from IL-2 or the IL-2 receptor. IL-4 induces a low proliferative response in thymocytes and peripheral blood lymphocytes, but the response was considerably enhanced by preactivation of the thymocytes or peripheral blood T cells. Both T4+ and T8+ antigen-specific proliferative and cytotoxic T cell clones and T3 natural killer clones proliferated in response to IL-4. But one of six T4+ and one of four T8+ T cell clones were consistently found to be unresponsive. The proliferative responses to IL-4 were always lower than those obtained with IL-2. Most of the T cell clones generally became unresponsive to IL-4 10 days after stimulation, but still responded well to IL-2. These results indicate that the responsiveness to IL-4 is relatively short lasting and is regulated by activation signals. Interestingly, IL-4 acted in synergy with IL-2 in promoting the growth of T cell clones. Our results establish that IL-4 can act as a T cell growth factor independently of IL-2.     

Spontaneous production of a suppressor factor by the human macrophage-like cell line U937. I. Suppression of interleukin 1, interleukin 2, and mitogen-induced blastogenesis in mouse thymocytes

The human macrophage-like cell line U937 spontaneously produced a nondialyzable factor that inhibited interleukin 1 (IL 1), interleukin 2 (IL 2), and phytohemagglutinin (PHA)-induced blastogenesis in mouse thymocytes. The suppression by U937 supernatant factor occurred independently of the concentration of IL 1 or PHA, indicating that it was noncompetitive. The U937 suppressor factor was not cytotoxic for thymocytes, nor did it affect the spontaneous proliferation of T lymphoblastoid cell lines and U937. Physicochemical characterization showed that the U937 suppressor factor was nondialyzable, partially inactivated by heat treatment (56 degrees C), ammonium sulfate (67% saturation) precipitable, sensitive to pH 2.5, and resistant to freeze-thawing. Molecular weight of the factor inhibiting co-mitogenic IL 1 activity was approximately 85,000, as estimated by gel filtration. The U937 cell line may provide a model for the study of mechanisms and mediators of immunosuppression by mononuclear phagocytes.     

Activation via TCR or IL-2 receptor of a CD8+ suppressor T cell clone: Effect on IL-10 production and on proliferation of the suppressor T cell

In a previous study, we established CD8⁺ suppressor T cell (Ts) clone 13G2 which produced the suppressive lymphokine, interleukin-10 (IL-10). In this study, we examined what physiological activator could induce both production of IL-10 from 13G2 and the proliferation of 13G2. Both the antigenic stimulation mimicked by the anti-CD3 antibody and the T cell growth factor interleukin-2 (IL-2) induced IL-10 production from the 13G2 clone equally well. 13G2 cells proliferated remarkably with IL-2 stimulation, while anti-CD3 only slightly induced proliferation of the clone. 13G2 cells also produced IL-10 in the presence of hydroxyurea which blocked transit of cells from G₁ to S phase. However, cycloheximide blocked the production of IL-10 from the Ts clone. The study demonstrates that both the anti-CD3 antibody and IL-2 induced IL-10 synthesis of the Ts clone equally well, and the proliferative response of Ts cells was induced more by IL-2 than by anti-CD3. IL-2 proved to be a good stimulator for Ts cells to produce suppressive lymphokine and to multiply their population.Abbreviation Ts suppressor T cell - Th helper T cell - Ag antigen - APC antigen presenting cell - IL interleukin - TCR T cell receptor - mAb monoclonal antibody     

A monoclonal antibody to human B lymphoblastoid cells activates human and murine T lymphocytes

A B lymphoblastoid cell line can provide a comitogenic, accessory signal for mitogen-treated T cells. In a study evaluating the antigenic determinant of such cells that mediate this effect, a monoclonal antibody (I57) was raised against the Daudi cell line. This antibody was found to interact with a 30-kDa protein on these cells and had agonistic properties. It enhanced the B lymphoblastoid accessory cell and interleukin 1 (IL-1)-dependent stimulation of PHA-treated murine thymocytes. The stimulatory effect of I57 on PHA-treated thymocytes was more pronounced at high, supraoptimal concentrations of the lectin. This was in contrast with the effect of IL-1 that failed to stimulate these cells treated with PHA at high concentrations. I57 also enhanced stimulation of thymocytes treated with IL-2 alone or with both PHA and IL-2. I57 exhibited by itself mitogenic activity for human T cells. These cells, treated with IL-2, were further stimulated by I57. I57 seems to be different from other agonistic antibodies that have been described so far.     

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.     

B cell stimulatory factor-2 is involved in the differentiation of cytotoxic T lymphocytes

The induction of cytotoxic T lymphocytes (CTL) from precursor T cells requires both antigen and lymphokine signals. Previous work from our laboratory has indicated that three lymphokines are required for the induction of CTL from murine thymocytes; interleukin 2, interferon-gamma (IFN-gamma), and a partially characterized factor referred to as cytotoxic differentiation factor (CDF). While attempting to clone CDF from the human T cell line C10-MJ2, we found that a gene encoding CDF-like activity is identical to the gene encoding the factor known variously as B cell stimulatory factor-2 (BSF-2), IFN-beta 2, and 26-kDa protein. We report here that BSF-2 can induce the differentiation of Ly-2+ CTL from murine thymocytes in the presence of interleukin 2 and that the level of cytotoxicity is augmented by the addition of murine IFN-gamma. Serine esterase, a marker for cytotoxic granules in CTL, was induced only in the presence of BSF-2, and the level of serine esterase activity correlated with the level of serine esterase activity correlated with the level of cytotoxicity. These data suggest that BSF-2 is a differentiation factor for CTL and that it functions in part by inducing proteins required for mediating target cell lysis.     

Augmentation of effector CD8+ T cell generation with enhanced granzyme B expression by IL-27

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation. We have recently demonstrated that IL-27 has a potent antitumor activity, which is mainly mediated through CD8+ T cells, and also has an adjuvant activity to induce epitope-specific CTL in vivo. In this study, we further investigated the in vitro effect of IL-27 on CD8+ T cells of mouse spleen cells. In a manner similar to CD4+ T cells, IL-27 activated STAT1, -2, -3, -4, and -5, and augmented the expression of T-bet, IL-12Rbeta2, and granzyme B, and slightly that of perforin in naive CD8+ T cells stimulated with anti-CD3. IL-27 induced synergistic IFN-gamma production with IL-12 and proliferation of naive CD8+ T cells. Moreover, IL-27 enhanced proliferation of CD4+ T cell-depleted spleen cells stimulated by allogeneic spleen cells and augmented the generation of CTL. In STAT1-deficient naive CD8+ T cells, IL-27-induced proliferation was not reduced, but synergistic IFN-gamma production with IL-12 was diminished with decreased expression of T-bet, IL-12Rbeta2, granzyme B, and perforin. In T-bet-deficient naive CD8+ T cells, IL-27-induced proliferation was hardly reduced, but synergistic IFN-gamma production with IL-12 was diminished with decreased expression of IL-12Rbeta2, granzyme B, and perforin. However, IL-27 still augmented the generation of CTL from T-bet-deficient CD4+ T cell-depleted spleen cells stimulated by allogeneic spleen cells with increased granzyme B expression. These results suggest that IL-27 directly acts on naive CD8+ T cells in T-bet-dependent and -independent manners and augments generation of CTL with enhanced granzyme B expression.     

Identification and characterization of a B cell activation factor (BCAF) produced by a human T cell line

A human T cell line, Peer, that expresses the T cell helper phenotype produces discrete activation and growth factors for tonsillar B cells. The B cell activation factor produced by Peer is biochemically and physiologically distinct from other lymphokines known to enhance B cell proliferation, namely, interleukin 1, interleukin 2, interferon, and previously characterized B cell growth factors (BCGF). The BCGF produced by Peer is functionally similar to previously described BCGF but has a m.w. of approximately 30,000 daltons. The identification and characterization of a T cell-derived activation factor that can induce apparently resting (Go phase) B cells to enter S phase in the absence of an exogenous first signal has important implications in the additional dissection of the complex steps in the human B cell cycle.     

Further evidence for a human B cell activating factor distinct from IL-4

Supernatants from activated human T cell clones were previously shown to contain B cell-activating factor (BCAF), an activity which results in polyclonal resting B cell stimulation. In the present study, we investigate the relationship between this activity and human interleukin-4 which was also shown to act on resting B cells. The supernatant of the T cell clone TT9 contains IL-4 but anti-IL-4 antiserum does not affect the response of B cells as measured by thymidine uptake or cell volume increase. Furthermore, IL-4 induces Fc epsilon-receptor (CD23) expression on 30% of unstimulated human B cells, whereas BCAF-containing supernatants from clone P2, that do not contain detectable amounts of IL-4, promote B cell proliferation without inducing CD23 expression. Our results therefore establish that IL-4 and BCAF are distinct activities and suggest that they trigger different activation pathways in human B cells. In addition, culture of B cells with T cell supernatants for 72 hr induces a three- to fourfold increase in the expression of HLA-DR, -DP, and -DQ antigens in 50% of B cells. The addition of inhibiting concentrations of anti-IFN-gamma, LT, or IL-4 antisera to the cultures does not change these results. Finally, 30% of B cells cultured with T cell supernatants leave the G1 phase of the cell cycle and 20% reach mitosis. Taken together, our findings further support the existence of a B cell-activating factor responsible for the activation of resting human B cells.     

Stimulation of EBV-activated human B cells by monocytes and monocyte products. Role of IFN-beta 2/B cell stimulatory factor 2/IL-6

EBV infects human B lymphocytes and induces them to proliferate, to produce Ig, and to give rise to immortal cell lines. Although the mechanisms of B cell activation by EBV are largely unknown, the continuous proliferation of EBV-immortalized B cells is dependent, at least in part, upon autocrine growth factors produced by the same EBV-infected B cells. In the present studies we have examined the influence of monocytes on B cell activation by EBV and found that unlike peripheral blood T cells and B cells, monocytes enhance by as much as 30- to 50-fold virus-induced B cell proliferation and Ig production. Upon activation with LPS, monocytes secrete a growth factor activity that promotes both proliferation and Ig secretion in EBV-infected B cells and thus reproduces the effects of monocytes in these cultures. Unlike a number of other factors, rIFN-beta 2/B cell stimulatory factor 2 (BSF-2)/IL-6 stimulates the growth of human B cells activated by EBV in a manner similar to that induced by activated monocyte supernatants. In addition, an antiserum to IFN-beta that recognizes both IFN-beta 1 and IFN-beta 2 completely neutralizes the B cell growth factor activity of activated monocyte supernatants. These findings demonstrate that IFN-beta 2/BSF-2/IL-6 is a growth factor for human B cells activated by EBV and suggest that this molecule is responsible for B cell growth stimulation induced by activated monocyte supernatants. We have examined the possibility that IFN-beta 2/BSF-2/IL-6 might also be responsible for B cell growth stimulation by supernatants of EBV-immortalized B cells and thus may function as an autocrine growth factor. However, IFN-beta 2/BSF-2/IL-6 is not detectable in supernatants of EBV-immortalized B cells by immunoprecipitation. Also, an antiserum to IFN-beta that neutralizes IFN-beta 2/BSF-2/IL-6 fails to neutralize autocrine growth factor activity. This suggests that autocrine growth factors produced by EBV-immortalized B cells are distinct from IFN-beta 2/BSF-2/IL-6. Thus, the continuous proliferation of EBV-immortalized B cells is enhanced by either autocrine or paracrine growth factors. One of the mediators with paracrine growth factor activity is IFN-beta 2/BSF-2/IL-6.     

Human B cell proliferation in response to IL-4 is associated with enhanced production of B cell-derived growth factors

To investigate the capacity of human IL-4 to function as a B cell growth factor (BCGF), we studied its ability to promote proliferation of a selected B cell line. We show that the cell line, designated A4, proliferated in response to IL-4 in a dose-dependent manner. The A4 cells also proliferated in response to their own B cell derived growth factor (B. BCGF), suggesting autocrine-mediated growth. The ability of IL-4 to induce proliferation of the A4 cell line was dependent on the level of autocrine growth. At low cell density, IL-4 induced marked dose-dependent proliferation. However, as A4 cell density increased, the ability of IL-4 to induce proliferation was diminished. The possibility that IL-4 may be mediating the autocrine growth of A4 cells was ruled out, because A4 cell-derived BCGF failed to induce CD23/low affinity receptors for the Fc region of IgE on activated tonsillar B cells and anti-IL-4 antibody did not block B. BCGF activity. We found that IL-4 stimulation of A4 cells and activated tonsillar B cells is associated with enhanced production of B. BCGF. These data indicate that human IL-4 has the capacity to promote proliferation of the B cell line A4, and that the ability of IL-4 to function as BCGF is associated with enhanced autocrine growth of activated B cells.     

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.     

Recombinant T cell replacing factor (interleukin 5) acts with antigen to promote the growth and differentiation of single hapten-specific B lymphocytes

The role of murine recombinant T cell replacing factor (rTRF) (interleukin 5) in the early activation, proliferation, and antibody-forming cell (AFC) clone formation of single fluorescein (FLU)-specific B cells was examined in vitro. FLU-specific B cells were selected by their adherence to FLU-gelatin and then cultured in 10-microliters wells with or without rTRF in the presence or absence of the T-independent antigen FLU-polymerized flagellin (FLU-POL). rTRF acting alone was unable to induce early B cell activation as assessed by significant cell enlargement after 24 hr in culture. When acting in the presence of FLU-POL, however, a greater number of B cells were induced to enlarge than with FLU-POL alone. When FLU-specific B cells were cultured in the presence of FLU-POL, the addition of rTRF markedly increased the frequencies of both proliferating clones and AFC clones above that induced by FLU-POL alone. Furthermore, in the presence of FLU-POL, the activity of rTRF was comparable to that seen with the mixture of B cell growth and differentiation factors contained within the supernatant from concanavalin A-stimulated EL4 cells. However, rTRF exerted little activity when acting alone in contrast to the medium conditioned by concanavalin A-stimulated EL4 cells which showed some activity in the absence of FLU-POL. rTRF acting with FLU-POL also promoted AFC clone development among single B cells stimulated in the presence of 3T3 fibroblast filler cells. Thus rTRF can be added to the list of B cell active factors (including recombinant murine interleukin 1 and recombinant human interleukin 2) that act in the concomitant presence of antigen to induce both growth and differentiation among single hapten-specific murine splenic B cells. This stands in contrast to the activity seen with interleukin 4 (formerly termed B cell stimulatory factor 1) which acts to promote early activation and proliferation but not IgM secretion.     

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.     

Interleukin secretion by B cell lines and splenic B cells stimulated with calcium ionophore and phorbol ester

A B cell lymphoma A20.2J and splenic B cells produced an active material to support the proliferation of an interleukin 2 (IL-2)-dependent T cell line, CTLL-2, by stimulation with both calcium ionophore A23187 and phorbol myristate acetate (PMA). Although the production of the active material was induced by stimulation with A23187 alone in A20.2J cells, both A23187 and PMA were essential for the stimulation of splenic B cells. Neither A20.2J cells nor splenic B cells produced the active material by stimulation with PMA alone. The production was inversely proportional to the concentration of fetal calf serum in culture medium. The active material produced by B cells was indicated to be IL-2 and not B cell-stimulating factor 1 (BSF-1) for the following reasons: 1) the proliferation of CTLL-2 cells in the presence of active material was inhibited by the inclusion of anti-IL-2 receptor or anti-IL-2 in culture medium but not by anti-BSF-1; 2) the material showed no co-mitogenic activity to purified splenic B cells with anti-immunoglobulins and did not support the proliferation of FDC-P2 which are known to grow in the presence of BSF-1; and 3) IL-2 mRNA could be detected in A20.2J and splenic B cells stimulated with A23187 and PMA in Northern blot analysis. Some B cell hybridomas were also shown to produce IL-2 by similar stimulation to A20.2J. Splenic B cells as well as A20.2J cells were able to produce IL-2 by stimulation with anti-immunoglobulins. These results suggest that under certain conditions IL-2 can be produced by splenic B cells, at least some subsets of B cells, and B cell lines.     

Interleukin 2 induces proliferation of normal "resting" human T cells in the absence of other known external stimulation

Interleukin 2(IL-2) is known to stimulate the progression of activated T cells from G1 through the rest of the cell cycle. We have demonstrated that addition of purified recombinant human IL-2 (rIL-2) to fresh normal human peripheral blood mononuclear cells (PBM), which were IL-2 receptor (Tac) negative by FACS analysis, stimulated marked proliferation of the PBM. IL-2-induced proliferation was also observed with umbilical cord blood mononuclear cells. Monocyte depletion of PBM resulted in a marked reduction of rIL-2-induced proliferative response which could be restored by adding back autologous irradiated monocytes but not by interleukin 1. The T cells preincubated with rIL-2 showed a five to six times enhanced autologous mixed-lymphocyte reaction (AMLR) compared to controls. The rIL-2-induced proliferative response of PBM was inhibited in a concentration-dependent fashion by preincubation of PBM with an anti-HLA-DR framework monoclonal antibody. The proliferating cells were shown by two-color flow cytometric analysis to be primarily Leu-1+ and Leu-4+ T cells (both leu-3+ and Leu-2+ subsets); however, 6 to 19% of responding cells had surface markers for B cells or NK cells. The data demonstrate that rIL-2 can induce proliferation of "resting" human T cells. The phenomenon may be related to a monocyte-dependent AMLR which induces IL-2 receptors and IL-2 responsiveness in a subset of T cells.