A soluble factor produced by bone marrow natural suppressor cells blocks interleukin 2 production and activity

We previously reported that a population of Fc gamma-receptor+ (Fc gamma R+) suppressor cells present in normal unstimulated rabbit bone marrow inhibited the growth of autologous rapidly proliferating bone marrow cells devoid of Fc gamma R. It is now reported that the Fc gamma R+ bone marrow cells produced a soluble, nondialyzable suppressor factor(s) (SF) which blocked the proliferation of Fc gamma R- bone marrow cells. In addition, the Fc gamma R+ cells and SF significantly inhibited spleen cell proliferation in response to concanavalin A (Con A), phytohemagglutinin, and pokeweed mitogen. The bone marrow SF exhibited a dose-dependent suppression of the growth of IL-2-dependent T lymphocytes in the presence of IL-2. SF also completely blocked the production or release of IL-2 by Con A-stimulated T cells. Thus, these bone marrow natural suppressor cells produced a soluble factor, which regulated the growth of rapidly proliferating bone marrow cells and also regulated T cell reactivity by modulating IL-2 production and activity.     

Lymphocyte function in human bone marrow. III. Isotype commitment, metabolic and secretory characteristics of immunoglobulin producing cells

We have examined the functional and metabolic properties of immunoglobulin (Ig)-secreting cells in adult (rib) bone marrow, the tissue which provides the major proportion of serum Igs. In the absence of polyclonal activators, high rate Ig production (1-2 micrograms/day/10(6) marrow mononuclear cells) was sustained from the beginning of culture throughout 2 weeks and then declined. Ten percent of the Ig secreted was of the IgM isotype and IgG/A made up the remainder at equal proportions. Infection of marrow cells with Epstein-Barr virus (EBV) induced the production of large amounts of IgM, but virtually all IgG/A-committed cells were refractory to stimulation with EBV. Both EBV-induced and the "spontaneous" Ig production was inhibited by cycloheximide, but only EBV-induced IgM production was blocked by hydroxyurea and gamma-irradiation. The polyclonal activators PHA and PWM induce suppressor-T-cell activity in marrow cultures. This suppressor function involves nonproliferating cells which acquire suppressive activity 3-4 days after mitogenic activation. Prednisolone and cyclosporine A modulate Ig production in cultures of peripheral lymphocytes but had no effect on Ig secretion in marrow cell cultures. This observation was reminiscent of the absent or at best marginal short-term effects on in vivo serum Ig levels which is typical for these drugs. Our observations suggest that the marrow Ig-producing B-lymphoid cell compartment shows major differences to other tissue sites with respect to properties of the Ig-secreting cells the immunoregulatory activities able to control their function, and the response of these cells to clinically important drugs.     

Regulation of IFN-gamma induction in human peripheral blood cells by exogenous and endogenously produced interleukin 2

Interferon (IFN)-gamma production, stimulated by the addition of exogenous interleukin (IL) 2, T cell mitogens, or tuberculin purified protein derivative (PPD) was studied in cultures of separated human mononuclear cells or unseparated peripheral blood leukocytes (PBL). IFN-gamma was induced by the addition of IL 2 to cultures of otherwise unstimulated cells. The minimal concentration of exogenous IL 2 required to cause a reproducible stimulation of IFN-gamma was about 10 U/ml, i.e., approximately 50 times the minimal concentration required to stimulate proliferation in an IL 2-dependent murine cytotoxic T cell line. Approximately 500 to 1000 IL 2 U/ml were required to produce maximal stimulation of IFN-gamma production in otherwise unstimulated cultures. Monoclonal antibody anti-Tac, specific for an epitope associated with the IL 2 receptor (IL 2 R), inhibited IFN-gamma induction by exogenous IL 2 less strongly than induction by phytohemagglutinin (PHA) or concanavalin A (Con A). The highest degree of inhibition was exerted by anti-Tac on IFN-gamma production stimulated with PPD. Stimulation of IFN-gamma induction by exogenous IL 2 and the inhibitory action of anti-Tac on IFN-gamma production were also seen in cultures of irradiated (2000 R) cells. Treatment of cells with subinducing doses of Con A or phorbol myristate acetate increased IFN-gamma induction by exogenous IL 2. Taken together, the data suggest that endogenously generated IL 2 is a major mediator of IFN-gamma induction in PBL cultures stimulated with antigens or T cell mitogens.     

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.     

Role of interleukin 2, interleukin 4 and interleukin 5 in the T helper cell-driven B cell polyclonal differentiation in the elderly.

There is evidence for an impaired T cell-mediated B cell response during senescence. In thirty aged donors, pokeweed mitogen (PWM)-driven immunoglobulin (Ig) synthesis by B cells co-cultured with autologous enriched CD4+ lymphocytes and low amounts of monocytes, was evaluated. Under such experimental conditions, elderly cultures displayed a reduced IgG and/or IgM production when compared with the younger counterpart. Moreover, interleukin (IL)-2 and/or IL-5 addition to cultures led to an enhancement of Ig release. In contrast, IL-4 supplementation failed to positively modulate B cell differentiation. At the same time, aged cells cultured in the presence of IL-2 + IL-5 exhibited an increased Ig synthesis, while the addition of IL-2 + IL-4 or IL-4 + IL-5 mixtures did not induce any significant effect in comparison with homologous untreated samples. The results suggest a critical role for IL-2, IL-4 and IL-5 in the modulation of T helper cell-driven B cell polyclonal responsiveness in the elderly.     

Induction and suppression of immunoglobulin synthesis in cultures of human lymphocytes: effects of pokeweed mitogen and Staphylococcus aureus Cowan I

The synthesis and secretion of immunoglobulins by human peripheral blood mononuclear cells in cultures stimulated with pokeweed mitogen or Staphylococcus aureus Cowan I were evaluated by enumeration of cells containing cytoplasmic immunoglobulins and cells actively secreting immunoglobulins, and by quantitation of immunoglobulins released into culture supernatants. The two mitogens caused comparable stimulation of immunoglobulin production; however, in contrast to pokeweed mitogen, S. aureus was active in cultures depleted of T lymphocytes, and its stimulatory effects were resistant to the influence of suppressor T cells generated by co-stimulation with concanavalin A or by preincubation without mitogenic stimulus. These results indicate distinct pathways of induction and suppression of immunoglobulin synthesis for these two polyclonal B cell activators, and suggest that stimulation by S. aureus is less thymus dependent than that induced by pokeweed mitogen.     

Stimulation of immunoglobulin biosynthesis in human B cells by wheat germ agglutinin. I. Evidence that WGA can produce both a positive and negative signal for activation of human lymphocytes

Wheat germ agglutinin (WGA), previously regarded strictly as a nonmitogenic or anti-mitogenic lectin, can under appropriate conditions markedly stimulate in vitro synthesis and secretion of immunoglobulin (Ig) by human B lymphocytes. Stimulation of Ig production by WGA is 1) confined to a narrow lectin dose range (2 to 10 micrograms/ml; 2) abrogated by the simple sugar N-acetyl-D-glucosamine but not by a variety of other monosaccharides; 3) effective only after early additions of WGA within the initial 72 hr of 12-day cultures; 4) detected in the presence of B and T cells but not B cells alone; and 5) polyisotypic in nature, as indicated by augmented synthetic rates of Ig in each of 3 major classes (IgG, IgA, and IgM). With few exceptions, WGA produces equivalent or greater rates of Ig production as obtained in cultures activated with pokeweed mitogen (PWM), a well-recognized T-dependent polyclonal activator of human B cells. Furthermore, periperal blood lymphocytes from select individuals that respond weakly to PWM are markedly stimulated with WGA. In contrast to these stimulatory effects of WGA on Ig production by lymphocytes exposed to low lectin concentrations, addition of WGA in amounts greater than 15 micrograms/ml to PWM-stimulated human lymphocyte cultures produces marked suppression of the expected level of Ig synthesis. These data indicate that varying doses of WGA can produce contrasting stimulatory and inhibitory effects on human B cell metabolism.     

Suppression of pokeweed mitogen-stimulated immunoglobulin production in patients with rheumatoid arthritis after treatment with total lymphoid irradiation

Patients with intractable rheumatoid arthritis (RA) were treated with total lymphoid irradiation (TLI, 2000 rad). We previously reported long-lasting clinical improvement in this group associated with a persistent decrease in circulating Leu-3 (helper subset) T cells and marked impairment of in vitro lymphocyte function. In the present experiments, we studied the mechanisms underlying the decrease in pokeweed mitogen stimulated immunoglobulin (Ig) secretion observed after TLI. Peripheral blood mononuclear cells (PBL) from TLI-treated patients produced 10-fold less Ig (both IgM and IgG) in response to pokeweed mitogen than before radiotherapy. This decrease in Ig production was associated with the presence of suppressor cells in co-culture studies. By using responder cells obtained from normal individuals (allogeneic system), PBL from eight of 12 patients after TLI suppressed Ig synthesis by more than 50%. In contrast, PBL from the same patients before TLI failed to suppress Ig synthesis. Suppression by post-TLI PBL was also demonstrated in an autologous system by using responder cells cryopreserved before TLI. Again, only cells obtained after TLI were suppressive in four of seven patients. PBL with suppressive activity contained suppressor T cells, and the latter cells bore the Leu-2 surface antigen. In 50% of the patients studied, suppressor cells were also found in the non-T fraction and were adherent to plastic. Interestingly, the Leu-2+ cells from TLI-treated patients were no more potent on a cell per cell basis than purified Leu-2+ cells obtained before TLI. Additional experiments suggested that the suppression mediated by T cells after TLI is related to the increased ratio of Leu-2 to Leu-3 cells observed after radiotherapy.     

Effect of recombinant IL 2 and gamma-IFN on proliferation and differentiation of human B cells

The effects of IL 2 and gamma-IFN on the activation of human B cells was studied with recombinant IL 2 and gamma-IFN. BCDF-responsive B lymphoblastoid cell lines and highly purified human B cells were employed as target B cells. IL 2 or gamma-IFN did not induce any IgG or IgM secretion in the B cell lines CESS and SKW6-CL4, in which IgG and IgM were inducible with conventional T cell factor(s). IL 2 alone did not induce the optimum production of Ig, but did induce proliferation in the SAC-stimulated B cell population. No Leu-1-, Leu-4-, or Leu-7-positive cells were detected in B cell populations that had been stimulated with SAC for 3 days. FACS analysis showed that a portion of the SAC-stimulated B cells (30%) were in the G2 or M stages by IL 2 stimulation. The addition of gamma-IFN together with IL 2 induced IgM and IgG secretion in SAC-stimulated B cells that was comparable with that induced by a conventional T cell factor(s). IL 2 induced proliferation not only in SAC-stimulated B cells but also in an anti-mu-stimulated B cell population. Stimulation of T cell populations with anti-mu and IL 2 did not induce significant proliferation, suggesting the direct effect of IL 2 on B cells. Double staining of anti-mu-stimulated B cells with anti-Ig and anti-Tac antibodies demonstrated that anti-mu stimulation induced an increased expression of Tac antigen on surface Ig-positive B cells. All of these results strongly supported the notion that IL 2 was one of the growth factors for B cells, and gamma-IFN was one of the differentiation factors for B cells.     

The potentiation of B lymphocyte responses through CD2/LFA-3 interactions involving erythrocytes is IL2 independent

The response of human B cells to pokeweed mitogen (PWM) stimulation is potentiated when autologous erythrocytes (E) are added to peripheral blood mononuclear cell (PBMC) cultures. This potentiation has been previously shown to be dependent on interactions between the CD2 molecule on T cells and the lymphocyte function-associated antigen 3 (LFA-3) expressed by autologous erythrocytes. Since in other experimental systems the activation of T cells by CD2/LFA-3 interactions has resulted in increased secretion of interleukin 2 (IL2), we were interested in studying the role of IL2 in PBMC cultures stimulated with PWM and autologous E. The addition of autologous E significantly depressed IL2 levels in PWM-stimulated PBMC cultures. This effect was not secondary to increased expression of IL2 receptors by activated cells, since the addition of anti-TAC antibodies did not result in a significant increase in measurable levels of IL2. The addition of anti-IL2 to PBMC failed to abrogate the potentiating effect of E and it actually further enhanced the production of IgM and IgG from cultures stimulated with PWM + E. These results suggest that the potentiation of B cell function induced by autologous E is not mediated by IL2, either directly or indirectly. It is possible that the effect of autologous E either is mediated by other interleukins or is dependent on cell-to-cell contact with directed release of IL2 and/or other lymphokines without detectable secretion to the extracellular compartment.     

Stimulation of Immunoglobulin Production from Human B Lymphocytes by Staphylococcus aureus: Effects of Monocytes and Con A-Induced Suppressor Cells

Significant immunoglobulin (Ig) production by human peripheral blood lymphocytes was induced in vitro by stimulating the cells with pokeweed mitogen (PWM) and Staphylococcus aureus Cowan I (SpA CoI). IgG, IgM, and IgA were determined by a combination of the latex fixation test and radioimmunoassay. High levels (1,000 to 5,000 μg/ml) of IgG and IgM and a lesser amount of IgA were constantly produced during 7 to 8 days of incubation with both stimulants. Ig production induced by SpA Col stimulation was independent of the presence of T cells, while Ig production induced by PWM required T cells exclusively. Depletion of monocytes in the culture caused but a slight decrease in Ig production (particularly in the case of IgG). While the addition of a small number of monocytes enhanced IgG induction by both stimulants, coculture with an excess number of monocytes inhibited Ig induction (particularly IgG) by PWM stimulation but not by SpA CoI stimulation. Marked suppression of Ig production (IgG, IgM, and IgA) was observed in cocultures with Con A-activated T cells. The phenomena of suppression were observed in both the SpA Col-stimulated and PWM-stimulated lymphocytes. These data indicate that Ig production from B cells stimulated with a polyclonal B cell activator, SpA CoI, was independent of T cells and relatively of independent of monocytes, but could be subjected to the regulation of the Con A-induced suppressor T cells.     

Separable helper factors support B cell proliferation and maturation to Ig secretion

The 24-hr culture supernatant of Con A-activated spleen cells (SN) contains helper factors that enable maturation to high-rate polyclonal Ig secretion and enhance proliferation in cultures of mouse B cells activated with the F(ab')2 fragment of class-specific rabbit antimouse IgM antibody (anti-Ig). When interleukin 2 (IL 2), also called T cell growth factor, is removed from SN by absorption with an IL 2-dependent cell line at either 4 degrees C or 37 degrees C, all the helper activity for anti-Ig-activated B cells is also removed. Partial removal of IL 2 results in partial removal of helper activity for B cells. However, the IL 2-depleted SN appears to contain another helper factor, TRF, that enables anti-Ig-activated B cell cultures to mature to high-rate Ig secretion. This TRF activity is revealed by adding purified human IL 2 or an IL 2-containing supernatant of a cloned, lectin-activated T cell hybridoma line (FS6-14.13) to Il 2-depleted SN, which restores the polyclonal antibody response to anti-Ig. The hybridoma supernatant by itself supports proliferation of anti-Ig-activated B cell cultures, as measured by an increase in cell number, but not maturation to Ig secretion. This proliferative response is likewise IL 2 dependent, although purified IL 2 with anti-Ig is not sufficient. These experiments define separable combinations of factors acting on anti-Ig-activated B cell cultures, one of which (SN) results in both proliferation and maturation to high-rate Ig secretion, whereas the other (hybridoma supernatant) results in proliferation only. IL 2 appears to be an essential component of both combinations, although the target cell for IL 2 action in this system remains to be determined.     

Two phenotypically distinct suppressor T cell subpopulations inhibit the induction of B cell differentiation by phytohemagglutinin

Human B lymphocytes can be induced to differentiate into antibody-secreting plasma cells by Leu-3+ T lymphocytes stimulated with pokeweed mitogen (PWM), a polyclonal T cell activator. In contrast, other polyclonal T cell mitogens, such as phytohemagglutinin (PHA), also activate Leu-3+ T cells but are relatively ineffective inducers of B cell differentiation. We have performed a series of experiments to investigate the mechanism underlying this apparent paradox. When human B cells were cultured with unfractionated T cells and PWM or PHA, only PWM was able to induce plasma cell formation and immunoglobulin (Ig) secretion. However, when the T cells were treated with mitomycin C (MMC) before culture, both PWM and PHA were able to induce significant B cell differentiation. These data indicated that both mitogens were able to activate the helper T cells required for B lymphocyte differentiation and suggested that MMC-sensitive suppressor T cells were responsible for inhibiting the induction of antibody-secreting cells by MMC-untreated T cells stimulated with PHA. Phenotypic analysis of the T cells capable of suppressing PHA-induced B cell differentiation revealed that small numbers of either Leu-2+ or Leu-3+ T cells could profoundly suppress the B cell differentiation induced by PHA. In contrast, significant suppression of PWM-stimulated B cell differentiation was observed only with relatively large numbers of Leu-2+ T cells. These data confirm previous reports that OKT4+/Leu-3+ T cells can suppress human B cell differentiation and indicate that the difference in B cell differentiation induced by PWM and PHA with MMC-untreated T cells is largely a reflection of the relative potency of these mitogens to activate these phenotypically distinct suppressor T cell subpopulations.     

Differential regulation of activation, clonal expansion, and antibody secretion in human B cells

Limiting dilution analysis, hemolytic plaque assay, and ELISA procedures were used to study the recruitment, clonal expansion, and antibody secretion in human TNP-specific B cells activated in the presence of TNP-ovalbumin (TNP-OA), pokeweed mitogen (PWM), or regulatory T cells. TNP-OA-responsive, hapten-specific PFC precursor cells occupy approximately 0.5% of all sIgM+/sIgD+ B cells in cord blood, bone marrow, peripheral blood, and tonsil. The PWM-responsive, hapten-specific PFC precursor pool is 70 to 90% smaller and does not express sIgD. Antigen-reactive B cells go through a minimum of three divisions in culture (six to nine PFC per clone), and antibody secretory rates of about 10(4) molecules IgM/cell/hr are achieved. In contrast, PWM-induced clone sizes were at least 60 PFC per clone, with antibody secretory rates of approximately 6 to 7 X 10(4) molecules IgM/cell/hr. Addition of high-dose carrier-primed suppressor T cells to limit dilution cultures reduced PFC precursor cell recruitment by up to 99%. However, in the few clones escaping from suppression, both clonal expansion and antibody secretory rates were much higher than in suppressor cell-free cultures, generating 30 to 60% of the antibody secreted in controls but with consequently much more restricted clonal diversity. When limiting dilution cultures were compared with standard microcultures of 2 X 10(5) cells, both clonal expansion and antibody secretory rates were much lower than expected, with a culture efficiency calculated to be 10 to 20% of that in low-density cultures. Our data suggest that the B cell subsets activated by antigen and by mitogen differ in their abilities for clonal expansion and antibody secretion. The hapten-specific and -responsive B cell family is expressed early in ontogeny, and in adults it is distributed evenly throughout the body. These limiting dilution experiments revealed that the primary effect of regulatory T cells is a drastic reduction in clonal diversity, and much less a mere reduction in overall response magnitude.     

Heterogeneity among T helper cells. Interleukin 2 secretion and help for immunoglobulin secretion

T lymphocytes are thought to provide "help" for B cells by activating them from the resting state, by secretion of antigen-nonspecific lymphokines that promote B cell differentiation and maturation, and by providing signals that induce isotype switching. To clarify the extent to which these different forms of helper activity could be carried out by individual T cells, we set up cultures in which B cells activated, and were in turn themselves stimulated by, limiting numbers of T cells through differences at the H-2 or Mls loci. At T cell doses at which responses were likely to represent the activity of individual helper T cells (or their immediate clonal progeny), we found that some T cells were able both to produce interleukin 2 (IL-2) and to induce secretion of both IgM and IgG, whereas others induced immunoglobulin (Ig) secretion without detectable IL-2 production, and still others made IL-2 but did not promote antibody secretion. We could not detect B cell stimulatory factor 1 production by alloantigen-stimulated T cells, and the addition of antibodies to B cell stimulatory factor 1 did not prevent Ig production. Two results, however--higher Ig accumulation in those wells that received an IL-2-producing cell, and inhibition by anti-IL-2 receptor antibodies of B cell but not T cell function--are consistent with a direct stimulatory effect of IL-2 on B cells in this system. The pattern of helper functions exhibited by T cells freshly isolated from mice differs from that inferred from studies of cloned lines of T cells in long term cultures.     

Inhibition of N-linked oligosaccharide trimming mannosidases blocks human B cell development.

Deoxymannojirimycin (dMM) or swainsonine (SW), which block conversion of high-mannose to complex-type N-linked glycans, strongly inhibited the production of immunoglobulin (Ig) when added to cultures of human lymphocytes together with the polyclonal B cell activators pokeweed mitogen (PWM) and Staphylococcus aureus (SAC). To obtain the inhibitory effect, inhibitor had to be present during the first 36 h of culture. Addition at later timepoints was less effective and showed that neither inhibitor interfered with rate of production or secretion of Ig as such. Viability and proliferation of the lymphocytes, as defined by cell number and rate of DNA synthesis, were not influenced by the presence of dMM or SW, and no changes in the relative number of helper (T4+) or suppressor (T8+) cells were observed. Thus, for normal differentiation of human B lymphocytes into Ig secreting (plasma) cells in response to PWM and SAC, conversion of high-mannose to complex N-linked glycans is essential.     

Regulation of immunoglobulin production in human peripheral bood leukocytes: cellular interactions.

The intercellular influences regulating immunoglobulin (Ig) synthesis by normal human peripheral blood leukocytes (PBL) were investigated in cells stimulated by pokeweed mitogen (PWM). This system was shown to be totally T lymphocyte dependent as purified B lymphocytes (less than or equal to 1% T lymphocytes) failed to make significant amounts of Ig. No evidence was obtained for an Ig class switch as all classes of Ig (IgM, IgG, IgA) were shown to be produced in increasing amounts over a 6-day time period. T lymphocytes demonstrated maximum helper effect when mixed with equal numbers of B cells. This helper effect was mediated through the dual mechanisms of increasing the number of B lymphocytes containing cytoplasmic Ig and by increasing the maturity of these B lymphocytes as demonstrated by an increasing Ig production per B lymphocyte. When present in higher numbers, T lymphocytes were also capable of suppressing Ig production. This T-mediated suppression was first evident as a decrease in the Ig produced per B lymphocyte (decreased maturity). With maximum T suppression Ig-containing B lymphocyte numbers were also diminished. T lymphocyte help was relatively independent of macrophages (phagocytic cells) and did not require DNA synthesis for expression. Both T help and suppression were shown to cross allogeneic barriers. Immature T lymphocytes (thymocytes) were incapable of mediating either activity. Normal human PBL contain T lymphocytes campable of mediating both T help and suppression and the Ig produced by PBL was shown to be the balance of these activities. This balance probably represent the participation of distinct T lymphocyte subpopulations analogous to the T helper (Ly 1+) and T suppressor (Ly 2+, 3+) populations in the mouse.     

Stimulation of immunoglobulin secretion in human B lymphocytes as a direct effect of high concentrations of IL 2

In certain human IgM and IgG cell lines, immunoglobulin (Ig) secretion is highly stimulated by a B cell inducing factor (BIF) that is free of interleukin 2 (IL 2). BIF also induces Ig secretion in purified peripheral blood B cell populations that have been mitogenically stimulated by Staphylococcus aureus bacteria. Low concentrations of IL 2 (less than 20 U/ml) are not active in these systems. We now show that IL 2 at concentrations above 100 U/ml can induce Ig secretion in these blood B cells and B cell lines. Both conventional IL 2, purified from the human JURKAT and gibbon MLA-144 cell lines, and recombinant IL 2 are active. Very high concentrations approaching 10(4) U/ml are optimal for Ig secretion. Antibody to the T cell IL 2 receptor, anti-Tac, did not inhibit stimulation of the IgM cell line SKW6.4 by IL 2, and no Tac antigen was detected on the cells. The 9B11 monoclonal anti-IL 2 antibody that neutralizes T cell growth activity also abrogates stimulation of Ig secretion by conventional and recombinant IL 2 in the SKW6.4 cell line. However, the 1H11 monoclonal anti-(conventional thr3-glycosylated IL 2), which does not neutralize T cell growth activity, does inhibit induction of Ig secretion by the corresponding IL 2 in the B cell line. These results suggest that IL 2 stimulates B cells via a low-affinity interaction with a receptor different from the Tac receptor identified on T cells, and that the active site on the IL 2 molecule for B cells differs from that for T cell targets. If IL 2 promotes Ig secretion by binding with a low affinity to the B cell BIF receptor, IL 2 and BIF could be homologous proteins.     

Immunoregulatory human T lymphocytes triggered as a consequence of viral infection: clonal analysis of helper, suppressor inducer and suppressor effector cell populations

The regulatory functions of a series of human T cell clones specific for an autologous Epstein-Barr virus transformed B lymphoblastoid cell line were examined. Two T4+ T cell clones, termed AT4II and AT4IV, and one T8+ clone, AT8III, were maintained in culture for greater than or equal to 9 months and were characterized in detail. Both T4+ clones provided helper function for autologous B cell immunoglobulin production when added to unstimulated peripheral blood mononuclear cells. In addition, these same clones produced soluble inducer factors after specific antigenic stimulation. However, when AT4II, AT4IV and their subclones were tested on pokeweed mitogen stimulated peripheral blood mononuclear cells, it was found that AT4IV provided help for immunoglobulin production whereas AT4II cells were strongly suppressive. This suppression by AT4II was indirect and required the presence of fresh, autologous, unirradiated T8+ cells. In contrast, the T8+ AT8III clone markedly inhibited Ig production by autologous B cells in the absence of any additional T8+ cells from peripheral blood and produced a soluble suppressor factor upon specific antigenic triggering. Thus, after stimulation with autologous Epstein-Barr virus transformed cells, at least three discrete regulatory human T cell populations can be defined at the clonal level: helper, inducer of suppression and suppressor effector clones.     

Control of mitogen-induced transformation: characterization of a splenic suppressor cell and its mode of action.

The present study demonstrates that mouse spleen cells contain a population of glass wool adherent T lymphocytes which exhibit the capacity to suppress non-glass adherent lymphocyte responses to mitogens. These suppressor cells are stimulated by both low and high doses of PHA¹ and high doses of con A. The suppressor cell effect is observed when UNA, but not RNA or protein synthesis, is studied. This glass-adherent suppressor cell population is characterized as being the primary DNA synthesizing cells during the early (0–8 hr) stages of culture. Suppression still occurs when the suppressor cells are treated with mitomycin C, actinomycin D or cycloheximide. This implies that new macromolecular synthesis may not be required for suppression to occur. Suppression is blocked by inhibiting synthesis of prostaglandin and is mimicked by Bu₂cAMP. This suggests that mitogen activated suppressor cells regulate T cell responses via production of prostaglandin which modulates the concentration of intracellular cyclic nucleotide levels.