Mitogenic Activity of a Water-Soluble Adjuvant (Bu-WSA) Obtained from Bacterionema matruchotii

Butanol-extracted water-soluble adjuvant (Bu-WSA) obtained from Bacterionema matruchotii was cultured with peripheral blood mononuclear cells (PBM) in the presence of sub- and/or supra-optimal mitogenic concentrations of concanavalin A (Con A). The addition of Bu-WSA resulted in increased tritiated thymidine incorporation above that produced by Con A alone. Bu-WSA by itself is not mitogenic for PBM and in fact produced a decrease in thymidine uptake compared to the control. We investigated the response of subpopulation(s) of PBM to Bu-WSA, Con A and a mixture of Bu-WSA and Con A. Separation of PBM into purified T cells, B cells and macrophages showed that cell-cell cooperation of T cells with B cells or macrophages is necessary for the observed synergistic effect of Bu-WSA with Con A. A marked increase in thymidine incorporation by the mixture of T and B cell populations occurred, while only a small amount of thymidine was incorporated when the B cell population was absent. Mitomycin treatment revealed that the response could be ascribed to the T-cell response with a B-cell helper effect. Moreover, Con A and Bu-WSA appeared to act on the same T cell population. This model may provide unique information about the activation of human peripheral blood T cells compared with the activation of these cells by other mitogens.     

B cell helper factors. II. Synergy among three helper factors in the response of T cell- and macrophage-depleted B cells

The concanavalin A- (Con A) stimulated supernatant of normal spleen cells (normal Con A SN) was shown to contain a set of helper factors sufficient to allow T cell- and macrophage- (M phi) depleted murine splenic B cells to produce a plaque-forming cell response to the antigen sheep red blood cells (SRBC). The activity of normal Con A SN could be reconstituted by a mixture of three helper factor preparations. The first was the interleukin 2- (IL 2) containing Con A SN of the T cell hybridoma, FS6-14.13. The second was a normal Con A SN depleted of IL 2 by extended culture with T cell blasts from which the 30,000 to 50,000 m.w. factors were isolated (interleukin X, IL X). The third was a SN either from the M phi tumor cell line P388D1 or from normal M phi taken from Corynebacterium parvum-immune mice. The combination of all three helper factor preparations was required to equal the activity of normal Con A SN; however, the M phi SN had the least overall effect. The M phi SN and IL 2 had to be added at the initiation of the culture period for a maximal effect, but the IL X preparation was most effective when added 24 hr after the initiation of culture. These results indicate that at least three nonspecific helper factors contribute to the helper activity in normal Con A SN.     

Effects of aging on cell cooperation and lymphocyte responsiveness to cytokines

Functional activities and cell cooperation of macrophages (Mphi), T cells, and B cells of young and old Lewis rats were compared. Splenic M phi from young and old rats provided accessory help for T cell mitogenesis and B cell mitogenesis, provided accessory help for generation of PFC, and produced IL 1 equally well as measured in costimulator assays. Splenic T cells of aged Lewis rats, however, were poorly responsive in mitogen assays and did not respond to supplemental IL 2 and antigen with blast transformation and with increased help for B cells to produce PFC. "Old" B cells did not respond in vitro to mitogens with help from M phi and T cells, nor did they respond to B cell helper factor with increased PFC. The data indicate that hyporesponsiveness of the immune system, especially of B cells, in aged rats is due in part to defective reactivity to interleukins and cytokine(s) and to defective cell-cell cooperation.     

The anti-T cell monoclonal antibody 9.3 (anti-CD28) provides a helper signal and bypasses the need for accessory cells in T cell activation with immobilized anti-CD3 and mitogens

CD28 is an antigen of 44 kDa which is expressed on the membrane of the majority of human T cells. The present study examines the functional effects of an anti-CD28 monoclonal antibody (mAb 9.3) on T cell activation induced with immobilized anti-CD3 mAb OKT3 or with mitogens, in the absence of accessory cells. To this end, we used blood resting T cells that were completely depleted of accessory cells (monocytes, B cells, and natural killer cells), and consequently did not respond to recombinant interleukin-2 (rIL-2), to immobilized OKT3, to PHA, or to Con A. Addition of mAb 9.3 to the cultures enhanced IL-2 receptor expression (Tac antigen) on PHA- or immobilized OKT3-stimulated T cells and induced IL-2 receptors on Con A-stimulated T cells. Moreover, addition of mAb 9.3 to cultures of T cells stimulated with PHA, Con A, or immobilized OKT3 resulted in IL-2 production. Soluble mAb 9.3 was a sufficient helper signal for T cell proliferation in response to PHA or immobilized OKT3. Crosslinking of mAb 9.3 by culture on anti-mouse IgG-coated plates enhanced the helper effect and was an essential requirement for the induction of T cell proliferation in response to Con A. No other anti-T cell mAb (anti-CD2, -CD4, -CD5, -CD7, -CD8) was found to provide a complete accessory signal for PHA or Con A stimulation of purified T cells. T cell proliferation induced by the combination of PHA and mAb 9.3 was strongly inhibited by the anti-IL-2 receptor mAb anti-Tac. In conclusion, mAb 9.3 can provide a signal bypassing monocyte requirement in T cell activation with immobilized OKT3, PHA, and Con A, resulting in an autocrine IL-2-dependent pathway of proliferation.     

IL 2 restores memory B cell activation by antigen-specific T cell clone variants

It has recently been demonstrated that there are at least two separate pathways by which a single keyhole limpet hemocyanin (KLH) reactive T cell clone can induce B cell differentiation. With the use of the high-dose antigen-driven system (10 micrograms/ml trinitrophenyl (TNP)-KLH), a KLH-specific T cell clone was able to induce a primary anti-TNP response in unprimed B cells. In the presence of aliquots of the same T cell clone, a low-dose of antigen (5 X 10(-2) micrograms/ml TNP-KLH) induced an immunoglobulin (Ig)G response in primed B cells. It has also been demonstrated that there are variant subclones of such KLH-specific helper T cell clones that are unable to provide antigen-specific help in the presence of low-dose antigen but maintain the high-dose antigen-driven helper response. This study was undertaken to investigate whether interleukin 2 (IL 2) had some activity in the low-dose, antigen-driven response induced by the T cell clone. With the use of a variant T cell clone (which lost low-dose, antigen-driven helper activity), it was demonstrated that IL 2 was capable of reconstituting the low-dose, antigen-driven helper activity. To investigate whether accessory cells were required in this system, we removed the adherent cell population from the primed spleen cells added to culture. Interestingly, removal of the G10-adherent cells eliminated the low-dose, antigen-driven response induced by IL 2. Additionally by add-back experiments, we were able to demonstrate that the necessary adherent cell population did not require major histocompatibility complex (MHC) restriction for reconstitution of the IL 2-dependent, low-dose, antigen-driven response. Furthermore, 1% concanavalin A (Con A) supernatant (Sn), but not interleukin 1 (IL 1), could replace this adherent cell function. These data suggest that in this system, IL 2 bypasses the MHC-restricted interaction between T cells and antigen-charged adherent cells; B cells can present antigen to cloned helper T cells efficiently for primary responses but need an added factor(s) to induce IgG production; and adherent cells are essential for IgG production in primed B cells, possibly through the release of soluble factor(s) included in Con A Sn.     

Heterogeneity of human monocyte subsets in the promotion of B cell colonies and the role of interleukin 1

The abilities of human peripheral blood mononuclear-phagocyte (M phi) subpopulations and of interleukin 1 (IL 1) to support human B cell colony formation in semisolid cultures stimulated by staph protein A were analyzed. Human M phi subsets enriched for complement receptors (CR) effectively functioned as accessory cells supporting colony growth, whereas the responses obtained with CR-depleted M phi were 4.6-fold less. In experiments analyzing IL 1 production, CR-enriched M phi secreted four to 12 fold greater amounts of basal and stimulated IL 1 than CR-depleted M phi. Also, the addition of IL 1 to CR-depleted M phi resulted in a fourfold increase of colony numbers. The responses of cultures containing CR-depleted M phi plus IL 1, however, remained 30% less than those observed for cultures supplemented with CR-enriched M phi. Other studies showed that IL 1 was unable to substitute for M phi; the responses of cultures containing IL 1 and B cells were reduced 10-fold compared to cultures supplemented with autologous M phi. These findings indicate that human M phi subsets exist that differ in their ability to function as accessory cells. Although IL 1 can collaborate with certain M phi subsets to restore their accessory cell function, it cannot replace intact M phi. Thus, it is possible that other monokines or lymphokines play a role in M phi accessory cell function.     

The role of interleukin 1 in human B cell activation: inhibition of B cell proliferation and the generation of immunoglobulin-secreting cells by an antibody against human leukocytic pyrogen

The role of factors released by monocytes (M phi) in the activation of human B lymphocytes was examined by studying the effect of an antiserum against human leukocytic pyrogen (LP) on mitogen-stimulated B cell proliferation and the generation of immunoglobulin-secreting cells (ISC) by peripheral blood mononuclear cells (PBM). Antiserum against LP was obtained from rabbits immunized with LP-containing human M phi supernatants. The globulin fraction of this antiserum inhibited pokeweed mitogen- (PWM) stimulated B cell proliferation and the generation of ISC in a concentration-dependent manner, with 50% inhibition of responsiveness observed with 10 micrograms/ml. By contrast, PWM-induced T cell [3H]thymidine incorporation was not inhibited by concentrations of anti-LP as great as 2000 micrograms/ml. The F(ab')2 fraction of anti-LP also inhibited the generation of ISC in response to both PWM and formalinized Staphylococcus aureus, but required 50 micrograms/ml to achieve 50% inhibition. Anti-LP inhibited the generation of ISC only if present during the first 24 hr of a 6 to 7-day incubation; later addition was not inhibitory. Inhibition was more marked in cultures partially depleted of M phi than in whole PBM cultures. Whereas absorption of the anti-LP with PBM failed to remove the capacity to inhibit the generation of ISC, anti-LP-mediated inhibition of responsiveness could be reversed by the addition of crude M phi culture supernatants or a variety of highly purified interleukin 1 (IL 1) preparations, but not by T cell supernatants. These results indicate anti-LP inhibits human B cell activation by removing the requisite M phi-derived factor IL 1 and also confirm that IL 1 plays an essential role in B cell proliferation and the generation of ISC in man.     

Analysis of the activation of lymphoid cells by mitogens in vitro with limiting dilution methods: adherent peritoneal cells suppress B-cell activation and synergize with WEHI-3 in the activation of T cells by Con A

Adherent peritoneal cells (APC) have often been used as a pure and effective macrophage population. Using partition analysis and small numbers of lymphoid cells activated by mitogens (concanavalin A for T cells (in the presence of TCGF) and LPS + DxS for B cells) we found that APC were accessory cells for T cell activation and growth but were not effective for B cells. Although APC were effective in assisting T-cell mitogenesis, they were not especially efficient. However, when APC were mixed with irradiated WEHI-3 cells (a tissue culture line previously shown to exhibit accessory cell activity in vitro for mitogenic activation T and B cells), the APC and WEHI-3 showed apparent synergy. One reason for failure of APC to assist B-cell mitogenesis was traced to the presence of a suppressor cell population which overcame the accessory cell help given by irradiated WEHI-3 cells to LPS-DxS stimulated murine B cells. It is thus possible to find "helper" effects (synergy of APC and WEHI-3 assisting the mitogenesis of T cells), as well as suppressor effects within the range of cells found in adherent accessory cells.     

OT-CLL: a human T cell chronic lymphocytic leukemia that produces IL 2 in high titer

This report identifies and describes a human T cell chronic lymphocytic leukemia, OT-CLL, which can be triggered by selected mitogens (either PHA or Con A) to produce IL 2 in high titer. Optimal IL 2 production requires culturing OT-CLL cells at 2 to 5 X 10(6)/ml for 24 hr in the presence of 1 to 2% PHA-M. Under these conditions, the titer of IL 2 generated is greater than 20-fold that obtained from conventional sources, e.g., from mitogen-activated tonsillar lymphocytes. Two lines of experimental evidence suggest that the tumor cell product(s) is IL 2. First, in functional assays, suprenatants derived from cultures of PHA-activated OT-CLL cells trigger the proliferation and long-term growth of IL 2-dependent human TCL cells. Second, a partial biochemical purification of the active moiety(ies) derived from OT-CLL demonstrates marked similarity to conventional human IL 2. Thus, the biologically active material(s) precipitates in 50 to 70% saturated (NH4)2 SO4 solutions; elutes from DEAE-Sepharose in the presence of 0.04-0.08 M NaCl; and has an apparent m.w. of approximately 14,000, as determined by Sephadex G-100 gel filtration. In addition, analysis of OT-CLL cells by indirect immunofluorescence, utilizing a panel of monoclonal antibodies, confirms not only that these tumor cells are of T cell lineage but that they display surface antigens that define the normal human peripheral T cell subset subserving helper or inducer function: OKT3+, OKT4+ , OKT8-.     

Concanavalin A triggers T lymphocytes by directly interacting with their receptors for activation

Anti-HLA-DR antibodies did not inhibit concanavalin A-(Con A) induced T cell proliferation or the generation of suppressor cells capable of inhibiting immunoglobulin synthesis in autologous mononuclear cells after pokeweed mitogen stimulation. Nylon-wool purified T cells (pretreated with anti-HLA-DR antibody and C) exposed to Con A acquired responsiveness to interleukin 2 (IL 2) and were able to absorb this growth factor, whereas nonlectin-treated cells did not respond to IL 2 and could not absorb it. In the presence of interleukin 1 (IL 1), Con A stimulated the synthesis of IL 2 in purified OKT4+ lymphocytes but not OKT8+ cells. However, in the absence of IL 1, neither resting OKT4+ nor Con A-treated OKT4+ cells produced IL 2. Con A by itself did not directly stimulate macrophages to synthesize IL 1, although it could do so in the presence of OKT4+ but not OKT8+ lymphocytes. In addition, Con A induced proliferation of purified T cells provided IL 1 was supplied to the cultures. Cyclosporin A rendered Con A-treated T cells unresponsive to IL 2, made lectin-stimulated OKT4+ lymphocytes unable to respond to IL 1, and inhibited the synthesis of IL 2. Furthermore, this drug abrogated the Con A-stimulated synthesis of IL 1 by acting on OKT4+ lymphocytes and not on macrophages. Finally, cyclosporin-A suppressed the proliferative response and the generation of suppressor T cells induced by Con A. The following are concluded: 1) HLA-DR antigens do not seem to play any role in the triggering of T cells by Con A, and macrophages participate in lectin-induced activation of T cells mainly by providing IL 1. 2) Cyclosporin-A inhibits activation of T cells by interfering with the mechanism by which Con A stimulates T lymphocytes. 3) Con A triggers T lymphocytes by directly interacting with their receptors for activation.     

Functional analysis of human T cell subsets defined by monoclonal antibodies. III. Regulation of helper factor production by T cell subsets

In the present report we extended our previous studies demonstrating that obligatory T-T interactions are important in regulating human immune responses in vitro. Functionally distinct human T cell subsets were isolated by complement-mediated lysis using the monoclonal antibodies OKT4 and OKT8. Evidence was obtained that during allogeneic interactions, OKT4+, but not OKT8+, responder T cells are required to generate helper factor(s) capable of polyclonally activating human B cells independent of additional T cell help. Importantly, the alloantigen-induced helper factor(s) production and/or release was found to be suppressed by addition of graded numbers of radiosensitive OKT8+ cells. On the other hand, no evidence was obtained that supernatant derived from alloactivated OKT8+ cells could counterbalance the helper activity generated in the presence of supernatant from alloactivated OKT4+ cells. Furthermore, OKT8+ cells, known to suppress PWM-driven B cell differentiation in the presence of OKT4+ cells, do not suppress B cell differentiation induced by preformed helper factor even in the presence of OKT4+ cells. These data further underscore the importance of functional T-T interactions in immunoregulation in vitro and support the idea that the target of suppression of B cell differentiation, induced either by alloantigen-triggered helper factor or PWM, are OKT4+ cells and not B cells themselves.     

Cellular origin and interactions involved in gamma-interferon production induced by OKt3 monoclonal antibody

OKT3 monoclonal antibody, a human T cell mitogen, induced interferon production by cultured mononuclear cells at 10(-11) M concentrations. Interferon was secreted only under conditions wherein OKT3 was mitogenic, and production was correlated with cell proliferation. Thus, like mitogenesis, interferon secretion reached a peak 3 days after OKT3 stimulation, was inhibited by a factor(s) in human serum, and required 1000 times higher concentrations of Fab and F(ab')2 fragments of OKT3 for induction. The interferon was most likely of "gamma" (immune) type, because pH 2 and 56 degrees C treatments denatured it, whereas anti-alpha or -beta interferon antibodies did not. Mononuclear cells were fractionated into subpopulations that contained OKT4+ cells (helper/inducer T cells), OKT8+ cells (cytotoxic/suppressor T cells), and OKM1+ cells (monocytes) by combining sheep red blood cell rosetting and complement-mediated lysis using monoclonal antibodies against specific cell types. Both OKT4+ and OKT8+ cells proliferated upon OKT3 stimulation with the absolute requirement of OKM1+ cells. However, OKT4+ cells plus OKM1+ cells were necessary for the secretion of interferon. Studies with selective pretreatments with mitomycin C suggested that gamma-interferon was secreted by the OKT4+ cells and that the OKM1+ population subserved an accessory function.     

T cells produce TRF in response to Con A and factors in T cell hybridoma supernatants

In recent experiments, a soluble factor (TRF) that mediates the differentiation of anti-immunoglobulin (Ig)-activated B cells to Ig-secreting cells has been identified. TRF works in concert with a growth factor, probably IL 2, in the induction of activated B cells. In previous studies, TRF was identified in culture supernatants of activated T cells and accessory cells, and thus the cellular source (T cell or accessory cell) of the factor was not determined. In the present studies, we succeeded in inducing the production of TRF by T cell populations from which accessory cells had been vigorously depleted. Lymph node cells were depleted of accessory cells by nylon wool adherence and anti-Ia and complement treatment; these cells were activated with Con A and a T cell hybridoma supernatant that contains IL 2. Supernatants from these activated T cell cultures supported the differentiation of anti-Ig-activated B cells to Ig secreting cells. These results show that T cells produce the differentiation factor, and further that they do so in response to ligand (Con A) plus a T cell-derived factor.     

cAMP is an essential signal in the induction of antibody production by B cells but inhibits helper function of T cells

Dibutyryl cAMP and IL 1 were found to stimulate antigen-specific and polyclonal antibody production when added together to cultures of highly purified B cells. We propose that IL 1 and an elevation in cytoplasmic cAMP represent minimal signal requirements for B cell activation. In contrast to its effect on B cells, dibutyryl cAMP inhibited helper T cell activity. Cyclic AMP suppressed the production of IL 2 and T cell replacing factor (TRF) by T cells and thus abrogated the ability of helper T cells to enhance SRBC-specific antibody production by B cells. Cyclic AMP did not inhibit the generation by T cells of B cell growth factor (BCGF). BCGF, not normally detected in Con A supernatant, was found in the culture supernatant of spleen cells that were stimulated with Con A in the presence of cAMP. Our findings indicate that cAMP blocks the production of an inhibitor of BCGF activity. cAMP had no effect on the production by macrophages of IL 1.     

The murine IL 2 receptor. III. Cellular requirements for the induction of IL 2 receptor expression on T cell subpopulations

The accessory cell requirements for the induction of the IL 2 receptor by the lectin Con A on murine T cell subsets were directly assayed with anti-IL 2 receptor monoclonal antibodies. Substantial levels of IL 2 receptor expression were induced on T lymphocytes of the MHC class I-restricted, suppressor/cytotoxic phenotype (L3T4-, Ly-2+) in the presence and absence of accessory cells. In contrast, high levels of IL 2 receptor expression could only be induced on T cells of the MHC class II-restricted, helper/inducer phenotype (L3T4+, LY-2-) in the presence, but not in the absence, of accessory cells. Ia- cells such as the P388D1 macrophage line or cultured fibroblasts (DAP X 3) were as efficient as the Ia+ B cell hybridoma LB in providing accessory cell function for the L3T4+, Ly-2- subset. PMA, but not purified human IL 1, could substitute for accessory cells for both IL 2 receptor expression and IL 2 secretion by the L3T4+, Ly-2- subset. These data suggest that IL 2 receptor induction on the L3T4+, Ly-2- subset is complex, possibly requiring a T cell-accessory cell interaction, whereas the lectin may directly trigger IL 2 receptor expression on L3T4-, Ly-2+ T cells.     

NK cell function in severe combined immunodeficiency (SCID): evidence of a common T and NK cell defect in some but not all SCID patients

The immunologic work-up of eight infants with the clinical diagnosis of severe combined immunodeficiency (SCID) was performed with special emphasis on natural killer (NK) cell function and ontogeny. Contrary to previous reports, our study shows that not all SCID patients lack NK activity; some may even express very high NK- and antibody-dependent cellular cytotoxicity (ADCC). The present group of eight SCID infants was homogeneous with respect to normal levels of the purine metabolism enzymes adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP). They all had low serum Ig levels and were defective for specific antibody formation against BSA and diphtheria toxin (DiT). None of the infants' peripheral blood mononuclear cells (PBMC) proliferated significantly upon in vitro stimulation with PHA, concanavalin A (Con A), pokeweed mitogen (PWM), and irradiated allogeneic lymphocytes. Seven of eight patients, however, responded significantly to mitogenic factors present in a lectin-free interleukin 2 (IL 2) preparation, and two exhibited a positive costimulation as well with simultaneous exposure to IL 2 + Con A. The lymphocyte marker analysis revealed high percentages of OKT10+ cells in seven of eight infants, whereas peripheral T cells (OKT3+) with suppressor/killer (OKT8+) or helper/inducer (OKT4+) phenotypes were abnormally low in all infants with one exception. The PBMC of two patients formed low to normal percentages of E rosettes but expressed no B cell markers (B-/SCID). The six other infants had high percentages of B cells (B+/SCID) but lacked E rosette-forming cells. High NK and ADCC activity was found in the two B-/SCID patients. The B+/SCID infants either totally lacked NK and ADCC function (four of six) or expressed low to normal NK activity together with some T cell markers as revealed by monoclonal antibody staining but not by E rosette formation (two of six). From the data presented, an ontogenic model is proposed that assumes the status of an independent cell lineage in between T cells and monocytes for human NK cells, or that places these cells in close proximity to early differentiation steps of the T cell lineage. In any case, NK cell function clearly constitutes an additional parameter of heterogeneity in the immunologic analysis of SCID.     

Accessory cell-T cell interactions involved in anti-CD3-induced T4 and T8 cell proliferation: analysis with monoclonal antibodies

The effect of monoclonal antibodies (Mab) directed at T cell and accessory cell (AC) surface molecules on OKT3-induced T4 and T8 cell proliferation was examined. Mab directed at nonpolymorphic class I (W6/32, MB40.5) and class II (L243) major histocompatibility complex (MHC)-encoded gene products, an epitope common to LFA-1, CR3, and the p150, 95 molecule (60.3), and a heterodimer present on monocytes (M phi) and activated T cells (4F2) inhibited M phi-supported OKT3-induced proliferation of both T4 and T8 cells. Moreover, an Mab directed at the CD4 molecule (66.1) inhibited OKT3-induced T4 but not T8 cell proliferation, whereas an Mab directed at the CD8 molecule (OKT8) inhibited T8 but not T4 cell responses. With the exception of 66.1, each inhibited OKT3-induced T cell proliferation when added as late as 15 hr after the initiation of culture. Inhibition could not be explained by competition for Fc receptors on the AC. A variety of other Mab including OKT11 and those directed at other HLA-DR and DQ determinants were not inhibitory. The inhibitory Mab were found to diminish T4 cell IL 2 production and IL 2 receptor expression. Consequently, IL 2 reversed some but not all of the Mab-mediated inhibition of T cell proliferation. In contrast to the effects noted with M phi-supported responses, 60.3 and 66.1 but neither L243 nor 4F2 inhibited OKT3-induced T4 cell proliferation supported by Ia- or IFN-gamma-treated Ia+ endothelial cells. None of the Mab tested inhibited T cell proliferation induced by the AC-independent stimuli OKT3 and phorbol myristate acetate (PMA) or calcium ionophore and PMA in the presence or absence of added AC. The data therefore suggest that the Mab inhibit OKT3-induced activation of T4 and T8 cells by preventing necessary interactions between AC and T cell surface proteins. Moreover, the results suggest that different arrays of interaction molecules are involved in OKT3-induced T cell proliferation depending on the nature of the AC and the responding T cell subset.     

Signals involved in T cell activation. I. Phorbol esters enhance responsiveness but cannot replace intact accessory cells in the induction of mitogen-stimulated T cell proliferation

The role of accessory cells (AC) in the initiation of mitogen-induced T cell proliferation was examined by comparing the effect of intact macrophages (M phi) with that of 4-beta-phorbol 12-myristate 13-acetate (PMA). In high-density cultures, purified guinea pig T cells failed to proliferate in response to stimulation with phytohemagglutinin (PHA), concanavalin A (Con A), or PMA alone. The addition of M phi to PHA or Con A but not PMA-stimulated cultures restored T cell proliferation. The addition of PMA to high-density T cell cultures stimulated with PHA or Con A also permitted [3H]thymidine incorporation, but was less effective than intact M phi in this regard. This action of PMA was dependent on the small number of AC contaminating the T cell cultures as evidenced by the finding that PMA could not support mitogen responsiveness of T cells that had been depleted of Ia-bearing cells by planning, even when these cells were cultured at high density. When PMA was added to T cell cultures supported by optimal numbers of M phi, catalase-reversible suppression of responses was noted. Even in cultures containing catalase, PMA failed to enhance responsiveness above that supported by optimal numbers of M phi. A low-density culture system was used to examine in greater detail the possibility that PMA could completely substitute for M phi in promoting T cells activation. In low-density cultures, mitogen-induced T cell proliferation required intact M phi. PMA could not support responses even in cultures supplemented with interleukin 1-containing M phi supernatants or purified interleukin 2 alone or in combination. Similar results were found in high-density cultures of T cells depleted of Ia-bearing cells. These results support a model of T cell activation in which AC play at least two distinct roles. The initiation of the response requires a signal conveyed by an intact M phi, which cannot be provided by either a M phi supernatant factor or PMA. The response can be amplified by additional M phi or M phi supernatant factors. PMA can substitute for M phi in this regard and can provide the signal necessary for amplification of T cell proliferation supported by small numbers of intact AC.     

Role of interleukin 2 on enhancement of concanavalin A-induced human peripheral blood lymphocyte proliferation by murine B cell mitogens

Murine B cell mitogens such as bacterial lipopolysaccharide (LPS), butanol-extracted water soluble adjuvant (Bu-WSA), dextran sulfate (DS), synthetic muramyl dipeptide (MDP), and its analog MDP-Lys (L18) do not show any mitogenic ability in vitro on human peripheral blood lymphocytes or mixed cell populations of purified T and B cells obtained from the lymphocytes in an ordinary culture system. However, these mitogens are capable of enhancing the mitogenic effect of concanavalin A (Con A) in the cultures. In the presence of one of these mitogens, the activity of interleukin 2 (IL 2), but not interleukin 1, in the supernatants obtained from cultures containing Con A-stimulated T cell and B cell populations was higher than that of control cultures. The role of the newly produced IL 2 in the synergistic effect of the mitogens in human lymphocyte cell cultures was discussed.     

Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins

A panel of antigen-specific mouse helper T cell clones was characterized according to patterns of lymphokine activity production, and two types of T cell were distinguished. Type 1 T helper cells (TH1) produced IL 2, interferon-gamma, GM-CSF, and IL 3 in response to antigen + presenting cells or to Con A, whereas type 2 helper T cells (TH2) produced IL 3, BSF1, and two other activities unique to the TH2 subset, a mast cell growth factor distinct from IL 3 and a T cell growth factor distinct from IL 2. Clones representing each type of T cell were characterized, and the pattern of lymphokine activities was consistent within each set. The secreted proteins induced by Con A were analyzed by biosynthetic labeling and SDS gel electrophoresis, and significant differences were seen between the two groups of T cell line. Both types of T cell grew in response to alternating cycles of antigen stimulation, followed by growth in IL 2-containing medium. Examples of both types of T cell were also specific for or restricted by the I region of the MHC, and the surface marker phenotype of the majority of both types was Ly-1+, Lyt-2-, L3T4+, Both types of helper T cell could provide help for B cells, but the nature of the help differed. TH1 cells were found among examples of T cell clones specific for chicken RBC and mouse alloantigens. TH2 cells were found among clones specific for mouse alloantigens, fowl gamma-globulin, and KLH. The relationship between these two types of T cells and previously described subsets of T helper cells is discussed.