Induction of antibody responses to influenza virus in human lymphocyte cultures. I. Role of interleukin 2

The in vitro T cell-dependent antibody response of human lymphocytes to influenza virus X31 was used to study the role of T cell-derived lymphokines in antigen-specific responses. Supernatant from cultures of phytohaemagglutinin-stimulated, pooled human tonsil cells (PHA-MLR) was capable of replacing T cells and inducing T-depleted tonsil cells to secrete influenza-specific antibody. The T cell-replacing activity of PHA-MLR supernatant co-purified with interleukin 2 (IL 2) on Ultrogel AcA54 gel filtration and reversed phase-high performance liquid chromatography. PHA-MLR supernatant and IL 2 also enhanced B cell proliferation induced by anti-mu or Staphylococcal aureus strain Cowan I (SAC). A murine monoclonal antibody directed against the human IL 2 receptor (Mab 2A3) was used to completely block the enhancement of influenza-specific antibody production mediated by PHA-MLR supernatant, purified IL 2, and recombinant human IL 2. Mab 2A3 did not affect the T-independent B cell proliferation induced by anti-mu or SAC, but abrogated the enhancing effect of the PHA-MLR supernatant and IL 2 in this culture system. Immunofluorescence studies failed to demonstrate binding of Mab 2A3 to B cells activated by the X31 influenza virus and IL 2, or by SAC. By using Mab 2A3 to mask out IL 2 effects in the influenza-specific culture system, no other B cell differentiating activities were revealed in supernatants from lymphocytic cultures stimulated with a variety of mitogens. Thus, our results indicate that the production of influenza-specific antibodies by T-depleted human lymphocyte cultures is absolutely dependent on the presence of both antigen and IL 2.     

The role of IL 1 in the antigen-specific activation of murine class II-restricted T lymphocyte clones

The role of IL 1 in the antigen-specific activation of class II-restricted T lymphocytes was examined by using a model system consisting of cloned WEHI 5 B lymphoma accessory cells and class II-restricted, soluble antigen- or alloantigen-reactive T cell clones. The addition of exogenous recombinant IL 1 to the T cell cultures resulted in a significant enhancement of the antigen-specific T cell proliferation response, but at best, only small increases in IL 2 release. Goat IgG anti-IL 1 antibodies were added to the T cell cultures to assess their effect on T cell activation. The IL 1 enhancement of the T cell proliferation response was inhibited by the anti-IL 1 antibodies in a dose-dependent manner. In contrast, only modest levels (10 to 25%) of proliferation inhibition were observed in T cell cultures containing either WEHI 5 or splenocyte accessory cells but no exogenous IL 1. When the anti-IL 1 antibodies were added to primary mixed lymphocyte cultures stimulated by WEHI 5 cells in the absence of exogenous IL 1, no significant inhibition of proliferation was observed. A small but statistically significant proliferation inhibition was observed when anti-IL 1 antibodies were added to mixed lymphocyte reaction cultures stimulated by splenocytes. Two-color cytofluorometric analysis of the effects of IL 1 on antigen-activated T cell clones demonstrated that under suboptimal stimulation conditions, IL 1 stimulated a small but significant increase in the number of T cells bearing IL 2 receptors. In the presence of optimal numbers of WEHI 5 accessory cells, IL 1 enhanced T cell proliferation in the absence of a detectable increase in the number of T cells bearing IL 2 receptors, the number of IL 2 receptors per T cell, or the levels of IL 2 released. Finally, exogenous IL 1 can be added as late as 18 to 24 hr after culture initiation without significantly reducing its ability to enhance the T cell proliferation response. These data indicate that IL 1 has pleiotropic effects on murine T lymphocytes and can function to enhance T cell activation at multiple points during the activation sequence.     

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.     

Mitogenic stimulation of human B lymphocytes by the mannose-specific adhesin on Escherichia coli type 1 fimbriae

Escherichia coli type 1 fimbriae contain in association with the major structural protein a lectin-like adhesin moiety that mediates attachment of E. coli to mannose-containing receptors on the surface of host cells. We have investigated the lymphocyte mitogenic activity of this mannose-specific adhesin by comparing the ability of purified wild type type 1 fimbriae containing the adhesin and mutant type 1 fimbriae lacking the adhesin to stimulate proliferation in human lymphocytes. Both fimbriae stimulated a peak of proliferation at 8 days whereas only the wild type fimbriae stimulated an additional peak of proliferation occurring at 3 days. Proliferation at 3 days but not at 8 days could be blocked by the addition of alpha-methyl-D-mannoside. Neonatal lymphocytes from umbilical cord blood responded to both wild type and mutant fimbriae in a fashion similar to adult cells. Stimulation of separated T and non-T cell populations indicated that the proliferation seen at 3 days was solely due to non-T cells whereas the 8-day response was due to T cell proliferation. The addition of gamma-irradiated T cells did not appear to enhance the 3-day response of the non-T cells. However, the 8-day response by T cells was dependent on the presence of gamma-irradiated non-T cells. In cultures of unseparated cells, wild type fimbriae stimulated more than 75% of the B cells to enter the S and G2 phase at 3 days whereas at 8 days cycling T cells were present in both wild type and mutant fimbriae-stimulated cultures. Taken together, our observations suggest that the adhesin molecule stimulates a polyclonal mitogenic response in B cells that peaks at 3 days, and other structural components of the fimbriae are responsible for evoking an 8-day (probably immune) response in T cells.     

In vitro response of subpopulations of human tonsil lymphocytes. I. Cellular collaboration in the proliferative response to PHA and Con A.

Human tonsil lymphocytes have been separated into three subpopulations of cells: purified B cells and two subsets of purified T cells (F1 and F2). B cells were obtained by rosetting with neuraminidase treated SRBC. F1 and F2 were separated by filtration on a nylon wool column using different speeds of elution. Purified B cells contained less than 5% T cells, the T cells preparations contained less than 5% B cells for F1 and 10 to 15% for F2, respectively. A significant contamination in cells not identified by any B or T marker was observed in purified B cells and in F1. Adherent cells enhanced the response of each lymphochte population to PHA and Con A. This explained the paradoxically low responsiveness of the purified T cells. Purified B cells did not respond to these mitogens in different culture conditions. However, a small B cell response was observed when they were cultured in the presence of mitomycin-treated T cells. Striking was the enhancing effect of B cells on the T cell response to PHA and Con A. This enhancing effect was observed even when B cells were treated with mitomycin or depleted in adherent cells. The comparison of the F1 and F2 response suggested that they contained distinct types of T cells.     

The novel IgD binding protein from Moraxella catarrhalis induces human B lymphocyte activation and Ig secretion in the presence of Th2 cytokines

Moraxella IgD binding protein (MID) is a novel bacterial outer membrane protein with IgD-binding properties. MID was purified from the respiratory pathogen Moraxella catarrhalis and is here shown to have B cell stimulatory properties. Purified MID in the range of 0.01-0.1 microg/ml was optimal to induce a proliferative response in human PBL. MID coupled to Sepharose and formalin-fixed M. catarrhalis preparations induced similar proliferative responses in PBL cultures. MID or MID-Sepharose stimulated purified human peripheral B cells as measured by proliferation. In contrast, MID or MID-Sepharose did not activate T cells. Preincubation of purified B cells with anti-IgD Abs inhibited MID-Sepharose-induced B cell proliferation. The addition of IL-4 specifically induced IL-6 production in MID-Sepharose-activated B cells. IgM secretion was detected in B cell cultures stimulated with MID or MID-Sepharose and IL-2 for 10 days. Secretion of IgG and IgA was efficiently induced in cultures from purified B cells stimulated with the combination of MID or MID-Sepharose and IL-4, IL-10, and soluble CD40 ligand, suggesting that Th2-derived cytokines were required for optimal plasma cell generation. Taken together, MID has properties that make it an important tool to study IgD-targeted activation of B cells.     

Plasmodium falciparum and P. vivax gametocyte-specific exoantigens stimulate proliferation of TCR gammadelta+ lymphocytes

Immune modulation of Plasmodium vivax and P. falciparum gametocytes occurs over the course of erythrocytic infection. The response is linked to proliferative and inflammatory responses, which may be stimulated by stage-specific gametocyte proteins. Stage-specific exoantigens were purified from supernatants of P. falciparum and P. vivax gametocyte cultures, and either primary or secondary postinfection lymphocytes were stimulated for proliferation. Five of 25 exoantigens purified from P. falciparum gametocyte cultures and 6 of 28 exoantigens isolated from P. vivax were gametocyte stage specific. Metabolic labeling of soluble P. falciparum gametocyte proteins confirmed synthesis and secretion of 5 stage-specific exoantigens, with molecular masses of 118, 62, 52, 37, and 33 kDa. Purified gametocyte exoantigens within the range of 50 to 100 kDa stage-specifically stimulated proliferation of lymphocytes from postprimary P. falciparum infections, and from postprimary and secondary P. vivax infection patients with homologous purified exoantigens. T-cell receptor (TCR)gammadelta+, and CD3+ CD8+ and CD3+ CD4- CD8- T cells were specifically upregulated from P. falciparum primary- and P. vivax secondary-infection lymphocytes, respectively, using gametocyte stage-specific exoantigens. CD25+ was the major activation marker expressed by CD3+ and gammadelta T cells when stimulated with gametocyte exoantigens. None of the T cell markers was significantly upregulated using gametocyte stage-specific exoantigens with primary-infection P. vivax lymphocytes.     

Both cloned interleukin 2 and purified interleukin 1 are required for optimal growth of purified L3T4+ and Lyt 2+ lymphocytes initiated by concanavalin A

Concanavalin A (Con A), cloned interleukin 2 (IL-2), purified interleukin 1 (IL-1) or two different crude preparations containing IL-1 activity alone, did not induce proliferation of rigorously accessory cell (AC)-depleted splenic L3T4+ or Lyt 2+ lymphocytes. Con A together with saturating concentrations of cloned IL-2 (100 U/ml) promoted less than 40% of the proliferative responses observed in AC-supplemented L3T4+ and Lyt 2+ T-cell cultures. The three preparations of IL-1 used supported minimal proliferation of Con A-treated purified L3T4+ or Lyt 2+ lymphocytes. However, all these IL-1 preparations promoted significant growth of the T-cell populations if AC (1%) were included in the cultures. Cloned IL-2 combined with purified IL-1 promoted proliferation of Con A-treated L3T4+ and Lyt 2+ lymphocytes achieving approximately 75% of the responses observed in AC-supplemented T-cell cultures. The additive effect of IL-1 was apparent in the presence of saturating concentrations of cloned IL-2. Finally, Con A alone induced a detectable number of both L3T4+ and Lyt 2+ lymphocytes to express IL-2 receptors as determined with the anti-mouse IL-2 receptor antibody 7D4 by immunofluorescence and FACS analysis. Purified IL-1 neither induced detectable number of L3T4+ or Lyt 2+ T cells to express IL-2 receptors nor increased the number of Con A-treated T cells bearing IL-2 receptors. We have interpreted these findings to indicate the following: Con A alone is sufficient to induce highly purified L3T4+ and Lyt 2+ lymphocytes to express IL-2 receptors. Cloned IL-2 and purified IL-1 are required for optimal growth of L3T4+ and Lyt 2+ lymphocytes and these cytokines together efficiently replace AC in growth of T cells initiated by Con A. IL-1 alone does not replace AC in Con A-induced activation of mouse T cells. IL-1 exerts potentiation on IL-2-driven growth of Con A-treated L3T4+ and Lyt 2+ lymphocytes. The additive activity of IL-1 on growth of normal T cells is not due to increased production of IL-2 in the cultures or induction of normal T cells to expression of IL-2 receptors by IL-1. We propose that IL-1 optimizes the action and/or interaction of IL-2 with its receptors on the T-cell membrane (by, i.e., increasing affinity of the IL-2 receptor for its ligand and/or stabilizing the IL-2 receptor).     

Endotoxin-induced T lymphocyte proliferation

The lymphocyte response to endotoxin (LPS) has been attributed largely to the action of this agent as a polyclonal activator of B lymphocytes. In this study we found that a cloned murine interleukin 2-dependent cytotoxic T cell line, CT 6, proliferates in response to LPS, thus providing the first evidence that T cells can be stimulated directly by LPS. The response was dose and time dependent and was blocked by polymyxin B, an inhibitor of LPS-induced mitogenesis. The fact that this is a cloned T cell line, free of other potentially contaminating lymphoid cell types, precludes the possibility that this proliferation is due to contaminating B lymphocytes or is mediated by macrophage-derived products such as interleukin 1. Moreover, highly purified splenic T lymphocyte populations (purified by negative/positive selection or by a rigorous column purification procedure) contain a small subpopulation (approximately 3%) of T cells that proliferate in response to LPS. This population is missing in the endotoxin-hyporesponsive C3H/HeJ mouse. As was observed in the CT 6 line, proliferation of splenic T cells in response to LPS was inhibited by polymyxin B. Furthermore, treatment of LPS-stimulated T cells with anti-T cell antibodies plus complement blocks the uptake of 3H-thymidine by these cultures. Exogenous interleukin 1 failed to stimulate the T cell cultures comparably to LPS and therefore cannot account for the degree of stimulation observed. These findings support and extend previous findings that suggested a role for an endotoxin-sensitive T cell population in the induction of certain responses, such as LPS-induced adjuvanticity of the lymphocyte-dependent LPS induction of macrophage procoagulant activity.     

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro. Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBL's) and 50% of the spleen cells while passage over NW yields 40% of the applied PBL's and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBL's respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBL's that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.     

Inhibition of human B cell responsiveness by prostaglandin E2

The capacity of prostaglandin E2 (PCE2) to modulate human peripheral blood B cell proliferation and the generation of immunoglobulin-secreting cells (ISC) stimulated by Cowan 1 strain Staphylococcus aureus and mitogen-stimulated T cell supernatant was examined. PGE2 significantly inhibited both responses, whereas PGF2 alpha had no inhibitory effect. Responses of highly purified B cells obtained from spleen, lymph node, and tonsil were also inhibited. In addition PGE2 suppressed B cell responses supported by recombinant interleukin 2 rather than T cell supernatant. PGE2-mediated inhibition was mimicked by forskolin, a direct activator of adenylate cyclase. Kinetic studies indicated that PGE2 inhibited B cell responses by a progressively greater increment as cultures were prolonged. Evaluation by flow cytometry after staining with acridine orange or mithramycin indicated that PGE2 had no effect on initial B cell entry into the G1 phase of the cell cycle, passage through G1, and entry into S, G2, and M. Rather, PGE2 inhibited responses of postdivisional daughter cells. PGE2 inhibited responses in cultures stimulated by the calcium ionophore ionomycin and T cell supernatant but had minimal effects in cultures stimulated by the combination of ionomycin and phorbol myristate acetate. Moreover, phorbol myristate acetate reversed PGE2-mediated inhibition of proliferation stimulated by S. aureus or S. aureus + T cell supernatant. These results indicate that PGE2 suppresses the continued growth and differentiation of human B cells, although it has no effect on initial B cell activation and suggest that PGE2 may play a role in regulating human B cell responses in vivo.     

Relation between sister chromatid exchange,cell proliferation and proportion of B and T cells in human lymphocyte cultures

Summary Human B and T lymphocytes differ in the rate of cell proliferation and frequency of sister chromatid exchange (SCE) when cultured separately in short-term cultures. This difference could theoretically be responsible for part of the variation in the SCE-frequency previously observed among healthy subjects since there is individual variation in the proportion of B and T cells in the peripheral blood. We have therefore studied cell proliferation and SCE-frequency in conventional short-term cultures of lymphocytes from 28 healthy subjects with different proportions of B and T cells. The percentage, of B or T lymphocytes did not correlate with the SCE-frequency, nor with the rate of cell proliferation in culture. However, a significantly higher SCE-frequency was found in slowly proliferating cultures than in cultures with a high rate of turn over. Thus, the rate of cell proliferation appears to be an important determinant of the SCE-frequency in conventional lymphocyte cultures. Although the data do not exclude attribution of the difference in SCE-frequency between rapidly and slowly growing cultures to differences in subpopulations of lymphocytes, it appears less likely that B and T cells constitute these tentative subpopulations.     

Alloreactive T cells from individual soft agar colonies specific for guinea pig Ia antigens. II. Cellular and molecular requirements for optimal proliferative responses

We have investigated the cellular and molecular requirement for optimal proliferative responses of several alloreactive T cell lines that were derived from individual soft agar colonies and were specific for guinea pig Ia antigens. Optimal proliferation of several colonies was observed in cultures containing purified allogeneic macrophages and growth factor(s) present in supernatant fluids of Con A-activated T cells (Con A-S). Significant proliferative responses of these alloreactive T cell colonies were also routinely detected in cultures only supplemented with unfractionated irradiated allogeneic peritoneal exudate cell (PEC). The T cell component of the stimulator cell population was crucial for these responses by producing necessary growth factor(s) endogenously in the culture. Thus, 2 signals, allogeneic Ia antigens and growth factor(s), were required for optimal proliferative responses of these alloreactive T cell colonies. Furthermore, macrophage-associated Ia antigen was more efficient than B cell-associated Ia for these responses. The requirement for allogeneic Ia antigen was not absolute, since the colonies could easily be expanded when the cultures were supplemented with irradiated syngeneic PEC and the T cell mitogens, Con A or PHA. The effect of the mitogen was mediated via the T cells in the irradiated PEC, since removal of the T cells from these PEC markedly reduced the responses. Thus, it is likely that a nonspecific signal(s) presumably from T cells can promote proliferation of alloreactive T cell colonies in the absence of allogeneic Ia antigen. These results suggest 2 mechanisms of activation of these alloreactive T cells.     

Lymphocyte responses to syngeneic antigens. I. Enhancement of the murine autologous mixed lymphocyte response by polyethylene glycol

The normally weak murine T-cell proliferative response against autologous non-T stimulator cells (the autologous mixed lymphocyte culture (MLC) was enhanced markedly by inclusion of the hydrophilic polymer, polyethylene glycol (PEG), into the culture medium. Potentiation of the autologous MLC was indicated on the basis of increased [³H]TdR incorporation by responding cells, as well as by the numbers of viable cells recovered from mixed cell cultures. PEG is not a polyclonal activator of T and/or B lymphocytes, since nylon wool nonadherent lymphoid cells (T cell-enriched fraction), nylon wool adherent cells (B cell-enriched fraction) and T cell-deficient “nude” spleen cells were not stimulated into DNA synthesis when cultured separately with PEG. Inclusion of 4% PEG into the culture medium was found to optimally enhance autologous MLC, although concentrations between 2 and 5% also significantly elevated responsiveness. At a responder/stimulator ratio of 1:2, autologous MLC yielded peak [³H]TdR incorporation after 5 days of culture. At lower ratios (1:1 and 2:1), however, Δ cpm of autologous MLC continued to increase over a culture period of 7 days. Enhanced responsiveness in the presence of PEG was observed in strains of mice representing a variety of H-2 haplotypes, indicating that at least the potential for autoreactivity of this type is a naturally occurring and widespread characteristic of murine species. An absolute requirement for purified T responder cells was necessary in the autologous MLC, since unseparated lymphoid cell responder LN or spleen cells demonstrated marked proliferation when cultured alone in medium containing PEG. The proliferation of T cells to autologous non-T cells within the same unseparated lymphoid cell preparation appears to be responsible for this phenomenon. Ia antigens expressed by the stimulator cells are involved in the induction of T-cell response, since anti-Ia sera added directly to the cultures inhibited the autologous MLC, but did not affect other T-cell responses to alloantigens or mitogens. Despite the marked proliferation observed in the autologous MLC performed in the presence of PEG, there was no generation of cytotoxic effector cells. Thus, PEG does not appear to add, or alter determinants on stimulator cells to an extent that they are recognized as foreign by precursor cytotoxic T cells. Although the mechanism of enhancement of autologous MLC by PEG is not totally defined, it appears, at least functionally, to promote cellular interactions that occur normally between T cells, B cells, and macrophages. In this respect, PEG will be a powerful and useful probe to dissect the cellular interactions that take place in autologous responses.     

Regulation of murine T cell proliferation by B cell stimulatory factor-1

The proliferation of mitogen-activated primary T cells, antigen-activated memory T cells from mixed leukocyte culture, and antigen-dependent alloreactive T cell clones in response to purified murine recombinant B cell stimulatory factor-1 (also known as interleukin 4) was examined. We found that B cell stimulatory factor-1 (BSF-1) stimulated optimal proliferation of these T cells only after their recent activation by antigen or mitogen. Analysis of cell surface BSF-1 receptor expression indicated that although T cell activation is accompanied by a small increase in BSF-1 receptor expression, the cells also express BSF-1 receptors prior to activation at a time when they do not proliferate in response to BSF-1. BSF-1 was as effective a stimulus as interleukin 2 for inducing proliferation of the Lyt-2+ subpopulation of concanavalin A-activated murine spleen cells and an alloreactive cytolytic T cell clone. However, the L3T4+ subpopulation of concanavalin A-activated spleen and an alloreactive helper T cell clone were less responsive to BSF-1 than to interleukin 2. Taken together, the data indicate an important role for BSF-1 in the regulation of normal T cell proliferation.     

Anti-proliferative effect of IFN-gamma in immune regulation. II. IFN-gamma inhibits the proliferation of murine bone marrow cells stimulated with IL-3, IL-4, or granulocyte-macrophage colony-stimulating factor

A biphasic dose response curve was observed when the bone marrow-derived cell line FDCP1, used as an indicator line for IL-3 bioassays, was exposed to supernatants from some activated T cell clones but not others. The active component which inhibited proliferation at the higher supernatant concentrations appeared to be IFN-gamma, based on the following observations. 1) Only those culture supernatants which contained IFN-gamma gave a biphasic dose response curve; 2) with these supernatants, an anti-IFN-gamma mAb augmented the proliferation of FDCP1 cells at the higher supernatant concentrations; and 3) rIFN-gamma profoundly inhibited the proliferation of FDCP1 cells stimulated with rIL-3 or rIL-4. rTNF-alpha inhibited FDCP1 proliferation only to a modest extent, yet the combination of rTNF-alpha + rIFN-gamma provided greater inhibition than each agent alone. The proliferation of a second bone marrow-derived cell line, DA1, was not inhibited by rIFN-gamma or rIFN-gamma + rTNF-alpha when stimulated with rIL-3 or recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF). Fresh bone marrow cells also showed a suboptimal proliferative response when stimulated with T cell supernatants containing IFN-gamma, and this response was augmented considerably upon the addition of anti-IFN-gamma mAb. Bone marrow cell proliferation was observed upon exposure to rIL-3, rIL-4, or rGM-CSF, and these responses were inhibited by rIFN-gamma; rTNF-alpha also produced a synergistic effect with these cells. Bone marrow cell colony formation stimulated by rIL-3 or rGM-CSF also was inhibited by rIFN-gamma. Colony formation in bone marrow cell cultures was not observed in response to rIL-4. Collectively, these results suggest that Th1 cells, which in addition to IL-3 and GM-CSF also produce IFN-gamma, may regulate hemopoietic cell proliferation and colony formation differently from the way Th2 cells do, which do not produce IFN-gamma.     

Monoclonal antibodies that inhibit activation and proliferation of lymphocytes. II. Requisite role of the monoclonal antibody-defined antigen systems in activation and proliferation of human and rat lymphocytes

A murine monoclonal antibody (MoAb) B3 to rat cells and MoAb HBJ127 and HBJ98 to human cells were found previously to recognize the homologous antigen systems (gp130 in the rat and gp125 in the human) which are predominantly distributed on the cell surface of proliferating cells of the respective species, and the expression of the antigen systems in lymphocytes were indicated previously to correlate closely with the activation and proliferation of the lymphocytes. In this respect, the in vitro effects of these MoAb on the nucleic acid synthesis, cell cycles, or proliferation of stimulated rat and human lymphocytes were examined by use of T cell-enriched and B cell-enriched cell populations. The addition of B3 MoAb to cultures diminished Con A-induced or allogeneic mixed lymphocyte culture-induced rat T cell proliferation and lipopolysaccharide-induced rat B cell proliferation, whereas B31 MoAb, which is unreactive with the gp130 antigen, did not inhibit these lymphocyte responses. Similarly, both HBJ127 and HBJ98 MoAb could inhibit the human lymphocyte proliferation in vitro, although HBJ127 MoAb showed about eight times greater inhibitory activity than did HBJ98 MoAb; HBJ127 MoAb almost completely inhibited the DNA synthesis of the Con A-stimulated lymphocytes at concentrations higher than 13 micrograms/ml. The flow cytometric analysis of the cellular nucleic acid contents with acridine orange-stained cells showed that when B3 MoAb and Con A were simultaneously added to unstimulated rat T cells, progression of the cell cycle was blocked at the G0 to G1 transition. In this culture condition, the appearance of the B3-defined antigen was arrested in a moderate level, as determined with fluorescein-stained cells. On the addition of B3 MoAb to the culture of the T cells after 24-hr Con A stimulation, the MoAb also strongly inhibited the cellular DNA synthesis, but it did not arrest the cell cycle at a certain phase and did not modulate the corresponding antigen. These data suggest that the B3 MoAb-defined antigen on the rat lymphocytes and the HBJ127/HBJ98 MoAb-defined antigen on the human lymphocytes may play some requisite roles not only in lymphocyte activation but also in the subsequent progression through the cell cycle to proliferate.     

A phorbol ester (TPA) can replace macrophages in human lymphocyte cultures stimulated with a mitogen but not with an antigen

A culture system was developed in which human peripheral blood mononuclear cells (PBM) depleted of macrophages did not proliferate in response to the lectin mitogen PHA or to the soluble antigen of tetanus toxoid. These cells were able to respond to both mitogen and antigen if purified autologous macrophages were added back to the culture. The response to PHA was partially restored by supplementing the cultures with supernatants from LPS-stimulated macrophages or with partially purified human interleukin 1 (IL 1). The response to tetanus was not restored by reconstitution with these materials. The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), has been shown to have IL 1-like effects in other species and is a polyclonal activator of human T and B lymphocytes. In this study, we tested the ability of TPA to replace macrophages in human lymphocyte cultures stimulated with mitogen or with antigen. Small doses of TPA (50 ng/ml) completely replaced macrophages in the PHA-stimulated cultures; however, in doses of up to 400 ng/ml, TPA was not able to replace macrophages in cultures stimulated with tetanus. Thus, TPA appears to mimic the macrophage-replacing ability of soluble factors (IL 1, macrophage supernatants) in the triggering of human lymphocytes.     

T lymphocyte-enriched murine peritoneal exudate cells. I. A reliable assay for antigen-induced T lymphocyte proliferation.

The in vitro activation of murine thymus-derived (T) lymphocytes by soluble protein and synthetic antigens has been difficult to assess because of the lack of a specific and reliable proliferation assay. The present report describes the development of an assay system which overcomes these problems by making use of a population of nylon wool column-purified T lymphocytes obtained from thioglycollate-induced peritoneal exudates of immunized mice. PETLES (peritoneal exudate, T lymphocyte-enriched cells) were composed mainly of T lymphocytes, eosinophils and small numbers of macrophages. Contamination with bone marrow-derived (B) lymphocytes averaged only 2%. When PETLES from immunized mice were stimulated in microtiter cultures with the immunizing antigen, large degrees of proliferation ensued as measured by incorporation of 3H-methyl-thymidine 5 days after initiation. As few as 1.25 x 10(4) cells and as little as 50 ng/ml of antigen gave significant stimulation. Maximum responses were obtained witn a series of 10 experiments under these optimal conditions, gave a mean incorporation of 70,900 cpm while the controls cultured without antigen showed only 3,600 cpm. PETLES from nonimmunized mice or from mice immunized to other antigens did not respond to DNP5OVA although they did respond to mitogens. The antigen-induced proliferation was shown to require the presence of immune T lymphocytes by two criteria: elimination of the response by treatment with anti-Thy 1.2 serum plus complement and failure to reconstitute the response when the few remaining immune B lymphocytes left after anti-Thy 1.2 treatment were added to nonimmune T lymphocytes. In addition, the system exhibited carrier specificity. Because of the paucity of B lymphocytes in the population, their contribution to the overall magnitude of the proliferative response was negligible as demonstrated by the small response to B cell mitogens. Thus, the assay appears to be a quantitative as well as a qualitative assay for one aspect of T lymphocyte function. This technique should prove useful for the study of murine T lymphocytes in vitro.     

Characterization and partial purification of a specific interleukin 2 inhibitor

To investigate the mechanisms that regulate the action of interleukin 2 (IL 2) and possibly limit its activity, we screened supernatants of mouse spleen cell cultures which had been stimulated with concanavalin A (Con A) for their ability to inhibit IL 2-mediated proliferation of a cloned IL 2-dependent line. Inhibitory activities with m.w. of 10,000 to 12,000 and 60,000 to 80,000 daltons could be identified in supernatants of both L3T4+ and Ly-2+ T cells, but not in supernatants of Con A or lipopolysaccharide-stimulated B cells. Maximal inhibitory activity was observed after 3 to 4 days of stimulation, and this inhibitory activity could be overcome by increasing the stimulatory concentration of IL 2. When the factor was further purified by reverse-phase high pressure liquid chromatography, it eluted as a single peak with an m.w. of 11,000 to 12,000 daltons which inhibited IL 2- but not IL 3-dependent proliferation. The mechanisms by which this new lymphokine might play in the control of the clonal expansion of T lymphocytes are discussed.