A T cell activity that enhances polyclonal IgE production and its inhibition by interferon-gamma

The addition of concanavalin A-stimulated supernatants of the helper T cell clone, D9.1, to cultures of lipopolysaccharide (LPS)-stimulated T-depleted mouse spleen cells caused more than a 100-fold increase in immunoglobulin (Ig) E production. These supernatants cause a 10-fold to 15-fold increase in IgG1, a fivefold to 10-fold increase in IgA, and a fivefold to 10-fold decrease in IgG3. These effects are optimal when the supernatants are added 1 to 2 days after stimulation with LPS. Cells from mouse strains that normally give little or no IgE response in vivo give normal IgE levels in response to LPS plus the supernatant of Concanavalin A-stimulated D9.1 cells in vitro. The enhancement of both IgE and IgG1 can be completely inhibited by relatively low concentrations of interferon-gamma (IFN-gamma). Both the IgE-enhancing activity and IFN-gamma act directly upon purified B cells.     

Synergy between T cell-replacing factor and bacterial lipopolysaccharides (LPS) in the primary antibody response in vitro: a model for lipopolysaccharide adjuvant action.

Unfractionated spleen cells, B cells from normal mice, and nu/nu spleen cells respond to the addition of bacterial lipopolysaccharide (LPS) and T-cell-replacing factor (TRF) by production of plaque-forming cells (PFC) in excess of the number expected from the addition of LPS and TRF separately. This synergistic activity is dependent on the presence of the antigen, SRBC. Supernatants of both allogeneic spleen cell mixtures and spleen cells cultured with Con A are effective and synergize best at concentrations suboptimal for their ability to act as TRF alone. Culture supernatants of unstimulated normal or fractionated cell populations are ineffective. Synergy is not dependent on the presence of macrophages in the cultures. Purified LPS free from active contaminants, as well as commercially available LPS, show synergy with TRF. Synergy was seen when TRF was added at initiation of culture or 24 hr later. It is suggested that synergy is the equivalent of LPS adjuvant activity, that the role of T cells in LPS adjuvanticity is that of a conventional cooperating cell, and the LPS acts as an adjuvant by inducing B cells to become more sensitive to T cell helper factors.     

Generation by lipopolysaccharide of a late-acting soluble suppressor of antibody synthesis.

Polyclonal stimulation of normal mouse spleen cells by lipopolysaccharide (LPS) from Salmonella typhimurium resulted in the generation of a factor which was capable of suppressing the humoral immune response in vitro. LPS effectively induced the release of the inhibitory material into the supernatants of these cultures within 24 hr. The suppressive mediator, which was similar in properties to the antigen-generated, transiently-acting soluble suppressor (TASS) reported earlier, partially abrogated (by 30–80%) the anti-sheep erythrocyte plaque-forming cell response when added to test cultures ~20 hr prior to assay for direct hemolytic plaques. Although LPS, in submitogenic doses, also was effective in depressing the in vitro hemolysin response, the inhibitory activity of residual mitogen present in the test supernatants, and that of the LPS-induced factor, were shown to be different. By use of antisera and complement treatment to selectively deplete spleen cell populations of T or B lymphocytes, it was demonstrated that B cells were essential for production of the suppressive mediator.     

Helper factors for hapten-self cytotoxic T cells occur in NZB culture supernatants despite deficient AMLR

The ability of helper T cells from NZB mice to produce non-interleukin 2 (IL-2) lymphokines in the autologous mixed lymphocyte reaction (AMLR) was examined. Factors present in normal AMLR culture have been previously reported to mediate the development of a cytotoxic T-cell response to trinitrophenyl (TNP)-modified syngeneic thymocytes. Young NZB mice, like the normal strains, were able to produce the helper factors in the AMLR and to utilize these mediators in the cytotoxic induction system. Old autoimmune NZB mice demonstrated a poor proliferative response in the AMLR and were unable to activate hapten-specific cytotoxic cells in the presence of AMLR culture supernatant from either young or old mice. This was not due to a lack of cytotoxic precursors, nor was it a normal consequence of aging, but may be related to decreased IL-2 production by helper T cells. Interestingly, supernatant from AMLR proliferation deficient old NZB mice contained normal amounts of the AMLR helper factor. These data suggest that AMLR helper factor production is not directly related to the proliferative response and that two different helper-T-cell subpopulations may be responsible for these activities. The production of these mediators in mice which cannot utilize them raise questions about their role in autoimmunity.     

Suppression of ACTH-induced steroidogenesis by supernatants from LPS-treated peritoneal exudate macrophages

The results from a number of clinical and experimental studies have suggested that during endotoxemia, suppression of adrenocortical steroidogenesis may occur. We have examined the possibility that macrophages are the source of a factor that suppresses adrenocortical steroidogenesis. Resident and peptone-elicited peritoneal exudate macrophages (PEM) from C3HeB/FeJ mice were incubated for 4 hr at 37 degrees C in the presence or absence of T cell hybridoma-derived lymphokine (LK) that contained high concentrations of MAF activity (assessed by induction of nonspecific tumoricidal activity in PEM). The LK was removed by rinsing, and fresh medium was added, followed by Salmonella minnesota R595 LPS (final concentration 10 micrograms/ml). After 18 hr at 37 degrees C the PEM supernatants and control medium from flasks without cells were harvested and stored at -20 degrees C. Explanted rabbit adrenocortical cells in 96-well plates were exposed to 30 microliters of PEM supernatant or control medium and ACTH (10 or 100 mU/ml) in a final volume of 120 microliters for 3 consecutive days. The adrenocortical cell supernatants were harvested each day, followed by replenishment of medium, PEM supernatant, and ACTH. Fluorogenic steroid production in wells that received control medium or supernatants from PEM not treated with LPS was normal (0.22 microgram +/- 0.010 (SD) per 5 X 10(4) cells). However, as much as 75 to 95% suppression of steroidogenesis was observed in wells that received supernatants from PEM treated with LK and LPS, compared to 40% suppression in wells that received supernatant from PEM treated with LPS alone. Continued exposure (over 3 days) of adrenocortical cells to supernatants from LPS-treated PEM resulted in progressively decreasing response to ACTH. Comparable suppressive activity was observed in supernatants from LPS-treated bone marrow-derived macrophages. In further experiments, suppression was observed in wells that were pretreated (22 hr) with the appropriate PEM supernatant, and evidence was obtained that the suppressive activity was not due to carry-over LPS. Finally, results from control experiments demonstrated that suppressive PEM supernatants neither inactivate ACTH nor interfere with the assay of fluorogenic steroids. Thus, these results suggest that during endotoxemia, products from LPS-stimulated macrophages may suppress adrenocortical function.     

CD4+ Leu-8+ T cell supernatant activity that inhibits Ig production.

The subpopulation of CD4+ T cells that expresses the Leu-8 peripheral lymph node homing receptor suppresses PWM-stimulated Ig synthesis. To determine the mechanism of this suppression, the immunoregulatory activity of culture supernatants obtained from peripheral blood CD4+ Leu-8+ T cells cultured with anti-CD3 mAb and PMA (Leu-8+ supernatant) was determined. Leu-8+ supernatant suppressed PWM-stimulated Ig synthesis in cultures containing non-T cells and CD4+ Leu-8- T cells. In contrast, the supernatant from CD4+ Leu-8- T cells did not suppress Ig synthesis. The inhibitory activity of CD4+ Leu-8+ T cell supernatants could not be accounted for by a deficiency or excess of IL-2, IL-4, IFN-gamma, IL-6, or PGE2. In studies examining the effect of CD4+ Leu-8+ supernatant on T cells, the supernatant did not alter either mitogen-induced proliferation or the helper function of CD4+ Leu-8- T cells. In studies examining the effect of CD4+ Leu-8+ supernatant on B cells, the supernatant inhibited Staphylococcus aureus Cowan I strain-induced B cell Ig secretion but not B cell proliferation. The suppressor activity of Leu-8+ supernatant was eliminated by protease treatment and was eluted by HPLC in two main peaks, with molecular sizes of 44 and 12 kDa. In summary, these studies indicate that supernatants from activated CD4+ Leu-8+ T cells directly suppress B cell Ig production.     

Interferon-gamma induction by lipopolysaccharide: dependence on interleukin 2 and macrophages

Bacterial lipopolysaccharide (LPS) induced fresh murine splenocytes to produce interferon (IFN)-alpha/beta presumably by stimulation of the B lymphocytes and macrophages. However, when the splenocytes were "aged" for 24 to 72 hr in culture before addition of the LPS, the IFN response was significantly increased and was determined to be predominantly IFN-gamma. Because low levels of interleukin 2 (IL 2) were found to be spontaneously produced by the unstimulated splenocytes during the "aging" process, the effect of IL 2 on IFN induction by LPS in fresh splenocytes was examined. The addition of LPS to freshly prepared splenocyte cultures that were treated with human IL 2, either native or recombinant, before exposure to the LPS resulted in the LPS inducing large amounts of IFN-gamma. IL 2 alone induced little if any IFN in the splenocyte cultures. Depletion of T cells and large granular lymphocytes (LGL) from the cultures by anti-Thy-1.2 antibodies plus complement abrogated IFN-gamma production, and the addition of polymyxin B to "aged" splenocyte cultures resulted in loss of IFN production in response to LPS. Cultures that were enriched for T cells and LGL by passage through nylon wool produced significant amounts of IFN-gamma in response to LPS only if first treated with IL 2. Furthermore, the addition of splenic adherent cells to purified nylon wool-non-adherent (NWNA) cells augmented IFN-gamma production, whether or not the NWNA cells were pretreated with IL 2. This enhancement appeared to require direct contact between adherent cells and NWNA cells, because physical separation abrogated IFN production. The addition of recombinant IL 1 or LPS-conditioned supernatants of macrophage cultures did not replace adherent cell activity. These data demonstrate that LPS, which predominantly induces IFN-alpha/beta in fresh murine splenocytes, is able to stimulate T lymphocytes to produce IFN-gamma if the T cells are first exposed to endogenously produced or exogenously applied IL 2. Because IFN-gamma is a potent activator of the bactericidal and cytocidal potential of macrophages, the induction of IFN-gamma by bacterial LPS may play an important role in resistance/recovery mechanisms against bacterial infections.     

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.     

Augmentation of B-cell responsiveness by a tumor-activated T-cell factor

The growth of the P815 mastocytoma in syngeneic DBA/2 mice led to an activation of Ly1+2- T cells. These T cells produced a soluble factor or factors in culture which, when added to normal spleen cells or B cells in the presence of syngeneic Ly1 cells, caused a genetically unrestricted augmentation of the plaque-forming response toward sheep red blood cells (SRBC). The culture supernatant of the activated T cells did not support the proliferation of an interleukin-2 (IL-2)-dependent cell, nor exhibit properties of late-acting TRF. Active supernatants appeared to affect directly B cells during the first 48 hr of culture with SRBC in such a way as to make them more responsive to antigen-specific Ly1-cell help.     

Differential effect of interferon-gamma and interleukin-2 on the induction of IgA and IgM anti-dextran responses

Supernatants from T-cell lines and T-cell hybridomas can substitute for T cells in the induction of the anti-alpha(1,3) Dextran B1355 plaque-forming cell response in culture. The present study sought to define the lymphokines required for the induction of IgA and IgM anti-alpha (1,3) dextran responses. Recombinant Interferon-gamma (IFN-gamma) supported the induction of low levels of IgA anti-alpha(1,3) dextran plaque-forming cells in splenic B-cell cultures. IgA responses were substantially increased when cultures containing IFN-gamma were supplemented with an interleukin 2 (IL-2)-containing supernatant from the murine T-cell hybridoma BW.Mls, purified murine IL-2, or recombinant human IL-2. In striking contrast, IgM anti-alpha(1,3) dextran plaque-forming cells were not produced in cultures containing IFN-gamma alone or in combination with purified or recombinant IL-2. However, substantial IgM responses could be produced in cultures containing IFN-gamma and BW.Mls supernatant. This data indicates that there may be different lymphokine requirements for the induction of IgA and IgM anti-alpha(1,3) dextran B cells, or alternatively, such B cells may be in different stages of differentiation and therefore, not respond to the same lymphokines.     

Modulation of the mitogenic PHA response of carp, Cyprinus carpio L., by extracellular products of Aeromonas salmonicida

The influence of bacteria-free supernatants from cultures of atypical virulent (V234/81, auto-agglutinating. A-layer positive) and avirulent (126/68, non-agglutinating, A-layer negative) strains of A. salmonicida , obtained after different culture times in yeast-tryptone broth at 20°C, was tested on the PHA response of carp pronephric leucocytes in vitro . Supernatants from virulent cultures modulated the response, whereas avirulent supernatants had no effect. The response was enhanced (400%) by supernatant from early virulent cultures (20 h), but severely depressed (<3%) by later ones (96 h). The effects were dose-dependent. Inhibitory activity of 96-h supernatant was lost by heating (70°C, 30 min), suggesting that the inhibiting factors are all proteinaceous.
Heated 96-h supernatant was as stimulatory as early supernatant. Stimulation of leucocytes also occurred in the absence of PHA with early and heat-treated 96-h supernatants, but at a tenth of the level, suggesting that only stimulated cells (blasts) might respond to the substance(s) present in supernatants. Membrane fragments from virulent and avirulent bacteria, and purified LPS from virulent bacteria, were stimulatory with or without PHA. Endotoxin-free, heat-treated, 96-h culture supernatants were also stimulatory, suggesting that an additional mitogenic factor(s), other than LPS, is present in the supernatants. The modulating in vitro effects of extracellular products from A. salmonicida might explain the immunosuppression seen during later stages of erythrodermatitis in vivo.     

The allogeneic effect: the mechanism of allosuppression by Lyt-1, Ia- T cells

The kinetics of allohelp mediated by diffusable factors revealed that help by nonirradiated T cells (TOR) peaked at 48 to 72 hr, followed by a sharp decline if the T cells remained in the cultures. The temporal decrease in help after 72 hr was not mediated by suppressor lymphokines because mixtures of early (24 to 48 hr) and late (120-hr) allogeneic supernatants enhanced help synergistically. Lyt-1, Ia- T cells mediated the temporal decline in help and suppressed allogeneic B cell activation in co-cultures, and this "down-regulatory" activity (allosuppression) was radiosensitive. Help by irradiated T cells (T1000R) increased gradually until it plateaued between 96 and 120 hr. The helper activities of the allogeneic supernatants were directly proportional to their T cell growth factor (TCGF) activities. In addition, their kinetics were identical, and the removal of TCGF from 48-hr allogeneic supernatants by adsorption with TCGF-dependent HT-2 cells depleted both helper and TCGF activities. Help was restored to depleted 48-hr and 120-hr allogeneic supernatants by preparations of TCGF obtained from concanavalin A (Con A)-stimulated FS6-14.13 hybridoma cells that were adsorbed with lipopolysaccharide (LPS)-activated B cells or normal spleen cells (NS), but not with HT-2 cells. These results indicate that allohelp is dependent on TCGF. Moreover, help was dependent on at least one factor in addition to TCGF, because a high level of synergy occurred between TCGF and the "help-deficient" 120-hr allogeneic supernatant. In conclusion, the mechanism whereby Lyt-1, Ia- T cells regulated B cell activation with positive and negative allogeneic effects was through the production and subsequent exhaustion of TCGF, respectively. The production of TCGF and help was radioresistant, but exhaustion of TCGF and suppression was radiosensitive.     

Effect of L-ornithine on proliferative and cytotoxic T-cell responses in allogeneic and syngeneic mixed leukocyte cultures

The effect of L-ornithine on cytotoxic and proliferative responses in mixed leukocyte cultures has been analyzed. The activation of cytotoxic T lymphocytes (CTL) was strongly inhibited when 9 X 10(-3) M L-ornithine was added at the initiation of the cultures. The CTL precursor cells were not completely and irreversibly inactivated, however, since the cells generated normal cytotoxic activity if resuspended after 6 days in fresh culture medium together with a fresh set of stimulator cells. Experiments in microcultures with nylon-wool-nonadherent T-cell-enriched spleen cells as responder cells and "plastic adherent cells" as stimulator cells revealed that the cytotoxic responses were almost completely suppressed if ornithine was added within the first 20 hr but were only partially suppressed if ornithine was added after 48 hr. Also, ornithine had only a mild suppressive effect on proliferative responses in allogeneic and syngeneic mixed leukocyte cultures. The strong suppressive effect of the cytotoxic response was therefore not explained by a general toxic effect of L-ornithine on the responding cells in the culture. The addition of interleukin 2 (IL-2)-containing EL-4 supernatants did not prevent but rather enhanced the suppressive effect of L-ornithine. This indicated that the inhibitory effect was not (exclusively) expressed at the level of the IL-2-producing helper T cells. Since activated macrophages have been reported to secrete arginase, it appears that L-ornithine may be part of a regulatory circuit that selectively regulates the development of cytotoxic effector T cells.     

Suppression of a pokeweed mitogen-stimulated plaque-forming cell response by a human B lymphocyte-derived aggregated IgG-stimulated suppressor factor: suppressive B cell factor (SBF)

The mechanisms whereby formed immune complexes (IC) or immunoglobulin aggregates can suppress further antibody production were explored by culturing normal human peripheral blood mononuclear leukocytes (PBL) with heat-aggregated IgG (HAIgG) and collecting the culture supernatants at 24 hr. These supernatants were found to suppress a pokeweed mitogen (PWM)-induced rheumatoid factor plaque-forming cell (RF-PFC) response in normal individuals. PWM-induced anti-trinitrophenylated sheep red blood cell (TNP-SRBC) PFC were also inhibited by suppressor supernatants from HAIgG-stimulated PBL, suggesting that the polyclonal PFC response was inhibited by a suppressor factor. The suppressor factor inhibited PWM stimulated RF-PFC throughout the culture period, but suppression was maximal at the peak of the RF-PFC response. Suppressor factor was only effective at the initiation of cultures, suggesting that it inhibited early events in the PWM-stimulated RF-PFC response. Molecular weight determination of the suppressor factor by differential membrane fractionation suggested a m.w. range of 30,000 to 50,000, and chromatography on Sephadex G-100 showed a peak activity at an approximate m.w. of 32,000. Studies suggested the factor was not an interferon. Depletion of T lymphocytes by E rosetting and macrophages/monocytes by G-10 adherence did not affect the generation of suppressor factor. Depletion of T lymphocytes (OKT4, OKT8) and NK cells (Leu-11b) by antibody-dependent, complement-mediated cytotoxicity also did not affect the generation of suppressor factor. Depletion of B lymphocytes with OKB7 resulted in the generation of significantly less suppressor factor. Suppression produced by unstimulated purified B lymphocytes was approximately one-half that seen when B lymphocytes were stimulated with HAIgG. Differential membrane fractionation studies suggested that only HAIgG-stimulated B cell cultures contained peak activity in the 30,000 to 50,000 m.w. fraction. Supernatants from unstimulated purified T cells also generated suppression, which was approximately one-half of that seen with HAIgG-stimulated B cells, but no increase in suppressor activity was seen in T cell cultures after incubation with HAIgG. These studies demonstrate that HAIgG is capable of stimulating B lymphocytes to produce a lymphokine, suppressive B cell factor (SBF), which is capable of suppressing a polyclonal PFC response. SBF may be important in feedback control of human immunoglobulin production.     

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.     

Systemic suppression of delayed-type hypersensitivity by supernatants from UV-irradiated keratinocytes. An essential role for keratinocyte-derived IL-10.

Exposing murine keratinocyte cultures to UV radiation causes the release of a suppressive cytokine that mimics the immunosuppressive effects of total-body UV exposure. Injecting supernatants from UV-irradiated keratinocyte cultures into mice inhibits their ability to generate a delayed-type hypersensitivity reaction against allogeneic histocompatibility Ag, and spleen cells from mice injected with supernatant do not respond to alloantigen in the in vitro MLR. A unique feature of the immunosuppression induced by either total-body UV-exposure or injecting the suppressive cytokine from UV-irradiated keratinocytes is the selectivity of suppression. Although cellular immune reactions such as delayed-type hypersensitivity are suppressed antibody production is unaffected. Because the selective nature to the UV-induced immunosuppression is similar to the biologic activity of IL-10, we examined the hypothesis that UV exposure of keratinocytes causes the release of IL-10. Keratinocyte monolayers were exposed to UV radiation and at specific times after exposure mRNA was isolated or the culture supernatant from the cells was collected. IL-10 mRNA expression was enhanced in UV-irradiated keratinocytes. The secretion of IL-10 by the irradiated keratinocytes was determined by Western blot analysis. A band reactive with anti-IL-10 mAb was found in supernatants from the UV-irradiated but not the mock-irradiated cells. IL-10 biologic activity was determined by the ability of the supernatants from the UV-irradiated keratinocytes to suppress IFN-gamma production by Ag-activated Th 1 cell clones. Anti-IL-10 mAb neutralized the ability of supernatants from UV-irradiated keratinocytes to suppress the induction of delayed-type hypersensitivity in vivo. Furthermore, injecting UV-irradiated mice with antibodies against IL-10 partially inhibited in vivo immunosuppression. These data indicate that activated keratinocytes are capable of secreting IL-10 and suggest that the release of IL-10 by UV-irradiated keratinocytes plays an essential role in the induction of systemic immunosuppression after total-body UV exposure.     

Induction of CTL responses to alloantigens by a Db-specific T helper clone

A T cell helper clone was derived 2 yr ago from a mixed lymphocyte culture. This clone, referred to as clone 9, was propagated in interleukin 2 (IL 2)-containing medium in the presence of irradiated stimulator and irradiated syngeneic spleen cells. Clone 9 was of H-2d origin and was found to be Thy-1+ and Lyt-1-2-. Clone 9, as well as supernatant factor(s) derived from it, were able to enhance the primary cytotoxic responses of Db responder cells to alloantigens. Furthermore, clone 9 cells or its factor(s) were only active when added during the first 24 hr of a 5-day culture period. When a low stimulator cell dose (10(4) cells per 0.2 ml culture) was used, it was possible to demonstrate that clone 9 also required a source of irradiated allogeneic splenic accessory cells to exert its helper action. Under these conditions, clone 9 or its factor(s) could also synergize with IL 2-containing medium in mounting cytotoxic responses to alloantigens. Synergy between IL 2-containing medium and clone 9 or its factor(s) was observed only when Db responder cells were used. The helper activity in clone 9 supernatant was also specifically absorbed out by Con A-stimulated Db spleen cell blasts. Preincubation with clone 9 supernatant for 1 hr at room temperature also led to enhanced cytotoxic responses of Db responder cells to alloantigens. Clone 9 supernatant was also found to be devoid of detectable IL 2 activity. Thus, clone 9 or its helper factor(s) appear to exert its helper activity by an early interaction with Db cytotoxic T lymphocyte precursors (CTL-P).     

Production of IL 2 and IFN-gamma by T cells from malaria patients in response to Plasmodium falciparum or erythrocyte antigens in vitro

T cells from patients acutely infected with malaria exhibit a disease-related stimulation of DNA synthesis in response to Plasmodium falciparum antigen in vitro. This response is weak and short-lived, suggestive of induction of suppressor mechanisms. Exogenous T cell growth factor (IL 2) that was added to antigen-stimulated T cell cultures enhanced proliferation in antigen-responsive cultures, indicating that the lymphocytes expressed IL 2 receptors. In contrast, the addition of IL 2 to cultures that did not respond to antigen had no effect. Antigen-responsive cultures contained endogenous IL 2 as well, and the antigen-induced lymphocyte proliferation was correlated with IL 2 production. However, the results suggested that IL 2 production by the patients' T cells was insufficient or actively shut off, and that this was responsible for the premature cessation of their DNA synthesis. Supernatants from 60% of the T cell cultures treated with malaria antigen and from 30% treated with RBC ghost antigen contained interferon-gamma (IFN-gamma), as determined by a cytopathic effect inhibition assay combined with acid treatment and antibody neutralization or by an IFN-gamma-specific ELISA. There was no obvious correlation between antigen-induced lymphocyte proliferation and the presence of IFN-gamma in the culture supernatants. A high IFN-gamma activity was also seen in antigen-treated cultures from P. falciparum-immune donors living in highly endemic malaria areas. In contrast, no IFN-gamma was found in supernatants of antigen-treated T cells from healthy donors or patients with Plasmodium vivax malaria. Thus, the IFN-gamma activity of these cultures appears to reflect the presence of antigen-reactive T cells and may be useful as a sensitive indicator of cellular immunity in P. falciparum malaria.     

Analysis of the negative allogeneic effect using B cells and helper T cell lines as an indicator system: B cells identified as target cells for suppression

The target cells for H-2b T lymphocytes mediating a negative allogeneic effect in vitro were analyzed by using carrier-specific helper T cell lines of H-2b, H-2d, or F1 origin and hapten-primed T-depleted spleen cells also expressing one or both of these haplotypes. The helper T cell lines were shown to be carrier specific and H-2b or H-2d restricted. Most importantly, the lines derived from H-2b homozygous mice were devoid of alloreactivity against H-2d and vice versa. Titration of naive H-2b T lymphocytes to the indicator cultures resulted in suppression of the secondary anti-DNP response of the indicator cells whenever the B cells expressed H-2d antigens. The lack of suppression observed in mixtures in which only the helper T cell lines expressed H-2d antigens was not reversed by the increased addition of naive H-2bxd cells, indicating that an insufficient amount of H-2d antigens present on the low number of helper T cells used did not account for this finding. Moreover, the polyclonal plaque-forming cell responses of F1 spleen cells to LPS were also suppressed by naive parental T cells. From these findings it is concluded that the suppressor T cells directly recognize and inhibit allogeneic B cells without the involvement of helper T cells. In addition, it was shown that the suppression of secondary anti-hapten responses by naive allogeneic T cells is blocked by monoclonal anti-Lyt-2 antibody added at the onset of culture. Addition late in culture had no effect, pointing to a functional role of the Lyt-2-bearing structure at an early stage of the suppressive events resulting in the negative allogeneic effect.     

Distinction of B cell maturation factors from lymphokines affecting B cell growth and viability

Supernatants from S26.5 helper T cells, autoimmune viable motheaten (mev/mev) mouse spleen cells, EL4 lymphoma cells, and recombinant DNA-derived interferon gamma (IFN-gamma), all of which display B cell maturation factor (BMF) activity, were assayed for effects on B cell proliferation alone and with Dextran Sulfate (DxS) and anti-immunoglobulin antibodies (alpha-Ig). Both EL4 and S26.5 supernatants showed BCGF-II (DxS co-stimulator) activity, whereas only EL4 supernatant had BCGF-I (alpha-Ig co-stimulator or BSF-I) activity. Supernatants from mev/mev spleen cells and recombinant DNA-derived IFN-gamma showed no activity in either assay. Fractionation of S26.5 supernatant by chromatofocusing showed a divergence of BMF activity (BMF-T, pIa of 6.0) from BCGF-II activity (pIa of 5.4), providing evidence for their physical nonidentity. IFN-gamma, which decreases B cell viability in culture, was separable from BMF-T by phenyl-Sepharose chromatography. BMF-T from S26.5 supernatant was separated from IFN-gamma and BCGF-II and was shown to induce B cell maturation without affecting B cell proliferation. The molecular characteristics of the purified BMF-T were pIa 6.0, Mr 55,000 by G-75 gel filtration, and Mr 16,000 by SDS-PAGE. These data demonstrate that several lymphokines (BMF) exist that mediate the maturation of B cells to active Ig secretion without stimulating B cell proliferation.