Cloned T cells with "co-helper" activity. I. Biologic activities of the clone

Clones of sheep erythrocyte-(SRBC) specific helper T cells with the surface phenotype Thy-1+, Ly-1+, Ly-2- have been derived that grow in vitro in the absence of exogenous antigen or added growth factors. The IL 2-independent clone, 101.6 has been shown to produce a supernatant factor that augments the primary anti-SRBC but not anti-burro RBC responses of whole spleen cells or Ly-1 T plus B cell cultures. The supernatant does not help B cells directly. This augmenting activity is terminated "co-helper" because the enhancement requires the presence of normal Ly-1 T cells. The supernatant of 101.6 was not shown to contain IL 2; co-helper activity was distinguishable from IL 2 activity by absorption with SRBC but not with Con A blasts, and we observed that co-helper activity does not act on spleen cells that differ at the major histocompatibility complex.     

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.     

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.     

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.     

Origin of autoreactive T helper cells. I. Characterization of Thy-1+, Lyt-, L3T4- precursors in the spleen of normal mice

A new population of dull Thy-1+, Ly-1-, Lyt-2-, L3T4- PNA- cells, resistant to a double cytotoxic treatment by monoclonal antibodies to these T cell markers plus complement, has been isolated from the spleen of normal adult BALB/c and DBA/2 mice (Tkr cells). These cells exhibit no spontaneous autoreactivity or alloreactivity but can be activated with concanavalin A (Con A). Once activated, they differentiate into bright Thy-1+, Ly-1+, Lyt-2-, L3T4+ PNA- T lymphocytes. Con A-activated Tkr cells also strongly proliferate in the presence of allogeneic or syngeneic dendritic cells in secondary cultures. Moreover, contrary to other Con A-stimulated T cell populations, they induce B lymphocytes to proliferate and to differentiate into Ig-secreting cells at a very high level. Con A-activated Tkr cells are therefore very potent polyclonal B cell activators. Restimulated of Tkr cells by syngeneic dendritic cells can be inhibited by anti-L3T4 or anti-class II monoclonal antibodies. The results suggest that Tkr cells are the precursors of class II-specific autoreactive T helper cells. Tkr cells are absent in the spleen of B6 animals. This indicates that their expression might be genetically controlled. It also suggests that Tkr cells may not be the unique splenic precursors of autoreactive T cells. Con A activation of Tkr cells in Click's medium is 2-mercaptoethanol dependent and highly sensitive to pCO2, like the response of thymocytes. Tkr cells are also absent in the spleen of nude mice. We conclude that Tkr cells represent splenic precursors of autoreactive T helper cells equivalent to Thy-1+, Ly-2-, L3T4- PNA- cortical thymocytes.     

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.     

Class-specific regulation of anti-DNA antibody synthesis and the age-associated changes in (NZB x NZW)F1 hybrid mice

Investigations of regulatory helper and suppressor T cells in the in vitro anti-DNA antibody synthesis in NZB x NZW (B/W) F1 hybrid mice were initiated by the development of an in vitro system in which G10-passed B cells from B/W F1 mice were cocultured with mitomycin C-treated T cells in the presence of Con A and either in the presence or in the absence of LPS. It was revealed that each IgG and IgM anti-DNA antibody synthesis was under the regulation of separate L3T4+ helper and Ly-2+ suppressor T cells. The function of these class-specific regulatory T cells was age-dependent. Although the helper effect of L3T4+ T cells on IgG antibody synthesis increased, the effect of L3T4+ T cells on IgM antibody production decreased in B/W F1 mice with aging. The IgG anti-DNA antibody production in the cocultures of L3T4+ T cells and B cells was suppressed by addition of Ly-2+ T cells from young but not aged B/W F1 mice, whereas the production of IgM anti-DNA antibodies was suppressed by Ly-2+ T cells from aged but not young B/W F1 mice. We also found that although IgM anti-DNA antibody-producing B cells were already present in 2-mo-old mice, B cells producing IgG antibodies under the influence of L3T4+ T cells appeared in mice at 7 mo of age. These data clearly indicate that separate class-specific regulatory T cells are involved in the production of IgM and IgG anti-DNA antibodies and that the total serum level of the antibodies is reflected by both their age-associated changes and the generation of antibody-forming B cells in B/W F1 mice.     

Role of Ly-1:Qa1- and Ly-1:Qa1+ inducer T cells in activation of Ly-23 effectors of suppression of antibody production in mice

Ly-2+ effectors of T cell-mediated suppression require inducing signals from antigen and a helper cell bearing the Ly-1+:Qa1+ surface phenotype. In this report, we have further examined the helper cell requirements for suppressor cell induction of antibody production in mice. By using the T cell subset education procedure in vitro, we have activated T cells to sheep red blood cells (SRBC) antigens and then purified Ly-2 cells before testing for suppressor activity in assay cultures of defined T and B cell subsets. We have confirmed our previous observations that Ly-1+:Qa1+ cells are required for activation of T suppressors, but have found that under the appropriate conditions, there is not a strict requirement for the Ly-123 subset of T cells. Furthermore, if Ly-23 cells are stimulated in the presence of Ly-1+:Qa1- T cells, effective suppressors can be obtained only if a source of Ly-1:Qa1+ inducers is added to the assay culture. If Ly-23 cells are activated by antigen in the absence of Ly-1 cells, subsequent exposure to the Ly-1+:Qa1+ subset under the conditions tested here is not sufficient to activate suppressors. These results show that effectors of suppression, like B cells and cytotoxic T lymphocytes, may respond to two helper cells.     

Thy-1+ epidermal cells proliferate in response to concanavalin A and interleukin 2

Mouse epidermal cells (EC) are composed of at least two phenotypically discrete populations of cells that in epidermal sheets have a dendritic morphology: Ia+ Langerhans cells (LC) and dendritic, bone marrow-derived, Ia- cells that express Thy-1 antigen (Thy-1+ dEC). Thy-1+ dEC lack other typical T cell markers such as L3T4, Lyt-1, and Lyt-2; however they do express Ly-5 and asialo GM1 in common with NK cells and certain other leukocytes. To investigate the functional capabilities of Thy-1+ dEC in vitro, cell suspensions prepared from trypsin-disaggregated sheets of mouse body wall epidermis were first enriched to 8 to 20% Ia+ and 20 to 40% Thy-1+ cells by centrifugation over Isolymph and then were cultured for 2 to 10 days with Concanavalin A (Con A) and/or partially purified rat IL 2. Con A-induced proliferation of EC was readily seen, with the maximal response occurring at a Con A concentration of 2.5 micrograms/ml on day 5 of culture. Con A responses were significantly enhanced by the continuous presence of 1 microgram/ml indomethacin. Responses both in the presence and absence of Con A were significantly enhanced by the addition of 5 to 10 U/ml of partially purified rat IL 2; proliferation in cultures stimulated by both Con A and IL 2 continued to increase throughout the 10-day culture period. Culture of fluorescence-activated cell sorter (FACS)-separated EC suspensions revealed that Thy-1-depleted EC and irradiated Thy-1+ EC failed to proliferate in response to Con A and IL 2, whereas unirradiated purified Thy-1+ EC gave enhanced Con A- and IL 2-induced responses compared with the unseparated population. Finally, to distinguish between the proliferation of small numbers of mature peripheral T cells and that of Thy-1+ dEC, antibody and complement-depletion studies were conducted with an unusual monoclonal anti-Thy-1 reagent, 20-10-5S, and with the anti-T cell reagents, anti-L3T4 and anti-Lyt-2. Thy-1+ dEC, but not LC, express the 20-10-5S determinant; furthermore, in CBA (Thy-1.2) mice 20-10-5S reacts with Thy-1+ dEC, thymocytes, and peripheral T cells, whereas in AKR/J (Thy-1.1) mice, it reacts only with Thy-1+ dEC and thymocytes and not with peripheral T cells. Pretreatment of AKR/J EC with 20-10-5S and complement abolished the capacity of such cells to respond to Con A and to IL 2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phenotypic, functional, and molecular genetic comparisons of the abnormal lymphoid cells of C3H-lpr/lpr and C3H-gld/gld mice

Lymph node cells from C3H mice homozygous for lpr and gld were compared for expression of cell surface antigens, lectin-binding sites, functional characteristics, expression of ecotropic MuLV, and organization of Ig and T cell receptor (TcR) beta-chain genes. The abnormal cells (Ly-2-/L3T4-) populating nodes of both mutant strains were specifically purified by using plate separation techniques. The purified abnormal cells were shown to express the beta-chain of the TcR, to exhibit rearrangements of the beta-chain genes, and to express TcR beta and alpha gene mRNA, demonstrating the T cell origin of these populations. FMF analyses of the separated abnormal cells showed them to be Thy-1+, Ly-1+, Ly-2-, L3T4-, Ly-5(B220)+, Ly-6+, Ly-22+, Ly-24+, sIg-, ThB-, Ia-, HSA-/+, and PC.1+ and to bind at high levels lectins that normally bind preferentially to B cells. These cells did not proliferate or generate CTL in response to stimulation with alloantigens, and supernatants of cells stimulated with Con A were devoid of IL 2. These characteristics do not correspond to those of any known immature or mature population of normal T cells. The findings that the abnormal T cells of lpr and gld homozygotes are indistinguishable for each parameter examined support the suggestion that these mutations may affect different enzymes in a common metabolic pathway of major importance to T cell differentiation and function.     

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

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

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.     

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.     

Differentiation of thymocytes in fetal organ culture: analysis of phenotypic changes accompanying the appearance of cytolytic and interleukin 2-producing cells

At the 14th day of gestation, embryonic thymocytes+ are large, functionally incompetent cells with H-2K+ Thy-1+ B14- Ly-2- L3T4- phenotype, some of which express TL antigen. Differentiation of these cells in organ culture is characterized by: 1) appearance of cells expressing Ly-2 and L3T4 molecules, first among the population of large cells, after 2 days of culture; 2) appearance of small H-2K- Thy-1+TL+B14+Ly-2+L3T4+ and H-2K-Thy-1+TL+B14-Ly-2+ L3T4+ cells between days 2 and 4; 3) accumulation of small H-2K- Thy-1+ TL+ B14- Ly-2+ L3T4+ (but not H-2K- Thy-1+ TL+ B14+ Ly-2+ L3T4+) cells until day 5 of culture, and their subsequent gradual disappearance which is paralleled by an increase of the proportion of medium-sized H-2K+ Thy-1+ TL- B14- cells with various Ly-2 L3T4 phenotypes; 4) appearance and subsequent accumulation of cytolytic and IL 2-producing cells between days 4 and 6. Comparison of these results with the data from similar in vivo studies shows that differentiation of organ-cultured thymocytes rather closely follows the in vivo development only during the first week of culture and shows significant deviations thereafter. Precursors of cytolytic cells and cytolytic effector cells as well as IL 2-producing cells are found among both Ly-2+ and Ly-2- populations of thymocytes, indicating that there is no clear association between Ly-2 phenotype and the ability to kill or to secrete IL 2.     

Dexamethasone stimulates thyrotropin-releasing hormone production in a C cell line

The hypothalamic peptide hormone TRH is also found in other tissues, including the thyroid. While TRH may be regulated by T3 in the hypothalamus, other regulators of TRH have not been identified and the regulation of TRH in nonhypothalamic tissues is unknown. We recently demonstrated the biosynthesis of TRH in the CA77 neoplastic thyroidal C cell line. We studied the regulation of TRH by dexamethasone in this cell line because glucocorticoids have been postulated to inhibit TSH secretion by decreasing TRH in the hypothalamus. Furthermore, TRH in the thyroid inhibits thyroid hormone release. Thus by regulating thyroidal TRH, glucocorticoids could also directly affect thyroid hormone secretion. Treatment of CA77 cells for 4 days with dexamethasone produced dose-dependent increases in both TRH mRNA and cellular and secreted TRH. Increases in TRH mRNA and peptide levels could be seen with 10(-9) M dexamethasone. A 4.8-fold increase in TRH mRNA and a 4-fold increase in secreted peptide were seen with 10(-7) M dexamethasone. Dexamethasone treatment did not increase beta-actin mRNA levels or cell growth. These results suggest that glucocorticoids may be physiological regulators of TRH in normal C cells. In addition to their inhibitory effects on TSH, glucocorticoids may decrease thyroid hormone levels by increasing thyroidal TRH. Since the glucocorticoid effects on C cell TRH are the converse of what is expected for hypothalamic TRH, glucocorticoid effects in these two tissues may be mediated by different regulators.     

Interleukin 2 inhibits antigen-stimulated lymphokine synthesis in helper T cells by inhibiting calcium-dependent signalling

Certain L3T4+, Lyt-2- cloned murine helper T lymphocytes (HTL), when cultured with a high concentration of interleukin 2 (IL 2), become temporarily unresponsive to antigenic stimulation, as indicated by failure to proliferate and by reduced secretion of lymphokines when challenged with antigen. Exposure of cloned HTL to IL 2 also renders these cells less responsive to concanavalin A (Con A). Here we demonstrate that antigen-unresponsive HTL also accumulate reduced levels of lymphokine mRNA, thus indicating a pretranslational block of the response to antigen. However, HTL which had been pretreated with IL 2 and were unresponsive to antigen responded strongly to antigen + A23187 or to A23187 + PMA but failed to respond to antigen + PMA. With HTL made unresponsive to antigen or to Con A by exposure to IL 2, increases in intracellular calcium ion levels stimulated by Con A also were reduced. Thus, for mouse HTL clones, the IL 2-induced state of unresponsiveness to antigen or Con A appears to reflect an inability of such HTL to increase intracellular free calcium to a level sufficient for activation of lymphokine genes.     

Modulation of T-cell functions: I. Effect of 2-mercaptoethanol and macrophages on T-cell proliferation

The in vitro effects of 2-mercaptoethanol (2-ME), macrophages (MØ), and concanavalin A (Con A) on the proliferation of normal spleen cells (NSC), MØ-depleted spleen cells (DSC), T cells, T-cell subpopulations, and B cells were assessed by [³H]thymidine incorporation. 2-ME alone was consistently shown not to be mitogenic for purified T cells; however, 2-ME enhanced the early (Days 1 and 2) Con A (2 μg/ml)-induced response of NSC, DSC, and T-cell preparations, but depressed the late response (Days 4 and 5). 2-ME alone was mitogenic for purified B-cells, as reported previously; and the 2-ME-induced B-cell response was inhibited by Con A. Preincubation of T cells with 2-ME was sufficient for enhanced Con A responsiveness; however, if 2-ME was added 24 hr after the initiation of culture, no alteration of the Con A-induced response was observed. Ly-2,3⁺ T cells were unresponsive to Con A (0.3–20 μg/ml), but the addition of 2-ME or peritoneal cells enhanced the Con A responsiveness of Ly-2,3⁺ T cells over 200-fold. Ly-1⁺ T cells responded with a similar doseresponse and kinetic profile as unselected T cells. Although Ly-1⁺ T cells responded to Con A, unlike Ly-2, 3⁺ T cells, extensive removal of MØ significantly reduced the Con A-induced responsiveness of the Ly-1⁺ T cells. The reactivities of Ly-1⁺ and Ly-2,3⁺ DSC could be reconstituted by the addition of MØ or 2-ME; however, the kinetic response of Ly-1⁺ T cells peaked on Day 2–3, and Ly-2,3⁺ T cells had a delayed response which peaked on Day 4–5. The results indicated that (i) 2-ME and/or MØ accelerate the response kinetics of T-cells to Con A; (ii) T-cell subpopulations have differential requirements for MØ and/or 2-ME in the response to Con A; (iii) T-cell subpopulations exhibit differential dose responsiveness to Con A; and (iiii) 2-ME alters Con A responsiveness by a direct effect on T cells.     

Interleukin 2-mediated immune interferon (IFN-gamma) production by human T cells and T cell subsets

Human interleukin 2 (IL 2, or T cell growth factor), which was free of lectin and interferon activity (IFN), induced human peripheral T lymphocytes to produce immune IFN (IFN-gamma). In contrast, non-T cells and macrophages did not produce IFN-gamma in response to IL 2. IL 2 acted directly on unstimulated T cells to induce IFN-gamma production, and also acted in synergy with a suboptimal dose (2 micrograms/ml) of concanavalin A (Con A) to enhance IFN-gamma production. The IFN-gamma-inducing activity of partially purified IL 2 was absorbed along with the IL 2 activity by murine IL 2-dependent CT-6 cell line cells. This further supports the view that IFN-gamma-inducing activity is identical to IL 2. When T cells were separated further into helper/inducer T4+ and suppressor/cytotoxic T8+ subsets by negative selection with monoclonal antibody and complement, both T4+ and T8+-enriched cells produced significant levels of IFN-gamma in response to IL 2. Complete removal of macrophages from purified T lymphocyte populations by treatment of OKM1 plus complement consistently reduced IFN-gamma production in response to IL 2 to a limited degree; readdition of macrophages restored IFN-gamma production by both T cell subsets. This observation that IL 2 contributes to the production of IFN-gamma by human lymphocytes suggests that a cascade of lymphocyte-cell interactions participates in human immune responses.     

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).