Lymphokine-mediated induction of cytolytic activity in a T cell hybridoma

Functionally inducible CTL hybridomas were constructed by fusing alloantigen-specific T cells (C57BL/6 alpha-DBA/2) with cells from the rat thymoma line W/FU (C58NT)D. A cloned hybridoma line (KSH.4.13.6) that was specifically cytolytic in the presence of activated rat spleen cell supernatant fluid (rat Con A SN) lost activity when transferred to normal medium. However, a cytolytic activity could be reinduced by culturing KSH.4.13.6 cells in medium containing rat Con A SN or secondary mixed leukocyte culture SN. By using various sources of SN, it was found that cytolytic induction required two different factors. PMA-induced EL-4 SN and SN from antigen-activated cloned T cells, neither of which were capable of inducing cytolytic activity alone, were able to synergize in the cytolytic induction of KSH.4.13.6 IFN-gamma and IL 1 failed to induce cytolytic activity even in the presence of EL-4 SN. Furthermore, this hybridoma produced macrophage activating factor (MAF) upon culture in rat Con A SN, although MAF production could not be induced by either specific antigen or lectins. The kinetics of induction and loss of cytolytic activity mediated by rat Con A SN were similar to those of the induction of MAF production. However, EL-4 SN, which by itself was incapable of inducing cytolytic activity, was able to induce MAF production in the KSH.4.13.6 hybrid to an extent similar to that induced by rat Con A SN. These results suggest that the induction of cytolytic activity and of MAF production in this cloned hybridoma cell line are regulated by different mechanisms. Such a functionally inducible T cell hybrid may provide a tool for biochemical and molecular analysis of T cell function and regulation, and of the characterization of cytokines required for CTL differentiation.     

Three different signals are required for the induction of cytolytic T lymphocytes from resting precursors

The requirement for the signals in induction of cytolytic T lymphocytes (CTL) has been investigated. C57BL/6 X CBA/T6 F1 spleen cells stimulated with the lectin leukoagglutinin (L-A) failed to show CTL activity in a PHA-facilitated assay, although L-A-activated splenic T cells were able to respond to T cell growth factor (TCGF). Concanavalin A (Con A) on the other hand was able to induce cytolytic activity from CTL-P, as well as to render splenic T cells responsive to TCGF. Furthermore, L-A-activated splenic T cells could generate cytolytic activity upon subsequent culture in secondary mixed leukocyte culture supernatant (2 degrees MLC SN). In contrast, EL-4-derived SN (EL-4 SN) was unable to induce cytolytic activity from L-A-activated spleen cells. In addition, proliferation of L-A-activated spleen cells cultured in EL-4 SN was similar to those cultured in 2 degrees MLC SN. Nonactivated spleen cells were totally unresponsive to both SN in proliferation and generation of CTL. Analysis of T cell clones for the production of a factor necessary for induction of cytolytic activity revealed that both cytolytic and noncytolytic T cell clones were able to produce a factor(s) for the generation of cytolytic activity from L-A-activated T cells. On the other hand, SN from certain antigen-stimulated T cell clones produced factors capable of inducing cytocytic activity by L-A-activated T cells only in the presence of EL-4 SN. Neither EL-4 SN nor cloned T cell SN alone had such a capacity. The nature of the necessary lymphokines in the SN from the clone cells or from the EL-4 is unknown. In the case of the EL-4 SN, it is not known whether the presence of TCGF plays a role or whether that role is perhaps more differentiative than proliferative. This study provides evidence that the induction of CTL from CTL-P can be dissociated into activation, which is required to render T cells responsive to second signals, and differentiation, which is mediated by two different factors.     

Generation of cytotoxic T lymphocytes from thymocyte precursors to trinitrophenyl-modified self antigens. I. Requirement of both killer-helper factor(s) and interleukin 2 for CTL generation from a subpopulation of thymocytes

The requirement for signals in the induction of cytotoxic T lymphocytes (CTL) from thymocyte precursors has been investigated. Either unfractionated or peanut agglutinin-binding (PNA+) C3H/He thymocytes were stimulated with mitomycin C(MMC)-treated, 2,4,6-trinitrophenyl(TNP)-modified syngeneic spleen cells in the presence of a variety of lymphokine preparations. Cellfree supernatant (CFS) from purified protein derivatives(PPD-CFS) stimulated Mycobacterium tuberculosis (Tbc)-primed cells, or partially purified interleukin 2 (IL 2) mediated strong cytotoxic responses in unfractionated thymocytes, whereas only PPD-CFS at final concentrations beyond 30% was active for CTL generation in PNA+ thymocytes. Neither IL 2 at concentrations of below 60 U/ml nor a low concentration of PPD-CFS (at final below 10%) had such a capacity. The addition of monoclonal anti-IL 2 receptor antibody completely blocked CTL generation induced by PPD-CFS in PNA+ thymocytes. In contrast, anti-immune interferon (IFN-gamma) antibody showed a marginal effect. PPD-CFS (10%) and IL 2 (10 U/ml) could synergistically trigger PNA+ thymocytes to induce CTL generation. These results suggested that both IL 2 and "helper" factors other than IL 2 are required for CTL generation from PNA+ thymocytes. We refer to these kinds of helper factors as killer helper factors (KHF). Partially purified IL 2-free KHF show two peaks of activities at apparent m.w. 14,000 to 34,000 and 44,000 to 90,000, and are heterogeneous with respect to isoelectric point, which is between 4.5 and 5.1. Cultures that received TNP-modified syngeneic cells and KHF on day 0 and IL 2 on day 2 generated potent CTL responses, whereas the addition of IL 2 on day 0 followed by the addition of KHF on day 2 to the culture was ineffective, suggesting that KHF is required in the early phase of the culture to achieve optimal CTL responses.     

B cell stimulatory factor-2 is involved in the differentiation of cytotoxic T lymphocytes

The induction of cytotoxic T lymphocytes (CTL) from precursor T cells requires both antigen and lymphokine signals. Previous work from our laboratory has indicated that three lymphokines are required for the induction of CTL from murine thymocytes; interleukin 2, interferon-gamma (IFN-gamma), and a partially characterized factor referred to as cytotoxic differentiation factor (CDF). While attempting to clone CDF from the human T cell line C10-MJ2, we found that a gene encoding CDF-like activity is identical to the gene encoding the factor known variously as B cell stimulatory factor-2 (BSF-2), IFN-beta 2, and 26-kDa protein. We report here that BSF-2 can induce the differentiation of Ly-2+ CTL from murine thymocytes in the presence of interleukin 2 and that the level of cytotoxicity is augmented by the addition of murine IFN-gamma. Serine esterase, a marker for cytotoxic granules in CTL, was induced only in the presence of BSF-2, and the level of serine esterase activity correlated with the level of serine esterase activity correlated with the level of cytotoxicity. These data suggest that BSF-2 is a differentiation factor for CTL and that it functions in part by inducing proteins required for mediating target cell lysis.     

Bioassays for the detection of a cytokine that enhances the development of cytolytic T lymphocytes

Interleukin 2 (IL-2) is an important growth factor for cytolytic T lymphocytes (CTL). Other factors here termed cytolytic differentiation factor(s) (CDF) may be required for CTL responses, but it has been difficult to identify suitable bioassays. We report a polyclonal assay in which lytic activity develops after 2 days of culture with lectin and lymphokines. CDF is required for the development of Thy-1+, CD8+, CD4- CTL in this assay. The responsive T cells are thymocytes from certain strains of mice (A, Swiss NCS, B6.H-2k) or peripheral T cells cultured in the presence of high doses of hydrocortisone acetate. If these precautions are taken, little or no lytic activity develops in the presence of rIL-2 alone. CDF is present in the media of mitogen-stimulated mouse spleen or human blood mononuclear cells, but not in the conditioned media of a number of mouse and human cell lines. It is immunologically distinct from IL-2, but only acts in concert with IL-2 to induce CTL. A large panel of available mouse and human cytokines do not synergize with Il-2 in the bioassay. These include interferons, colony-stimulating factors, lymphotoxin/tumor necrosis factor, and IL-1, -3, and -4. Therefore a distinct CDF(s) seems essential for the induction of at least some CTL. The assays described here should be useful in purifying the molecule responsible for this biological activity.     

Interleukin 2-mediated induction of lytic activity in a cloned murine CTL line

We have analyzed the ability of interleukin 2 (IL 2) to induce lytic activity within a cloned murine H-2Dd-specific CTL line. Weakly lytic CTL harvested 6 to 7 days after previous stimulation with irradiated DBA/2J spleen cells and conditioned medium from secondary MLC (MLC SN) could be reactivated to high antigen-specific lytic activity with highly purified gibbon IL 2 or E. coli-produced human recombinant DNA IL 2. Dose-response curves with IL 2 and MLC SN suggest that IL 2 may be the principal detectable activity in MLC SN that is active on these CTL. Doses of IL 2 or MLC SN that were saturating for the induction of lytic activity were suboptimal for the expression of DNA synthesis measured by 3HTdR incorporation. This is consistent with a mechanism in which different threshold IL 2 concentrations are required to induce these two biologic responses. Finally, we show that IFN-gamma has little effect on the expression of lytic activity either alone or in combination with IL 2 in this bioassay.     

Requirement for delivery of signals by physical interaction and soluble factors from accessory cells in the induction of receptor-mediated T cell proliferation. Effectiveness of IFN-gamma modulation of accessory cells for physical interaction with T cells

We analyzed the mechanism by which accessory cells support the induction of the proliferation of human peripheral blood T cells by a monoclonal anti-CD3 antibody, OKT3. Cross-linking of T cell receptor/CD3 complex by anti-CD3 coupled to latex beads and the addition of IL-1 are not enough to induce the IL-2 production and proliferation of T cells extensively depleted of accessory cells, while the addition of both the culture supernatant of macrophages or a monoblastic cell line, U937 cells, and the paraformaldehyde-fixed macrophages or U937 cells which had been precultured with interferon-gamma before fixation into the culture of the T cells with anti-CD3-latex did induce the T cell proliferation. Lack of the addition of either one of these did not induce the response. These results indicate that the signal(s) delivered by soluble factors released from the accessory cells and that delivered by the physical interaction between accessory cells and T cells are both required for the induction of IL 2 production and proliferation of T cells by anti-CD3-latex. Importantly, the macrophages or U937 cells had to be cultured with Con A-stimulated lymphocyte culture supernatant or IFN-gamma prior to fixation with paraformaldehyde, suggesting that a molecule(s) inducible on accessory cells surface by IFN-gamma or other lymphokine is necessary for the effective accessory cell-T cell interaction to induce the T cell response. It was further revealed that the activity of the culture supernatant of accessory cells may be mediated synergistically by IL 1 and a certain other factor(s) and was actually shown to be replaced by the combined addition of rIL-1 and rIL-6 but not by rIL-1 alone. The experimental system described here will be very useful for dissecting the accessory functions for T cell activation.     

Distinct signals are required for proliferation and lymphokine gene expression in murine T cell clones

Experiments were performed to assess the capacity of lectin (Con A), ionomycin, phorbol ester (PMA), and recombinant IL 2 to mediate proliferation as well as the expression of cell surface IL 2 receptors, two lymphokine genes, IL 2 and IFN-gamma, and the c-myc proto-oncogene in cloned T cell populations. Stimulation of T cell clones with recombinant IL 2 resulted in proliferation and sustained expression of the c-myc cellular proto-oncogene, but did not induce the expression of mRNA for the lymphokines IFN-gamma and IL 2. In contrast, stimulation of cloned T cells with lectin alone induced significant IFN-gamma and IL 2 mRNA expression, up-regulation of the number of cell surface IL 2 receptors, and transient c-myc expression. Ionomycin alone was not a sufficient signal for lymphokine mRNA induction. The phorbol ester PMA alone induced neither proliferation nor lymphokine gene expression but potentiated lectin and ionomycin-mediated signals. We also performed experiments to examine whether the T cell response to extracellular stimuli was a function of the activation state of the cell. Reexposure of 48-hr antigen-activated cloned cells to identical stimuli revealed several differences. Low but significant levels of IFN-gamma mRNA were now also reinduced in activated clones cells in response to IL 2 or PMA alone. Activated cells were refractory to reinduction of IL 2 mRNA by any stimulus, which may reflect a physiologic mechanism to limit clonal expansion after antigenic stimulation. This could be partially reversed by restimulation with lectin in the presence of cycloheximide, suggesting a role for a labile protein repressor in the down-regulation of IL 2 mRNA expression. PMA alone induced an IL 2-independent proliferative response. We demonstrate that distinct signals are required for lymphokine gene expression vs cellular proliferation in cloned T lymphocyte populations, and that the capacity of extracellular stimuli to reinduce expression of lymphokine genes or to mediate cell proliferation is altered by prior activation.     

Interleukin-6 is constitutively produced by human CTL clones and is required to maintain their cytolytic function

Maturation of cytolytic T lymphocytes from nonlytic precursors requires cytokines in addition to IL2. Interleukin-6 is the principal cytokine that cooperates with IL2 in the induction of CTL differentiation from murine and human thymocyte precursors. However, a cytotoxic differentiation factor (CDF) role of IL6 for mature T cells is challenged by data indicating that IL2 alone is sufficient for CTL generation. The aim of this study was to identify a model system in which IL6 acted as a CDF for human peripheral T cells. We noted that IL6 was endogenously produced by CTL clones in the course of their expansion with APC, lectin, and IL2. The majority of several hundred T-cell clones, both CD4+ and CD8+, produced IL6 in response to relatively high doses of IL2. Other experiments that compared the cytolytic function of CTL clones cultured in the presence of IL6 with that of the same clones cultured in the absence of IL6 demonstrated that IL6 contributes to the cytolytic ability of the majority of human CTL clones. Our data suggest that IL6 acts in an autocrine fashion to support CTL differentiation in human T-cell clones.     

Antigen-independent activation of memory cytotoxic T cells by interleukin 2

Culture supernatants from mitogen- or antigen-activated murine spleen cells are capable of causing reexpression of specific cytolytic activity from inactive memory cytotoxic T lymphocytes (CTL) in the absence of the original priming antigen. We have demonstrated that memory CTL from cytolytically inactive day 14 MLC cells are induced to reexpress high levels of specific cytotoxic activity after incubation with IL 2. Highly purified IL 2 was shown to induce levels of lytic activity comparable with that induced by supernatants from secondary mixed lymphocyte cultures (secondary MLC SN), suggesting that only IL 2 is necessary for the reactivation process. Moreover, only Lyt-2+ cells are necessary for reactivation inasmuch as inactive MLC cells depleted of Lyt-1+ cells by treatment with antibody and complement, followed by FACS selection of Lyt-2+ cells, were efficiently reactivated by IL 2. Because IL 2 is considered a proliferative signal, we examined whether proliferation was requisite for reactivation of memory CTL by IL 2. In the presence of cytosine arabinoside, which effectively inhibited proliferation, IL 2 was capable of reactivating memory CTL as efficiently as antigen, thus implying a differentiative role for IL 2 in secondary CTL activation. Reactivation of CTL by IL 2 and antigen appear to be functionally distinct events, because antigen but not IL 2 could trigger immune interferon release, although either IL 2 or antigen induced high levels of cytotoxicity. We propose that resting, memory CTL retain a heightened level of expression of IL 2 receptors as compared with naive CTL precursors, and thus are able to respond directly to exogenous IL 2. The consequences of this are proliferation and reexpression of specific killing activity, but this signal is not sufficient to induce immune interferon secretion. Rather, it appears that a signal via the antigen receptor is necessary for release of this lymphokine.     

Retrovirus-mediated immunosuppression. I. FeLV-UV and specific FeLV proteins alter T lymphocyte behavior by inducing hyporesponsiveness to lymphokines

Murine splenocytes were used to study the in vitro immunosuppressive effects of UV-inactivated feline leukemia virus (FeLV-UV). FeLV-UV blocks both alloantigen (DBA/2)-induced and Con A-induced proliferation of C57BL/6 splenocytes in a dose-dependent manner. Furthermore, C57BL/6 anti-DBA/2 mixed lymphocyte cultures containing FeLV-UV fail to develop detectable DBA/2-specific cytolytic activity, although FeLV-UV has no effect on the cytolytic activity of preformed C57BL/6 anti-DBA/2 cytolytic T cells (CTL). Disruption of lymphocyte proliferation and CTL generation by FeLV-UV could not be overcome by the addition of exogenous lymphokines. These data suggest that FeLV-UV can interfere with the lymphokine reactivity of alloactivated lymphocytes. In fact, FeLV-UV blocks the lymphokine-induced proliferation of the murine IL 2-dependent cell line CTLL-20. The CTLL-20 cells were subsequently used to study the mechanism(s) by which retroviruses alter T lymphocyte function. Normally, CTLL-20 cells undergo significant proliferation when cultured in EL4 SN, an IL 2-containing culture supernatant from PMA-stimulated EL4 cells. This lymphokine-induced CTLL-20 proliferation is abrogated in a dose-dependent manner by UV-inactivated murine leukemia virus (MuLV-UV), FeLV-UV, and a purified 15,000 dalton viral protein, p15, derived from FeLV. Suppression of CTLL-20 proliferation requires only brief contact (6 hr) with FeLV-UV or with p15, but is most efficient after prolonged (24 hr) contact with these agents. Furthermore, suppression of CTLL-20 proliferation by FeLV-UV and p15 is reversible, because CTLL-20 cells which have been pretreated for 24 hr with FeLV-UV or p15 are equally as efficient at responding to EL4 SN as untreated CTLL-20. Additional studies indicate that CTLL-20 cells continue to remove IL 2 activity from EL4 SN in the presence of suppressive concentrations of FeLV-UV, and that suppressive concentrations of FeLV-UV do not remove IL 2 activity from EL4 SN. This suggests that FeLV does not block CTLL-20 proliferation by absorbing or inactivating IL 2, or by occluding IL 2 receptors, and that T lymphocytes develop an insensitivity to lymphokines after contact with FeLV-UV, which may be caused by a metabolic, rather than an immunologic, defect. Because lymphokines are requisite signals for T cell function, considerable immunosuppression would be associated with acquired lymphokine insensitivity.     

Cytolytic T lymphocyte clones that proliferate autonomously to specific alloantigenic stimulation. II. Relationship of the Lyt-2 molecular complex to cytolytic activity, proliferation, and lymphokine secretion

Previous analyses of the inhibitory effects of anti-Lyt-2 monoclonal antibodies (mAb) on cytolytic activity suggested that Lyt-2/3 antigens expressed on the surface of murine cytolytic T lymphocytes (CTL) are involved in antigen recognition. In the present study, we investigated the effects of anti-Lyt-2 mAb (in the absence of complement) on the functional activities of H-2K/D-specific Lyt-2+ CTL clones that proliferate to antigenic stimulation in the absence of helper T cells or added interleukin 2 (IL 2) and secrete lymphokines. For those clones that were inhibited in cytolysis by anti-Lyt-2 mAb, a parallel inhibition of antigen-dependent proliferation and lymphokine secretion (interferon, macrophage-activating factor) was observed. Inhibition of proliferation or lymphokine secretion could be overcome by the addition of IL 2 or lectin, respectively. Collectively, these results would strongly suggest that anti-Lyt-2 mAb were inhibiting CTL antigen recognition. Not all CTL clones, however, were inhibited in cytolysis by anti-Lyt-2 mAb, in which case proliferation and lymphokine secretion were similarly unaffected. This heterogeneity of Lyt-2+ CTL clones in their susceptibility to inhibition of cytolytic activity, proliferation, and lymphokine secretion by anti-Lyt-2 mAb is discussed in the context of a model proposing that Lyt-2/3 molecules function to stabilize the interaction between CTL receptors and the corresponding target/stimulating cell antigens. Such a stabilization may be required by CTL possessing few and/or low affinity receptors.     

IL-10: a novel cytotoxic T cell differentiation factor

A previous report concluded that a new cytokine, designated IL-10, is a growth cofactor for thymocytes, spleen, and lymph node cells. In this report, we have focused on the effects of IL-10 on CD8+ spleen T cells. We first observed that IL-10 enhances the growth of CD8+ T cells to IL-2. We then investigated the effect of murine rIL-10 on the induction of murine effector CTL from CTL precursors (CTL-p) using both bulk and filler cell-free limiting-dilution cultures. IL-10 alone could not induce Con A-activated FACS-sorted CD8+ T cells either to proliferate or to generate effector CTL. In combination with IL-2, however, IL-10 augmented the cytolytic activity of effector CTL generated from Con A-activated spleen CD8+ T cells in bulk cultures incubated for 5 days. In limiting-dilution cultures (using solid-phase anti-CD3 mAb as stimulus), IL-10, in combination with IL-2, substantially increased the CTL-p frequency and augmented the cytolytic activity per clone expanded from one CD8+ T cell when compared with cells cultured in IL-2 alone. Kinetic studies showed that IL-10 is required at both early and late culture stages for optimal generation of effector CTL. The potentiating effects of IL-10 on CTL function were neutralized by an anti-IL-10 mAb. These results indicate that IL-10 has direct effects on mature T cells, and suggest that IL-10 also functions as a cytotoxic T cell differentiation factor, which promotes a higher number of IL-2-activated CTL-p to proliferate and differentiate into effector CTL. In contrast, IL-10 did not enhance significantly the lymphokine-activated killer cell activity of IL-2-grown CD8+ cytotoxic T cells.     

Reversal of phorbol ester-mediated reduction of cloned T lymphocyte cytolysis by interleukin 2

During previous studies on the regulation of cloned T lymphocyte function, we observed that murine cytotoxic T lymphocyte (CTL) clones progressively lose the ability to lyse appropriate target cells during prolonged (24 to 48 hr) incubation with the tumor promoter phorbol myristic acetate (PMA). We further observed that the cytolytic function of PMA-treated CTL clones can be restored by incubation with secondary MLC supernatant (2 degrees MLC SN), a potent source of cytokines. We now report our observations on the nature of the cytokine(s) responsible for recovery of CTL activity. Like 2 degrees MLC SN, the lectin-induced SN from a cloned helper T cell and the lectin-induced SN from a T cell hybridoma can restore cytolytic activity to cloned CTL treated with PMA. In contrast, supernatants from L929 cells, WEHI-3 cells, and P388D1 cells fail to restore cytolytic activity to similarly treated cloned CTL. These data suggest that IL 2 and/or gamma-IFN, but not CSF-1, CSF-GM, IL 3, or IL 1, can influence expression of cytolysis by cloned CTL. Furthermore, highly purified IL 2 can restore cytolytic activity, even when cytosine arabinoside is present to inhibit clonal expansion. Our studies indicate that cytolysis is a reversible function of cloned CTL, and that cytolysis may not necessarily represent an end-stage feature of CTL maturation. Our studies further show that IL 2 is both necessary and sufficient for resumption of cytolytic function by "deactivated" CTL. As such, these observations suggest that IL 2 can regulate not only T cell proliferation but also the expression of cytolysis by some cytolytic T cell populations.     

Differential sensitivity of cytotoxic T lymphocytes and lymphokine-activated killer cells to inhibition by L-ornithine

The selective inhibition of murine cytotoxic T lymphocyte (CTL) differentiation in C57B1/6 (B6) anti-DBA/2 mixed leukocyte cultures (MLC) by the amino acid L-ornithine (Orn) could not be reversed by addition of up to 1000 U/ml IL-2. Analysis of the effects of Orn on induction of lymphokine-activated killer (LAK cells), using dosages of IL-2 from 10-1000 U/ml and measuring cytolytic activity against two tumor targets (P815 and YAC-1) over the course of 5 days, indicated that LAK cells were not suppressed by Orn. LAK precursors and effector cells were CD8- and ASGM1+, indicating that they were derived from natural killer (NK) cells. We also found that the growth and maintenance of cloned CTL lines were not sensitive to inhibition by Orn; nor was their acquisition of nonspecific cytolytic activity in the presence of high lymphokine concentrations. Thus, induction of naive CTL shows differential susceptibility to Orn inhibition relative to LAK and LAK-like activities by NK and cloned CTL lines in response to IL-2.     

IL-2 and IFN-gamma are two necessary lymphokines in the development of cytolytic T cells

A filler cell-free limiting-dilution microculture system has been developed for the expansion and differentiation of a high proportion of single CD4-CD8+ T cells into cytolytic T cell (CTL) clones. The stimulus used was PMA together with the calcium ionophore ionomycin. The growth and differentiation factors were rIL-2, together with either a Con A-stimulated spleen cell supernatant (CAS) or rIFN-gamma. CTL activity was monitored by an autoradiographic 111In-release assay. With CAS and rIL-2 present, 50% of all potential precursors (CTL-p) produced cytolytic clones. Substitution of rIFN-gamma for CAS gave a similar efficiency with up to 42% of CTL-p producing cytolytic clones. rIL-2 alone allowed only a small proportion (6%) of CD4-CD8+ T cells to become cytolytic clones. Addition of rIL-2 and rIFN-gamma at various stages of the culture demonstrated that IL-2 was required throughout, but exogenous IFN-gamma was required only during the early stages. It is concluded that for at least 40% of all CTL-p, the lymphokines IL-2 and IFN-gamma are essential and act in synergy to induce proliferation and differentiation into CTL.     

Interleukin 2 and concanavalin A stimulate interferon-gamma production in a murine cytolytic T cell clone by different pathways

We identified a variant murine cytolytic T lymphocyte (CTL) clone which, in contrast to the parent clone and all other murine T cell populations tested, was found to have acquired spontaneously the ability to produce interferon-gamma (IFN-gamma) in response to recombinant interleukin 2 (rIL-2). IFN-gamma production in response to concanavalin A (Con A), which was characteristic of all T cell populations tested, was preserved in this variant. The IFN produced by the variant in response to either stimulus was active in both a macrophage-activating factor assay and an anti-viral assay. Both activities induced by either stimulus could be blocked by monoclonal anti-IFN-gamma antibodies. Upon Northern blot analysis using an IFN-gamma-specific cDNA probe, the IFN-gamma RNA isolated from variant cells stimulated with Con A or IL-2 were found to migrate equivalently. The unusual pattern of responsiveness in this variant CTL was exploited to compare the mechanisms involved in induction of IFN-gamma production by Con A or IL-2. Striking differences were observed. Unlike IFN-gamma production induced by Con A, IFN-gamma production induced by IL-2 was not accompanied by an elevation of intracellular Ca2+ levels, did not require physiologic extracellular Ca2+ levels, and was not inhibited by the immunosuppressive agent cyclosporin A. Thus, in this variant CTL clone, conditions that have ordinarily been associated in an obligate manner with lymphokine gene expression were found instead to be related to the specific mode of stimulation.     

Monoclonal antibodies to interferon-gamma inhibit interleukin 2-dependent induction of growth and maturation in lectin/antigen-reactive cytolytic T lymphocyte precursors

In this study we tested the effect of monoclonal antibodies (moAb) AN-18 to murine IFN-gamma on the generation of cytolytic T cells (CTL) from a homogeneous population of precursor cells (CTL-P). As responder cells, highly purified Lyt-2+ C57BL/6 lymph node T cells were used that had been positively selected by flow cytofluorometry on a cell sorter. Lyt-2+ cells were set up in bulk culture or in limiting dilution (LD) either with Con A or with P815 tumor cells as antigen and recombinant human interleukin 2 (rec.hIL 2) in the presence or absence of moAb AN-18 and tested for growth and development of CTL. The results show that moAb AN-18 but not the unrelated moAb AN-37 diminished or abrogated proliferative and cytolytic responses of Lyt-2+ lymphocytes to lectin and rec.hIL 2 in a dose-dependent manner. The inhibitory activity of the antibodies could be abolished by neutralizing moAb AN-18 with recombinant murine IFN-gamma (rec.mIFN-gamma) before their addition to culture. Kinetic analysis shows that the inhibitory effect of moAb AN-18 is only optimal when added at the beginning of culture or up to 48 hr after initiation. The frequencies of CTL-P responding either to Con A or to P815 tumor cells and rec.hIL 2 were reduced up to 10-fold in the presence of moAb AN-18. The inhibitory capacity of moAb AN-18 was also operative in cultures containing on the average one antigen-specific CTL-P. Together with the finding that activated CTL-P secrete IFN-gamma in response to rec.hIL 2 in a dose-dependent manner, the data suggest that endogenous IFN-gamma collaborates with exogenous IL 2 in the induction of CTL-P. The generation of CTL may therefore represent a case of autocrine growth regulation of normal lymphocytes, in which the same cell synthesizes and responds to its own factor.     

Ontogeny of proliferative and cytotoxic responses to interleukin 2 and concanavalin A in murine fetal thymus

The ontogeny of proliferative and cytotoxic responses to concanavalin A (Con A) and interleukin 2 (IL 2) in C57BL/6J (B6) fetal thymus (FT) was investigated. Embryonic thymocytes were either taken from embryos at different times of gestation or from 14 day B6 FT that were maintained as organ cultures for various times. It was found that the B6 FT could proliferate to Con A and EL4 SN (an IL 2 containing culture supernatant) in a synergistic fashion. This synergy between Con A and EL4 SN was first observed at the 16th to 17th day of gestation. A similar differentiation process took place in 14-day FT that had been maintained as organ cultures; the synergy between Con A and EL4 SN was first observed after 3 days in organ culture. This synergy increased with increasing time of organ culture, and was most evident after 10 days. The synergy between Con A and EL4 SN was also observed when the EL4 SN was replaced with IL 2 which had been purified from crude EL4 SN to apparent homogeneity. B6 FT could also form cytotoxic T lymphocytes (CTL) on stimulation with Con A and EL4 SN. Con A-activated CTL (polyspecific) were detected by including phytohemagglutinin in the assay medium. CTL response was first detected in the 17-day fetal thymus by using this assay. In organ cultures, CTL responses were first detected after 4 days in organ culture, and reached peak levels after 12 to 14 days. The CTL precursor (CTL-P) frequencies in the B6 FT after 2, 5, 10, and 14 days in organ culture were less than 1/10,000, 1/2232, 1/297, and 1/70, respectively; the corresponding CTL-P frequency in adult thymus was 1/60. After 6 days in organ culture, B6 FT could also form CTL in response to Con A and pure IL 2. This finding suggests that the ability to synthesize other differentiation factors that are required for CTL responses is acquired at an early time of thymic differentiation.     

Induction of allogeneic cytolytic T lymphocytes by partially purified membrane glycoproteins

It has been previously shown that P815 (H-2^d) purified plasma membranes can induce cytolytic activity from primed C57BL/6 (H-2^b) spleen cells. The secondary cytolytic T lymphocyte (CTL) inducing activity is retained when these P815 plasma membranes are solubilized in deoxycholate. Evidence is now presented that the cell surface antigens responsible for CTL induction can be partially purified in active form and these antigens can be incorporated into reconstituted membranes and phospholipid vesicles. The active antigens have the properties expected for H-2 molecules on lentil lectin chromatography and gel filtration.