Generation of alloreactive cytotoxic T lymphocytes: production of T cell and macrophage helper factors in addition to IL 1 and IL 2 by peritoneal cells from mice immunized to Listeria monocytogenes

We investigate the production and biological activity of soluble helper factors produced by peritoneal T cells and macrophage derived from mice primed in vivo with Listeria monocytogenes. Supernatant fluids from co-cultures of these immune T cells and activated macrophages contained Interleukin 1 (IL 1) and Interleukin 2 (IL 2), and had the ability to assist the generation of cytotoxic T lymphocytes (CTL) from a population of nylon wool nonadherent spleen cells sensitized to allogeneic heat-treated thymocytes. The ability to assist CTL development involved T cell and macrophage factors in addition to IL 1 and IL 2. Immune T cells cultured alone produced a factor, devoid of significant IL 2 activity, that assisted CTL development only if adherent cells were present in the responding population. Activated macrophage produced a 38,000 dalton component, distinct from IL 1 on the basis of m.w., that assisted the development of CTL from nylon wool nonadherent splenic cells. Supernatants fluids from co-cultures of immune T cells and allogeneic, nonactivated macrophage contained a CTL helper factor but did not contain IL 1 or IL 2 activities. In contrast, supernatant fluids from co-cultures of immune T cells and syngeneic, nonactivated macrophage contained all 3 activities. This suggests a genetic restriction for the production of IL 1 and IL 2 that does not restrict the production of a CTL helper factor. These results demonstrate that T cell- and macrophage-derived helper factors distinct from IL 1 and IL 2 participate in the development of CTL.     

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.     

Regulation of the cytotoxic activity of alloreactive cytotoxic T lymphocytes by helper cells and lymphokines

The cytotoxic activity of alloreactive cytotoxic T lymphocytes (CTL) was maintained and augmented by transferring cells from a 5-day mixed lymphocyte culture MLC into a host culture (HC) containing indomethacin, freshly explanted normal spleen cells, and peritoneal cells which were syngeneic to the MLC cells. The MLC cells used in the transfer experiments were generated by culturing untreated H-2b splenic responders with irradiated H-2d stimulators, or were generated by culturing Lyt-2-depleted H-2b splenic responders with irradiated H-2d stimulators. The allo-CTL were found to be derived from the donor MLC (first culture) when unfractionated MLC cells were transferred into a host (second) culture and incubated for 5 days. In contrast, the allo-CTL were derived from host culture cells when Lyt-2-depleted MLC cells were transferred and the combined cultures incubated for 5 days. In the former case, the augmentation of MLC-derived cytotoxicity did not result from nonspecific expansion of all donor T cells; instead it was mediated by lymphokine(s), distinct from IL-2, produced by helper T cells generated in host culture, which appeared to selectively expand the antigen-specific CTL or to increase the cytotoxic activity of these CTL. The helper T cells were Thy-1+, L3T4+, and Lyt-2-. These findings indicate that antigen-nonspecific help was provided by helper cells or helper factors (lymphokines) generated in the host culture, which maintained and augmented the cytotoxic activity of the fully generated allo-CTL. This helper effect was also seen in the induction of primary allo-CTL responses which could be generated with fewer stimulating cells and with a stronger cytotoxic response at different R/S ratios tested. The generation of allo-CTL in second culture following transfer of Lyt-2-depleted MLC cells to host cultures appears to have involved antigen carryover from the MLC; however, antigen carryover alone was not sufficient. It appears that in the absence of Lyt-2+ suppressor T cells, antigen-specific help might be generated in donor cultures (Lyt-2-depleted MLC) which promoted or recruited the generation of antigen-specific CTL in host culture.     

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.     

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.     

Augmentation by serum-induced helper cells of T cell-mediated cytotoxic response against tumor associated antigens and alloantigens

Summary In vitro T cell-mediated cytotoxic responses to tumor associated antigens or alloantigens can be augmented by the addition of small amounts (0.1 to 1%) of syngeneic (mouse) or xenogeneic (rabbit) serum in the standard lymphocyte culture medium. Further studies showed that the augmentation is mediated by helper cells, which are induced by culturing the spleen cells or lymph node cells in the presence of these sera. In the syngeneic system performed with mixed lymphocyte tumor cell cultures (MLTC), the serum-induced helper cells are found to be resistant to the lysis of anti-Thy 1.2 antibody and are radioresistant; thus they have the characteristics of macrophages. In the allogeneic system performed with mixed lymphocyte culture (MLC), the serum-induced helper cells are also found to be resistant to the lysis of anti-Thy 1.2 antibody but are radiosensitive. In the latter case, however, removal of T cells abolishes the helper cell generation and only the T cell-enriched fraction provides for the generation of helper cells, indicating that the helper cells for MLC are probably derived from T cells but lose their susceptibility to anti-Thy 1.2 antibody lysis upon culturing in vitro. A study of the mode of action of the helper cells for MLC showed that they are probably needed at a later stage of cytotoxic response for the amplification of the killing efficiency of the T effector cells whereas the helper cells for MLTC are needed in the early induction phase of the immune response. These results indicate that although serum can augment the cytotoxic responses both in the syngeneic and in the allogeneic systems, the mechanism for the augmentation differs: macrophagelike helper cells are responsible for the augmentation of cytotoxic response to tumor associated antigens, whereas augmentation of cytotoxic response to alloantigens appears to be mediated by a subpopulation of T helper cells. Supported by a grant from the Japan Society for the Promotion of Science (T. I.).     

Macrophage T lymphocyte interactions in the anti-tumor immune response: a mathematical model

In this paper we present a model of the macrophage T lymphocyte interactions that generate an anti-tumor immune response. The model specifies i) induction of cytotoxic T lymphocytes, ii) antigen presentation by macrophages, which leads to iii) activation of helper T cells, and iv) production of lymphoid factors, which induce a) cytotoxic macrophages, b) T lymphocyte proliferation, and c) an inflammation reaction. Tumor escape mechanisms (suppression, antigenic heterogeneity) have been deliberately omitted from the model. This research combines hitherto unrelated or even contradictory data within the range of behavior of one model. In the model behavior, helper T cells play a crucial role: Tumors that differ minimally in antigenicity (i.e., helper reactivity) can differ markedly in rejectability. Immunization yields protection against tumor doses that would otherwise be lethal, because it increases the number of helper T cells. The magnitude of the cytotoxic effector cell response depends on the time at which helper T cells become activated: early helper activity steeply increases the magnitude of the immune response. The type of cytotoxic effector cells that eradicates the tumor depends on tumor antigenicity: lowly antigenic tumors are attacked mainly by macrophages, whereas large highly antigenic tumors can be eradicated by cytotoxic T lymphocytes only.     

Retrovirus-mediated immunosuppression. II. FeLV-UV alters in vitro murine T lymphocyte behavior by reversibly impairing lymphokine secretion

Murine splenocytes and cloned murine T cells were used to study the in vitro immunosuppressive effects of UV-inactivated feline leukemia virus (FeLV-UV) on lymphokine secretion. FeLV-UV can significantly depress the accumulation of IL 2 in cultures of Con A-stimulated C57BL/6 splenocytes and in cultures containing the alloreactive helper T cell clone B6D/2-2m plus Con A. Inhibition of lymphokine accumulation in these cultures could not be attributed to absorption or inactivation of IL 2 by the FeLV-UV or to the FeLV-UV-induced production of substances which interfere with the IL 2 bioassay. Thus, FeLV-UV appears to block production and/or secretion of IL 2 by a direct inhibitory effect on IL 2-secreting murine T lymphocytes. Additional studies indicate that FeLV-UV impairs IL 2 production only if added very soon after lymphocyte contact with lymphokine-inducing agents and that IL 2 secretion resumes when FeLV-UV is removed from the culture. FeLV-UV also impairs accumulation of MAF (interferon-gamma?) in cultures of Con A-stimulated C57BL/6 splenocytes and in cultures containing the alloreactive cytotoxic T lymphocyte clone B6D/2-7c plus Con A. The latter observation again suggests that FeLV-UV impairs lymphokine secretion by a direct effect on lymphokine-producing T lymphocytes. Furthermore, it suggests that FeLV-UV does not selectively impair production of IL 2 nor does it have selective inhibitory effects on helper T cells. Rather, FeLV-UV appears to have a general inhibitory effect on lymphokine production by T lymphocytes. Finally, concentrations of FeLV-UV which suppress MAF production by the CTL clone have little influence on the cytolysis mediated by the same cloned T cell population. Thus, the immunosuppressive influence of FeLV-UV is selective for phenomena associated with induction of new T lymphocyte functions, such as lymphokine secretion, and spares other immune functions already expressed by the same cells.     

Studies on cytotoxicity generated in human mixed lymphocyte culture

Summary High levels of cytotoxic activity against the natural killer (NK) cell-sensitive target K562 and the NK-resistant target UCLA-SO-M14 (M14) can be generated in vitro either by mixed lymphocyte culture (MLC) or by culture of lymphocytes in interleukin 2 (IL2) (lymphokine activated killer (LAK) cells). The purpose of this study was to identify similarities and differences between MLC-LAK and IL2-LAK cells and allospecific cytotoxic T cells. Induction of cytotoxicity against K562 and M14 in both culture systems was inhibited by antibodies specific either for IL2 or the Tac IL2 receptor. Like NK effector cells, the precursors for the MLC-LAK cells were low density large lymphocytes. However these precursors differed from the large granular lymphocytes that mediated NK cytolysis in sensitivity to the toxic lysosomotropic agent L-leucine methyl ester (LME). The resistance of the MLC-LAK precursors to LME indicated that the precursors included large agranular lymphocytes. Although interferon-gamma (IFN-gamma) is produced in MLC and in IL2 containing cultures, it is not required for induction of either type of cytotoxic activity. Neutralization of IFN-gamma in MLC-and IL2-containing cultures with specific antibodies had no effect on the induction of cytotoxic activities. Both allospecific cytotoxic T lymphocyte (CTL) and LAK activities were enhanced by IL2 and IFN-gamma at the effector cell stage. However, the mechanism of cytolysis was different in the two systems. NK- and MLC-induced LAK activities were independent of CD3-T cell receptor complex while CTL activity was blocked by monoclonal antibodies specific for the CD3 antigen. These results suggest that NK and the in vitro induced LAK cytotoxicities are a family of related functions that differ from CTL. Furthermore, MLC-induced and IL2-induced cytotoxicities against K562 and M14 appear to be identical.This work was supported by NIH grant CA34442     

Interleukin 2 regulates immune interferon (IFN gamma) production by normal and suppressor cell cultures

A Lyt-2+ lymphocyte is responsible for immune interferon (IFN gamma) production in mitogen-stimulated mouse spleen cell cultures. A Lyt-1+ helper cell is required for the induction of IFN gamma. Interleukin 2 (IL 2) can specifically replace the Lyt 1 cell requirement. IL 2 will also abrogate suppressor cell inhibition of IFN gamma production. IFN gamma production appears to be regulated by a dynamic interaction between helper cells (source of IL 2), suppressor cells (absorption of IL 2?) and IFN gammna-producer cells.     

Production of T cell differentiation factor in syngeneic lymphocyte macrophage culture for cytotoxic T lymphocyte generation

This study demonstrated that T cell differentiation factor (TCDF) was produced in syngeneic lymphocyte-macrophage cultures. Conditioned medium containing TCDF and interleukin 2 (IL 2) induced the differentiation of leukoagglutinin (LA)-activated cytotoxic T lymphocyte precursors (CTLp) into cytotoxic T lymphocyte (CTL) effectors. The production of TCDF and IL 2 peaked at day 4 to 5 in cultures containing normal spleen cells, syngeneic peritoneal macrophages, and indomethacin. Macrophages and T cells with Thy-1+, L3T4+, and Lyt-2- phenotype were needed for TCDF production. There was no requirement for xenogeneic serum in the culture medium; thus, TCDF could be produced in a syngeneic system. Recognition of self Ia molecules appeared to be essential for TCDF production, which was completely abolished by the addition of monoclonal anti-Ia antibody. In our experiments, removal of IL 2 from conditioned medium containing TCDF abolished its ability to generate LA-activated CTL. However, the cytotoxic response could be restored by the addition of a small amount (5 U/ml) of purified human recombinant IL 2 (HRIL 2), which alone was unable to generate LA-activated CTL at this dose. The generation of LA-activated CTL by high dose HRIL 2 (greater than 50 U/ml) was likely due to the endogenous production of TCDF. The bulk of TCDF could be separated from IL 2 by gel filtration in a Sephadex G-100 column. The peak of TCDF activity was concentrated at a m.w. of 16K dalton, and there was very little IL 2 activity in these fractions. When added alone to the LA-activated lymphocyte cultures, these active fractions were unable to induce CTL; supplementation of exogenous IL 2 was needed to restore the cytotoxic responses. Our findings indicate that both IL 2 and TCDF, which are needed in CTL generation. are produced in syngeneic cultures in the absence of antigenic or mitogenic stimulation.     

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.     

A human helper T cell clone secreting both killer helper factor(s) and T cell-replacing factor(s)

A human helper T cell clone (d4), which showed its helper effect on the differentiation of both T and B cells, was established by MLC reaction of normal T cells against a B lymphoblastoid cell line (CESS) followed by cloning in the presence of IL2 and x-irradiated CESS and autologous non-T cells. d4 cells helped the induction of cytotoxic T cells against UV-treated CESS cells. Antigen-stimulated d4 cells secreted helper factor(s) involved in the induction of cytotoxic T cells (killer helper factor(s), KHF), and KHF activity could be separated into two fractions, one with the m.w. of 15,000 to 20,000 and the other with the m.w. of 45,000 to 50,000. The factor with 15,000 to 20,000 m.w. showed IL 2 activity; the other factor showed gamma-interferon activity without IL 2 activity, suggesting that both IL 2 and gamma-interferon exerted KHF activity. d4 cells or their culture supernatant showed helper activity in the induction of IgG in a B cell line (CESS). The helper activity of the supernatant (TRF) was absorbed with CESS cells but not with IL 2-dependent CTLL, whereas KHF activity was absorbed with IL 2-dependent CTLL but not with CESS cells. The results showed that TRF and KHF were distinct molecules and a single helper T cell clone could secrete helper factors for both B and T cells.     

A helper factor needed for the generation of mouse cytolytic T lymphocytes is made by tumor cell lines, cloned T cells, and spleen cells exposed to a variety of stimuli

A helper factor termed cytolytic T lymphocyte helper factor (CHF) that is needed for the generation of allospecific mouse cytolytic T lymphocytes (CTL) in vitro was produced by mouse spleen cells 3 to 4 days after the time when interleukin 2 (IL 2) had reached its maximal production. These kinetics were observed by stimulation of immune spleen cells with allogeneic tumor or spleen cells, with Sendai or influenza viral peptides, with virus infected cells, or with concanavalin A (Con A). CHF produced by rat spleen cells was able to help in the generation of mouse CTL, indicating that this cytokine was not restricted genetically. CHF could also be made by WEHI-3 and EL4 cell lines, as well as cloned cytolytic and helper T cells. The production of CHF by WEHI-3 cells argues that CHF is not IL 2. In addition, if CHF was not present early in the in vitro stimulation no CTL were generated, suggesting that CHF participated in the activation of CTL precursors. The addition of IL 2-containing conditioned medium to the CHF assay resulted in no substantial CTL generation, although significant cellular proliferation was observed. In contrast, CHF-containing conditioned medium allowed the generation of CTL in the absence of the same level of proliferation.     

Limiting dilution analysis of alloreactive cytotoxic precursor cells in aging mice

The quantitative minimal estimate of the frequency of alloantigen specific cytotoxic T lymphocyte precursor cells (CTL-p) was determined in young and old C57BL/6 mice by limiting dilution analysis. Supernatant from phorbol myristate acetate-induced EL-4 cells was used as a source of IL 2 in these assays, which therefore were independent of the presence of the Lyt-2-, IL 2-producing cells known to be deficient in aging mice. These studies showed that 24-mo-old mice had approximately 10-fold fewer CTL-p than their young counterparts. Comparison of the limiting dilution assay (LDA) with IL 2-supplemented primary MLC shows that estimates of the frequency of CTL-p do not consistently agree with cytotoxic activity detected in the higher cell density primary MLC, and that the LDA is most likely a better estimate of the effect of age on the development of CTL.     

H-2K/H-2D and Mls and I region-associated antigens stimulate helper factor(s) involved in the generation of cytotoxic T lymphocytes

This study examines the antigen that stimulate production or release of a soluble helper factor(s) involved in development of cytotoxic T lymphocytes (CTL). Antigens associated with the Mls locus, I and K/D regions of the MHC were all capable of stimulating responder cells in MLC to produce helper factor. These supernatant fluids were all capable of providing "help" for the generation of cytotoxic T lymphocytes in MLC in which spleen cells are stimulated by allogeneic heat-treated thymocytes or splenocytes. Previous reports from our laboratory as well as others have shown that heat-treated cells do not stimulate a cytotoxic response. Heat-treatment of Mls, I, and H-2K/H-2D region incompatible stimulatory cells in MLC eliminated their ability to induce responder cells to produce helper factor, suggesting this is the mechanism whereby heat-treatment reduces the ability of cells to stimulate cell-mediated lympholysis (CML). The inability of supernatant fluids, from MLCs in which heat-treated cells were the stimulators, to assist in the generation of cytotoxic T cells did not appear to be the result of any suppressive factor induced by such treatment. Further, the antigens that stimulate pre-killer cells appear functionally distinct from those heat labile antigens (Mls, I, H-2K/H-2D associated) that stimulate helper factor production since heat-treated allogeneic cells served as stimulators of cytotoxicity provided helper activity was added to the MLC.     

Regulation of interleukin 2 receptor expression on murine cytotoxic T lymphocyte clones

We have previously reported that influenza virus-specific cytotoxic T lymphocyte (CTL) clones require antigen and exogenous growth factors for continued proliferation in culture. In this report we show that after stimulation with specific antigen, cloned CTL are capable of limited proliferation in response to interleukin 2 (IL 2) alone but with time these large blast-like cells revert to smaller, quiescent cells that are no longer responsive to IL 2. The IL 2-unresponsive CTL can not be driven to proliferate by supra-optimal concentrations of IL 2, and unresponsiveness correlates with decreased ability to absorb IL 2 from conditioned medium at 0 degrees C, suggesting that unresponsiveness is due to diminished IL 2 receptors. Stimulation of the unresponsive CTL with antigen leads to re-expression of the IL 2 receptor. Decreased absorbing capacity of the unresponsive cells could not be accounted for by their smaller surface area, and the IL 2-unresponsive cells seemed not to down-regulate all their immune functions, as they remained cytotoxic. These results provide a basis for the role of specific antigen in maintaining CTL clones in vitro. Furthermore, these results suggest that antigen-dependent CTL lines can be regulated and that antigen and IL 2 both play a role in their regulation.     

The role of macrophages in the generation of T helper cells. V. Evidence for differential activation of short-lived T1 and long-lived T2 lymphocytes by the macrophage factors GRF and NMF.

The generation of T helper cells in vitro requires macrophages or macrophage-derived factors such as genetically related macrophage factor (GRF) or nonspecific macrophage factor (NMF). However, there is a basic difference of T helper cell induction when using particulate antigens. The present study demonstrates that this difference is based on the activation of two different T cell subsets. GRF activates short-lived 'T1' cells which amplify the induction of T2 cells, which are the helper cell precursors. Thus, the genetic restriction of T helper cell induction seen with soluble antigen or GRF lies on the level of macrophage or GRF interaction with T1 cells. NMF (or macrophages) and particulate antigens directly activate the helper cell precursor (T2) indicating no requirement for T1-T2 cooperation. The direct activation of the helper cell precursor with particulate antigens does not require histocompatible macrophages or NMF from histocompatible macrophages. The present results may explain some of the discrepancies reported in the literature concerning the genetic requirements and specificity of T cell activation.