Interleukin 3 enhanced interleukin 2-dependent maturation of NK progenitor cells in bone marrow from mice with severe combined immunodeficiency

Human recombinant interleukin 2 (IL 2) and highly purified murine interleukin 3 (IL 3) were tested for their ability to generate NK activity in bone marrow cells from mice with severe combined immunodeficiency. IL 2 alone could dose dependently induce NK activity in marrow cells as determined by cytotoxicity against YAC-1 target cells. It was demonstrated that IL 3 had dual effects on the generation of NK activity in this culture system. The addition of IL 3 resulted in inhibition of NK cell activity seen at high concentrations of IL 2. In contrast, when IL 3 was added together with low concentrations of IL 2, the generation of NK cells as judged by cytotoxicity assay as well as the appearance of cells with NK phenotypes was markedly augmented. In some experiments, mice were treated with 5-fluorouracil (5-FU) to eliminate relatively differentiated NK precursors from bone marrow cells. It was noted that the residual immature marrow cells from 5-FU-treated mice showed little NK activity even after the culture with high concentrations of IL 2. Importantly, IL 3 could induce the generation of NK activity from 5-FU-treated marrow cells in the presence of IL 2. Kinetic studies indicated that NK activity was appreciably generated from 5-FU-treated marrow cells when preincubated with IL 3 at least for 12 hr and subsequently cultured with IL 2. The cells bearing IL 2 receptors appeared in 5-FU-treated marrow cells, even though cultured only with IL 3, which implied that IL 3 could support the development of very primitive NK cells from IL 2-unresponsive to IL 2-responsive states. These results suggested that IL 3 might play a crucial role for the IL 2-induced generation of NK cells in bone marrow through promoting the expression of IL 2R on NK progenitor cells.     

Augmentation of the generation of cytotoxic T lymphocytes against syngeneic tumor cells by recombinant human tumor necrosis factor

In order to clarify the effect of recombinant human tumor necrosis factor (rHu-TNF) on the antitumor T cell immune response, we examined the effect of rHu-TNF on the generation of cytotoxic T cells (CTL) against syngeneic tumor cells. Spleen cells from X5563 plasmacytoma-transplanted mice were stimulated in vitro with mitomycin C-treated X5563 cells in the presence or absence of rHu-TNF. The generation of CTL was augmented in a dose-dependent manner by the addition of rHu-TNF. The augmenting effect of rHu-TNF was more marked when indomethacin was added to the culture. The augmenting effect was observed only when rHu-TNF was added at the early stage of the generation of CTL. The cell surface phenotype of CTL generated was L3T4- and Lyt2+. The augmentation was shown not only by the chromium-51 release assay but also by the Winn assay. As to the specificity, the augmentation of CTL generation was observed by the addition of rHu-TNF when responder-primed spleen cells were stimulated with the tumor cells in vitro. On the other hand, augmentation was not observed when responder spleen cells were not stimulated with the tumor cells in vitro, or when responder spleen cells were obtained from normal mice. The CTL generated was not cytotoxic against other tumor cells of the same haplotype. Thus, rHu-TNF augmented the generation of CTL against syngeneic tumor cells in an antigen-specific manner. The in vivo effect of rHu-TNF was examined by administering rHu-TNF into X5563-bearing mice. The spleen cells of rHu-TNF-injected mice generated a much higher CTL activity against X5563 cells in vitro than did the spleen cells of uninjected mice. From these results, a possibility can be considered that in some cases, rHu-TNF may exert its antitumor activity by stimulating the immune system.     

Abrogation of anti-Pichinde virus cytotoxic T cell memory by cyclophosphamide and restoration by coinfection or interleukin 2

Previously, we demonstrated that memory cell-mediated immune responses can be generated in Pichinde virus (PV)-primed mice after secondary challenge in vivo with homologous virus. Further, treatment of mice with cyclophosphamide (CY) before primary infection with PV abrogated the generation of H-2-restricted, virus-specific cytotoxic T lymphocytes (CTL), and rechallenge of these mice was followed by neither a primary nor a secondary CTL response. Here, we demonstrate that this CY-induced block in memory anti-PV CTL generation was not due to establishment of a persistent infection. Interestingly, this CY-induced block in memory anti-PV CTL generation was overcome by secondarily coinfecting mice with PV and lymphocytic choriomeningitis virus (LCMV) or PV and Tacaribe virus. Secondary infection with LCMV or Tacaribe virus alone did not elicit anti-PV CTL. Coinfection resulted in the generation of a PV-specific memory CTL response as judged by maximal activity on day 4 after rechallenge. Co-infection with PV and vesicular stomatitis virus, an unrelated rhabdovirus, did not efficiently restore memory anti-PV CTL responses. Memory anti-PV CTL responses were also restored when interleukin 2 (IL 2)-containing supernatants were injected i.p. after rechallenge of CY-treated mice with PV. To demonstrate that IL 2 was the responsible lymphokine in these preparations, highly purified IL 2 was added to in vitro cultures of spleen cells from CY-treated PV-primed mice. In the presence of PV-infected syngeneic macrophages, addition of purified IL 2 resulted in a dose-dependent restoration of H-2-restricted anti-PV CTL activity. The CTL precursor (CTLp) frequency of CY-treated PV-primed mice was markedly decreased from that of normal PV-primed mice. Thus, the long-lasting block in the ability to generate a PV-specific memory CTL response after CY treatment appears to be due to both a lack of helper T cell activity and a significant reduction of CTLp. However, this block may be overcome by coinfecting with viruses that cross-react at the helper T cell level or by exogenous treatment with highly purified IL 2.     

IL-6/BSF-2 functions as a killer helper factor in the in vitro induction of cytotoxic T cells

rIL-6/B cell stimulatory factor 2 was found to augment CTL generation from mature as well as immature human T cells stimulated with UV-treated allogeneic cells. rIL-6 also acted on peanut agglutinin-positive murine thymocytes and Lyt-2-positive splenic T cells to give rise to CTL. rIL-6 alone could not induce CTL generation, the presence of IL-2 during the early phase of culture period was found to be essential for the IL-6 activity in the induction of CTL. The effect of rIL-6 was not mediated by the induction of IL-2 inasmuch as rIL-6 did not augment IL-2 production in MLC and anti-IL-2 antibody could not neutralize IL-6 activity. rIL-6 augmented CTL generation even when added 72 h after the initiation of cultures. The enhancing activity of rIL-6 could be neutralized with anti-IL-6 antibodies even when added 72 h after the initiation of cultures. The present data indicate that IL-6 acts in the late phase of CTL generation.     

Secondary in vitro generation of CTL specific for MM antigen by stimulation with somatic tumor hybrid, but not with parental tumor cells

Somatic tumor hybrid cells (L-FM3A#2), obtained by hybridization of MM tumor cells (FM3A/R) with HGPRT-less L cells, could induce CTL directed against MM antigen, a tumor-associated transplantation antigen that is expressed on some ascitic mammary tumor cell lines of C3H/He mice; parental tumor cells (FM3A/R) could not produce such CTL in syngeneic mice. In this study, the mechanisms of the generation of CTL by stimulation with L-FM3A#2 hybrid cells were investigated. In the secondary in vitro stimulation system, L cell component(s) that were introduced into hybrid cells by cell fusion play a role in the induction of CTL, as shown by the fact that stimulation with a mixture of L and FM3A/R cells could induce MM antigen-specific CTL, and that the killer helper effect of L cells could be replaced by cell free culture supernatants obtained from co-cultures of unprimed C3H spleen and L cells. IFN activity, but no IL 2 activity, was detected in the culture supernatants. Both IFN and killer helper activity were lost with pH 2 treatment; furthermore, CTL were generated by stimulation of primed spleen cells with FM3A/R cells in the presence of mouse beta-IFN, but not in its absence. These results suggest that IFN liberated by the helper cells that recognize L cell component(s) on the surfaces of tumor hybrid cells plays an essential role in the generation of CTL specific for MM antigen.     

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.     

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.     

IL-4-supported induction of cytolytic T lymphocytes requires IL-2 and IL-6

Previous work indicated that a CTL response can be generated by the combination of IL-2 plus IL-6 or IL-4 alone. Because of the ubiquitous production of IL-6 and its apparent ability to induce IL-2, we explored the interdependence of these lymphokines in supporting a CTL response from murine thymocytes. For thymocytes cultured in IL-4, further addition of IL-6 enhanced thymocyte proliferation. In addition, a role for IL-6 in thymocyte activation was indicated by the ability of anti-IL-6 mAb to block both IL-4-directed proliferation and the cytotoxic response found in the presence of IL-4. The addition of IL-2 to limiting doses of IL-4 augmented the CTL response; however, the response to high levels of IL-4 was not augmented by addition of IL-2. Consistent with this apparent involvement of IL-2 in the IL-4-mediated response we found: (a) that mAb to IL-2 significantly reduced the CTL response generated in the presence of IL-4; (b) that IL-2 activity was present in culture supernatant following incubation of thymocytes with high levels of IL-4; and (c) that enhanced IL-2 receptor expression found in the presence of IL-4 was blocked with the addition of anti-IL-2 antibody to the thymocyte culture. In contrast to the data for proliferation, anti-IL-4 mAb had no effect on the generation of CTL in the presence of IL-2 + IL-6 but readily blocked the CTL response to IL-4. These results indicate that, for thymocyte responders, the CD8+ CTL generated in the presence of IL-4 require both IL-2 and IL-6.     

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.     

Requirement for three distinct lymphokines for the induction of cytotoxic T lymphocytes from thymocytes

The induction of cytotoxic T lymphocytes (CTL) from CTL precursors requires a combination of antigen and lymphokine signals. To investigate lymphokine requirements for CTL generation, we used an assay in which helper T cell and accessory cell-depleted spleen cells or whole thymocytes were cultured with lectin (Con A) and lymphokines. This culture was followed by assessment of lectin-dependent cytolysis. High concentrations of recombinant interleukin 2 (R-IL 2) (100 U/ml) alone were not sufficient for lectin-mediated CTL induction from thymocytes, whereas 20 to 100 U/ml of R-IL 2 alone could induce a significant lectin-mediated CTL response from accessory cell-depleted spleen cells. Using thymocytes as responders, we found purified or recombinant interferon-gamma (IFN-gamma) did not cause cytolytic activity either in the absence of or in the presence of R-IL 2. However, supernatant from Con A-stimulated rat spleen cells (rat Con A SN) in combination with R-IL 2 could induce cytolytic activity, suggesting that several factors are required for CTL induction. Con A SN was fractionated by gel filtration and the fractions were tested for ability to induce CTL. In the presence of a low level of R-IL 2 (5 U/ml), fractions with a Mr of approximately 31,000 could induce CTL, and this activity was referred to as CTL differentiation factor (CDF). The peak fractions containing CDF activity did not have detectable IL 1, IL 2, IFN-gamma, or CSF activity. However, by add-back experiments and the use of blocking antibodies, a monoclonal antibody against the IL 2 receptor or antibodies against murine IFN-gamma, we demonstrated that CTL induction from mature thymocytes (L3T4-, Lyt-2+) requires CDF activity in addition to IL 2 and IFN-gamma.     

Synergy between recombinant interleukin 2 (rIL 2) and IL 2-depleted lymphokine-containing supernatants in facilitating allogeneic human cytolytic T lymphocyte responses in vitro

Supernatants from human mixed leukocyte cultures or lectin-depleted supernatants from cultures of PHA-activated human peripheral blood leukocytes were depleted of IL 2 by passage over an anti-human rIL2 immunoadsorbent column. The column eluates were concentrated, dialyzed, and tested for their ability to synergize with human rIL 2 in facilitating human cytolytic T lymphocyte (CTL) responses to allogeneic, uv-irradiated HT144 melanoma cells in vitro. CTL were generated in the presence of 1 X 10(-4) M hydrocortisone sodium succinate in order to minimize the generation of nonspecific lymphokine-activated killer (LAK) cells. IL 2-depleted lymphokine-containing supernatant (LKS), alone or in the presence of less than or equal to U/ml rIL 2 did not stimulate significant CTL responses. Recombinant IL 2 at greater than 2 U/ml stimulated weak CTL responses in the absence of LKS. However, strong synergistic CTL responses were observed when both IL 2-depleted LKS and greater than 2 U/ml rIL 2 were added to the cultures. CTL generated in these cultures could be distinguished from nonspecific LAK cells on the basis of their i) specificity, ii) T3 phenotype, and iii) kinetics of generation. Nevertheless, rIL 2 and IL 2-depleted LKS were sometimes observed to synergize in facilitating the generation of nonspecific LAK cells as well as the generation of specific CTL. When the times at which rIL 2 and IL 2-depleted LKS were added to the cultures were varied, IL 2 was found to be required early in CTL responses, whereas the synergistic factor(s) in LKS seemed to act later. Recombinant human interferon-gamma was unable to replace LKS in synergizing with rIL 2 to elicit CTL responses. In summary, these experiments suggest that LKS contains a late-acting factor(s), antigenically distinct from IL 2, which synergizes with IL 2 in facilitating human CTL responses.     

Suppression of alloimmune cytotoxic T lymphocyte (CTL) generation by depletion of NK cells and restoration by interferon and/or interleukin 2

By using rabbit antiserum to a glycolipid, ganglio-n-tetraosylceramide (ASGM1), the accessory effect of natural killer (NK) cells on the generation of alloimmune CTL in mice was investigated. When normal C3H/He mice were immunized with C57BL/6 or BALB/c spleen cells, they generated alloimmune CTL with a surface marker phenotype of Thy-1+ Lyt-1-2+ ASGM1-, preceded by early augmentation of cytotoxic activity of NK cells with a Thy-1-Lyt-1-2-ASGM1+ phenotype. Administration of anti-ASGM1 (10 microliters) in mice resulted in a complete depletion of NK activity and ASGM1+ cells in the spleen even 1 day after injection, but no changes in the proportions of T (Thy-1+) cells and their Lyt-1 and Lyt-2 subsets as revealed by an immunofluorescence analyzer (FACS) and phagocytic cells. When these anti-ASGM1-treated mice were immunized with allogeneic cells, they showed neither augmented NK activity nor generation of alloimmune CTL, and spleen cells isolated from these anti-ASGM1-treated mice produced no CTL response to alloimmunization in vitro. Normal spleen cells treated with the antiserum and complement in vitro also showed a complete NK depletion without any deterioration of T cells and their Lyt-1 and Lyt-2 subsets, and when stimulated with allogeneic cells they generated no CTL. Spleen NK (ASGM1+) cells were purified by Percoll-gradient centrifugations followed by complement-dependent killing of T cells with the use of anti-Thy-1 monoclonal antibody, and were further purified by panning methods with anti-ASGM1, giving a preparation consisting of greater than 90% ASGM1+, Ly-5+ cells, and less than 0.5% of Thy-1+, Lyt-1+, and Lyt-2+ cells. These purified ASGM1+ Thy-1- cells alone generated no alloimmune CTL in response to alloantigens, suggesting that ASGM1+ NK cells contained no precursors of alloimmune CTL. When added into NK-depleted spleen cells, they restored the normal alloimmune CTL response of the spleen cells, indicating that ASGM1+ fractions contained cells to provide an accessory function for CTL generation. Lyt-1+ cells purified by panning methods did not restore the CTL response of NK-depleted spleen cells. These results indicate that ASGM1+ NK cells, but not Lyt-1+ helper T cells contaminating ASGM1+ fractions at undetectable levels, are responsible for the accessory function. When these purified ASGM1+ Thy-1- cells were stimulated with allogeneic cells, they produced IL 2 and IFN.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

Contra-IL 2; a suppressor lymphokine that inhibits IL 2 activity

Suppressive activity of culture supernatant of AS-9 (AS-9 CS), a T cell hybridoma line that was derived from fusion of BW5147 thymoma and splenic T cells of anti-lymphocyte serum-treated C3H mice, was analyzed. AS-9 CS inhibited allogeneic cytotoxic T lymphocyte (CTL) generation as well as T cell proliferation to alloantigens and mitogens, but failed to inhibit B cell response to lipopolysaccharide or growth of tumor and fibroblast cells. Although addition of AS-9 CS to the allogeneic sensitization culture as late as on day 2 of incubation resulted in maximal inhibiton of CTL generation, removal of AS-9 CS on day 3 of incubation abolished its inhibitory effect. Addition of purified IL 2 together with AS-9 CS to the allogeneic sensitization cultures only partially abrogated the suppression. Experiments with IL 2-dependent cytotoxic T cell line (CTLL) showed that AS-9 CS suppressed the IL 2-induced proliferation of CTLL. Preincubation of AS-9 CS with CTLL removed its inhibitory effect on CTL generation. These results indicate that AS-9 CS interferes with the mechanism of T cell activation by IL 2. On this basis, AS-9 CS was named contra-IL 2.     

Natural killer activity in cloned cytotoxic T lymphocytes: regulation by interleukin 2, interferon, and specific antigen

It has previously been shown that monoclonal antigen-specific mouse CTL lines can be induced to express cytolytic activity with the same specificity as that of splenic natural killer (NK) cells following culture in high concentrations of concanavalin A-induced spleen cell supernatants. In the present experiments, we made use of this in vitro system to explore the regulation of NK activity at the clonal level. Interferon-alpha and interferon-beta and interleukin 2 (IL 2) were potent inducers of NK activity in CTL, demonstrating that these substances can activate NK functions directly without the participation of other cell types. By comparison, IFN-gamma was a poor activator of NK activity in CTL (and also in fresh spleen cells). Three major differences between induction of NK activity by IFN-alpha,beta and IL 2 were noted: IFN induced NK activity selectively without affecting specific cytolysis, whereas IL 2 also enhanced specific killing; IFN acted much more rapidly than IL 2; and IFN did not induce the cells to enter the cell cycle nor were there any obvious morphologic changes. Specific antigen was also a strong inducer of NK activity in CTL, but studies with antisera against the various classes of IFN revealed that this effect was mediated, at least in part, via the release of IFN-beta. By contrast, the same antisera had no effect on NK induction by crude TCGF or by highly purified IL 2, indicating that the regulation of NK activity by IL 2 occurs at the clonal level in an IFN-independent manner. Although, IL 2, IFN, and Ag could apparently act alone to induce NK activity, much greater (synergistic) induction was obtained by various combinations of these regulators, suggesting that the delivery of two (or more) signals to the responder cell was required for full expression of the NK state. As with fresh splenic NK cells, the induced NK state in cloned CTL was intrinsically labile as revealed by its rapid decay in the absence of inducers, but it could nonetheless be maintained indefinitely at very high levels in the continued presence of inducers. This clonal system thus displays a responsiveness to regulatory signals exactly analogous to that of splenic NK cells and provides a unique and exciting opportunity to evaluate the biochemistry of the regulation of NK activity.     

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.     

Interleukin 3 augments the murine primary cytolytic T lymphocyte response to allogeneic tumor cells

The primary anti-H-2k allospecific cytolytic T lymphocyte (CTL) response by BALB/c (H-2d) spleen cells in vitro to x-irradiated RDM4 (H-2k) tumor cells is weak. This response has been shown to be augmented by CTL helper factor (CHF), a factor present in supernatants of spleen cells cultured with Sendai virus (SC-CM). Conditioned medium from WEHI-3 cells (WEHI-CM) also contains activity that augments the BALB/c anti- RDM4 CTL response. Attempts to separate the CHF activity from interleukin 3 (IL 3), also present in WEHI-CM, were unsuccessful. Purified IL 3 was then tested, and was found to increase the BALB/c anti- RDM4 CTL response by five- to 10-fold. IL 3 is apparently the only material in WEHI-CM that is active in this assay. The response is apparently a classical CTL response because: 1) the effector cells are sensitive to monoclonal anti-Thy-1.2 antibody plus C; 2) the response is dependent on antigen stimulation, and it peaks on day 5 or 6 of culture; and 3) the effector cells are specific for H-2k targets. IL 3 must be added very early during the in vitro culture period for maximal augmentation of the response, consistent with possible action of IL 3 as a differentiation factor.     

Inhibition of alloantigen-induced cytolytic T lymphocytes in vitro with (2R,5R)-6-heptyne-2,5-diamine, an irreversible inhibitor of ornithine decarboxylase

The objective of the present investigation was to examine the effect of a new potent irreversible inhibitor of ornithine decarboxylase, (2R,5R)-6-heptyne-2,5-diamine (MAP) (MDL 72,175), on the induction of functionally reactive T-cell populations in vitro. We examined alloantigen-activated cytolytic T lymphocytes (CTL) and T-helper (TH) lymphocytes generated during a one-way mixed-leukocyte culture (MLC). The addition of MAP (1 mM) at the initiation of cell culture reduced intracellular putrescine, spermidine, and spermine levels by 81.9, 82.4, and 55.8% respectively. MAP reduced CTL induction 93.8, 78.4, and 37.5% when added at 0, 24, or 48 hr of culture, respectively. A dose-dependent inhibition of CTL induction and polyamine levels was observed following MAP treatment. In direct comparison with another ODC inhibitor, alpha-difluoromethylornithine (DFMO), MAP was five- to sixfold more potent in reducing CTL induction. CTL generation is dependent upon the endogenous production of the TH-cell product interleukin 2 (IL-2). MAP treatment reduced detectable IL-2 activity in a MLC by 54.8%. These results indicate that MAP is a potent inhibitor of alloantigen-activated CTL in vitro and deserves further investigation as a potential immunosuppressive agent.     

Active suppression of host-vs-graft reaction in pregnant mice. VI. Soluble suppressor activity obtained from decidua of allopregnant mice blocks the response to IL 2

The mammalian fetus expresses a variety of antigens against which the maternal immune system can react and which in an allogeneic mating bears paternal transplantation antigens. Although these antigens may be expressed on the fetal trophoblast cells that contact maternal uterine decidua, the "fetal allograft" is not usually rejected. Previous studies have demonstrated the presence of nonspecific non-thymus-derived suppressor cells in the lymph nodes draining the uterus and in decidua of laboratory mice undergoing first allogeneic pregnancy. These suppressor cells appeared to be small lymphocyte cells that inhibit the generation of cytotoxic T lymphocytes (CTL) in vitro and in vivo and elaborate a nonspecific non-MHC-restricted soluble suppressor activity when cultured for 48 hours at 37 degrees C in vitro. We now report that soluble suppressor activity obtained from the decidua (DS) of allopregnant C3H/HeJ mice inhibits both the primary and secondary (memory) CTL response in vitro but does not inhibit lysis of target cells by preformed CTL. DS did not suppress the proliferation of YAC lymphoma cells, P-815 cells, or a C3H placental trophoblastoma line. Suppressor activity was obtained from anti-thy-1.2 + complement-resistant cells in the decidua, could also be obtained from the decidua of allopregnant CD1 nu/nu mice, and was associated with a single peak of activity of approximately 100,000 daltons on Sephacryl 200 chromatography. Suppression could not be overcome by adding either crude or HPLC-purified IL 2 to the mixed lymphocyte cultures in vitro, and both crude and column-purified suppressor factor inhibited the IL 2-dependent proliferation of H-Y cells (a cloned T cell line with NK activity). Furthermore, DS inhibited the IL 2-dependent generation of cytotoxic effector cells in vitro in the absence of allogeneic stimulator cells. Thus, a soluble suppressor factor obtained from non-T cells present in the decidua of successfully allopregnant mice could block the response to IL 2 and inhibit the generation of both specific and nonspecific cytotoxic effector cells. The significance of this inhibition with respect to survival of the "fetal allograft" is discussed.