"Anergy" of TH0 helper T lymphocytes induces downregulation of TH1 characteristics and a transition to a TH2-like phenotype.

Mature CD4+ helper T lymphocytes have been categorized into two major functional phenotypes, TH1 and TH2, which produce distinct arrays of lymphokines and which are thought to arise from a pluripotential precursor cell termed TH0. Clonal anergy can be induced in TH1 clones by stimulating via the T cell receptor (TCR) complex in the absence of a costimulator molecule; however, anergy has been difficult to demonstrate in TH2 clones. We show here that treatment of cloned TH0 lines with anergizing stimuli results in the selective loss of TH1 characteristics and retention of a TH2 phenotype. Treated cells exhibit a substantial reduction in interleukin 2 (IL-2) production and antigen-specific cytolytic activity, but retain comparable IL-4 and IL-5 production in response to restimulation via the TCR complex. TH0 clones exposed to anergizing stimuli also increase in size, thus morphologically resembling TH2 cells. The signaling characteristics of these cells also are altered, in that they exhibit an elevated basal level of intracellular free calcium which fails to increase significantly with subsequent restimulation, reminiscent of the signaling characteristics of TH2 cells. "Anergized" TH0 clones thus share several functional, morphologic, and physiologic properties with cells of the TH2 phenotype, suggesting that TH2 cells may arise when TH0 cells are stimulated via the TCR complex in the absence of a putative costimulator molecule.

A clone-specific monoclonal antibody that inhibits cytolysis of a cytolytic T cell clone.

Monoclonal antibody 384.5 specifically inhibited cytolysis of P-815 target cells by cloned L3 cytotoxic T lymphocyte (CTL) effector cells. The lytic activity of other cloned CTL that have other distinct specificities was not affected. Antibody 384.5 did not inhibit the cytolytic activity of bulk populations of C57BL/6 mixed lymphocyte culture (MLC) cells. Concanavalin A-facilitated cytolysis by T cell clone L3 but not T cell clone B18 was inhibited by antibody 384.5, whereas phytohemagglutinin-facilitated cytolysis by L3 cells was not strongly inhibited. Antibody 384.5 binds specifically to L3 cells but not to several other T lymphocytes clones, or to a detectable portion of populations of primary MLC cells, normal spleen, thymus, lymph node, or bone marrow cells. In contrast, C57BL/6 anti-B10.A(5R) secondary MLC cells (genetically enriched for reactivity against the H-2Dd region gene products) contained a small population which reacted with the antibody 384.5. The determinant detected by antibody 384.5 was susceptible to trypsin treatment, and was reexpressed after overnight incubation. These results suggest that the monoclonal antibody 384.5 detects an endogenously synthesized clone-specific determinant associated with the cytolytic activity of the L3 CTL clone. These properties make antibody 384.5 an attractive candidate for an antibody that reacts with the antigen-recognition site of a cytolytic T cell antigen receptor.

An antigen receptor-driven, interleukin 2-independent pathway for proliferation of murine cytolytic T lymphocyte clones.

Proliferation of T lymphocytes can be induced by IL-2, either through an autocrine pathway in which the responding cell produces its own IL-2 or through an exocrine pathway in which IL-2 secreted by Th stimulates proliferation of IL-2-dependent CTL. However, proliferation of at least some CTL clones, such as CTL L3 and CTL dB45, also can be induced by stimulation of the antigen receptor in the absence of IL-2. Stimulation of these cloned CTL with T cell-depleted allogeneic spleen cells, allogeneic tumor cells, or immobilized mAb reactive with the T cell antigen receptor (TCR) induced thymidine incorporation, entry into cell cycle, and secretion of macrophage activating factor, but these stimuli did not induce the secretion of IL-2. Several observations indicated that such proliferation of cloned CTL induced by stimulation of the TCR was independent of IL-2; IL-2 could not be detected in supernatants from stimulated CTL cells. mAbs reactive with the murine IL-2-R efficiently blocked IL-2-mediated thymidine incorporation in cloned CTL and Th, but had no inhibitory effect on TCR-driven thymidine incorporation in the CTL clones. TCR-driven thymidine incorporation in cloned Th L2 cells was profoundly inhibited by these antibodies, indicating the operation of an IL-2-mediated autocrine pathway for proliferation in this cloned Th. When antibodies to the TCR were used to stimulate cloned CTL and Th, IFN-gamma mRNA was easily shown in the cloned CTL and Th. Although IL-2 mRNA could be detected in the cloned Th, it was never observed in the cloned CTL. These findings provide evidence for the existence of a TCR-mediated, IL-2-independent pathway for induction of cellular proliferation in cloned murine CTL.

Enhanced growth of primary tumors in cancer-prone mice after immunization against the mutant region of an inherited oncoprotein.

One major objective of tumor immunologists is to prevent cancer development in individuals at high risk. (TG.AC x C57BL/6)F1 mice serve as a model for testing the feasibility of this objective. The mice carry in the germline a mutant ras oncogene that has an arginine at codon 12 instead of glycine present in the wild-type, and after physical (wounding) or chemical promotion, these mice have a high probability for developing papillomas that progress to cancer. Furthermore, F1 mice immunized with Arg(12) mutant ras peptide in complete Freund's adjuvant (CFA) develop T cells within 10 d that proliferate in vitro on stimulation with the Arg(12) mutant ras peptide. Within 14 d, these mice have delayed-type hypersensitivity to the peptide. Immunization with CFA alone or with a different Arg(12) mutant ras peptide in CFA induced neither response. To determine the effect of immunization on development of tumors, mice immunized 3 wk earlier were painted on the back with phorbol 12-myristate 13-acetate every 3 d for 8 wk. The time of appearance and the number of papillomas were about the same in immunized and control mice, but the tumors grew faster and became much larger in the mice immunized with the Arg(12) mutant ras peptide. Thus, the immunization failed to protect against growth of papillomas. The peptide-induced CD4(+) T cells preferentially recognized the peptide but not the native mutant ras protein. On the other hand, mice immunized with Arg(12) mutant ras peptide and bearing papillomas had serum antibodies that did bind native mutant ras protein. Together, these studies indicate that active immunization of cancer-prone individuals may result in immune responses that fail to eradicate mutant oncogene-expressing tumor cells, but rather induce a remarkable enhancement of tumor growth.

Requirements for activation of CD8+ murine T cells. I. Development of cytolytic activity.

Cytolytic effector function fails to develop if proliferation of allospecific cytolytic T lymphocyte precursors is inhibited, but the requirements for generation of cytolytic activity have not been fully defined. In contrast, the cytolytic effector function of cytolytic T lymphocyte clones does not change during the cell cycle, and the level of cytolytic activity is independent of cellular proliferation. The requirement for proliferation by primary responding populations may reflect the need for clonal expansion of a few inherently cytolytic effector cells in order to reach a threshold number which can readily be detected in conventional cytolytic assays. Alternatively, proliferation may be required for cytolytic T lymphocyte precursors to differentiate into mature, functional cytolytic cells. Using CD8+ T cells which express an antigen-specific transgenic alpha/beta T cell receptor, we have studied the requirements for acquisition of cytolytic capacity. Stimulation of the T cell receptor alone appears to be sufficient to render naive, CD8+ transgenic T cells sensitive to the growth effects of interleukin-2 (IL-2), and in some circumstances to interleukin-4 (IL-4), but not to induce either lymphokine production or cytolytic activity. Costimulatory molecules expressed by allogenic stimulating cells appear to be required for lymphokine production, and CD8+ transgenic T cells initially appear to secrete only IL-2 and interferon-gamma. Stimulation of the T cell receptor of naive, CD8+ transgenic T cells appears to induce cytolytic activity only if cell proliferation occurs, either in response to IL-2 produced by the stimulated cells themselves when costimulatory molecules are present, or to IL-2 or IL-4 from exogenous sources if costimulatory molecules are absent.

Contributions of T cell clones to an understanding of the T cell receptor for antigen.

Cloned populations of T cells have been used to derive clone-specific monoclonal antibodies. These antibodies influence the specific immunological functions of the cloned T cells, suggesting that these antibodies react with the T cell receptor for antigen. Immunoprecipitates have been obtained with some of these antibodies, and the characteristics of the polypeptides from different cells are surprising similar. At present, little is known about this molecular complex other than that the two polypeptides are disulfide-linked and have apparent molecular weights of about 45,000 daltons and 43,000 daltons. This molecular complex, at least in human T cells, is associated with, but not covalently linked with, another molecular complex (Leu4/T3). However, modern techniques of cellular and molecular immunology should provide information very soon about the actual role of these molecules in T cell recognition of antigen. Indeed, cDNA clones that may encode genes for one of the peptide chains have been obtained.

Astrocyte cytolysis by MHC class II-specific mouse T cell clones.

The brain is "immunologically privileged," in part because class I and II MHC antigens are not normally present on glia or neurons. However, under certain conditions such as transplantation, glial cells express MHC proteins at levels sufficient for glia to become targets of immune responses. Cultured astrocytes expressing very low levels of MHC class I protein are killed efficiently by MHC class I antigen-specific CTL. Mouse brain allografts, however, are rejected by CD4+ T cells that are likely to be class II MHC-specific. The level of expression of MHC class II antigen needed to trigger specific killing of astrocytes by CD4+ T cells, independent of exogenous antigen, has not been studied. We examined the role of glial class II MHC in the lysis of cultured neonatal mouse astrocytes by an alloreactive murine CD4+ CTL alone. IFN-gamma induced functionally relevant increases in MHC class II antigen on target cells. Astrocytes were lysed by the CD4+ clone only when class II MHC antigens reached levels readily detectable by flow cytometry. MHC class II expression and lysis increased when astrocytes were coincubated with IFN-gamma and TNF-alpha. Conversely, lysis decreased when class II expression was downregulated by IFN-alpha/beta or dbcAMP. Cytolysis by CD4+ clones was blocked by antibodies to CD4 and LFA-1 on T cells, and to ICAM-1 and class II molecules on astrocytes. The role of LFA-1 in CD4+ cell-mediated lysis was greater than that of LFA-1/ICAM-1 in CD8+ T cell-mediated lysis. CD4+ cells may lyse activated astrocytes when the immune privilege of the brain is compromised as in transplantation, tumors, and inflammatory diseases.

Cytolytic T lymphocytes: an overview of their characteristics.

Cloned T cells have been useful for assessing the lytic potential of distinct T cell subsets and for determining the relative contribution of different effector mechanism involved in the lytic process. Alloreactive CD8+ murine T cell clones and cloned murine CD4+ TH1 and TH2 T cells reactive with nominal antigen (ovalbumin) lysed nucleated target cells bearing antigen or coated with anti-CD3 monoclonal antibody in a short term 51Cr-release assay. These clones were also evaluated for their ability to lyse efficiently sheep erythrocyte (SRBC) target cells coated with anti-CD3 mAb by a mechanism (presumably involving membrane damage) that does not involve nuclear degradation. Three patterns of lysis were observed: CD8+ and some CD4+ TH2 effector cells lysed efficiently nucleated target cells and anucleated SRBC coated with anti-CD3 mAb. However, CD4+ TH1 (and a few TH2) T cells which lysed nucleated target cells bearing antigen or coated with anti-CD3 mAb did not lyse efficiently the SRBC coated with anti-CD3 mAb. One CD4 bearing TH2 cell failed to lyse efficiently either nucleated target cells or anucleated SRBC coated with anti-CD3 mAb. These results indicate that both TH1 and TH2 clones have lytic capabilities. Furthermore, they suggest that some but not all TH2 murine T cell clones have lytic characteristics similar to those of conventional CD8+ CTL. However, it is not certain how these patterns of lysis of target cells in vitro relates to the capacity of CTL to lyse such target cells in vivo.

Accessory molecules involved in antigen-mediated cytolysis and lymphokine production by cytotoxic T lymphocyte subsets. I. Identification of functions for the T cell surface molecules Ly-6C and Thy-1.

The murine T cell surface molecules Ly-6C and Thy-1 are genetically and structurally distinct, yet they share two interesting properties: both are attached to the plasma membrane through a glycophosphatidylinositol linkage, and some mAb reactive with these molecules can activate T cells. Although mAb for Ly-6C and Thy-1 appear to mimic the function of physiologic ligands, direct evidence for the existence of these putative ligands has not been presented. In this report, we describe CTL clones that use Ly-6C and Thy-1 as accessory molecules for activation of cytolysis and the production of IFN-gamma based on inhibition of these functions with mAb. These studies were facilitated by the derivation of a nonactivating hamster IgM mAb specific for Ly-6C. CTL clones that use Ly-6C and Thy-1 as accessory molecules include a subpopulation of the previously described CD8+ alloreactive CTL that are not inhibited by mAb reactive with CD8, a CD8+ TCR-alpha/beta+ T cell clone specific for HSV glycoprotein D, and a CD4-CD8- TCR-gamma/delta+ T cell clone specific for HSV glycoprotein I. The role of Ly-6C and Thy-1 in target cell recognition is to some degree tissue-specific with respect to the APC/target cell. A mAb specific for Ly-6C appears to inhibit activation by prevention of adhesion between the effector cells and the target cells. This is the most direct evidence to date of a functional ligand for Ly-6C.

Mechanisms of lysis by cytotoxic T lymphocyte clones. Lytic activity and gene expression in cloned antigen-specific CD4+ and CD8+ T lymphocytes.

Cloned murine Th having properties of either Th1 or Th2 cells as well as CD8+ CTL were tested for the capacity to lyse: 1) nucleated target cells bearing Ag or coated with anti-CD3 mAb, or 2) SRBC target cells coated with anti-CD3 mAb in a short term 51Cr-release assay. The lysis of SRBC occurs by a mechanism that does not involve nuclear degradation but presumably does involve membrane damage. Three patterns were observed: CTL and some Th2 cells lysed efficiently nucleated target cells and SRBC coated with anti-CD3 mAb. Th1 and some Th2 T cells lysed nucleated target cells but did not lyse efficiently the SRBC coated with anti-CD3 mAb. Finally, some Th2 cells failed to lyse efficiently either nucleated or SRBC targets. We also examined these clones for their expression of N-alpha-benzyloxycarbonyl-L-lysin thiobenzyl esterase activity, and for the expression of perforin or CTLA-1 (granzyme B) mRNA. Total N-alpha-benzyloxycarbonyl-L-lysin thiobenzyl esterase activity expressed by CTL and Th2 clones tended to be higher than that of Th1 cells. Perforin mRNA and CTLA-1 mRNA were readily detectable in CTL and some Th2 clones. Expression of perforin and CLTA-1 mRNA correlated well with the capacity of these clones to lyse SRBC coated with anti-CD3 mAb. Our results show that some but not all Th2 clones have lytic characteristics similar to those of CD8+ CTL. Two mechanisms appear to contribute to their lytic process, one mechanism of lysis involves membrane damage that correlates with the expression of perforin mRNA; a second mechanism involves the induction of DNA degradation in the target cells. In contrast, some CD4+ effector cells appear to lack the capacity to lyse efficiently via the mechanism involving membrane damage and may only have the lytic activity associated with the capacity to induce DNA degradation.

Requirements for triggering of lysis by cytolytic T lymphocyte clones.

Cloned murine cytolytic T lymphocytes (CTL) having defined specificity were triggered by the phorbol ester together with a calcium ionophore (either A23187 or Ionomycin) to lyse syngeneic or third party target cells efficiently. Neither phorbol 12-myristate 13-acetate (PMA) nor calcium ionophore alone induced efficient lysis. The characteristics of the lytic process induced by these signals are similar to those of antigen-specific or lectin-facilitated lysis by CTL. Lysis is calcium and temperature dependent and shows kinetics which are not grossly different from lysis mediated via the antigen receptor. Two helper T lymphocyte clones were not induced to lyse efficiently EL-4 target cells by concanavalin A or PMA + ionophore. Triggering of lysis induced with PMA plus ionophore by the CTL clone L3 differed from antigen-mediated lysis in specificity and in the susceptibility to inhibition by cytochalasin B. Properties of the target cell determine which cell surface associative recognition structures are important in the efficient lysis of these cells. Anti-LFA-1 monoclonal antibodies inhibited efficiently both antigen-mediated and PMA + ionophore-induced lysis of P-815 or EL-4 target cells which are of hematopoietic origin. However, anti-LFA-1 antibodies do not inhibit antigen-mediated, lectin-facilitated, or PMA + Ionomycin-induced CTL cytolysis of target cells derived from the L cell fibroblast line. We conclude that two intracellular signals, which can be provided by the combination of PMA + ionophore, are required for efficient lysis by antigen-specific murine CTL clones. When the T cell receptor for antigen is bypassed using PMA + ionophore to trigger lysis, we show that Lyt-2 and LFA-1 molecules may be required for efficient lysis. These associative recognition structures appear to play an important role in postactivation steps leading to efficient delivery of the lethal hit to the target cell.

The requirements for triggering of lysis by cytolytic T lymphocyte clones. II. Cyclosporin A inhibits TCR-mediated exocytosis by only selectively inhibits TCR-mediated lytic activity by cloned CTL.

TCR-mediated granule exocytosis, as measured by the release of serine esterase activity, has been implicated in the lytic process of Ag-specific CTL. Exocytosis appears to be the mechanism of release of other lysis-relevant molecules including cytotoxic lymphokines and proteins that have the capacity to induce membrane lesions as measured by the hemolysis of non-nucleated SRBC. In the studies presented here, we assessed the contribution of exocytosis and lymphokine production in CTL lysis of nucleated and non-nucleated target cells by using a panel of murine CTL clones. Ag-mediated activation of cytolysis, lymphokine production, and exocytosis could be mimicked by mAb against the TCR/CD3 complex, or by stimulation with the combination of PMA + calcium ionophore, which appear to bypass the TCR (neither PMA nor calcium ionophore alone induced these functions efficiently in our CD8+ CTL clones). Although lysis, IFN-gamma production and exocytosis of N-alpha-benzyloxycarbonyl-L-lysin esterase (BLTE) activity were induced by either stimulus, we were able to identify distinct activation requirements for each of these functions. We found that lymphokine production, exocytosis, and cytolysis could be selectively inhibited. Cycloheximide inhibited IFN-gamma production, but did not inhibit exocytosis of BLTE activity or cytolysis. In addition we showed that cyclosporine A (CsA) profoundly inhibited IFN-gamma production as well as exocytosis induced by stimulation through the Ag receptor or by PMA + calcium ionophore. In contrast, CsA had little or no effect on lysis of nucleated target cells that bear the relevant Ag. These findings indicate that our CTL clones can lyse target cells by a mechanism independent of exocytosis or (de novo) lymphokine production. To directly assess the capacity of our CTL clones to lyse target cells without inducing nuclear damage we developed a system of coating non-nucleated SRBC with anti-CD3 mAb for use as stimuli and as targets for lysis. We found that our cloned CTL were indeed activated to produce IFN-gamma by SRBC that were coated with anti-CD3 mAb, and, furthermore, they were able to lyse the SRBC in a short term cytolytic assay. Thus our CD8+ CTL are capable of lysing certain target cells by a mechanism independent of DNA degradation, presumably by inducing a membrane lesion. In addition, CsA did inhibit lysis of the non-nucleated SRBC targets as well as exocytosis of BLTE activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytolytic activity of murine CD4+ T cell clones correlates with IFN-gamma production in mouse strains having a BALB/c background.

CD4+ murine T cell clones were derived from various strains of mice, and their pattern of lymphokine secretion and cytolytic activity was compared. Limiting dilution cultures were established with lymph node cells from mice sensitized with OVA. Alloreactive CD4+ T cell clones also were derived in limiting dilution cultures prepared with naive BALB/c-H-2dm2 lymph node cells stimulated with irradiated BALB/c splenocytes. A total of 24 days elapsed between establishment of cultures and analysis of lymphokine production and cytolytic activity. Cytolytic capacity was assessed by using target cells that had been pulsed with Ag or coated with anti-CD3 mAb. We observed that: 1) the frequency of OVA-reactive T cells from various mouse strains was approximately the same; 2) both Th1 and Th2 cells as well as cells not encompassed within these categories could be lytic if derived from DBA/2, B10.D2, B10.A, C57BL/10, or C57BL/6 mice; and 3) the vast majority of CD4+ cloned T cells derived from BALB/c, BALB/c-H-2dm2, BALB.B, or BALB.K that did not produce IFN-gamma (including Th2 cells) did not exhibit cytolytic activity, whereas most clones derived from these strains that produced IFN-gamma were cytolytic. These observations indicate that both Th1 and Th2 cells from several mouse strains express cytolytic activity. Such cytolytic activity was not restricted to clones maintained in long term cultures. However, genes outside the MHC appeared to regulate the cytolytic activity of T cells. In particular, CD4+ T cell clones which did not produce IFN-gamma were not cytolytic when they were derived from BALB/c mice and mutant or MHC congenic inbred mice having a BALB background. Cytolytic activity of CD4+ T cells, in addition to the pattern of lymphokine production, may be important in graft rejection and in immune responses to infectious diseases.

Anergized T cell clones retain their cytolytic ability.

CD4+ T cells have been described to have both helper and lytic function. The helper function of Th1 cells in particular can be inactivated by inducing the T cell into a state of nonresponsiveness in which the T cell is no longer capable of producing IL-2 or proliferating in an autocrine way to a conventional antigenic stimulus. To determine whether the lytic ability of Th1 cells can also be rendered nonfunctional upon anergy induction, we induced Th1 clones into a nonresponsive state and tested their ability to lyse target cells in an Ag-specific and MHC class II-restricted manner. We show that cells newly induced into an anergic state were able to lyse target cells nonspecifically. This effect was short-lived and after resting in culture media, the cells regained their ability to lyse target cells in an Ag/MHC-specific manner, and this ability was comparable to normal resting T cells. In contrast, the helper function of these cells remained nonresponsive, and the cells were unable to proliferate or to secrete IL-2 in response to the same antigenic stimulus used for lysis. Therefore, the lytic pathway appears to be regulated separately from the proliferative/lymphokine pathway(s) and is not affected long-term by an anergic stimulus.

Cytolytic activity of murine IL-2-producing CD4+ and CD8+ T cell clones cycles in response to IL-2.

Alloreactive or OVA-reactive cloned murine CD4+ or CD8+ T cells that produce IL-2 exhibit greatly reduced cytolytic activity after being cultured with high concentrations of rIL-2. Furthermore, such cells fail to produce lymphokines or proliferate when stimulated with Ag. The duration of this unresponsiveness to Ag correlates with the concentration of rIL-2 to which the cells were exposed; higher concentrations of rIL-2 prolong the period of unresponsiveness. The presence of ionomycin during the cytolytic assay restores lytic activity to cells rendered unresponsive by exposure to rIL-2. These results suggest that rIL-2-induced unresponsiveness to Ag is a consequence of impairment of a calcium-dependent signal important for cytolysis, proliferation, and lymphokine production. Thus, IL-2 appears to be an important lymphokine that regulates T cell responses downward as well as upward.

Regulation of T lymphocyte subsets.

Patterns of cytokine secretion and functional differences distinguish T lymphocyte subsets. T lymphocyte subsets are also regulated differentially. Most established CD8+ lymphocyte clones secrete gamma-interferon (IFN-gamma) but not interleukin 2 (IL-2) or IL-4. Using murine T cells which express a transgenic, antigen-specific alpha/beta T cell receptor (TCR) specific for L(d) class I major histocompatibility complex antigen, we have found that CD8+ lymphocytes can be divided into functional subsets. Freshly isolated CD8+ T cells are not cytolytic, do not proliferate and do not proliferate and do not secrete cytokines. Stimulation of TCR alone does not induce cytokine secretion, but cells become responsive to exogenous IL-2 or IL-4. Stimulation of CD28 together with TCR induces secretion of IL-2 and IFN-gamma, and cells proliferate without exogenous cytokines. Proliferation is necessary for the development of cytolytic activity. If IL-4 is present during initial stimulation, IL-4 is secreted following restimulation. Upon stimulation, some IL-4-producing murine CD8+ T cell clones express CD40 ligand (CD40L), and they potentiate proliferation and immunoglobulin secretion by small resting B cells. Thus, the CD8+ T cell subsets T cytotoxic 1 (Tc1) and Tc2 are analogous to CD4+ T helper 1 (Th1) and Th2. IL-2 production by naive CD8+ cells requires co-stimulation. IL-4 production by CD8+ T cells requires the presence of IL-4 during initial stimulation. Some IL-4-producing CD8+ T cells express CD40L following TCR stimulation and provide help for B cells.

Immunosuppressive effects of cyclosporin A on cloned T cells.

The effects of the immunosuppressive agent Cyclosporin A (CsA) on the immune response of T lymphocytes are not clearly understood. Much of the previous data are conflicting, possibly due to the study of bulk populations of cells. Therefore, we studied the effects of CsA on cloned murine helper T lymphocytes (HTL) and cytolytic T lymphocytes (CTL) after stimulation with Con A or with monoclonal antibodies to the cells' antigen receptors. In the HTL L2, proliferative responses and production of the lymphokines interleukin 2, interleukin 3, and interferon-gamma were blocked to background levels when CsA was added to cultures at a concentration of 0.1 to 1 microgram/ml. This inhibition of lymphokine production was found to occur at a pretranslational level, because the mRNA for these proteins was not detected when the L2 cells were stimulated in the presence of CsA. In addition, when CsA was added to cultures 3 hr after the cells had first been stimulated with the lectin Con A, levels of mRNA for the lymphokines isolated from the L2 cells 3 hr later were reduced approximately twofold. In the CTL L3, production of lymphokine (interferon-gamma) was also inhibited by CsA at a pretranslational level. However, proliferative responses to maximal stimulation with the clonotypic antibody 384.5 were not inhibited. In both HTL and CTL, the proliferative responses to recombinant IL 2 were not affected. To test whether CsA affects expression and function of the antigen receptor, we studied the effects of the drug on binding of anti-antigen receptor antibodies KJ 16-133 and 384.5 to the cell surfaces and the ability of L3 cells to lyse P815 target cells. At dosages which inhibited lymphokine production, CsA did not compete with binding of KJ 16-133 to L2 cells or 384.5 to L3 cells, as measured by flow cytometry, and the ability of L3 cells to lyse targets was unaffected. We conclude the following. CsA inhibits production of interleukin 2, interleukin 3, and interferon-gamma by L2 cells and interferon-gamma by L3 cells. This appears to occur as a result of a block in the transcription of the lymphokine genes. This pretranslational inhibition of lymphokine production can be invoked after transcription has begun. CsA does not affect expression of the T cell receptor for antigen as measured by monoclonal antibodies reactive with the receptors and by cytolytic activities of cytotoxic lymphocytes. CsA does not affect proliferative responses of HTL and CTL induced by interleukin 2.(ABSTRACT TRUNCATED AT 400 WORDS)

Activation of lymphokine genes during stimulation of cloned T cells.

To study the regulation of lymphokine production by T lymphocytes, we have characterized the activation of lymphokine genes in T cells by measuring the levels of lymphokine mRNA in cloned murine T lymphocytes after stimulation. Lymphokine mRNA was not detected in cells taken after seven days of maintenance culture. Following stimulation of T helper lymphocytes L2 and AD9.1 with concanavalin A, lymphokine mRNA appeared, reached peak levels and disappeared over a 43-h time period. A single stimulation event resulted in the induction of mRNA for interleukin 2 (IL 2), IL 3 and interferon gamma. Maximal mRNA levels were generally found at 6 h in the T helper lymphocytes, but could occur as late as 18 h. The lymphokine genes were expressed coordinately; however, in these cloned cells, IL 2 mRNA levels appeared to be lower than the other two mRNAs. Lymphokine titers in the supernatant fluids paralleled the appearance of mRNA but IL 2 titers began to fall after 12 h probably because of utilization of this lymphokine by the activated cells. In the cytolytic T lymphocyte, L3, qualitatively similar kinetics were found after stimulation by lectin or a clonotypic antibody with peak mRNA levels occurring later (18 h) with the antibody. These studies indicate a single stimulating event activates the lymphokine genes of T cells in a coordinate manner; the appearance of the lymphokines in supernatant fluids represents de novo synthesis of these proteins but the levels of lymphokines measured in supernatant fluids reflects both production and utilization rates, and exposure to IL 2 at the time of stimulation is not essential for the production of other lymphokines.