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Productive T cell activation leading to cytokine secretion requires the cooperation of multiple signaling pathways coupled to the TCR and to costimulatory molecules such as CD28. Here, we utilized two pharmacophores, PD98059 and FK506, that inhibit, respectively, mitogen-activated protein (MAP) kinase kinase 1 (MEK 1) and calcineurin, to determine the relative role of the signaling pathways controlled by these enzymes in T cell activation. Although the two compounds had distinctive effects on CD69 induction, they both suppressed T cell proliferation induced by anti-CD3 mAb, in a manner reversible by exogenous IL-2, suggesting that PD98059, like FK506, affects the production of, rather than the responsiveness to growth-promoting cytokines. Accordingly, IL-2 production by T cells stimulated with anti-CD3 mAb in conjunction with PMA or with anti-CD28 mAb was inhibited by both compounds. However, these compounds differentially affected the production of other cytokines, depending on the mode of activation. PD98059 inhibited TNF-alpha, IL-3, granulocyte-macrophage (GM)-CSF, IFN-gamma, and to a lesser extent IL-6 and IL-10 production but enhanced IL-4, IL-5, and IL-13 production induced by CD3/PMA or CD3/CD28. FK506 suppressed CD3/PMA-induced production of all cytokines examined here but to a lesser extent IL-13. FK506 also reduced CD3/CD28-induced production of IL-3, IL-4, IL-10, TNF-alpha, and IL-6 but augmented that of GM-CSF, IL-5, IFN-gamma, and IL-13. Therefore, the biochemical targets of PD98059 and FK506 contribute differently to the production of various cytokines by T cells, which may have implications for the therapeutic manipulation of this production.

Rapamycin (RAP) inhibits several biologic responses in the YAC-1 T cell lymphoma, including the serum-driven proliferation and cyclin A mRNA expression, the induction of Ly-6E Ag expression by IFN, and the induction of IFN-gamma production by IL-1. RAP also suppresses the enzymatic activity of the 70 kDa S6 protein kinase (pp70s6k). To define the mechanistic relationship between these multiple effects of RAP, we have generated stable somatic mutants with altered sensitivities to this drug. A first series of mutants, represented by the R19, 4R16, and 10R13 clones, showed markedly reduced sensitivity to the inhibitory effect of RAP on all biologic responses tested and on pp70s6k activity. Two other mutant types, R103 and R125, were both highly sensitive to RAP-mediated suppression of proliferation, of IL-1-induced IFN-gamma production, and of pp70s6k activity but differed in their Ly-6E response. This response was not affected by RAP in the R125 clone and was enhanced in the R103 clone. Therefore, the inhibitory effects of RAP on proliferation and IL-1-mediated IFN-gamma induction both appear associated with the inhibition of pp70s6k activity, whereas the modulation of Ly-6E induction is independent from the latter. Moreover, the cellular binding of [3H]dihydro-FK-506 was found to be blocked by RAP in all mutant types to the same extent as in wild-type YAC-1 cells, suggesting that the altered sensitivity to the effects of RAP in these mutants is not due to an inability of the drug to enter the cells or to interact with FKBP. Further biochemical characterization of the mutant cells described here is expected to help clarify the mechanisms of RAP action.

The YAC T cell lymphoma normally does not express Ly-6E mRNA or Ly-6E surface molecules but can be induced to do so on incubation with either IFN-gamma or IFN-alpha/beta. This system afforded a model to assess the possible role of protein kinase C (PKC) in IFN-mediated Ly-6E induction. First, we used various pharmacologic agents known to interfere with the function of PKC or other kinases. The PKC inhibitors H-7 and phloretin were found to block Ly-6E induction by IFN-gamma or IFN-alpha/beta both at the mRNA and protein levels. In contrast, inhibitors of cyclic nucleotide-dependent kinases (HA1004), of myosin L chain kinase (ML-9, A-3) or of calmodulin (R24157, W-7) failed to suppress this induction. Next, we investigated the effects of the PKC activators PMA and mezerein (MEZ) on Ly-6E expression. Although neither PMA nor MEZ by themselves could induce Ly-6E in YAC cells, both agents enhanced by up to fivefold the induction of Ly-6 mRNA and Ly-6E surface expression triggered by IFN-gamma. However, the induction of Ly-6E expression caused by IFN-alpha/beta was only marginally increased by cotreatment of YAC cells with PMA or MEZ. Altogether, these observations demonstrate that PKC or a related kinase is involved in the transduction mechanisms that lead to Ly-6E induction. However, activation of PKC is not sufficient for this induction and requires other unidentified signal(s) provided by IFN. Our data also indicate that IFN-gamma and IFN-alpha/beta induce Ly-6E through overlapping but distinct intracellular pathways with different sensitivities to PKC activators.

The structurally related immunosuppressive macrolides FK-506 and rapamycin (RAP) were previously shown to inhibit T cell stimulation through different mechanisms. FK-506 acts similarly to cyclosporin A (CsA) and prevents IL-2 production and IL-2R expression. RAP has little or no effect on these events but markedly impedes the response to IL-2. The present study was initiated to examine the possibility of a complementation between the immunosuppressive actions of RAP and FK-506 or CsA on various murine T cell responses. RAP potentiated the effect of CsA on proliferation and IL-2R expression in T cells stimulated with ionomycin + PMA. However, in the same system, RAP acted as a potent antagonist of FK-506 suppression. RAP also blocked FK-506- but not CsA-mediated inhibition of IL-2 mRNA induction. By using model systems sensitive to inhibition by RAP but not FK-506 we further demonstrated that FK-506 reciprocally behaves as an antagonist of RAP. In one such model, the stimulation of splenic T cells with IL-2 + PMA, FK-506, but not CsA, reversed the suppressive effect of RAP on proliferation. FK-506 also antagonized RAP-mediated inhibition with respect to the induction of Ly-6E Ag expression by IFN in YAC cells. To explore further the competition between the two macrolides at the cellular level, we performed binding experiments with a radiolabeled derivative of FK-506. Both FK-506 and RAP, but not CsA, inhibited the binding of this probe in YAC cells. Taken together, these data demonstrate that FK-506 and RAP antagonize each other's biologic activity and physically interact with a common receptor site(s) in T cells. Moreover, CsA acts at a site distinct from the cellular target(s) of FK-506 or RAP.

FK-506 and the structurally related macrolide rapamycin (RAP) were investigated in comparison with cyclosporin A (CsA) for their immunosuppressive effects on murine T cells. All three agents suppressed the proliferation of splenic T cells triggered by lectins or antibodies to CD3 and Ly-6C. FK-506 or CsA also inhibited proliferation, IL-2 production, and IL-2R expression in splenic T cells activated with ionomycin + PMA. However, RAP minimally affected IL-2 production and IL-2R expression in these cells, although it reduced proliferation. Similarly, FK-506 and CsA, but not RAP, suppressed IL-2 production by activated DO.11.10 T hybridoma cells. In such a system, as well as in normal T cells stimulated with high ionomycin concentrations, FK-506 and CsA enhanced proliferation, indicating that they both abrogate negative signals associated with T cell activation. On the contrary, RAP diminished the autonomous proliferation of hybridoma cells, whereas FK-506 and CsA had little effect. The proliferative response induced in D10.G4 cells by IL-1 + ionomycin but not that induced by IL-1 + PMA was sensitive to inhibition by FK-506 and CsA. In contrast, RAP inhibited equally well both types of stimulation. Finally, T cell proliferation driven by IL-2 or IL-4 was found to be relatively resistant to FK-506 or CsA but sensitive to RAP. Altogether, these data demonstrate that FK-506 and CsA alter similar calcium-associated events of T cell activation and block T cell proliferation primarily by suppressing lymphokine production. RAP interferes with a different set of events and inhibits T cells by impairing their response to growth-promoting lymphokines.

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

In this report we describe the reactivity patterns of two monoclonal anti-Ly-6.2 antibodies toward lymph node (LN) cells of the autoimmune MRL/Mp-lpr/lpr (lpr) and the normal congenic MRL/Mp-+/+ (+/+) mouse strains. The monoclonal antibody 34-11-3, which has previously been found to detect a LN-associated Ly-6.2 antigen, reacted with both lpr and +/+ LN cells. Another monoclonal antibody, 34-10-7, which has been demonstrated to detect a bone marrow- (BM) associated determinant, was found to react with a small proportion of +/+ LN cells and unexpectedly with a high percentage of lpr LN cells. The expression of this BM determinant found in the LN of lpr mice was age dependent, and its presence on Thy-1.2+ cells increased with the expansion of a subset of Lyt-1+2- cells. In contrast, dual labeling experiments showed that in +/+ and young lpr mice a small subset of Lyt-1+2+ cells express this BM-associated Ly6.2 determinant. Therefore these findings demonstrate that the lpr gene-dependent T cell expansion in the LN results in both aberrant expression and association of lymphocyte cell surface markers.

The NK-1.1(-) mouse was constructed by weekly injections of monoclonal anti-NK-1.1 antibody from birth through adulthood. Spleen cells from these mice have decreased NK-1.1+ cells and null (Thy-1- and B220-) cells. Their splenic NK activity to YAC targets was low and was not enhanced by IFN-alpha or IFN-beta. Bone marrow (BM) of these NK-1.1(-) mice have normal precursors to NK cells: 1) NK activity could be generated from NK-1.1(-) BM cells cultured in rIL 2 for 5 to 6 days. These cultured BM cells expressed Qa-5, Thy-1, AsGm-1, and NK-1.1 antigens. The precursor cells of these BM cytotoxic cells are NK-1.1-; 2) transfer of BM cells from the NK-1.1(-) mice reconstituted the NK activity of irradiated, NK-depleted recipients. Lymphokine-activated killer cells could also be generated from spleens of these NK-1.1(-) mice. Therefore, the NK-1.1(-) mice were specifically depleted of mature cytotoxic NK cells, but not the NK-1.1- precursors of NK cells. This mouse model is valuable to study ontogeny and physiologic relevance of NK cells.

The murine Ly-6A.2 and Ly-6E.1 antigens, which can transduce triggering signals in T cells, have been shown to become highly expressed after mitogenic stimulation. It has recently been found that enhanced expression of Ly-6A/E antigens is also induced by interferon-gamma (IFN-gamma) in resting T cells. Here, the possibility is investigated that Ly-6A/E induction on activated T cells may be due to the IFN-gamma known to be secreted by these cells. A potent neutralizing anti-IFN-gamma monoclonal antibody (mAb) (H-22.10) was used. This mAb was found to abrogate the augmentation of Ly-6A/E antigens produced in resting T cells by supernatants from T cells stimulated with concanavalin A. When added directly into cultures of T cells stimulated with concanavalin A or by the combination of ionomycin with the protein kinase C activator phorbol myristate acetate (PMA), the H-22.10 mAb inhibited Ly-6A/E enhancement without affecting the blastogenesis or the emergence of interleukin 2 receptors and transferrin receptors. Such a selective effect of the anti-IFN-gamma mAb indicated that IFN-gamma is involved in the up-regulation of Ly-6A/E antigens during T cell activation. In determining whether other activation signals, in addition to IFN-gamma receptor occupancy, may contribute to Ly-6A/E enhancement, it was found that suboptimal stimulation of BALB/c T cells provided by a 3-hr pulse with ionomycin plus PMA or by culture with PMA alone potentiated by about twofold the increase of Ly-6E.1 induced by exogenous IFN-gamma. Therefore, Ly-6A/E augmentation in activated T cells reflects primarily an action of endogenous IFN-gamma that is amplified (in BALB/c mice) by a protein kinase C-dependent step.

This report is a description of the pattern of reactivity of a rat monoclonal antibody (MAb 9F3) directed to the abnormal T cells expanding in MRL/Mp-lpr/lpr (MRL/lpr) mice. By using single- and two-color flow cytofluorometry analysis, this MAb was found to stain brightly 90 to 98% of lymph node (LN) T cells from MRL/lpr, C57BL/6-lpr/lpr (B6/lpr), and C3H/HeJ-lpr/lpr mice, and approximately 10 to 50 times less intensely 55 to 70% of T cells from control congenic +/+ mice. The study of in vitro proliferative responses of sorted cells from MRL/+ mice demonstrated that 9F3- T cells react better to phytohemagglutinin and allogeneic cells but less well to concanavalin A (Con A) than 9F3+ T cells. Upon Con A-induced blastogenesis, 65% of 9F3- T cells became 9F3+ whereas all 9F3+ remained 9F3+. Although only 15% of thymocytes, which included hydrocortisone-resistant population, were 9F3+ in +/+ mice, up to 60% of bright 9F3+ cells were detectable in the thymus of lpr-bearing mice after the onset of lymphoproliferation. Moreover, in both lpr-bearing and normal mice, the 9F3 MAb stained the totality of resting or mitogen-activated B cells. The 9F3 MAb also reacted with 100% of bone marrow (BM) cells, irrespective of the lpr gene. However, a subset of bright 9F3+, large BM cells was increased in frequency in lpr-bearing mice compared to congenic controls. Although expressed on macrophages, granulocytes, and erythrocytes, the 9F3 antigen was not in liver, kidney, and brain tissue of MRL/+ mice. Because the cell and tissue distributions of the 9F3 antigen do not correspond to any previously described murine antigen, this antigen may represent a new surface marker that should prove useful for studying lymphohemopoietic cell differentiation in normal and lpr-bearing mice.

The Ly-6C.2 molecule was purified from K36 tumor cells by affinity chromatography and gel filtration. The electrophoretically homogeneous preparation, with m.w. 15,000, was tested with a panel of antibodies that confirmed the presence of the LY-6C.2 epitope. An N-terminal sequence of 39 amino acids was obtained showing 59% homology with the corresponding portion of the Ly-6A.2 polypeptide. Based on the least homologous (29%) 14 amino acid segment, an oligonucleotide probe was constructed, and Ly-6C.2 cDNA was cloned from a BW5147 cDNA library. A 794-base pair cDNA containing the entire coding region had 82% homology with Ly-6A.2 cDNA. The encoded polypeptide sequence of 131 amino acids containing a perfect correlation with the N-terminal sequence data was 63% homologous with that of Ly-6A.2. The greatest homology was in the leader, first 16 N-terminal and last 39 C-terminal amino acids. The latter are likely to be important in determining the attachment of glycophosphatidylinositol. Despite results indicating fewer disulfide constraints in the Ly-6C molecule, the predicted sequence contains 10 cysteine residues nearly perfectly matched with those predicted in Ly-6A.

The murine Ly-6 locus controls multiple cell surface antigenic specificities with distinct cellular and tissue distributions. Although the functions of Ly-6 antigens are unknown, several of these antigens represent interesting markers of T cell differentiation and activation. In this work we used a panel of monoclonal antibodies (MAb) in conjunction with flow cytofluorometry (FCF) analysis to investigate the effect of interferon (IFN) on the surface representation of T cell-associated Ly-6 antigens. It was found that in vitro treatment of purified T cells from both C57Bl/6 (Ly-6.2) and BALB/c (Ly-6.1) mice with 10 to 10(4) U IFN-alpha/beta/ml results in a dose-dependent enhancement of Ly-6 antigen expression. This effect was already detectable after 12 to 18 hr and culminated after 48 hr of incubation. Both frequencies and brightness of Ly-6 bearing cells were increased. The most dramatic shifts were observed for the Ly-6A, D, and E antigens, which were augmented by eightfold to 20-fold upon exposure to 10(4) U IFN alpha/beta/ml. Expression of Ly-6C antigens was enhanced by fourfold to sixfold under the same conditions. Immunochemical analyses and use of metabolic inhibitors additionally demonstrated that such IFN-alpha/beta-induced phenotypic alterations of T cells reflect augmented de novo biosynthesis of Ly-6 molecules. Comparison of purified IFN-alpha and IFN-beta revealed that both are equally active in influencing Ly-6. IFN-gamma also enhanced Ly-6 expression but less efficiently than IFN-alpha/beta. Additional experiments were carried out to determine the selectivity of IFN-alpha/beta action on T cell phenotype. These studies demonstrated that IFN-induced Ly-6 enhancement occurs without emergence of interleukin 2 or transferrin receptors. Expression of H-2 and beta 2m antigens, previously known to be sensitive to IFN, was increased but to a much lesser extent than Ly-6. Most other cell surface antigens examined were minimally affected by IFN-alpha/beta with the exception of Ly-11 and Ly-23. Augmentation of these latter markers was lower than for Ly-6 antigens, however. Therefore the Ly-6 locus appears to be preferentially activated by IFN-alpha/beta in resting T cells. Additional exploration of this phenomenon should provide insight into both the biological significance of Ly-6 antigens and the mechanism(s) by which IFN affect T cell functions.

Cross-linking of cell surface Ly-6C molecules with the 6C3 rat monoclonal antibody (MAb) followed by anti-rat immunoglobulin antibody acts in concert with phorbol myristate acetate (PMA) as a potent mitogenic stimulus for normal T cells. Specificity of this stimulation was demonstrated by its absence in T cells from NZB, NOD, or STb/J mice which lack the 6C3 determinant. In 6C3+ normal strains, the extent of 6C3-mediated stimulation varied, depending on the level of 6C3 antigen expression. Analysis of this stimulation in purified T cell subsets revealed that in Ly-6.1 strains (e.g., BALB/c, CBA/J), Lyt-2+ cells responded, but not L3T4+ cells, whereas in Ly-6.2 strains (e.g., C57BL/6, MRL-+/+), both subsets produced IL 2 and proliferated, although with different kinetics. Moreover, in adult MRL-+/+ mice, the minor Lyt-2-/L3T4- subset from the lymph nodes gave low responses to 6C3 cross-linking, whereas that from the thymus reacted strongly. Stimulation via Ly-6C therefore provides a pathway for differential activation of normal T cells. In contrast, the expanding population of Lyt-2-/L3T4- T cells from lpr/lpr or gld/gld mice did not proliferate in response to 6C3 antigen cross-linking plus PMA despite high levels of 6C3 antigen expression. Responsiveness of lpr/lpr T cells could not be restored with IL 1, IL 2, or both. These T cells also failed to be triggered by conjunction of PMA with either Thy-1 antigen cross-linking or concanavalin A. Moreover, they were not stimulated, in the presence of PMA, by doses of ionomycin that were optimal for normal T cells, but did respond to higher ionomycin concentrations (2 micrograms/ml), and this response was not altered by Ly-6C cross-linking. It is concluded that the Ly-6C pathway of T cell activation is not functional in the aberrant lpr/lpr (and gld/gld) T cells, and that this defect may reflect abnormalities of intracellular signaling.

Recently, we found that a novel murine cell surface antigen recognized by the 9F3 monoclonal antibody demonstrates T cell heterogeneity in normal mice and in congenic strains homozygous for the lymphoproliferation- and autoimmunity-inducing lpr gene. The objective of the present work was to further characterize this heterogeneity by studying the distribution of the 9F3 marker among Lyt-2+ T cells. By using dual parameter flow cytofluorometry analysis, at least two subsets of peripheral Lyt-2+ cells differing in 9F3 expression could be distinguished. In MRL/Mp-++, C57BL/6, and C3H/HeJ mice, 9F3- and 9F3+ cells accounted, respectively, for 65 to 80% and 20 to 35% of the whole Lyt-2+ population. Interestingly, in lpr/lpr-bearing congenic strains, a three- to fivefold selective increase in the frequency and absolute number of 9F3+ Lyt-2+ cells was found to take place during aging. Functional analysis of Lyt-2+ cells isolated by panning revealed that in +/+ mice, these cells respond better to phytohemagglutinin and concanavalin A than in lpr/lpr mice, indicating that phenotypic changes of Lyt-2+ cells correlate with functional changes. Further evidence for the functional relevance of 9F3-defined Lyt-2+ cell heterogeneity was provided by the study of sorted cells from +/+ mice, showing that 9F3- cells exhibit higher mitogenic reactivities than 9F3+ cells. This finding suggests that 9F3+ Lyt-2+ cells in +/+ and lpr/lpr mice are functionally homologous. Together, these data indicate that, in addition to the known expansion of a population of abnormal Lyt-2-T cells, mice of the lpr/lpr genotype have an alteration of their Lyt-2+ cell population, characterized by an imbalance of 9F3-defined subsets and decreased mitogenic reactivities.

The patterns of cellular and strain reactivity of a monoclonal antibody (6C3 MAb) derived from the fusion of SP2/0 cells with splenocytes from rats immunized against MRL/MpJ-lpr/lpr T cells were characterized by using flow cytofluorometry (FCF) analysis. This MAb was found to stain 70 to 90% of T cells of mice with the lpr/lpr genotype and 20 to 60% of T cells of congenic +/+ strains. Dual-parameter FCF analysis of Lyt-2 vs 6C3 expression revealed the existence of several Lyt-2- and Lyt-2+ T cell subsets, one of which (Lyt-2- bright 6C3+) was expanded in lpr/lpr-bearing mice. The 6C3 MAb stained only 2 to 5% normal thymocytes but reacted with 40 to 50% bone marrow (BM) cells. A strain survey demonstrated the expression of the 6C3 antigen on peripheral T cells (and BM cells) of all strains examined, with the exception of NOD, NZB/B1NJ, and ST/bJ. Interestingly, in the positive strains, two types of 6C3 staining patterns of T cells were observed: bimodal or trimodal. Study of BXH and CXB recombinant inbred (RI) strains demonstrated that the bimodal and trimodal 6C3 patterns are associated with the Ly-6.1 and Ly-6.2 phenotypes, respectively. Linkage of 6C3 expression with the Ly-6 locus was confirmed by using the congenic C3H.B6-Ly-6b strain. Moreover, the 6C3 staining of T cells in Ly-6.2 strains was reduced by preincubation with the H9/25 and SK-142-446 MAb, which are known to recognize Ly-6.2-associated antigens. Therefore, the 6C3 MAb appears to detect a frame-work determinant on an Ly-6-linked antigen that is absent from T cells of NOD, NZB, and ST/bJ mice. Analysis of (NZB x C58) NX8 RI strains demonstrated a correlation between the lack of 6C3 expression on T cells and unresponsiveness in autologous mixed lymphocyte reaction (a property of NZB/B1NJ mice). The 6C3 MAb should prove useful for further genetic and biochemical analysis of the Ly-6 locus and its product(s), and for the delineation of functional subsets of T cells and BM cells in normal and lpr/lpr-bearing mice.