CD2, CD3, and MHC class II have been demonstrated to stimulate lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18) mediated adhesion (Van Kooyk et al., 1989, Dustin and Springer, 1989; Mourad et al., 1990). Activation of LFA-1 may be mediated by different intracellular signals generated from these stimuli, since previous findings suggest that triggering of LFA-1 through CD2 or CD3 leads to sustained and transient cell adhesion respectively (Van Kooyk et al., 1989). We investigated the role of intracellular signalling pathways in more detail. The results demonstrate that, in addition to protein tyrosine kinase (PTK) and protein kinase C (PKC) mediated signalling, increase in cytosolic-free calcium ([Ca2+]i) levels play a major role in the activation of LFA-1. The calcium ionophore Ionomycin, which increases [Ca2+]i is capable of directly activating LFA-1. Furthermore, activation of LFA-1 by triggering through CD2, CD3 or MHC class II is associated with an increase in [Ca2+]i levels, with kinetics that directly correlate with cell adhesiveness. Moreover, entry of extracellular Ca2+ via Ca-channels is involved in both the CD3- and MHC class II, as well as part of the CD2 induced LFA-1 activation. Depletion of intracellular calcium results in unresponsiveness of LFA-1 to these stimuli, further demonstrating a regulatory role for [Ca2+]i in LFA-1 mediated adhesion.

T cell-derived cytokines IFN-gamma and IL-4 have different regulatory effects on two functionally important molecules on human monocytes: MHC class II Ag and the Fc receptor for monomeric IgG, Fc gamma RI (CD64). MHC class II Ag, and Fc gamma RI are both upregulated in the presence of IFN-gamma. IL-4 induces MHC class II Ag expression but reduces Fc gamma RI expression. Recently, we showed that the cytokine IL-10 also affects MHC class II Ag expression. Here, we demonstrate that in contrast to the down-regulation of MHC class II Ag expression, IL-10 stimulates Fc gamma RI expression on human monocytes comparable to the levels of Fc gamma RI expression induced by IFN-gamma. The IL-10-induced Fc gamma RI expression is specific because anti-IL-10 antibodies completely reverse the IL-10-induced surface expression of Fc gamma RI and correlate with an enhanced capacity to lyse anti-D-coated human rhesus-positive erythrocytes. IL-10 fails to induce the expression of Fc gamma RII (CD32) and Fc gamma RIII (CD16). Furthermore, we demonstrate that IL-10 is able to prevent down-regulation in surface membrane expression of all three Fc gamma R that can be found when monocytes are cultured in the presence of IL-4. In contrast to IFN-gamma, IL-10 does not restore the reduced antibody-dependent cellular cytotoxicity (ADCC) activity of IL-4-cultured monocytes. Together, these results show that, similar to IFN-gamma, IL-10 is capable of enhancing Fc gamma R expression and ADCC activity, and that IFN-gamma, IL-4, and IL-10 have different regulatory effects on both monocyte Ag-presenting capacity and ADCC activity.

Interleukin (IL-4) can induce CD8 expression on mature CD4+ T cells. To study this phenomenon in more detail, we characterized CD8 expressed on IL-4-induced CD4+ CD8+ (double positive) T cell clones in comparison with that on CD8+ T cell clones. Using 2ST8-5H7 mAb that detects CD8 beta expression, we found that double positive T cell clones isolated with IL-4 express CD8 alpha but not beta, in contrast to CD8+ CTL cell clones, which express both chains of CD8. Northern blot analysis revealed that these double positive clones expressed CD8 alpha but not beta mRNA, indicating that CD8 alpha and beta are independently regulated at the pre-translational level. Immunoprecipitation experiments showed that CD8 expressed on a representative IL-4-induced double positive T cell clone consists mainly of homodimers of a single 34 kd protein of CD8 alpha. The amount of multimers detected from this clone was much less than that from a CD8+ CTL clone. These results suggest that persistent expression of CD8 beta is specific for the CD8+ lineage and may be involved in polymerization and stabilization of CD8 which enhances the efficiency of class I-restricted antigen recognition.

Mycobacteria elicit a cellular immune response in their hosts. This response usually leads to protective immunity, but may sometimes be accompanied by immunopathology due to delayed type hypersensitivity (DTH). A striking example in man is tuberculoid leprosy, which is characterized by high cellular immunity to Mycobacterium leprae and immunopathology due to DTH. Skin lesions of patients suffering from this disease have the characteristics of DTH reactions in which macrophages and CD4+ T lymphocytes predominate. In animal models, it has been shown that DTH responses are associated with the presence of a particular subset of CD4+ T cells (T helper type 1 [Th1]) that secrete only certain cytokines, such as interleukin 2 (IL-2), interferon gamma (IFN-gamma), and lymphotoxin, but no IL-4 or IL-5. We studied the cytokine release of activated M. leprae-reactive CD4+ T cell clones derived from tuberculoid leprosy patients. These T cell clones, which were reactive with mycobacterial heat shock proteins, exhibited a Th1- like cytokine secretion pattern with very high levels of IFN-gamma. Half of these clones secreted low levels of IL-4 and IL-5, but the ratio of IFN-gamma to IL-4 and IL-5 was much higher than that of T cell clones reactive with nonmycobacterial antigens. A Th1-like cytokine secretion pattern was also observed for T cell clones and polyclonal T cell lines from control individuals that recognized both heat shock and other mycobacterial antigens. The levels of IFN-gamma secreted by these clones were, however, significantly less than those of patient-derived T cell clones. This Th1-like pattern was not found with T cell clones from the same patients and healthy individuals generated in the same manner, but reactive with nonmycobacterial antigens. Our data thus indicate that mycobacteria selectively induce human T cells with a Th1- like cytokine secretion profile.

In the present study culture conditions resulting in optimal IgE synthesis by mononuclear cells (MNC) isolated from peripheral blood, tonsils or spleens from healthy nonallergic donors were investigated. The highest rate of IgE synthesis was obtained in a two‐step culture system in which the MNC were preincubated with interleukin 4 (IL4; 200 U/ml) for 48 h, washed and subsequently incubated with IL 4 (200 U/ml) for 9 days. Despite these culture conditions, IL4‐induced IgE synthesis varied considerably (1‐150 ng/ml) and MNC from 16/70 donors failed to produce IgE. Kinetic studies indicated that IL4 was required at the onset of the incubation phase. IgE synthesis was reduced by > 95% when addition of IL 4 in the incubation period was delayed 24 h or more. IL 4‐induced IgE synthesis was blocked by interferon‐γ (IFN‐γ). This inhibition is most effective when IFN‐γ was added in the 48‐h preincubation step or during the first 48 h of the incubation period. Interestingly, IL 4 was found to block spontaneous and lectin‐ or factor‐induced IFN‐γ production by MNC, purified CD3 + , CD4 + or CD8 + Tcells. This down‐regulatory effect of IL 4 on IFN‐γ production occurred at the mRNA transcription level. Furthermore, it is shown that IL 4 induced the release of soluble CD23 and that recombinant soluble CD23 enhanced IL4‐ induced IgE synthesis, but only when IL 4 was present at suboptimal concentrations. Collectively, our data indicate that IL4 and IFN‐γ regulate the level of IgE synthesis by influencing each other's activities reciprocally during the first 3 days of the culture.

In the present study, we investigated the lymphokine production patterns in a series of CD4+ and CD8+ host-reactive T cell clones isolated from PBL of a SCID patient, who was immunologically reconstituted by two allogeneic fetal liver and thymus transplantations 13 years ago. We demonstrate that these donor-derived T cell clones, specifically reacting with the MHC Ag expressed on the recipient cells, do not produce IL-4 and do not express IL-4 mRNA upon Ag or polyclonal stimulations. In contrast, CD4+ tetanus toxin-specific T cell clones isolated from the same patient and having the same HLA phenotype produced normal amounts of IL-4 upon activation. These data suggest that the failure to produce IL-4 is a specific characteristic of these host-reactive clones and is not due to a genetic defect of the transplanted cells. Furthermore, different modes of activation resulted in simultaneous production of IL-5, IL-2, IFN-gamma, granulocyte/macrophage-CSF, and transcription of the TNF-beta gene by the host-reactive clones, indicating that the lack of IL-4 production is not related to the mode of activation. The finding that some of these clones produce significant levels of IL-5 but no IL-4 indicates that the IL-4 and IL-5 genes are not always coexpressed in activated human T cells.

We have isolated a chromosomal DNA segment of the human IL-4 gene based on homology with a human IL-4 cDNA sequence and determined its complete nucleotide sequence. The human IL-4 gene, which occurs as a single copy in the haploid genome, is mapped on chromosome 5. It is composed of four exons and three introns and is approximately 10 kilobase pairs in size. 5'-Flanking regions of human and mouse IL-4 genes share about 85% homology extending more than 500 base pairs upstream of a "TATA" like sequence. Several patches of sequences are found in the 5'-flanking region of the human IL-4 gene which are homologous to sequence in the 5'-flanking regions of the IL-2, IL-3, IL-5, and granulocyte-macrophage (GM)-CSF genes. The IL-4 gene is inducible after treatment of human T cell clone by phorbol-12-myristate-13-acetate (TPA) and calcium ionophore A23187. The 2.3-kb 5'-flanking region of the human IL-4 gene transiently transfected into Jurkat human T cell leukemia cells is activated efficiently in response to TPA and A23187 stimulation and, although less efficiently, by human T cell leukemia virus type I-encoded p40x or BPV-encoded E2 protein. Combination of TPA/A23187 and p40x or E2 protein further augmented the level of expression.

We have isolated cDNA clones encoding human IL-3 from libraries constructed in a modified pcD mammalian expression vector by using mRNA prepared from activated human T cell clones. Amino acid sequence of human IL-3 deduced from DNA sequence of these cDNA clones agrees with that predicted from genomic sequence except at amino acid position 27. Northern blotting analysis and S1 nuclease analysis show that almost all activated T cell clones express IL-3 mRNA with kinetics similar to that observed in mouse T cell clones. However, striking difference was found in the level of granulocyte-macrophage-CSF and IL-3 mRNA expressed in activated human T cells. In contrast to mouse T cell clones, granulocyte-macrophage-CSF mRNA is expressed at least two orders of magnitude more abundant than IL-3 mRNA. Yeast Saccharomyces cerevisiae carrying human IL-3 cDNA fused downstream to alpha-factor leader sequence expressed and secreted biologically active IL-3. Several different rat anti-peptide antisera have been used to confirm the presence of human rIL-3 immunochemically. The immunoreactive human IL-3 expressed in transiently transfected COS7 cells or in yeast was observed to be heterogeneous. Human rIL-3 expressed in COS7 cells has multipotential CSF activity in semisolid cultures of bone marrow cells, and selectively induced the proliferation of My-10+ marrow or cord blood cells in liquid cultures.

The effects of p40x, a product of an human T cell leukemia virus type I, on the activation of lymphokine genes were examined. The mouse GM-CSF and IL-3 genes were activated by cotransfection with a pX containing plasmid both in Jurkat and CV1 cells. Mouse GM-CSF gene was also activated by phytohaemagglutinin A (PHA)/phorbol myristate acetate (PMA) or PMA/calcium ionophore A23187 stimulation. The 5'-flanking region of the mouse GM-CSF gene which is required for activation by pX or mitogen was mapped within 226 bp upstream from the transcription initiation site. Action of pX was not restricted to T cells. pX activated exogenously added GM-CSF, IL-2, IL-3 and IL-4 genes in fibroblasts. Activation of the GM-CSF gene in fibroblasts appears to require the same regulatory region as in T cells. Similar results were obtained using bovine papilloma virus encoded E2 protein. We propose that pX or E2 protein, both in T cells and fibroblasts, activates cellular component(s) in the signal transduction pathway which results in the activation of lymphokine genes in the absence of extracellular stimuli.

The effect of rIL-4 on the expression of low affinity receptor for the Fc part of IgE (Fc epsilon R2/CD23) and class II MHC antigens on Burkitt's lymphoma (BL) cell lines was investigated. Some of the BL lines contained low percentages of CD23 and HLA-DQ-positive cells, but virtually all cells expressed HLA-DR. IL-4 induced CD23 and class II MHC Ag expression on 7 of 9 BL. Optimal CD23 and class II MHC expression was observed after 48-72 h of incubation. Induction of CD23 and class II MHC Ag in the BL cell line BL2 by IL-4 was confirmed at the specific mRNA level. Significant activation of HLA-DQ mRNA was obtained after 6 h of incubation with IL-4 and gradually increased during prolonged incubation. Maximal induction of mRNA transcription occurred after 48 to 72 h. Optimal induction of HLA-DR and CD23 transcription in BL2 was also observed after 48 to 72 h. The induction of CD23 and class II MHC Ag seems to be specific for IL-4, because rIL-1, rIL-2, rIFN-gamma, recombinant granulocyte-macrophage-CSF, and a commercial source of low m.w. B cell growth factor were ineffective. In addition, the expression of class I MHC Ag, the transferrin receptor, CD38, CD25, CD10, CD20, and CD21 were not affected by IL-4. Interestingly, IFN-gamma and PGE2 suppressed the IL-4-induced membrane expression of CD23 and class II MHC Ag in a dose-dependent way. IFN-gamma also blocked IL-4-induced CD23 mRNA transcription in BL2 completely, whereas PGE2 (10(-7) M) was partially inhibitory. The induction of CD23 and class II MHC Ag by IL-4 required intact protein synthesis as shown by its inhibition by cycloheximide. These results indicate that the induction of CD23 and class II MHC Ag by IL-4 is regulated in a coordinated way.

CD4 and CD8 antigens are simultaneously expressed on most of the cortical thymocytes, that weakly express the T-cell antigen receptor(TCR)/CD3 complex. Mature peripheral T cells, however, strongly express the TCR complex and are positive for either CD4 or CD8. Nevertheless, a small percentage of peripheral CD3+ T cells express CD4 and CD8 simultaneously. These mature, double positive cells could be intermediates between CD4+CD8+ thymocytes and mature, single positive T cells, or they may originate from single positive T cells that acquire either CD4 or CD8. Here we report that activation and culturing of cloned CD4+ T cells in interleukin-4 (IL-4), results in the acquisition of CD8 due to its de novo synthesis. The IL-4-induced co-expression of CD8 on CD4+ T cells is reversible, in that CD8 disappeared from double positive T-cell clones isolated in IL-4, when they were cultured in IL-2. CD8 induced by IL-4 can be functional as a monoclonal antibody to CD8 inhibited anti-CD3-mediated cytotoxicity by a double positive T-cell clone.

In this study the effect of anti-cluster designation (CD) 2 monoclonal antibodies (mAb) on the activation of a cloned human T cell line, HY837, after triggering the CD3/T cell receptor (TcR) complex by anti-CD3 or anti-TcR mAb is described. HY837, which reacts with a series of mAb directed at different epitopes on the TcR, could be induced to proliferation and interleukin 2 (IL-2) production by soluble mAb directed at the CD3/TcR complex in the absence of accessory cells. mAb directed at the CD2 epitope T11-1 were shown to block the IL-2 production by HY837, as well as the expression of the IL-2 receptor, induced by anti-CD3 mAb, resulting in the inhibition of the proliferative response. The effect of anti-CD2 mAb on the proliferative response of HY837, induced by anti-CD3 mAb, was not due to a competition for Fc binding sites. In contrast, the proliferative responses and IL-2 production of HY837, induced by mAb directed at the TcR, were shown to be enhanced by the action of the anti-CD2 mAb. These results indicate that effects mediated by anti-CD3/TcR mAb cannot always be extrapolated to antigen-mediated effects and show that anti-CD2 mAb may regulate the T cell response, induced by mAb directed at the CD3/TcR complex, depending on which part of this complex is triggered during activation.