Interleukin-1beta-converting enzyme is a member of a family of human cysteine proteases with specificity for aspartic acid, which have been named caspases. Within this family of enzymes, transcript X (TX) and transcript Y (TY) (caspases 4 and 5, respectively) are very similar to ICE (caspase 1) and form the ICE subfamily. Given the high degree of conservation in the sequences of these proteases (more than 50% amino acid identity in the mature enzymes), it was of interest to examine whether they shared similar substrate specificities. The three enzymes, ICE, TX and TY, were therefore expressed in baculovirus-infected insect cells, as 30-kDa proteins lacking the propeptide. Automaturation into p20 and p10 subunits occurred within the cells. Active ICE, TX and TY were collected in the cell culture supernatants. In addition, their production induced the activation of an endogenous 32-kDa putative cysteine protease (CPP32) like caspase. T7-tagged ICE, TX and TY were purified by immunoaffinity and tested for their catalytic efficiency on YVAD-containing synthetic substrates and on the ICE natural substrate, pro-interleukin-1beta. TX cleaved the same synthetic substrates as ICE (Km of 90 microM and k(cat) of 0.4 s(-1) for Suc-YVAD-NH-Mec, where Suc represents succinyl and NH-Mec represents amino-4-methylcoumarin) and could cleave pro-interleukin-1beta into the same peptides as ICE but less efficiently. On the other hand, TY showed very little efficacy on the different ICE substrates (Km of 860 microM for Suc-YVAD-NH-Mec). These results show that the ICE/TX/TY subfamily has functional heterogeneity and that ICE remains the preferred enzyme for pro-interleukin-1beta cleavage.

IL-1β converting enzyme (ICE) and ICE-related proteases (IRPs) have been suggested to play a central role in apoptosis. We report the use of peptidic ICE inhibitors to reassess the role of this enzyme in the apoptosis induced by Fas or TNFα receptor ligation in Jurkat cells, U937 cells or monocytes. Our results show that inhibition of IL-1β processing can be dissociated from inhibition of apoptosis. Indeed, two out of three com-pounds active on ICE are not inhibitory for apoptosis. This shows that ICE is not required for progression in the apoptotic pathway, but that one or several IRPs are necessary. In addition, Western blot analysis of cell lysates shows that both ICE and CPP32 precursors disappear rapidly after apoptosis induction, while ICH-1L precursor remains intact. Concomitant appearance of cleavage products can be visualized for CPP32, but not for ICE, suggesting that the former is proteolytically activated. In addition, this precursor cleavage can be blocked by an ICE inhibitor active on apoptosis. Altogether, our data support the hypothesis that one or several IRPs are necessary for apoptosis and are responsible for ICE and CPP32 cleavage during this process.

Cysteine proteases of the interleukin-1 beta-converting enzyme family have been implicated in the effector process of apoptosis in several systems. Among these, CPP32 has been shown to be processed to active enzyme at the onset of apoptosis. Here, we show that CPP32 precursor is cleaved into its active form during phytohaemaglutinin A activation of T lymphocytes. Maximal processing is observed between day 3 and day 4 following addition of mitogen and is a transient process. Precursor cleavage is associated with the appearance of a CPP32-like enzymatic activity in cell lysates. At this time in the culture, almost no apoptotic cell and no dead cell can be detected, and T lymphocytes are actively proliferating. CPP32 processing also occurs when lymphocytes are stimulated through an allogeneic primary mixed lymphocyte reaction, Our results suggest that proteolytic activation of CPP32 could be a physiological step during T lymphocyte activation. In addition, these data indicate that CPP32 activation can occur independently of programmed cell death in T lymphocytes.

We have generated a series of monoclonal antibodies (mAb) using recombinant interleukin (IL)-1 beta-converting enzyme (ICE) p20 and p10 subunits as immunogens. The mAb have been selected for further study based on their reactivity with ICE in transfected COS cells and their lack of cross-reactivity with TX, the closest ICE homolog known to date. Two anti-p20 and one anti-p10 mAb have been used to study ICE expression by Western blotting and immunodetection. In ICE-transfected COS cells, the mAb recognize the p45 ICE precursor and the maturation products (p20 or p10 subunits) for which they are specific. In monocytes and cell lines expressing ICE, only precursor forms are detected and intracellular immunostaining followed by confocal microscopy shows that they are located in the cytoplasm. Quantification experiments show that THP1 cells express approximately 67,000 molecules of ICE precursor per cell, with an estimated precursor to mature ratio of at least 100. In these cells as well as in monocytes, lipopolysaccharide stimulation did not change the pattern of ICE expression, although efficient secretion of mature IL-1 beta was measured. However, upon cell disruption, precursor maturation was observed. Our results, therefore, show that ICE is present in cells as a large pool of intracytoplasmic precursor, and that very limited amounts of mature ICE protein are present, but nevertheless sufficient to allow efficient IL-1 beta cleavage. Altogether, these observations suggest that post-translational maturation of the precursor protein could represent a specific step in the regulation of ICE enzymatic activity.

We have identified a novel cDNA encoding a protein (named TX) with > 50

T lymphocytes express either the alpha/beta or the gamma/delta receptor (TCR) in a mutually exclusive fashion. Both structures are associated on the cell membrane with the CD3 proteins which are thought to transduce signals resulting from antigen recognition. The CD3 complex is present in both alpha/beta and gamma/delta cells and includes at least five proteins (designated gamma, delta, epsilon, zeta and eta). We have developed here a novel mAb, anti-CD3.TCR1, which immunoprecipitates the CD3 molecules from both alpha/beta and gamma/delta cells lysates following solubilization with Triton X-100. While the SDS-PAGE migration profile of the material recognized by either anti-CD3.TCR1 or anti-OKT3 are superimposable in both cell types, this mAb recognizes viable untreated gamma/delta T lymphocytes exclusively. These findings further support the view that molecular interactions within the TCR/CD3 protein complex are distinct in the two T lymphocyte populations.

The lymphocyte activation gene 3 (LAG-3), expressed in human activated T and natural killer (NK) cells, is closely related to CD4 at the gene and protein levels. We report here the initial characterization of the LAG-3-encoded protein. We have generated two monoclonal antibodies after immunization of mice with a 30-amino acid peptide that corresponds to an exposed extra loop region present in the LAG-3 immunoglobulin-like first domain. The reactivity of these reagents is directed against LAG-3 since they recognize both membrane-expressed and soluble recombinant LAG-3 molecules produced in a baculovirus expression system. The two antibodies are likely to react with the same or closely related epitope (termed LAG-3.1) exposed on the LAG-3 first domain extra loop, as assessed in competition experiments on LAG-3-expressing activated lymphocytes. Cellular distribution analysis indicated that the LAG-3.1 epitope is expressed on activated T (both CD4+ and CD8+ subsets) and NK cells, and not on activated B cells or monocytes. In immunoprecipitation experiments performed on activated T and NK cell lysates, a 70-kD protein was detected after SDS-PAGE analysis. 45-kD protein species were also immunoprecipitated. Both the 70- and 45-kD proteins were shown to be N-glycosylated. In Western blot analysis, only the former molecule was recognized by the anti-LAG-3 antibodies, demonstrating that it is LAG-3 encoded. These anti-LAG-3 antibodies were used to investigate whether the LAG-3 protein interacts with the CD4 ligands. By using a high-level expression cellular system based on COS-7 cell transfection with recombinant CDM8 vectors and a quantitative cellular adhesion assay, we demonstrate that rosette formation between LAG-3-transfected COS-7 cells and human leukocyte antigen (HLA) class II-bearing B lymphocytes is specifically dependent on LAG-3/HLA class II interaction. In contrast to CD4, LAG-3 does not bind the human immunodeficiency virus gp120. This initial characterization will guide further studies on the functions of this molecule, which may play an important role in immune responses mediated by T and NK lymphocytes.

Previously we have shown that a small fraction of human peripheral T cells expresses a surface receptor recognized both by the BMA031 mAb, specific for a TcR alpha/beta framework epitope, and by the A13 mAb, putatively specific for an epitope encoded by the V delta 1 gene segment. An interleukin 2-dependent polyclonal cell line (termed T2) was derived from such A13+BMA031+ circulating lymphocytes. The molecular characterization of the TcR chains expressed by T2 cells demonstrated indeed that the V delta 1 gene (one of the two major V delta genes) was transcribed with the C alpha gene segment. In the T2 polyclonal cell line, distinct V delta 1/C alpha transcripts were all found to include the same J alpha segment suggesting the existence of "hybrid" TcR alpha/delta chains encoded by unique V delta 1/J alpha rearrangements. The present study was designed to characterize further the V delta 1/J alpha rearranged genes expressed in A13+BMA031+ cells. Three additional cell lines were generated from peripheral blood of distinct adult healthy donors. Using the anchored polymerase chain reaction, it was found that 17 different J alpha segments were used in the 20 V delta 1J alpha C alpha transcripts which have been studied. Together, these data indicate that V delta 1 is a "mixed" (i.e. alpha/delta) TcR V segment which can join with most (if not all) J segments in the alpha/delta locus. In addition, it can be definitely concluded that the A13 mAb recognizes a V delta 1-encoded antigenic determinant and not a V delta 1J epitope (i.e. it can be defined and used as an anti-V delta 1 mAb, as opposed to reagents such as for example delta-TCS-1).

It has been shown recently that one gamma/delta cell line, termed IDP2, derived from a immunodeficient patient recognizes the CD1c molecule on the surface of target cells. In light of these data, we have tested 43 cloned and 11 polyclonal gamma/delta cell lines derived from peripheral blood of 19 donors following nonspecific mitogenic stimulation. In this panel, which included lymphocytes expressing various combinations of gamma and delta chains, only one clone, termed J2B7, was found to interact with target cells via a CD1c-dependent recognition pathway. These J2B7 lymphocytes have, like IDP2, a delta chain which results from the frequent V1/J1 rearrangement while they use a distinct V gamma gene segment. The data support the view that the CD1c major histocompatibility complex "class I-like" gene product does not have a pivotal contribution to the repertoire of peripheral blood gamma/delta cells in adult individuals.

In the present study, we have characterized the reactivity of two mAbs that are directed at the human TCR-gamma/delta. These reagents, designated anti-A13 and anti-TiV delta 2, were found to recognize antigenic determinants encoded by the TCR V delta 1 and V delta 2 gene segments, respectively. Immunofluorescence analyses performed with the antibodies confirmed that, in the TCR-gamma/delta+ cell subpopulation, the expression of V delta 2+ delta chains is largely predominant, as compared with the V delta 1+ counterparts. However, these experiments led to an apparently discrepant finding. Indeed, the total number of cells recognized by the anti-A13 plus the anti-TiV delta 2 antibodies was often greater than that detected with anti-TCR-delta 1, a reagent specific for a constant epitope of the human delta chain. Further investigation showed that the presence of a sizeable peripheral lymphocyte subset coexpressing the BMA031 and the A13 epitopes. Because the former antibody is known to recognize an invariant antigenic determinant of the TCR-alpha/beta dimer, these results suggested that the V delta 1 gene segment may be expressed with either C delta or C alpha. This hypothesis was confirmed using T2, an IL-2-dependent BMA031+ A13+ polyclonal cell line developed from peripheral blood of a healthy adult donor. Indeed, T2 cells were found to have productively rearranged the V delta 1 gene. Together, results of Northern blot analysis and cDNA cloning indicated that V delta 1 was expressed in these cells as part of a 1.6-kb full-length message including J alpha-C alpha segments.

We have studied two gamma/delta T cell clones, E102 and E117, generated in a mixed lymphocyte culture using an allogeneic Epstein-Barr virus-transformed B cell line, E418. These clones were both found to express a molecular form of T cell receptor (TCR) infrequent in human peripheral blood, associating a V1-J1-C delta chain and a V3-JP2-C2 gamma chain. Functionally, they appeared as cytotoxic T lymphocytes (CTL) with non-major histocompatibility complex (MHC) (class I and II) requiring cytotoxicity, able to kill both the immunizing (i.e., E418) and unrelated (e.g., K562, REX, F601, and KAS) target cells. A monoclonal antibody, anti-10H3, able to selectively inhibit the cytotoxic activity of the clones has been produced. This reagent defines a 43-kD molecule, designated TCT.1, with broad distribution in the hematopoietic system, that appears to be distinct from class I MHC gene products. A series of functional experiments using various effector/target cell combinations strongly suggested that TCT.1 may represent a unique TCR ligand involved in the interaction between these particular CTL clones and certain of the target cells tested, while others were likely to be recognized and killed through a TCR-independent natural killer-like pathway. Although further experimentation will be needed to strengthen our interpretation of the present data, this study provides additional evidence that some T lymphocytes, in particular of the gamma/delta type, may interact specifically with target cells in a non-MHC class I/II-requiring fashion.

We have assessed the organization of T cell gamma rearranging genes (TRG) in circulating TcR gamma/delta+ lymphocytes which do not express V gamma 9-encoded Ti gamma A+ gamma chain. Following purification of the minor TcR gamma/delta+ Ti gamma A- fraction, cloned cell lines were developed from peripheral blood of 5 individuals. Out of the 26 clones studied, only 3 TcR gamma/delta+ Ti gamma A- cells were found to express a disulfide-linked C1-encoded gamma chain. The remaining 23 Ti gamma A- clones with a C2-encoded nondisulfide-linked receptor were found to display rearrangements of various V genes to J2 segments on both chromosomes; there was no predominance of a unique rearrangement even though the TRG-V3 and -V4 genes belonging to subgroup I were frequently employed. Together, these findings further strengthen the hypothesis that lymphocytes with a C gamma 1 encoded chain are produced earlier in T cell ontogeny than the C gamma 2 counterparts. The "non-major histocompatibility complex (MHC) requiring" (i.e., "natural killer-like") cytotoxicity mediated by many TcR gamma/delta+ Ti gamma A- cells appeared to be very low as compared to that of Ti gamma A+ clones. Yet, treatment by the OKT3 monoclonal antibody revealed a strong lytic potential in the Ti gamma A- lymphocytes with little, if any, natural killer-like activity. Thus, with respect to the latter function, a substantial heterogeneity is found in cells expressing distinct gamma chains. In an attempt to characterize undefined specificities of Ti gamma A- lymphocytes, they were screened against a panel of Epstein-Barr virus-transformed B cell lines homozygous for HLA-DR1 to DR10 determinants; one of the clones was found to recognize DR7. In light of reports from other groups describing class I-related specificities, it is apparent that TcR gamma/delta+ lymphocytes are able, like the TcR alpha/beta+, to recognize and kill target cells through either an MHC-dependent (with involvement of either class I or class II gene products) or a non-MHC-requiring pathway.