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With the discovery of a second type of T cells, those expressing T cell receptor (TCR) γδ, the question has arisen: “What contribution do these cells make to the immune system?” We review here how TCR diversity is generated at the molecular level and what this means for the capability of TCR αβ and TCR γδ to recognize a variety of antigenic peptides in the context of conventional and alternative presenting molecules. We will discuss how tissue distribution and repertoire selection of γδ T cells are in part developmentally regulated and how the repertoire may be further shaped by antigenic stimulation. Critical investigation of the causes and consequences of γδ T cell proliferation in human disease situations will hopefully provide insight into the normal functions of this elusive T cell population.

CD27 is a member of the nerve growth factor receptor family, that includes two types of tumor necrosis factor receptor, CD40 and Fas/Apo‐1. Human CD27 has been found only on lymphocytes. In T cells, its expression strongly increases in a transient fashion upon antigenic stimulation, suggesting that CD27 plays a role during T cell activation. To analyze the function of CD27, we have identified the murine CD27 at the cDNA and protein level. Murine CD27 shows an identity of 65% compared with human CD27. The amino‐terminal cysteine‐rich region, i.e. the putative ligand‐binding domain, and the carboxy‐terminal part of the cytoplasmic domain are approximately 80% identical in man and mouse. Murine CD27 has 29% identity to 4‐IBB, another lymphocyte‐specific member of the receptor family defined only at the cDNA level. Murine CD27 and 4‐1BB have 39% homology in the cysteine‐rich domain and share a conserved region in the cytoplasmic tail. Expression studies identified murine CD27 mRNA in thymus and spleen, but not in non‐lymphoid tissues, while 4‐IBB mRNA was not detected in any tissue tested. In resting T cells, only murine CD27 mRNA was found, while in activated T cells murine CD27 as well as 4‐1BB were present at high levels. Murine CD27 and 4‐1BB mRNA are expressed with different kinetics during T cell activation, suggesting that these molecules play different roles in this process. Peptide antisera identified murine CD27 as a 45‐kDa protein on thymocytes and activated T cells, while 4‐1BB was precipitated as a 35‐40‐kDa protein from activated T cells.

Attenuated non-replicating or weakly replicating viruses are being used as vaccines but they generally elicit poor T cell immunity and might not afford optimal protection. We show that the virulence of a virus leads to altered levels of T cell memory because of the differential use of stimulatory receptors. Persistently replicating virus results in selective engagement of the TNFR family molecules OX40 (CD134) and CD27. These molecules drive enhanced CD8 T cell memory, and determine the protective capacity of a virus when used as a vaccine. Therefore the length of exposure to viral antigen results in the immune system engaging novel protein interactions, which are otherwise inactive. Defining this link between virulence and immunogenicity will aid the design of safe but still effective T cell vaccines.

We have recently reported that on human B lymphocytes, membrane IgM (mIgM) associates with a heterodimer of 47- and 37-kD polypeptides, the 47-kD subunit being encoded by the mb-1 gene. We show here that expression of mb-1, both at the mRNA and the protein level, is not restricted to IgM+ B cells but can also be found in IgM- pre-B cells and mIgM-IgG+ B cells. Membrane forms of IgD and IgG, isolated from freshly isolated human B cells and B cell lines, are expressed together with heterodimeric protein structures biochemically similar to the mIgM-associated polypeptides, and these were shown to comprise the products of the mb-1 and B29 genes, or homologous genes. Finally, all three classes of antigen receptors are linked to protein kinases, capable of phosphorylating the Ig-associated heterodimers. Our findings provide insight in the structural organization of the different antigen receptors on human B cells and have implications for their function.

We investigated the capacity of T lymphocytes from a leukocyte adhesion-deficient (LAD) patient to respond to alloantigen. Leukocytes of this patient completely lacked LFA-1 surface expression due to the absence of mRNA coding for the LFA-1 beta chain. Despite the absence of LFA-1, T lymphocytes obtained from this patient, cultured with allogeneic stimulator cells (lymphoblastoid B cells JY), were capable of lysing JY cells. Furthermore, two T cell clones (one CD4+ and one CD8+), generated from this lymphocyte culture, specifically lysed the allogeneic lymphoblastoid JY cells. The cytolytic capacity of LFA-1-negative T lymphocytes and T cell clones was comparable to that of control LFA-1-positive T cells with allospecificity against JY. Detailed analysis of the CD4 positive and LFA-1-negative T cell clone demonstrated that it specifically recognized HLA-DQ. Antibody inhibition studies showed that the CTL/target cell interaction was mediated through the CD2/LFA-3 adhesion pathway. LFA-1 expressed by the target cells did not participate in the CTL/target cell conjugate formation and contributed only minimally to the cytotoxic activity. Moreover, when allogeneic LFA-1-deficient B cells, bearing the appropriate HLA-DQ alloantigen, were used as target cells, significant levels of specific cytotoxicity were measured, further excluding a role for LFA-1 in this interaction. The adhesion molecules, VLA-4, CD44 and L-selectin (LECAM1) were not involved. These results demonstrate that LFA-1-negative T lymphocytes can exert allospecific cytotoxicity and that CTL/target cell contact is mediated through the CD2/LFA-3 route. This observation may explain in part why in LAD patients viral infections, cleared largely by T cells, are less frequently observed than bacterial infections, in which phagocytic cells play a major role.

The pathogenesis of human inflammatory bowel disease (IBD) and most experimental models of IBD is dependent on the activation and expansion of CD4+ T cells via interaction with mucosal APCs. The costimulatory receptor CD70 is transiently expressed on the surface of conventional dendritic cells, but is constitutively expressed by a unique APC population in the intestinal lamina propria. We used two experimental IBD models to evaluate whether interfering the interaction between CD70 and its T cell ligand CD27 would affect the development of colitis. Adoptive transfer of naive CD27-deficient CD45RBhigh CD4+ T cells into Rag-1−/− mice resulted in significantly less disease than when wild-type CD45RBhighCD4+ T cells were used. Moreover, a monoclonal anti-CD70 Ab prevented the disease caused by the transfer of wild-type CD45RBhigh CD4+ T cells into Rag-1−/− mice and the same Ab also ameliorated an established disease. The colitis associated proinflammatory cytokines IL-6, TNF-α and IFN-γ were significantly reduced after anti-CD70 Ab treatment, suggesting an overall reduction in inflammation due to blockade of pathogenic T cell expansion. Anti-CD70 Ab treatment also suppressed trinitrobenzene sulfonic acid-induced colitis in SJL/J mice. Because anti-CD70 Ab treatment suppressed multiple proinflammatory cytokines, this may be a more potent therapeutic approach for IBD than blockade of individual cytokines.

The leukocyte function-associated molecule-1 (LFA-1) plays a key role in cell adhesion processes between cells of the immune system. We investigated the mechanism that may regulate LFA-1-ligand interactions, which result in cell-cell adhesion. To this end we employed an intriguing anti-LFA-1 alpha mAb (NKI-L16), capable of inducing rather than inhibiting cell adhesion. Aggregation induced by NKI-L16 or Fab fragments thereof is not the result of signals transmitted through LFA-1. The antibody was found to recognize a unique Ca2(+)-dependent activation epitope of LFA-1, which is essentially absent on resting lymphocytes, but becomes induced upon in vitro culture. Expression of this epitope correlates well with the capacity of cells to rapidly aggregate upon stimulation by PMA or through the TCR/CD3 complex, indicating that expression of the NKI-L16 epitope is essential for LFA-1 to mediate adhesion. However, expression of the NKI-L16 epitope in itself is not sufficient for cell binding since cloned T lymphocytes express the NKI-L16 epitope constitutively at high levels, but do not aggregate spontaneously. Based on these observations we propose the existence of three distinct forms of LFA-1: (a) an inactive form, which does not, or only partially exposes the NKI-L16 epitope, found on resting cells; (b) an intermediate, NKI-L16+ form, expressed by mature or previously activated cells; and (c) an active (NKI-L16+) form of LFA-1, capable of high affinity ligand binding, obtained after specific triggering of a lymphocyte through the TCR/CD3 complex, by PMA, or by binding of NKI-L16 antibodies.

With the discovery of T cell receptor (TCR) γδ, the question has arisen what contribution T cells expressing this receptor type make to the immune system. This question can also be formulated as follows: Do γδ T cells complement or overlap with αβ T cells with respect to 1) antigenic specificities (repertoire), 2) functions, and/or 3) sites of action within the body? In the murine and the human systems a great deal of data have been gathered regarding repertoire and tissue distribution of γδ T cells. In the mouse, there appears to be a connection between repertoire and tissue distribution, in that cells expressing TCR γδ characterized by certain Vγ and Vδ gene segments are preferentially localized in certain tissues. We have made an inventory of the tissue distribution of human γδ T cells and looked into the repertoire bias of peripheral human γδ T cells. Here, we would like to present a simplified picture of the species differences between man and mouse with respect to these two issues.

CD27 is a membrane glycoprotein expressed on human T lymphocytes. Antigen recognition by T cells transiently induces high CD27 RNA and protein levels. This is dependent on intracellular Ca2+ and cAMP concentrations, but not on protein kinase C activation. CD27 belongs to a newly defined gene family, that includes receptors for nerve growth factor and tumor necrosis factor. Homology is primarily located in the N-terminal cysteine-rich domain, thought to be involved in ligand binding. Certain members of this family share a role in induction of neuronal and/or lymphoid cell differentiation and survival. We speculate that CD27 is a receptor for a T cell growth and/or differentiation factor that acts as a progression signal during a late stage of T cell activation.

Dexamethasone treatment during the expansion phase sustains stemness of mesenchymal stem cells from human bone marrow

In human B cells, the molecules that, upon receptor occupancy, couple membrane immunoglobulin to intracellular signal transduction pathways have never been identified. We here describe two phosphoproteins as integral parts of the B cell antigen receptor complex. Membrane IgM is non-covalently associated with a disulfide-linked heterodimer of glycoproteins. These molecules can be demonstrated on B cell lines and freshly isolated polyclonal B cell populations and are subject to phosphorylation at serine residues. Identification of these constituents of the B cell receptor complex opens up the opportunity to study coupling of the B cell antigen receptor to the intracellular signal transduction machinery at the molecular level.

The CD27 membrane antigen is exclusively present on a large subset of human peripheral blood T lymphocytes and on mature thymocytes. Several observations point towards a specific role of this molecule on activated T cells. Upon T cell activation induced via the T cell receptor complex, CD27 expression greatly increases, while addition of anti-CD27 monoclonal antibodies amplifies the proliferative response. Within the CD4+ subset, only CD27+ T cells provide helper activity for B cell differentiation. Interestingly, CD27 expression differs not only quantitatively, but also qualitatively between resting and activated T cells. On resting cells, CD27 is a disulfide-linked homodimer with subunits of 55 kDa molecular mass. Upon activation, also a 55-kDa monomer seems to occur, while in addition a 32-kDa component is found. The relationship between these two proteins has been investigated. The 55-kDa and 32-kDa molecules do not seem to be physically associated. The two CD27 components are structurally highly homologous as determined by two-dimensional mapping of tryptic peptides. They both express the epitopes recognized by anti-CD27 antibodies, indicating that the 32-kDa molecule is also T cell specific. Both 55-kDa and 32-kDa molecules carry N-linked carbohydrate groups. However, they differ in other, not yet specified, post-translational modifications, and do not arise from a common precursor simply by alternative N-glycosylation. The 32-kDa form may be derived by proteolytic processing from the 55-kDa protein, but most likely not from a larger (common) precursor. Alternatively, both molecules may arise from a common gene by alternative mRNA splicing, or be derived from highly homologous genes. The dramatic change in molecular composition of CD27 suggests a newly acquired function for CD27 on activated T cells.

Classical T lymphocytes recognize foreign antigens in the context of self major histocompatibility complex (MHC) molecules by means of the T-cell receptor (TCR)alpha beta heterodimer. The genes for TCR beta-chains, like immunoglobulin genes, are subject to allelic exclusion. The introduction of a functional TCR-beta gene into the germline of mice prevents rearrangement of endogenous TCR-beta genes. Here we report that the introduction of a non-functional TCR-beta genes. Here we report that the introduction of a non-functional TCR-beta gene with a deletion of the major part of the variable region (delta V-TCR-beta), also inhibits endogenous TCR-beta gene rearrangement. This inhibition is mediated via the encoded protein because impairment of endogenous TCR-beta gene rearrangement is not found if a frameshift mutation is introduced into the DJ region of the delta V-TCR-beta transgene. The delta V-TCR-beta transgene can lead to two phenotypes, in which lymphoid development is perturbed. Phenotype A is characterized by a severe impairment of both T and B cell development as reflected by the complete absence of certain lymphoid organs. In phenotype B, lymphoid organs are macroscopically normal, but T cell differentiation is impeded. Virtually all thymocytes lack membrane expression of TCR-alpha beta, but nevertheless carry the CD4 and CD8 antigens (CD4+CD8+ phenotype); they do not, however, mature further. The defect in mice of phenotype B but not of phenotype A can be corrected by the introduction of a functional TCR-beta gene.

The human leukocyte function-associated (LFA-1) antigen, the monocyte differentiation antigen Mo-1 which is characterized as the C3bi receptor and the glycoprotein p150,95 are characterized biochemically. Immunoprecipitations carried out with 6 different monoclonal antibodies (mAb) against LFA-1 indicated that four mAb (SPV-L1, SPV-L5, SPV-L7 and SPV-L11) were directed against the alpha chain, whereas mAb CLB54 and MHM-23 were found to react with the common beta chain of LFA-1, Mo-1 and p150,95. LFA-1 and Mo-1 expressed on KG-1 cells or lymphocytes, monocytes and granulocytes from one donor were homogeneous. Interestingly the alpha chain of p150,95 showed heterogeneity. The molecular weight of the alpha chain expressed on monocytes was consistently higher than that of the alpha chain on granulocytes. The beta subunits of LFA-1 and Mo-1 (as detected by mAb Bear-1) are not only similar in molecular weight and isoelectric focusing patterns, but it is demonstrated here that they are also identically glycosylated and have similar protein backbones as judged by tryptic peptide mapping. In spite of their structural similarities. LFA-1 and Mo-1 differ completely in some of their biological functions. Anti-LFA-1 mAb strongly inhibited monocyte-dependent T cell proliferation induced by tetanus toxoid or Helix pomatia hemocyanin and pokeweed mitogen-driven specific antibody production in vitro, whereas the anti-Mo-1 antibody Bear-1 was ineffective. These results suggest that the differences in these biological functions of LFA-1 and Mo-1 may be related to their different alpha subunits, which may recognize specific counter structures.

Rhesus monkeys provide an excellent preclinical model to test the effect of monoclonal antibodies (mAb) in vitro and in vivo. So far, mostly mAb have been used which were originally raised against human cell surface antigens but cross-reacted reasonably well with homologous antigens on rhesus monkey cells. However, to optimize the model, it was necessary to produce mAb which react specifically with subsets of rhesus monkey lymphocytes. In this report, three mouse anti-rhesus monkey mAb are described, specific for different subsets of rhesus monkey T lymphocytes. None of the reagents cross-reacts with human lymphocytes. Characterization of these mAb was based upon indirect immunofluorescence, using a simultaneous staining technique, and immunoprecipitation of the specific target antigens. One antibody (GM9) reacts with the same subset as is recognized by mAb specific for human CD8+ cells. The second mAb (GM13) is specific for a subset of CD8+ cells. A third mAb (FN18) was of particular interest: it identifies a cell surface complex, RhT3, expressed on mature T lymphocytes, of which the polypeptide chains have a molecular mass of 22 and 27 kDa. The data strongly suggest that RhT3 is a CD3-like determinant, so far unidentified in the rhesus monkey.

Monoclonal antibodies allow for the detection of antigens which are specific for human thymus-derived lymphocytes. Among these antigens, the T3 complex is of particular interest since it is involved in several T cell functions. The main target antigen of the anti-T3 reagents is borne by a 20-kDa glycoprotein. In addition, glycoproteins of 25-28, 37, and 44 kDa are found in anti-T3 immunoprecipitates derived from surface-labeled cells. The four antigens appeared to be strongly associated with each other in detergent-containing solutions. Comparative studies of the four proteins, facilitated by the use of endo-beta-N-acetylglycosaminidase F, revealed that their polypeptide backbones have different molecular weights and pI values. Moreover, peptide maps of the 20-kDa T3 and the 25-28-kDa T3 were quite different. Metabolic labeling experiments suggested that the 25-28-kDa protein might become associated with the 20-kDa T3 antigen during biosynthesis. The 37-kDa and 44-kDa proteins could not, however, be detected and, therefore, might become associated with the 20-kDa T3 on the cell surface. Evidence has been found for the existence of a fifth member of the T3 complex, namely an unglycosylated 20-kDa T3 species.

Cloned blood lymphocytes that do not express the alpha- and beta-chains of the T-cell receptor show MHC-unrestricted cytotoxicity. These cells carry the gamma-protein, disulphide-linked either to another molecule or to itself, and associated with the CD3 complex. These observations may help to solve the mystery posed by the discovery of the gamma gene.

The T3/T-cell receptor complex on the surface of human thymus-derived lymphocytes consists of four glycoproteins: the alpha-chain of relative molecular mass (Mr) 40,000-50,000 (40-50K), the beta-chain (37-45K); the gamma-chain (25K) and the delta-chain (20K). The T3 alpha- and beta-chains have been identified as clonotypic T-cell receptors, but functionally the T3/T-cell receptor chains seem to form a single complex: monoclonal antibodies directed at the 20K T3 components are mitogenic for normal human T lymphocytes and, at higher concentrations, anti-clonotypic and anti-20K reagents block T-cell function. Recently, Zanders et al. showed that incubation of human T-helper clones with high concentrations of antigen abolishes antigen-specific proliferation and induces disappearance of T3 from the cell surface. Thus, the T3/T-cell receptor complex consists of two variable subunits, the T3 alpha- and beta-chains, which interact with antigen and the monomorphic 20K/25K T3 chains. Recently, the existence of a fifth polypeptide chain, the unglycosylated T3 epsilon-chain, has been postulated. Here we confirm that a 20K epsilon-chain does exist. The T3 epsilon-chain differs from the T3 delta-chain in primary structure as judged by N-terminal amino acid sequencing, peptide mapping and immunoblotting with anti-T3-delta and anti-T3-epsilon antibodies. Treatment with endoglycosidase F revealed two nonglycosylated T3 delta polypeptide backbone chains (16K and 14K) with identical amino termini. Together with previous pulse-chase experiments this observation suggests that the 14K T3 polypeptide is derived from the 16K T3 precursor by proteolytic processing near the C-terminus of the molecule.

In this report, we have undertaken the phenotypic, functional and molecular characterization of a minor (less than 5%) subpopulation of adult thymocytes regarded as the earliest intrathymic T-cell precursors. Pro-T cells were immunoselected and shown to express different hematopoietic cell markers (CD45, CD38, CD7, CD5) and some activation-related molecules (4F2, Tr, HLA class II), but lack conventional T cell antigens (CD2-1-3-4-8-). TCR-gamma RNA messages are already expressed at this early ontogenic stage, while alpha and beta chain TCR genes remain in germline configuration. In vitro analyses of the growth requirements of pro-T cells demonstrated the involvement of the IL-2 pathway in promoting their proliferation and differentiation into CD3+ CD4+ or CD8+ mature thymocytes. Moreover, during the IL-2-mediated maturation process rearrangements and expression of both alpha and beta chain TCR genes occurred, and resulted in the acquisition of alpha/beta as well as gamma/delta (either disulphide-linked or non-disulphide-linked) heterodimeric TCR among the pro-T cell progeny.

Human T cell receptor (TcR) gamma delta displays a variety of protein forms. Disulfide-linked (type 1) or non disulfide-linked (type 2) receptors occur, with gamma chains encoded by the C gamma 1 or the C gamma 2 gene segment, respectively. Exon 2 of C gamma 2 may either be duplicated or triplicated (type 2a or 2b receptors). TcR gamma chains differ in molecular mass and charge between type 1 and type 2 receptors. The delta chains as well as the gamma chains have different structural properties between receptor types. This cannot be due to the use of different C delta gene segments, since the genome encodes only one. To understand the genetic basis of this dichotomy in gamma/delta combinations, rearrangement and expression of V gamma, J gamma, C gamma and V delta gene segments were determined in TcR gamma/delta+ clones derived randomly from peripheral blood of normal donors. Most clones used C gamma 1, a minority C gamma 2. The different protein properties of receptor types could be explained by the non-random expression of V gamma (J gamma) and V delta gene segments. Type 1 receptors preferentially used gamma chains encoded by the V gamma 9 and J gamma 1.2 gene segments together with delta chains encoded by V delta 2. In type 2a receptors, V gamma 9 was not predominant; often other V gamma gene segments were employed, but then in high frequency in coordination with V delta 1. Reactivity of the clones with monoclonal antibodies anti-Ti gamma A, BB3 and delta-TCS-1 correlated with the expression of the V gamma 9, V delta 2 and V delta 1 gene segments, respectively. Therefore, V gamma and V delta use in TcR gamma/delta+ cells from peripheral blood of eight healthy individuals, including the two donors of the clones, could be determined tentatively by double immunofluorescence. Indeed, the V gamma 9-V delta 2 combination was predominant, while the V gamma 9-V delta 1 and particularly the V gamma 9-"V delta other" combination was rare. These data indicate that the TcR gamma delta repertoire in peripheral blood of normal individuals is largely dependent on junctional diversity and suggest that selection of receptors occurs.

The interaction between the TNF receptor family member CD27 and its ligand CD70 provides a costimulatory signal for T cell expansion. Normally, tightly regulated expression of CD70 ensures the transient availability of this costimulatory signal. Mice expressing constitutive CD70 on B cells had higher peripheral T cell numbers that showed increased differentiation toward effector-type T cells. B cell numbers in CD70 transgenic (TG) mice progressively decreased in primary and secondary lymphoid organs. This B cell depletion was caused by CD27-induced production of IFNγ in T cells. We conclude that apart from its role in controlling the size of the activated T cell pool, CD27 ligation contributes to immunity by facilitating effector T cell differentiation.

T-cell antigen receptors (TCR) are divided into common αβ and less common γδ types. In the murine skin, TCR γδ + cells have been reported to form the great majority of epidermal T lymphocytes. We have examined the relative contribution of TCR αβ + and TCR γδ + cells to the T-cell population in normal human skin. Serial sections of freshly frozen skin specimens were acetone fixed, incubated with anti-CD3, βF 1 (anti-TCR αβ), anti-TCR γδ-1 and anti-TCR δ1 (anti-TCR γδ) monoclonal antibodies (MoAb), and stained with a highly sensitive method. Over 90% of the T cells of normal human skin are localized around the postcapillary venules of the dermis, while less than 5% are present within the epidermis. In papillary dermis, TCR γδ + cells formed on average 7% (anti-TCR γδ-1) or 9% (anti-TCR δ1) of the total number of CD3 + cells, while TCR αβ + cells constituted up to 80%. In epidermis, these percentages were 18% and 29% for TCR γβ + cells, and up to 60% for TCR αβ + cells. It is concluded that there is no preferential immigration or in situ expansion of TCR γδ + T cells in normal human skin, because the relative percentages found for the TCR αβ + and TCR γδ + populations in skin are comparable to those found in lymphoid organs and peripheral blood. However, the percentage of TCR γδ + cells in epidermis seemed on average higher than in papillary dermis. Therefore, there may still be a difference in migration patterns of TCR γδ + v TCR αβ + cells, but this does not result in their preferential localization in human epidermis. The hypothesis that TCR γδ + T cells have a specialized function in immunosurveillance of epithelia may thus not be valid for human epidermis.

An ultrastructural analysis of human cytotoxic T lymphocyte-target cell (CTL-TC) interaction has been undertaken to enable a better understanding of the killing mechanism. Attention was focused on granules in the CTL, which are known to contain lethal compounds. Within the membrane-delimited cytotoxic granule an electron-dense core as well as numerous membrane vesicles were identified. In CTL-TC conjugates, specific membrane interactions take place, allowing the formation of intercellular clefts into which the granule cores and internal vesicles are released. T cell surface membrane molecules known to be involved in CTL-TC interaction (T cell receptor, CD3 and CD8) are present on the membranes of the granule cores and internal vesicles, facing outward. An explanation for this localization of the membrane may be found in the fact that the granule is connected with an endocytotic pathway. Moreover, the lumen of the granule is rich in the enzyme cathepsin D, which indicates an association with a lysosomal compartment. Exocytosed vesicles and cores are seen to adhere to the plasma membrane of the TC. Although the exact contents of the granule vesicles and core remain to be identified, we suggest that specific interaction of CTL membrane molecules on the cytolytic granule components with molecules on the plasma membrane of the TC may ensure the unidirectional delivery of the lethal hit.

CD3+ cells, isolated from peripheral blood of two patients with T cell acute lymphoblastic leukemia (T-ALL), did not react with the monoclonal antibody WT31, which is thought to recognize a framework determinant on the conventional T cell receptor (TcR), consisting of disulfide-linked alpha and beta chains. The T-ALL cells of neither patient synthesized TcR alpha mRNA; the cells of patient DD contained only truncated (D-J) TcR beta mRNA, while the cells of patient HZ contained truncated as well as mature (V-D-J) TcR beta mRNA. The leukemic cells of both patients made TcR gamma mRNA. At the cell surface, the T-ALL cells of patient DD expressed a CD3-associated disulfide-linked dimer, which contained the TcR gamma protein. On the leukemic cells of patient HZ the TcR gamma protein was present as a 41-44-kDa CD3-associated subunit in a noncovalently linked form. The TcR gamma genes in the T-ALL cells of patient DD were rearranged exclusively to the C gamma 1 locus, while in the T-ALL cells of patient HZ both C gamma 1 alleles were deleted and rearrangement to the C gamma 2 locus had occurred. The C gamma 1 gene segment, just like the TcR alpha and TcR beta gene segments, contains a cysteine codon in its second exon. This cysteine residue is involved in the formation of the interchain disulfide bond. The human C gamma 2 gene segment, however, does not contain a cysteine codon in its second exon. The absence of the cysteine residue in C gamma 2 encoded TcR gamma chains explains the lack of an interchain disulfide bond in the TcR on the T-ALL cells of patient HZ. The TcR gene configuration, as well as the expression of model for T cell differentiation in which the TcR gamma gene rearranges first to the C gamma 1 locus prior to or coinciding with D-J joining of the TcR beta gene, followed by rearrangement to the C gamma 2 locus and V-D-J joining of the TcR beta gene.

1. Chlorpropamide, carbamazepine and clofibrate have an antidiuretic action in patients with neurohypophyseal diabetes insipidus which is qualitatively similar to that of antidiuretic hormone (ADH). 2. An additive antidiuretic effect is produced by combination of chlorpropamide and carbamazepine with small dosages of ADH. 3. After an immediate and transient antidiuresis, a single intravenous bolus injection of lysine vasopressin given during treatment with chlorpropamide, chlorpropamide with a continuous intravenous infusion of lysine vasopressin, carbamazepine or clofibrate, resulted in increased water diuresis for 12–24 h or longer. 4. This paradoxical diuresis was not observed during treatment with chlorothiazide. 5. It is suggested that the antidiuretic action of chlorpropamide, carbamazepine and clofibrate is localized at the receptor site for ADH in the distal renal tubular cell.

Five Mab raised against the T cell antigen receptor of the human T cell line HPB-ALL which react with a subpopulation of normal peripheral blood T cells are described. Three Mab, 3D6, 1C1, and 1C2, react with 3 to 5% of normal PBL and stimulate proliferation of the cells with which they react. An increase in the number of cells which react with all five Mab occurs. Two Mab, 2D4 and 65, react with subsets of the cells which bind 1C1, 1C2, and 3D6 and divide the family into four subgroups, 2D4+ 65+, 2D4+ 65-, 2D4- 65+, and 2D4- 65-. Functional T cell clones in all four subfamilies have been observed. Cytolytic function can be correlated with the TcR phenotype expressed because all of the Mab which react with a particular clone inhibit its ability to lyse a specific target. The epitopes recognized by the panel are closely related because all five block each other's binding to HPB-ALL. In addition, the determinants recognized by 3D6, 1C1, and 1C2 on normal lymphocytes are probably very closely related because all clones examined react with all three Mab.

Eight monoclonal reagents, anti-T 1 , -T 3 , -T 4 , -T 5 , -T 6 , -T 8 , -T 9 , -T 10 , which define stages of differentiation of human thymus derived lymphocytes, were used for biochemical analysis of their target antigens. In comparing these cell surface antigens with murine markers, the following homologues are proposed: T 1 /Lyt-1, T 5 and T 8 /Lyt-2 and -3, T 6 /TL, and T 10 /Qa2. The antigens T 3 and T 8 are involved in functions which are specifically programmed in T lymphocytes (T 3 ) or the T cytotoxic subset (T 8 ). T 9 is involved in the receptor site for transferrin.

The human cell surface antigen T3 is involved in several T lymphocyte specific functions, as determined by the effect of monoclonal antibodies (OKT3, anti-Leu-4, UCHT1) directed at this molecular structure. The main target antigen of these monoclonal antibodies is a glycoprotein of 20 kDa. It is associated with four, less predominant, structurally distinct glycoproteins of 25-28 kDa, 37 kDa and 44 kDa. Of these molecules only the 20-kDa T3 antigen could be labeled with the hydrophobic reagent 5-iodonaphthyl-1-azide (INA). Here we present evidence that the main 20-kDa T3 antigen is comprised of, in fact, two structurally different molecules. One of these is a glycoprotein with a protein backbone of 14 kDa, the other is an unglycosylated protein of 20 kDa. This unglycosylated protein is labeled specifically with INA. Additional evidence for the existence of two different 20-kDa T3 antigens is provided by studies using the enzymes endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F and the drug tunicamycin. We hypothesize that the specific susceptibility to labeling with INA of the unglycosylated 20-kDa T3 form reflects a positioning in the lipid bilayer different from that of the glycosylated 20-kDa T3 form.

A second type of TCR molecule has been identified on human and murine T lymphocytes, which involves the protein products of the gamma and delta genes. T lymphocytes bearing this receptor may constitute a separate cell lineage with a distinct immune function. We have produced an mAb, which specifically detects human TCR-gamma/delta in native as well as denatured states, this in contrast to previously used anti-gamma chain peptide sera, which only reacted with denatured protein. The receptor occurs in different molecular forms, with or without interchain disulphide bonds, in which a delta chain may or may not be detected by cell surface iodination. The mAb is reactive with all these receptor forms. Therefore, this antibody could be used to determine the expression of TCR-gamma/delta on viable human T lymphocytes. In normal individuals, TCR-gamma/delta was found on a subset composing 2-7% of CD3+ lymphocytes in peripheral blood and 0.1-1.0% in thymus. The majority of these cells do not express the CD4 or CD8 antigens, although a significant percentage of CD8+ cells was found. TCR-gamma/delta+ cells in peripheral blood are resting lymphocytes, as judged by ultrastructural analysis. T cell clones with different receptor types can display MHC-nonrestricted cytolytic activity, which is shown to be induced by the culture conditions, most likely by growth factors such as IL-2. This strongly suggests that TCR-gamma/delta does not play a role in target cell recognition in MHC-nonrestricted cytotoxicity. The anti-TCR-gamma/delta antibody can specifically induce cytotoxic activity in clones expressing the receptor, but in addition inhibit growth factor induced cytotoxicity, which indicates a regulatory role of the TCR-gamma/delta/CD3 complex in MHC-nonrestricted cytotoxicity.

A monoclonal antibody, 3D6, identifies a public idiotope or allotope on the human T-cell receptor for antigen, since it not only reacts with the tumor line HPB-ALL, against which it has been raised, but also with 3–13% of peripheral blood T lymphocytes of normal donors. 3D6 + cells have been isolated from an allogeneic mixed lymphocyte culture and cloned by limiting dilution. In this way, allospecific clones were obtained both of the T4 + T8 − and the T4 − T8 + phenotype, which included proliferative as well as cytotoxic cells. Within a panel of 20 cytotoxic clones, different specificities for both class I and class II MHC antigens were found. The clones were tested for their reactivities with four additional anti-T-cell receptor antibodies raised against HPB-ALL. Two of these, 1C1 and 1C2, reacted with all 3D6 + clones. By means of two other antibodies, 2D4 and 65, the 3D6 + receptor family could be divided into four structurally distinct subfamilies. Biochemical analysis suggested that the 1C1, 1C2, 2D4, and 3D6 antibodies define epitopes on the beta chain of the receptor. Isoelectric focusing of receptor molecules isolated from cytotoxic clones with different specificites indicated that there are extensive structural differences in both alpha and beta chains of the receptors. No correlation could be found between the antigenic specificity of a clone and the structure of its receptor in this analysis. It is postulated that the 1C1, 1C2, and 3D6 epitopes may be encoded by a particular germline V beta segment, in analogy with similar, previously described findings in both the human and the murine system.

The complete amino acid sequence of one of the polypeptide chains of the human T3/T-cell receptor complex, the T3 glycoprotein (T3-delta-chain) of relative molecular mass 20,000, was deduced from cDNA clones derived from HPB-ALL cells and a human T-cell clone. Inspection of the 171-amino acid sequence reveals a signal peptide, a 79-amino acid extracellular domain, one transmembrane region and a 44-amino acid intracellular domain which show no sequence homology with members of the T-cell receptor/immunoglobulin/MHC multi-gene family. The T3 delta-chain is coded for by a single-copy gene the expression of which is restricted to T lymphocytes in humans and mice.

S5.7 recognizes a 20 kD cell surface protein which is present on T lymphocytes. S5.7 binds to a nonglycosylated protein, which can be labeled by cell-surface radioiodination and by a hydrophobic reagent [125I]-iodo-5-naphthyl-1-azide (INA). As the T-lymphocyte-specific T3 complex was found to contain a nonglycosylated 20 kD species, and since this 20 kD T3 form can be labeled preferentially by INA, a comparison between T3 and S5.7 was made. Isoelectric focusing experiments showed, however, that the two proteins are different. Moreover, the S5.7 monoclonal antibody does not block CML, is not mitogenic, reacts with immature cells of several hemopoietic lineages, and differs in that respect from anti-T3 monoclonal antibodies.

Monoclonal antibodies (mAb) reactive against the gamma/delta T cell antigen receptor (TcR) have been used to characterize the distribution and structural properties of gamma/delta TcR-bearing lymphocytes in blood and thymus. Consistent with prior reports the TcR gamma/delta-1 and delta-1 mAb react with all gamma/delta TcR+ T lymphocytes in blood and thymus. By contrast the TCS-delta mAb was found only to react with a subset of the gamma/delta TcR-bearing T cell population. Several lines of evidence suggest that this reagent preferentially reacts with the V delta 1 gene product. Using these reagents, it was observed that gamma/delta TcR+ T lymphocytes comprise 4.6 +/- 3.5% (range 1.0-16.3%) of peripheral blood lymphocytes. However, analysis of peripheral blood from normal adult donors revealed that in 29 of 32 the TCS-delta (possibly V delta 1)-bearing cells comprised less than 30% of the total gamma/delta-TcR+ population. Biochemical analysis demonstrated that the predominant form of the gamma/delta TcR in adult peripheral blood is a disulfide-linked heterodimer, indicating preferential use of the C gamma 1 gene. The delta TcR chain from these TcR-gamma/delta-1+/TCS-delta- T cells was remarkably basic in charge, as analyzed by nonequilibrium pH gradient electrophoresis. By contrast with peripheral blood the majority of freshly isolated and interleukin 2-cultured gamma/delta TcR+ thymocytes were predominantly TcR-gamma/delta-1+/TCS-delta +, and preferentially expressed V delta 1. Moreover, both disulfide-bonded and nondisulfide-bonded gamma/delta TcR heterodimers were expressed in all thymuses examined and both forms were contained within the TCS-delta + thymic subset. Similar to recent findings in the mouse, these studies suggest a possible bias in the structural form of gamma/delta TcR based on tissue location.

The progressive diversification of T-lymphocytes begins within the thymus gland. Since cell-cell interactions may play a major role in this process, the study of the expression of thymic surface markers would aid our understanding of thymic differentiation. On murine thymocytes, selective expression of genes coding for cell surface markers has been studied with alloantisera [l]. More recently, monoclonal antibodies have made possible the study of selective expression of cell surface glycoproteins on human thymocytes. Thus, as in the mouse, early and late events in the thymic differentiation have been recognized [2]. Although the precise function of these thymic differentiation antigens remains to be determined, it seems plausible that they may govern associative recognition among cooperative cells sets. One could therefore surmise that inappropriate expression of some of the thymic differentiation antigens may playa role in leukemogensis.

The cytotoxic reaction mediated by cytotoxic T lymphocytes (CTL) consists of three phases: first, the CTL binds to the target cell; next, the CTL is triggered to lyse the target cell; and in the third phase, the CTL detaches from the target cell which is lysed in the absence of the CTL. Recently, we obtained evidence that human alloreactive CTL clones initially adhere to target cells without the involvement of the interaction between the T cell receptor (Tcr) and its specific target antigen. In the present study, we investigated the effect of monoclonal antibodies specific for the Tcr on the cytotoxic reaction of three CD8+ HLA-A2-specific CTL clones, using a single cell assay in which the binding event can be distinguished from the post-binding (lytic) phase of the cytolytic reaction. It was found that monoclonal antibodies directed at a variable part of the Tcr do not affect the binding phase but strongly block the lytic phase of the cytotoxic reaction. An anti-constant region Tcr antibody and an anti-CD3 reagent had a similar effect on the two phases of the reaction as the anti-variable part Tcr antibodies. In contrast, antibodies specific for LFA-1 strongly blocked the adhesion phase but did not affect the lytic phase. Antibodies specific for CD-8 had intermediate effects. They could block both the adhesion as well as the lytic phase. The effect of anti-CD8 appeared to be dependent on the CTL clone tested. One clone was found to be inhibited in the adhesion phase, but not in the lytic phase, whereas anti-CD8 hardly blocked the adhesion phase of two other CTL clones, but affected the lytic step of those clones. Our data indicate that LFA-1 is a major adhesion molecule in the CTL reaction, whereas the Tcr/CD3 complex is implicated in a phase after the initial formation of conjugates. CD8 is associated with both steps in the cytolytic reaction. In addition to its minor role in the adhesion phase, our data suggest strongly that CD-8 is involved in the triggering phase of the cytolytic reaction.

We have analyzed T-cell receptor delta (TcR-delta) gene rearrangement and transcription in appropriately phenotyped mononuclear cells derived from 12 patients with T-cell acute lymphoblastic leukemia (T-ALL). The T-ALL cells were also analyzed for rearrangement and transcription of the T-cell receptor(TcR)-beta and gamma genes as well as for the presence of TcR-alpha gene transcripts. Four T-ALLs expressed TcR-gamma delta at the cell surface, while three expressed TcR-alpha beta. The other five T-ALLs did not express a TcR-CD3 complex on their cell membrane. The TcR-gamma delta + T-ALL had rearranged both TcR-delta gene alleles and contained mature 2.2 and 1.5 kb TcR-delta transcripts. In one case, immature 1.9 and 1.2 kb TcR-delta transcripts were also found. Furthermore they contained mature TcR-gamma mRNA, mature or immature TcR-beta mRNA, but no TcR-alpha mRNA. The three TcR-alpha beta + T-ALLs contained mature alpha and beta transcripts, but lacked TcR- delta transcripts as a result of deletion of both TcR-delta gene alleles. These data are in line with a mutually exclusive expression of TcR-alpha and -delta genes, which may be important to ensure the presence of only one type of TcR per T cell. One of the five CD3- T- ALLs had germline TcR-beta, gamma, and delta genes. The other four CD3- T-ALLs had rearranged their TcR-beta, gamma, and delta genes and contained immature 1.9 and 1.2 kb TcR-delta gene transcripts. Remarkably, one of these T-ALLs also contained TcR-alpha transcripts in addition to the immature TcR-delta transcripts, which was in line with the deletion of one TcR-delta gene allele and rearrangement of the other allele. This suggests that prevention of dual receptor expression may not only be regulated by the presence of germline TcR-alpha genes in TcR-gamma delta + cells or by deletion of both TcR-delta gene alleles in TcR-alpha beta + cells, but also via other regulation mechanisms. Finally, our data indicated that the combinatorial repertoire of the TcR-delta genes is limited, which has also been described for the TcR-gamma genes.