Delta Chain Research Articles

Molecular structures of cattle T-cell receptor gamma and delta chains predominantly expressed on peripheral blood lymphocytes.

Molecular structures of cattle T-cell receptor gamma and delta chains predominantly expressed on peripheral blood lymphocytes.

Malignant lymphoma originating from the earliest T‐lineage precursor cell

The earliest T-lineage precursor cells with a CD3-CD4+CD8- phenotype and germline-state T-cell receptor (TCR) genes have recently been identified in the murine thymus. We report a case of malignant lymphoma that was considered to originate from the human counterpart of this newly defined murine T-cell population. The surface phenotype of the lymphoma cells was CD2+CD3-CD4+CD8-CD25-CD34-CD44+HLA-DR+. TCR-beta, -gamma, -delta chain genes and immunoglobulin heavy and light chain genes were all in a germline state. These characteristics were markedly similar to those of the murine earliest T-lineage precursor cells. The present case thus strongly suggests the presence of the human counterpart of this novel murine T-cell population.

Direct sequence analysis of TCR V delta 2-D delta 3 rearrangements in common acute lymphoblastic leukaemia and application to detection of minimal residual disease.

T cell receptor delta chain (TCR delta) gene rearrangements were studied by Southern blot analysis in 36 patients with common acute lymphoblastic leukaemia, including 14 adults and 22 children. The majority of patients (68%) had either a rearrangement or deletion of one or more TCR delta genes. The most frequent rearrangement involved a partial recombination of V delta 2 to D delta 3 (55%). D delta 2-D delta 3 rearrangements were present in five patients (14%). To investigate the TCR delta rearrangement as a tumour marker in minimal residual disease studies, presentation samples from 18 patients were amplified by PCR and directly sequenced. Although the size of the V delta 2-D delta 3 junction varied by only 40 bp, sequence analysis showed extensive diversity. This was derived from four factors: deletion of the 5' end of D delta 3 gene (15/18) and 3' end of V delta 2 gene (16/18); the presence of D delta 2 sequences (6/18); insertion of N nucleotides (15/18); association of P nucleotides with intact V delta 2 and D delta 3 genes (5/18). N nucleotides were the major feature, contributing to 75% of the junction. D delta 1 sequences were not involved. Twenty base oligonucleotide probes, constructed from the junctional sequences, were capable of detecting residual tumour cells at the 10(-4) sensitivity level. Cross hybridization studies confirmed the probes to be clone specific. Longitudinal studies on patients undergoing treatment were capable of detecting tumour in remission samples.

Interaction of cutaneous stromal cells and γ/δ T cell receptor (TcR)‐positive cells. I. Vγ5−γ/γTcR+ T cells migrating from organ‐cultured murine skin proliferate by co‐culture with cutaneous stromal cells in the presence of interleukin‐2

It has been reported that Thy-1+CD3+CD4-CD8- cells as well as Langerhans cells migrate from organ-cultured murine skin into culture medium. We examined whether these Thy-1+ populations of migrating cells were derived from Thy-1+ dendritic epidermal T cells (Thy-1+ DEC) and found that they were Thy-1+CD3+CD4-CD8-gamma/delta TcR+ (gamma delta+T) cells but did not express V gamma 5TcR, which was used by a vast majority of Thy-1+ DEC. Recently, a unique interaction between stromal cells and lymphohemopoietic progenitors has been reported in bone marrow and thymus. In this study, we established fibroblastoid cutaneous stromal cell (CSC) lines and clones from murine skin and examined the interaction between CSC and gamma delta+T cells. When these gamma delta+T cells were co-cultured with CSC, a marked proliferation of small lymphoid cells was observed only in the presence of interleukin (IL)-2. Neither CSC alone nor IL-2 alone could induce a similar proliferation. Flow cytometry revealed that they were Thy-1+CD3+CD4-CD8-gamma/delta TcR+ but V gamma 5TcR-. Analysis of the major segments of their TcR by polymerase chain reaction demonstrated that V gamma 1, V gamma 2, V gamma 4 and all of the V delta chains from V delta 1 to V delta 7 were used without any predominant pattern. These data indicate the possible presence of gamma/delta+T cells other than V gamma 5TcR+Thy-1+ DEC in the murine skin and the unique capacity of the CSC to support the growth of these migrating gamma/delta+ T cells. The nomenclature of murine T cell receptor gamma chain is according to Reilly et al. (Nature 1986. 321:878). The relationship between the different nomenclature systems is summarized in Takagi et al. (J. Immunol. 1989. 141:2112).

Associations between subunit ectodomains promote T cell antigen receptor assembly and protect against degradation in the ER

The T cell antigen receptor (TCR) is an oligomeric protein complex made from at least six different integral membrane proteins (alpha beta gamma delta epsilon and zeta). The TCR is assembled in the ER of T cells, and correct assembly is required for transport to the cell surface. Single subunits and partial receptor complexes are retained in the ER where TCR alpha, beta, and CD3 delta chains are degraded selectively. The information required for the ER degradation of the TCR beta chain is confined to the membrane anchor of the protein (Wileman et al., 1990c; Bonifacino et al., 1990b). In this study we show that the rapid degradation of the TCR beta chain is inhibited when it assembles with single CD3 gamma, delta, or epsilon subunits in the ER, and have started to define the role played by transmembrane anchors, and receptor ectodomains, in the masking proteolytic targeting information. Acidic residues within the membrane spanning domains of CD3 subunits were essential for binding to the TCR beta chain. TCR beta chains and CD3 subunits therefore interact via transmembrane domains. However, when sites of binding were restricted to the membrane anchor of the TCR beta chain, stabilization by CD3 subunits was markedly reduced. Interactions between membrane spanning domains were not, therefore, sufficient for the protection of the beta chain from ER proteolysis. The presence of the C beta domain, containing the first 150 amino acids of the TCR ectodomain, greatly increased the stability of complexes formed in the ER. For assembly with CD3 epsilon, stability was further enhanced by the V beta amino acids. The results showed that the efficient neutralization of transmembrane proteolytic targeting information required associations between membrane spanning domains and the presence of receptor ectodomains. Interactions between receptor ectodomains may slow the dissociation of CD3 subunits from the beta chain and prolong the masking of transmembrane targeting information. In addition, the close proximity of TCR and CD3 ectodomains within the ER may provide steric protection from the action of proteases within the ER lumen.

Molecular cloning of porcine T cell receptor α, β, γ and δ chains using polymerase chain reaction fragments of the constant regions

Fragments of the constant regions of porcine alpha, beta, delta and two types of gamma T cell receptor (TcR) chains were obtained by reverse transcription and polymerase chain reaction (PCR). Screening of a porcine peripheral T cell cDNA library with these PCR fragments led to the isolation of porcine TcR alpha, beta, gamma and delta chain clones. Sequence analysis of these clones and the respective PCR fragments demonstrated the existence of one alpha, one beta, three gamma and one delta chain isotype. Comparisons of the deduced amino acid sequences of the constant region with other species revealed a significant homology. For two of the three identified porcine gamma chain insertions of 38 and 40 amino acids were found within the hinge region. In addition, our sequence data demonstrate a high variability in the cytoplasmic C gamma domain among the three porcine TcR gamma isotypes as well as between species, which might be of structural and/or functional significance. Comparison with biochemical data indicate the existence of four porcine TcR gamma isotypes.

Peripheral selection of antigen receptor junctional features in a major human gamma delta subset.

Recent studies demonstrating the existence of murine gamma delta T cell subsets with structurally identical T cell receptors (TcR) suggest that unlike alpha beta T cells, some gamma delta T cells are specialized in the recognition of a limited number of monomorphic antigens. However, this question still remains open in humans, since the TcR structural diversity of their peripheral gamma delta T cells was shown to be extensive. Here we have analyzed in detail the TcR chain genes expressed by human V gamma 9+V delta 2+ peripheral blood lymphocytes (PBL), a major peripheral gamma delta T cell subset in adults and present evidence for an antigen-driven peripheral selection of both TcR gamma and delta junctional motifs among these cells. First, it is shown that the proportion of V gamma 9+V delta 2+ cells expressing the V9JPC1 gamma chain is much higher among PBL than among thymus-derived clones, indicating that preferential use of this J gamma segment is not due to pairing or combinatorial constraints. Second, analysis of V9JPC1 gamma transcripts derived from V gamma 9+V delta 2+ PBL clones revealed a high prevalence of a unique V9JP gamma sequence with limited "N" nucleotide additions and VJ trimming, which could not be accounted for by enzymatic or antigen-independent structural limitations. Third, the TcR delta chain expressed by most V gamma 9+V delta 2+ PBL clones, though diverse in amino acid composition and length, carried a highly distinctive junctional motif, found at a much lower frequency among V2DJ delta sequences derived from V gamma 9-V delta 2+ PBL or V gamma 9+V delta 2+ thymocytes. Together, these results which demonstrate shared gamma and delta junctional features by cells using unique V gamma and V delta genes, suggest that in vivo selection of V gamma 9+V delta 2+ lymphocytes is mediated by a highly restricted number of nominal ligands.

Double-negative (CD4- CD8-) T cells from adult T-cell leukemia patients also have poor expression of the T-cell receptor alpha beta/CD3 complex

We present four patients with adult T-cell leukemia (ATL) derived from a novel T-cell subset (CD4-, CD8- [double-negative, DN], T-cell receptor [TCR] alpha beta+). In the ATL cells of these patients, neither gene nor surface expression of CD4 and CD8 antigens was detected. Clinical and laboratory data showed no difference between DN-ATL and CD4+ATL patients. In contrast to typical CD4+ATL cells, DN-ATL cells were shown to express the protein and messenger RNA (mRNA) for S100 beta in immunocytochemical assay and the reverse-transcription polymerase chain reaction assay. The mean fluorescence intensity of the TCR/CD3 complex was extremely low in all four DN-ATL patients as well as in typical CD4+ ATL. All four patients had TCR beta and gamma chain gene rearrangements, with deletion of TCR delta chain gene and mRNA expression for TCR alpha, beta, and CD3 delta but not for TCR gamma and delta chain genes. Thus, CD4- CD8- TCR alpha beta T cells are also a target for human T-cell lymphotrophic virus type I-induced leukemogenesis. In addition, expression of the TCR alpha beta/CD3 complex on the DN-ATL cells was further diminished by the addition of anti-CD3 or anti-TCR alpha beta monoclonal antibody. These results suggest that the decreased expression of the TCR alpha beta/CD3 complex by ATL cells plays a key role in the development of ATL, irrespective of CD4 expression.

Double-Negative (CD4– CD8–) T Cells From Adult T-Cell Leukemia Patients Also Have Poor Expression of the T-Cell Receptor αβ/CO3 Complex

Abstract We present four patients with adult T-cell leukemia (ATL) derived from a novel T-cell subset (CD4-, CD8- [double-negative, DN], T-cell receptor [TCR] alpha beta+). In the ATL cells of these patients, neither gene nor surface expression of CD4 and CD8 antigens was detected. Clinical and laboratory data showed no difference between DN- ATL and CD4+ATL patients. In contrast to typical CD4+ATL cells, DN-ATL cells were shown to express the protein and messenger RNA (mRNA) for S100 beta in immunocytochemical assay and the reverse-transcription polymerase chain reaction assay. The mean fluorescence intensity of the TCR/CD3 complex was extremely low in all four DN-ATL patients as well as in typical CD4+ ATL. All four patients had TCR beta and gamma chain gene rearrangements, with deletion of TCR delta chain gene and mRNA expression for TCR alpha, beta, and CD3 delta but not for TCR gamma and delta chain genes. Thus, CD4- CD8- TCR alpha beta T cells are also a target for human T-cell lymphotrophic virus type I-induced leukemogenesis. In addition, expression of the TCR alpha beta/CD3 complex on the DN-ATL cells was further diminished by the addition of anti-CD3 or anti-TCR alpha beta monoclonal antibody. These results suggest that the decreased expression of the TCR alpha beta/CD3 complex by ATL cells plays a key role in the development of ATL, irrespective of CD4 expression.

Divergent evolution of T cell repertoires: extensive diversity and developmentally regulated expression of the sheep gamma delta T cell receptor.

Sheep gamma delta T cells express an unprecedented repertoire of antigen receptors contributed by increased diversity in both variable and constant region gene segments. Variable region diversity results mainly from the utilization of a large family of duplicated V delta genes that have retained two distinct hypervariable segments comparable with the complementarity determining regions present in other antigen receptor V genes. This implies that sheep V delta chains have been intensely selected during evolution, probably at sites involved in ligand recognition. The sheep gamma delta heterodimer occurs in at least five isotypic variants formed by the association of a single C delta segment with one of five functional C gamma segments, each with distinctive hinge regions. Our analysis also shows that the establishment of a normal peripheral repertoire is both developmentally regulated and dependent on the continual presence of a functional thymus during ontogeny. The existence of an expanded V gene repertoire and multiple receptor isotypes together with the prominence of gamma delta T cells in the sheep immune system argues that this lineage of T cells has a more elaborate functional role in this evolutionary pathway.

Human fetal liver gamma/delta T cells predominantly use unusual rearrangements of the T cell receptor delta and gamma loci expressed on both CD4+CD8- and CD4-CD8- gamma/delta T cells.

Substantial numbers of both alpha/beta and gamma/delta T cells are present in human fetal liver, which suggests a role of the fetal liver in T cell development. The diversity of fetal liver T cell receptor (TCR) gamma and delta chain rearrangements was examined among both CD4+CD8- and CD4-CD8- gamma/delta T cell clones. In addition, TCR delta chain transcripts from three fetal livers were sequenced after polymerase chain reaction amplification of TCR delta chains with V delta 1 or V delta 2 rearrangements. Five of six fetal liver gamma/delta T cell clones had a V delta 2-D delta 3-J delta 3 gene rearrangement with limited junctional diversity; three of these clones had an unusual CD4+CD8- phenotype. V delta 2-D delta 3-J delta 3 gene rearrangements were also common among both in-frame and out-of-frame transcripts from three fetal livers, indicating that they are the result of an ordered rearrangement process. TCR gamma chain sequences of the fetal liver gamma/delta T cell clones revealed V gamma 1-J gamma 2.3, V gamma 2-J gamma 1.2, and V gamma 3-J gamma 1.1 rearrangements with minimal incorporation of template-independent N region nucleotides. TCR gamma chain rearrangements found in these fetal liver T cell clones were different from those that have been observed among early thymic gamma/delta T cell populations, while similar TCR delta chain rearrangements are found among gamma/delta T cells from both sites. These data demonstrate that the fetal liver harbors gamma/delta T cell populations distinct from those found in the fetal thymus, suggesting that the fetal liver is a site of gamma/delta T cell development in humans. These unusual T cell populations may serve a specific function in the fetal immune system.

Putative prethymic T cell precursors within the early human embryonic liver: a molecular and functional analysis.

Hematopoietic cells present in the liver in early human fetal life were characterized by phenotypic analysis using a broad panel of monoclonal antibodies. Expression of very late antigen 4 and leukocyte function-associated antigen 3 cell adhesion receptors and 4F2 cell activation molecules was found in all fetal liver hematopoietic cells before acquisition of T cell-, B cell-, or myeloid-specific surface markers, and before the time of intrathymic colonization. Molecular studies showed that expression of the interleukin 2 receptor beta (IL-2R beta) also occurred in the embryonic liver at this early ontogenic stage. In contrast, no expression of IL-2R alpha or IL-2 transcripts was found in fetal liver cells, whereas transcription of the IL-4 gene was detected in a small fetal liver cell subset. Putative T cell precursors were identified among the hematopoietic fetal liver cells by the expression of genes encoding the gamma, delta, epsilon, and zeta invariant chains of the CD3-T cell receptor (TCR) complex. However, no transcription of the polymorphic alpha and beta TCR genes was detected. Functional in vitro assays further demonstrated that fetal liver hematopoietic cells from those early embryos were capable of proliferating in response to T cell growth factors, including IL-4 and IL-2. However, whereas IL-4-induced proliferation paralleled the appearance in vitro of CD45+CD7-CD4dull cells expressing the CD14 myeloid antigen, as well as of CD34+ primitive hematopoietic progenitors, differentiation into CD45+CD7+CD8+CD3- immature T cells was observed when using IL-2. Moreover, coculture with thymic epithelial cell monolayers provided additional evidence that early fetal liver hematopoietic cells may include very primitive T cell precursors, which were able to differentiate in vitro into TCR alpha/beta+ mature T cells. Therefore, our results indicate that, after triggering of the T cell-specific maturation program in primitive fetal liver hematopoietic progenitors, specific signals provided intrathymically by epithelial cells may fulfill the requirements to drive terminal differentiation of prethymically committed T cell precursors.

The diversity of in-frame TCR delta chain transcripts in aging SCID mice.

From spleen, liver, bone marrow, and gut of old C.B-17 scid/scid (SCID) mice, TCR delta chain transcripts were amplified by the polymerase chain reaction (PCR) using V delta 1-, V delta 2-, V delta 3-, V delta 4-, V delta 5-, V delta 6-, and C delta-specific primers. Selectively amplified, TCR delta-chain-encoding cDNA from these organs of five old SCID mice was cloned, and 175 randomly selected clones were sequenced. In this panel, 44 distinct rearrangement events were detected, 82% (36/44) of which were in-frame. For V delta 2, V delta 4, V delta 5, and V delta 6, 34 in-frame transcripts were found in five old SCID mice. No potentially functional transcript containing V delta 3 was found and an unusual type of transcript containing the V delta 1 gene segment spliced directly in-frame to C delta was the predominant type of V delta 1 expression. Junctional diversity was evident in most sequenced clones indicating an extensive potential diversity of SCID-derived TCR delta chains. Identical transcripts were amplified from different organs of the same old SCID mouse. No organ-specific expression of V delta genes was evident. TCR delta chain transcripts could be neither amplified from young SCID mice nor from young SCID mice nor from young or old scid/scid nu/nu mice (bred on a BALB/c background). Hence, an apparently thymus-dependent development of oligoclonal populations of TCR delta+ cells is observed in old SCID mice.

Instability of assembled T-cell receptor complex that is associated with rapid degradation of zeta chains in immature CD4+CD8+ thymocytes.

The intracellular fate of newly synthesized T-cell receptor (TCR) chains was compared in CD4+CD8+ (double positive; DP) thymocytes and in CD4+CD8- or CD4-CD8+ (single positive; SP) thymocytes. Purified DP and SP thymocytes from normal adult mice were analyzed by pulse-chase metabolic labeling and immunoprecipitation with specific anti-TCR antibodies. Biosynthesis of invariant chains (CD3 gamma, -delta, -epsilon, and zeta) was comparable between DP and SP thymocytes, whereas DP thymocytes synthesized TCR alpha and TCR beta chains at lower and higher levels than SP thymocytes, respectively. These newly synthesized TCR chains were degraded at different rates in SP thymocytes based on their sensitivities for degradation as previously reported: TCR alpha, TCR beta, CD3 gamma, and CD3 delta chains were rapidly degraded and CD3 epsilon and zeta chains were stable. Although the degradation rates of clonotypic and invariant CD3 chains were similar in DP and SP thymocytes, the zeta subunit was rapidly degraded in DP thymocytes (t1/2, approximately 1.5 hr). Degradation of zeta was inhibited by NH4Cl, implicating lysosomes as the site of degradation. Comparison of TCR subunit assembly in DP and SP thymocytes demonstrated that, despite the same relative rate of formation of TCR complexes in a pulse period (30 min), complete complexes were unstable and degraded during the subsequent 6 hr of chase in DP thymocytes. This contrasted with the stability and a progressive increase in the levels of completely assembled complexes in SP thymocytes. Thus, these results demonstrate that a unique posttranslational regulation operates in the formation of TCR complexes in DP thymocytes and that lack of stability of complete TCR complexes is a crucial mechanism that may account for the limited surface TCR expression on this thymocyte subset.

Biochemical evidence of the physical association of the majority of CD3 delta chains with the accessory/co-receptor molecules CD4 and CD8 on nonactivated T lymphocytes.

The association of components of the CD3 complex with the accessory molecules CD4 and CD8 was studied by immunoprecipitation experiments followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Enhanced surface iodination was achieved by a water-soluble derivative of the Bolton-Hunter reagent. Using freshly isolated nonactivated splenic T cells, we find that antibodies to CD4 and to CD8 strongly co-precipitate a 28-30-kDa band identical in mobility to the delta chain of the CD3 complex. Components corresponding in mobility to the epsilon and gamma chains of the CD3 complex are also co-precipitated but to a much lesser extent. The identity of the co-precipitated 28-30-kDa material with the CD3 delta chain was ascertained by two-dimensional nonreducing/reducing SDS-PAGE, by two-dimensional non-equilibrium pH gradient electrophoresis/SDS-PAGE and by one-dimensional peptide mapping with three different proteases. The co-precipitated 28-30-kDa material was identical to the CD3 delta chain by all these criteria. Quantitative analyses by densitometric gel tracing revealed that the amounts of CD3 delta co-precipitated with anti-CD4 and anti-CD8 add up to those in anti-V beta precipitates and to an average of 90% of those in anti-CD3 epsilon precipitates. We conclude that the majority of CD3 delta chains are associated with the accessory/co-receptor molecules CD4 or CD8 on resting T cells, and that this association is independent of antigen-specific recognition by the T cell receptor.

Evidence for oligoclonality of T cell receptor delta chain transcripts expressed in rheumatoid arthritis patients.

The inflamed synovium of rheumatoid arthritis (RA) patients contains gamma/delta T cells which express predominantly T cell receptor (TcR) variable (V) delta 1 and V delta 2 chains. Such T cells may contribute to the pathogenesis of RA. To assess the extent of clonality among these T cell populations we sequenced the junctional regions of rearranged TcR V delta 1-C delta and V delta 2-C delta chain cDNA, after using the polymerase chain reaction (PCR) to amplify TcR delta chain transcripts isolated from synovial membrane mononuclear cells (SMC) of five RA patients. The sequences of these delta chain transcripts were compared with those found in peripheral blood mononuclear cells (PBMC) of the same patients and in PBMC of four healthy controls. In contrast to control PBMC, V delta 1 chain cDNA derived from PBMC of three patients showed a strong bias towards usage of the same V-joining (J) combination and junctional region sequences, although the specific sequences were unique in each patient. However, oligoclonality of the V delta 1 chain was less marked in SMC of two of these patients and absent in SMC of the other patients. For V delta 2, oligoclonality was detected in PBMC of two patients. In SMC of a single patient, a dominant V delta 2 transcript was detected that utilized the J delta 2 segment, which was rarely expressed in the normal TcR repertoire. These results indicate in vivo clonal expansion of V delta 1- and V delta 2-expressing gamma/delta T cells in the peripheral blood of RA patients and a synovial T cell infiltrate which consists largely of polyclonally expanded gamma/delta T cells, but shows clonal dominance in some patients. Our data strongly support a role for V delta 1+ and V delta 2+ gamma/delta T cells in the pathogenesis of RA, and, although the nature of the antigen(s) recognized by these cells remains elusive, this report suggests the potential involvement of antigen(s) specific for the V region and V-J junction.

Phenotypic Changes Induced by Interleukin-2 (IL-2) and IL-3 in an Immature T-Lymphocytic Leukemia Are Associated With Regulated Expression of IL-2 Receptor β Chain and of Protein Tyrosine Kinases LCK and LYN

We have previously reported the establishment of an interleukin-3 (IL-3)-dependent and phenotypically myeloid cell line (TALL-103/3), obtained by culturing cells from an immature T-lymphoblastic leukemia in the presence of IL-3. These cells differentiated into a T-lymphoid cell line (TALL-103/2) upon removal of IL-3 and incubation in IL-2. Despite the different phenotype, the two cell lines remained karyotypically and genotypically identical. Here, we have analyzed the phenotypic changes and the signaling events induced by these two lymphokines in TALL-103/3 cells by switching them to temporary growth in IL-2 and returning them to IL-3. All four sublines obtained (the myeloid in IL-3 and the lymphoid in IL-2) expressed RNA for CD3, IL-2 receptor (R) alpha, and T-cell receptor (TCR)-gamma and -delta chains. However, cells cultured in IL-3 failed to express detectable levels of the IL-2R beta chain at both the protein and RNA levels, whereas cells exposed to IL-2 always expressed IL-2R beta. In parallel with the changes in IL-2R beta expression, the SRC-like protein tyrosine kinase (PTK) p56 LCK could not be detected in IL-3-dependent cells, but was abundant in the IL-2-dependent cells and underwent markedly increased autophosphorylation in response to IL-2. In contrast, p53/p56 LYN was highly expressed in IL-3-dependent cells, and greatly decreased when these cells were switched to growth in IL-2. LYN kinase autophosphorylation modestly increased in response to IL-3. None of the other kinases in the SRC family that were tested underwent increased autophosphorylation after lymphokine stimulation, indicating the specificity of IL-2 for LCK and of IL-3 for LYN. The TALL-103 cell lines provide a unique system to study the interaction between lymphokines and SRC-family PTKs in signal transduction pathways leading to hematopoietic cell differentiation.

Phenotypic changes induced by interleukin-2 (IL-2) and IL-3 in an immature T-lymphocytic leukemia are associated with regulated expression of IL-2 receptor beta chain and of protein tyrosine kinases LCK and LYN

We have previously reported the establishment of an interleukin-3 (IL- 3)-dependent and phenotypically myeloid cell line (TALL-103/3), obtained by culturing cells from an immature T-lymphoblastic leukemia in the presence of IL-3. These cells differentiated into a T-lymphoid cell line (TALL-103/2) upon removal of IL-3 and incubation in IL-2. Despite the different phenotype, the two cell lines remained karyotypically and genotypically identical. Here, we have analyzed the phenotypic changes and the signaling events induced by these two lymphokines in TALL-103/3 cells by switching them to temporary growth in IL-2 and returning them to IL-3. All four sublines obtained (the myeloid in IL-3 and the lymphoid in IL-2) expressed RNA for CD3, IL-2 receptor (R) alpha, and T-cell receptor (TCR)-gamma and -delta chains. However, cells cultured in IL-3 failed to express detectable levels of the IL-2R beta chain at both the protein and RNA levels, whereas cells exposed to IL-2 always expressed IL-2R beta. In parallel with the changes in IL-2R beta expression, the SRC-like protein tyrosine kinase (PTK) p56 LCK could not be detected in IL-3-dependent cells, but was abundant in the IL-2-dependent cells and underwent markedly increased autophosphorylation in response to IL-2. In contrast, p53/p56 LYN was highly expressed in IL-3-dependent cells, and greatly decreased when these cells were switched to growth in IL-2. LYN kinase autophosphorylation modestly increased in response to IL-3. None of the other kinases in the SRC family that were tested underwent increased autophosphorylation after lymphokine stimulation, indicating the specificity of IL-2 for LCK and of IL-3 for LYN. The TALL-103 cell lines provide a unique system to study the interaction between lymphokines and SRC-family PTKs in signal transduction pathways leading to hematopoietic cell differentiation.

Role and environment of the conserved Lys27 of snake curaremimetic toxins as probed by chemical modifications, site-directed mutagenesis and photolabelling experiments.

The positive charge of Lys27 was suppressed by chemical means in two short-chain curaremimetic toxins, namely erabutoxin a (Ea) from Laticauda semifasciata and toxin alpha from Naja nigricollis. This modification leads to a decrease in the binding affinity of the toxins for the nicotinic acetylcholine receptor, which range 6-15-fold, as judged from both the data reported here and those previously described in the literature. A negatively charged glutamate residue has been introduced at position 27 of erabutoxin a by site-directed mutagenesis. This change provokes a 120-fold decrease in the affinity, which reflects a major alteration of toxin-receptor cognate events. Using toxin-alpha derivative harbouring a photoactive group at Lys27, we probed the toxin local environment in a receptor-bound state by photocoupling experiments. The delta chain was the predominant coupling target, in contrast to previous observations indicating that a photoactive probe on Lys47 predominantly labelled the alpha chain. The toxin derivative weakly labelled the alpha and gamma chains but not the beta chain. The toxin may therefore interact with subunits other than the alpha chain, at least in the vicinity of Lys27.

Molecular analysis of a pro-T cell clone transformed by Abelson-murine leukemia virus, displaying progressive gamma delta T cell receptor gene rearrangement and surface expression.

We present a molecular analysis of T cell differentiation in a set of clones derived from in vitro Abelson murine leukemia virus (A-MuLV) infection of fetal liver cells. The parental clone had partial rearrangement of the beta and gamma loci and spontaneously displayed progressive rearrangement of V gamma genes during in vitro culture. Further differentiation of these clones leading to delta gene rearrangement and CD4 expression, then CD8, CD3 and T cell receptor gamma delta chain surface expression was obtained after intrathymic transfer followed by in vitro co-culture with thymic tissue. These A-MuLV clones, therefore, appear to represent a powerful model system for studying the early molecular events of T cell development at the clonal level.