Role of the multiple T cell receptor (TCR)-zeta chain signaling motifs in selection of the T cell repertoire.

Immature thymocytes undergo a selection process within the thymus based on their T cell antigen receptor (TCR) specificity that results either in their maturation into functionally competent, self-MHC-restricted T cells (positive selection) or their deletion (negative selection). The outcome of thymocyte selection is thought to be controlled by signals transduced by the TCR that vary in relation to the avidity of the TCR-ligand interaction. The TCR is composed of four distinct signal transducing subunits (CD3-gamma, -delta, -epsilon, and zeta) that contain either one (CD3-gamma, -delta, -epsilon) or three (-zeta) signaling motifs (ITAMs) within their intracytoplasmic domains. A possible function for multiple TCR ITAMs could be to amplify signals generated by the TCR during selection. To determine the importance of the multiple TCR-zeta chain ITAMs in thymocyte selection, transgenes encoding alpha/beta TCRs with known specificity were bred into mice in which zeta chains lacking one or more ITAMs had been genetically substituted for endogenous zeta. A direct relationship was observed between the number of zeta chain ITAMs within the TCR complex and the efficiency of both positive and negative selection. These results reveal a role for multiple TCR ITAMs in thymocyte selection and identify a function for TCR signal amplification in formation of the T cell repertoire.

Commitment of immature CD4+8+ thymocytes to the CD4 lineage requires CD3 signaling but does not require expression of clonotypic T cell receptor (TCR) chains.

As a consequence of positive selection in the thymus, immature CD4(+)8(+) double-positive, [DP] thymocytes selectively terminate synthesis of one coreceptor molecule and, as a result, differentiate into either CD4(+) or CD8(+) T cells. The decision by individual DP thymocytes to terminate synthesis of one or the other coreceptor molecule is referred to as lineage commitment. Previously, we reported that the intrathymic signals that induced commitment to the CD4 versus CD8 T cell lineages were markedly asymmetric. Notably, CD8 commitment appeared to require lineage-specific signals, whereas CD4 commitment appeared to occur in the absence of lineage-specific signals by default. Consequently, it was unclear whether CD4 commitment, as revealed by selective termination of CD8 coreceptor synthesis, occurred in all DP thymocytes, or whether CD4 commitment occurred only in T cell receptor (TCR)-CD3-signaled DP thymocytes. Here, we report that selective termination of CD8 coreceptor synthesis does not occur in DP thymocytes spontaneously. Rather, CD4 commitment in DP thymocytes requires signals transduced by either CD3 or zeta chains, which can signal CD4 commitment even in the absence of clonotypic TCR chains.

CD5 expression is developmentally regulated by T cell receptor (TCR) signals and TCR avidity.

Recent data indicate that the cell surface glycoprotein CD5 functions as a negative regulator of T cell receptor (TCR)-mediated signaling. In this study, we examined the regulation of CD5 surface expression during normal thymocyte ontogeny and in mice with developmental and/or signal transduction defects. The results demonstrate that low level expression of CD5 on CD4(-)CD8(-) (double negative, DN) thymocytes is independent of TCR gene rearrangement; however, induction of CD5 surface expression on DN thymocytes requires engagement of the pre-TCR and is dependent upon the activity of p56(lck). At the CD4(+)CD8(+) (double positive, DP) stage, intermediate CD5 levels are maintained by low affinity TCR-major histocompatibility complex (MHC) interactions, and CD5 surface expression is proportional to both the surface level and signaling capacity of the TCR. High-level expression of CD5 on DP and CD4(+) or CD8(+) (single positive, SP) thymocytes is induced by engagement of the alpha/beta-TCR by (positively or negatively) selecting ligands. Significantly, CD5 surface expression on mature SP thymocytes and T cells was found to directly parallel the avidity or signaling intensity of the positively selecting TCR-MHC-ligand interaction. Taken together, these observations suggest that the developmental regulation of CD5 in response to TCR signaling and TCR avidity represents a mechanism for fine tuning of the TCR signaling response.

Knock-in mutation of the distal four tyrosines of linker for activation of T cells blocks murine T cell development.

The integral membrane adapter protein linker for activation of T cells (LAT) performs a critical function in T cell antigen receptor (TCR) signal transduction by coupling the TCR to downstream signaling pathways. After TCR engagement, LAT is tyrosine phosphorylated by ZAP-70 creating docking sites for multiple src homology 2-containing effector proteins. In the Jurkat T cell line, the distal four tyrosines of LAT bind PLCgamma-1, Grb2, and Gads. Mutation of these four tyrosine residues to phenylalanine (4YF) blocked TCR-mediated calcium mobilization, Erk activation, and nuclear factor (NF)-AT activation. In this study, we examined whether these four tyrosine residues were essential for T cell development by generating LAT "knock-in" mutant mice that express the 4YF mutant protein under the control of endogenous LAT regulatory sequences. Significantly, the phenotype of 4YF knock-in mice was identical to LAT(-/)- (null) mice; thymocyte development was arrested at the immature CD4(-)CD8(-) stage and no mature T cells were present. Knock-in mice expressing wild-type LAT protein, generated by a similar strategy, displayed a normal T cell developmental profile. These results demonstrate that the distal four tyrosine residues of LAT are essential for preTCR signaling and T cell development in vivo.

Function of CD3 epsilon-mediated signals in T cell development.

The T cell antigen receptor (TCR) and pre-TCR complexes are composed of multiple signal-transducing subunits (CD3 gamma, CD3 delta, CD3 epsilon, and zeta) that each contain one or more copies of a semiconserved functional motif, the immunoreceptor tyrosine-based activation motif (ITAM). Although biochemical studies indicate that individual TCR-ITAMs may bind selectively or with different affinity to various effector molecules, data from other experiments suggest that at least some ITAMs are functionally equivalent. In this study, we examined the role of CD3straightepsilon ITAM-mediated signals in T cell development by genetically reconstituting CD3 epsilon-deficient mice with transgenes encoding either wild-type or ITAM-mutant (signaling defective) forms of the protein. The results demonstrate that signals transduced by CD3 epsilon are not specifically required for T cell maturation but instead contribute quantitatively to TCR signaling in a manner similar to that previously observed for zeta chain. Unexpectedly, analysis of TCR-transgenic/CD3 epsilon-mutant mice reveals a potential role for CD3 epsilon signals in T cell survival.

A role for the Tec family tyrosine kinase Txk in T cell activation and thymocyte selection.

Recent data indicate that several members of the Tec family of protein tyrosine kinases function in antigen receptor signal transduction. Txk, a Tec family protein tyrosine kinase, is expressed in both immature and mature T cells and in mast cells. By overexpressing Txk in T cells throughout development, we found that Txk specifically augments the phospholipase C (PLC)-gamma1-mediated calcium signal transduction pathway upon T cell antigen receptor (TCR) engagement. Although Txk is structurally different from inducible T cell kinase (Itk), another Tec family member expressed in T cells, expression of the Txk transgene could partially rescue defects in positive selection and signaling in itk(-)(/)(-) mice. Conversely, in the itk(+/+) (wild-type) background, overexpression of Txk inhibited positive selection of TCR transgenic thymocytes, presumably due to induction of cell death. These results identify a role for Txk in TCR signal transduction, T cell development, and selection and suggest that the Tec family kinases Itk and Txk perform analogous functions.

Differential effects of zeta and eta transgenes on early alpha/beta T cell development.

The zeta-family dimers (zeta, eta, and gamma) are a group of structurally and functionally related proteins that are expressed in developing thymocytes and function as signal transducing subunits of the T cell antigen receptor (TCR) and certain Ig Fc receptors. Zeta, eta, and gamma each contain one or more copies of a conserved tyrosine-based activation motif (TAM) that is known to be required for signal transduction. To examine the developmental importance of multiple or individual TAM elements we generated transgenic mice that express: (a) full-length (FL) zeta-chain (3 TAMs); (b) eta-chain, a naturally occurring variant of zeta that is derived from alternative splicing (2 TAMs); or (c) truncated zeta-chain (CT108; 1 TAM), under the control of the human CD2 promoter and regulatory elements. Unexpectedly, we found that overexpression of the FL zeta chain caused premature termination of RAG-1 and RAG-2 expression, prevented productive rearrangement of the TCR-alpha and TCR-beta genes and blocked entry of thymocytes into the CD4/CD8 developmental pathway. In contrast, we found that overexpression of eta or CT108 had no effect on normal thymocyte maturation. These results suggest that an early signaling pathway exists in precursor TCR- thymocytes that can regulate RAG-1 and RAG-2 expression and is differentially responsive to individual members of the zeta-family dimers.

Delayed maturation of CD4- CD8- Fc gamma RII/III+ T and natural killer cell precursors in Fc epsilon RI gamma transgenic mice.

Fc epsilon RI gamma (gamma) is a member of a group of related proteins (the zeta-family dimers) that function as signal-transducing components of both Fc receptors and the T cell antigen receptor (TCR). Analysis of gamma expression during fetal thymus ontogeny revealed that it is expressed in early thymocytes, before the initiation of clonotypic TCR-alpha and TCR-beta gene rearrangement but is down-regulated in most adult thymocytes. To explore a possible role for gamma in thymocyte development, we generated transgenic mice in which this protein was overexpressed at all stages of ontogeny. Overexpression of gamma inhibited the maturation of T cells as well as natural killer (NK) cells. The developmental effects were transgene dose related and correlated with markedly delayed maturation of fetal CD4-CD8- FcRII/III+ thymocytes, cells thought to include the progenitors of both T and NK cells. These results suggest that the zeta and gamma chains serve distinctive functions in thymocyte development and indicate that Fc receptor(s) may play an important role in regulating the differentiation of early progenitor cells within the thymus.

RIBP, a novel Rlk/Txk- and itk-binding adaptor protein that regulates T cell activation.

A novel T cell-specific adaptor protein, RIBP, was identified based on its ability to bind Rlk/Txk in a yeast two-hybrid screen of a mouse T cell lymphoma library. RIBP was also found to interact with a related member of the Tec family of tyrosine kinases, Itk. Expression of RIBP is restricted to T and natural killer cells and is upregulated substantially after T cell activation. RIBP-disrupted knockout mice displayed apparently normal T cell development. However, proliferation of RIBP-deficient T cells in response to T cell receptor (TCR)-mediated activation was significantly impaired. Furthermore, these activated T cells were defective in the production of interleukin (IL)-2 and interferon gamma, but not IL-4. These data suggest that RIBP plays an important role in TCR-mediated signal transduction pathways and that its binding to Itk and Rlk/Txk may regulate T cell differentiation.

T cell development in mice lacking all T cell receptor zeta family members (Zeta, eta, and FcepsilonRIgamma).

The zeta family includes zeta, eta, and FcepsilonRIgamma (Fcgamma). Dimers of the zeta family proteins function as signal transducing subunits of the T cell antigen receptor (TCR), the pre-TCR, and a subset of Fc receptors. In mice lacking zeta/eta chains, T cell development is impaired, yet low numbers of CD4+ and CD8+ T cells develop. This finding suggests either that pre-TCR and TCR complexes lacking a zeta family dimer can promote T cell maturation, or that in the absence of zeta/eta, Fcgamma serves as a subunit in TCR complexes. To elucidate the role of zeta family dimers in T cell development, we generated mice lacking expression of all of these proteins and compared their phenotype to mice lacking only zeta/eta or Fcgamma. The data reveal that surface complexes that are expressed in the absence of zeta family dimers are capable of transducing signals required for alpha/beta-T cell development. Strikingly, T cells generated in both zeta/eta-/- and zeta/eta-/--Fcgamma-/- mice exhibit a memory phenotype and elaborate interferon gamma. Finally, examination of different T cell populations reveals that zeta/eta and Fcgamma have distinct expression patterns that correlate with their thymus dependency. A possible function for the differential expression of zeta family proteins may be to impart distinctive signaling properties to TCR complexes expressed on specific T cell populations.

Role of TCR zeta chain in T cell development and selection.

Signals mediated by the T cell receptor (TCR) are required for thymocyte maturation and selection. To examine the role of TCR zeta chain signals in development, TCR expression was restored in zeta-deficient mice with transgenic zeta chains that partially or completely lacked sequences required for signal transduction. The zeta chain played a role in thymic development by promoting TCR surface expression, but zeta-mediated signals were not essential because TCRs that contained signaling-deficient zeta chains promoted T cell maturation and transduced signals associated with thymic selection.

T cell development in mice that lack the zeta chain of the T cell antigen receptor complex.

The zeta subunit of the T cell antigen receptor complex is required for targeting nascent receptor complexes to the cell surface and for receptor-mediated signal transduction. To examine the significance of the zeta subunit in T cell development, mice deficient for zeta expression were generated by gene targeting. These zeta-/- mice had few CD4+CD8+ thymocytes, and the generation of CD4+ and CD8+ single positive T cells was impaired but not completely abrogated. Peripheral T cells were present but were unusual in that they expressed small amounts of CD5 and few T cell receptors. Thus, zeta chain expression influences thymocyte differentiation but is not absolutely required for the generation of single positive T cells.

TCR zeta chain in T cell development and selection.

Current data suggest that an important function of the multimeric structure of the TCR is to enable the assembly of structurally and functionally different forms of the TCR, the pre-TCR and alphabetaTCR complexes, at different stages in development. Four distinct TCR subunits (the CD3gamma, delta, and epsilon chains and the zeta chain) contain signal transducing motifs; however, the zeta chain is notable for containing three of these elements. These motifs, especially those within the zeta chain, function to amplify signals generated by the TCR, and this property is especially critical during thymocyte selection. The results of several recent experiments argue that positive and negative selection of thymocytes may involve activation of distinct downstream signaling pathways. The outcome of thymocyte selection can also be influenced, however, by quantitative effects such as changes in ligand concentration or direct alteration of the TCR signaling potential. Recent studies pertaining to the kinetics of TCR-ligand interactions may provide insight into how signaling through the TCR can be regulated either quantitatively or qualitatively.

LAT is required for tyrosine phosphorylation of phospholipase cgamma2 and platelet activation by the collagen receptor GPVI.

In the present study, we have addressed the role of the linker for activation of T cells (LAT) in the regulation of phospholipase Cgamma2 (PLCgamma2) by the platelet collagen receptor glycoprotein VI (GPVI). LAT is tyrosine phosphorylated in human platelets heavily in response to collagen, collagen-related peptide (CRP), and FcgammaRIIA cross-linking but only weakly in response to the G-protein-receptor-coupled agonist thrombin. LAT tyrosine phosphorylation is abolished in CRP-stimulated Syk-deficient mouse platelets, whereas it is not altered in SLP-76-deficient mice or Btk-deficient X-linked agammaglobulinemia (XLA) human platelets. Using mice engineered to lack the adapter LAT, we showed that tyrosine phosphorylation of Syk and Btk in response to CRP was maintained in LAT-deficient platelets whereas phosphorylation of SLP-76 was slightly impaired. In contrast, tyrosine phosphorylation of PLCgamma2 was substantially reduced in LAT-deficient platelets but was not completely inhibited. The reduction in phosphorylation of PLCgamma2 was associated with marked inhibition of formation of phosphatidic acid, a metabolite of 1,2-diacylglycerol, phosphorylation of pleckstrin, a substrate of protein kinase C, and expression of P-selectin in response to CRP, whereas these parameters were not altered in response to thrombin. Activation of the fibrinogen receptor integrin alpha(IIb)beta(3) in response to CRP was also reduced in LAT-deficient platelets but was not completely inhibited. These results demonstrate that LAT tyrosine phosphorylation occurs downstream of Syk and is independent of the adapter SLP-76, and they establish a major role for LAT in the phosphorylation and activation of PLCgamma2, leading to downstream responses such as alpha-granule secretion and activation of integrin alpha(IIb)beta(3). The results further demonstrate that the major pathway of tyrosine phosphorylation of SLP-76 is independent of LAT and that there is a minor, LAT-independent pathway of tyrosine phosphorylation of PLCgamma2. We propose a model in which LAT and SLP-76 are required for PLCgamma2 phosphorylation but are regulated through independent pathways downstream of Syk.

Cloning and characterization of PTP-K1, a novel nonreceptor protein tyrosine phosphatase highly expressed in bone marrow.

A novel nonreceptor protein tyrosine phosphatase (PTP), PTP-K1, was identified using a consensus polymerase chain reaction-based approach. The full length cDNA encompasses an open reading frame of 1362 base pairs, predicting a protein of 453 amino acid residues with a molecular mass of 54 kDa. The PTP domain is located in the N-terminal portion of the molecule and shares approximately 50% amino acid identify with two other nonreceptor PTPs: PEP and PTP-PEST. PTP-K1 is preferentially expressed in mouse bone marrow with transcripts of 1.7 kb, 1.9 kb and 3.5 kb. The 1.7 kb transcript was also detected in kidney, lung and ovary. The PTP domain of PTP-K1 was expressed as a fusion protein in bacteria and had intrinsic PTP catalytic activity. Indirect immunofluorescence microscopy in COS-7 cells showed that PTP-K1 was localized to the cytoplasm. Ptp-k1 was mapped to mouse chromosome 1, and was closely linked to the interleukin-1 receptor gene. The high level expression of PTP-K1 mRNA in bone marrow suggests that PTP-K1 may be involved in signal transduction in growth and differentiation of hematopoietic cells.

Murine txk: a protein tyrosine kinase gene regulated by T cell activation.

To identify genes involved in signal transduction pathways that regulate T cell activation and development, murine fetal thymocytes were screened for expression of protein tyrosine kinase family members by the polymerase chain reaction. Using this approach, a non-receptor protein tyrosine kinase, txk, was identified and cloned. Tsk is expressed in thymocytes as early as fetal day 13.5 and its expression at the mRNA level continues throughout development. Txk transcripts are present in thymocytes, peripheral T cells and mast cell lines, but are not detectable in B cell macrophage/monocyte cell lines or in non-hematopoietic fetal or adult tissues. In both thymocytes and T cells, txk transcripts are down-regulated after activation with PMA and ionomycin, concanavalin A or T cell receptor cross-linking. Sequence analysis indicates that txk contains SH2, SH3 and kinase catalytic domains and belongs to the tec family of cytoplasmic protein tyrosine kinases which includes tec, itk and btk. Its unique N-terminus contains a proline-rich region, but unlike the other tec family members, does not contain a pleckstrin homology domain. The restricted expression pattern of txk and its regulation by T cell activation make it an excellent candidate for involvement in signal transduction during thymocyte development.

Insights into T cell development and signal transduction provided by TCR-zeta chain deficient mice.

The T cell antigen receptor (TCR) transduces signals that mediate different responses depending on the stage of development of the T cell and the nature of the ligand it engages. The presence of multiple signal transducing subunits (CD3-gamma-delta,-epsilon and zeta chain) suggests the potential to control these responses by altering the subunit composition of the TCR. zeta chain represents an especially important signalling molecule as it contains multiple signalling motifs within its cytoplasmic tail. The generation and analysis of zeta deficient (zeta-/-) and zeta-transgenic mice has provided insight into the role of zeta as well as the CD3 subunits in TCR surface expression, T cell activation and thymocyte development. Herein, we discuss the results from such experiments which suggest distinct roles for zeta chain and the CD3 components at different stages of T cell development.

Induction of the Bacillus subtilis SOS-like response by Escherichia coli RecA protein.

A plasmid that expresses the Escherichia coli RecA protein partially restored DNA repair and recombination capability and induction of the SOS-like (SOB) response in a recE4 mutant of Bacillus subtilis. In the presence of DNA-damaging agents, the E. coli RecA protein induced din operon expression, Weigle-reactivation activity, and synthesis of a B. subtilis recombination protein (Recbs) analogous to RecA but was unable to stimulate prophage induction. In addition, the RecA protein was capable of inducing the SOB response in competent recE4 strains of B. subtilis, independent of exposure to DNA-damaging agents. The results suggest that (i) the SOS response of E. coli and the SOB response of B. subtilis are strikingly similar from both a phenotypic and a regulatory standpoint and that RecA and LexA protein analogs exist in B. subtilis, (ii) the Recbs protein is capable of regulating its own production, and (iii) SOS-inducing (RecA-activating) signals are generated in B. subtilis following either DNA damage or the development of physiological competence.

Antiphospholipid antibodies: anticardiolipin and the lupus anticoagulant in systemic lupus erythematosus (SLE) and in non-SLE disorders. Prevalence and clinical significance.

PURPOSE: To determine the prevalence of lupus anticoagulant and anticardiolipin in systemic lupus erythematosus (SLE) and in non-SLE disorders, and to evaluate the clinical significance of these autoantibodies as they relate to thromboembolic events, neuropsychiatric disorders, thrombocytopenia, and fetal loss. DATA IDENTIFICATION: A computer-assisted search of the literature (MEDLINE, 1966 to 1989) and review of the bibliographies of all identified articles. STUDY SELECTION: Series of ten or more subjects were included if the assays used for detecting lupus anticoagulant or anticardiolipin met the specified minimal criteria for validity. DATA EXTRACTION: Series were categorized according to antibody type and underlying disease. A systematic appraisal of patient selection methods, study design, and assay methods was done. RESULTS OF DATA ANALYSIS: An analysis of 29 published series (comprising over 1000 patients with SLE) yielded an average frequency of 34% for the lupus anticoagulant and 44% for anticardiolipin. Antiphospholipid antibodies are also prevalent in patients with various non-SLE disorders. In patients with SLE, a statistically significant association exists between the presence of either antibody and a history of thrombosis, neurologic disorders, or thrombocytopenia. The available data suggest, but do not firmly support, an association between antiphospholipid antibodies and history of fetal loss in women with SLE. Contrary to prevailing opinion, none of these associations have been shown conclusively in patients with non-SLE disorders. CONCLUSIONS: The results of predominantly retrospective series suggest that for certain persons (patients with SLE or closely related disorders) antiphospholipid antibodies may be important risk factors for thrombosis, neurologic disease, thrombocytopenia, and fetal loss. Standardized tests for lupus anticoagulant and anticardiolipin, as well as long-term, prospective clinical studies, are needed to determine the prognostic value of antiphospholipid antibodies.