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.

Fine tuning of TCR signaling by CD5.

Current data indicate that CD5 functions as an inhibitor of TCR signal transduction. Consistent with this role, thymocyte selection in TCR transgenic/CD5(-/-) mice is altered in a manner suggestive of enhanced TCR signaling. However, the impact of CD5 deletion on thymocyte selection varies depending on the transgenic TCR analyzed, ranging from a slight to a marked shift from positive toward negative selection. An explanation for the variable effect of CD5 on selection is suggested by the observation that CD5 surface expression is regulated by TCR signal intensity during development and CD5 surface levels on mature thymocytes and T cells parallel the avidity of the positively selecting TCR/MHC/ligand interaction. In this study, we generated mice that overexpress CD5 during thymocyte development (CD5-tg), and then examined the effect of CD5 overexpression or CD5 deletion (CD5(-/-)) on selection of thymocytes that express the same TCR transgenes. The results demonstrate that the effect on thymocyte selection of altering CD5 expression depends on the avidity of the selecting interaction and, consequently, the level of basal (endogenous) CD5 surface expression. Substitution of endogenous CD5 with a transgene encoding a truncated form of the protein failed to rescue the CD5(-/-) phenotype, demonstrating that the cytoplasmic domain of CD5 is required for its inhibitory function. Together, these results indicate that inducible regulation of CD5 surface expression during thymocyte selection functions to fine tune the TCR signaling response.

Critical relationship between TCR signaling potential and TCR affinity during thymocyte selection.

Whether a developing thymocyte becomes positively or negatively selected is thought to be determined by the affinity/avidity of its TCR for MHC/peptide ligands expressed in the thymus. Presumably, differences in affinity translate into differences in the potency of the ensuing TCR-mediated signals, and these differences in signal strength determine the outcome of thymocyte selection. However, there is little direct evidence establishing a relationship between TCR-ligand affinity and signal strength during positive and negative selection. The TCR complex contains multiple signaling motifs, known as immunoreceptor tyrosine-based activation motifs (ITAMs) that are required for T cell activation. To examine the effects of TCR signal strength on selection, the signaling potential of the TCR was modified by substituting transgenic TCR zeta-chains containing either three, one, or zero ITAMs for endogenous (3-ITAM) zeta-chain. These zeta-chain variants were then bred into different alphabetaTCR transgenic backgrounds. We report that reductions in TCR signaling potential have distinct effects on the selection of thymocytes expressing different TCRs, and that the requirement for zeta-chain ITAMs critically depends upon the specificity and apparently, affinity, of the TCR for its selecting ligand(s).

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.

LAT is essential for Fc(epsilon)RI-mediated mast cell activation.

The linker molecule LAT is a substrate of the tyrosine kinases activated following TCR engagement of T cells. LAT is also expressed in platelets, NK, and mast cells. Although LAT-deficient mice contain normal numbers of mast cells, we found that LAT-deficient mice were resistant to IgE-mediated passive systemic anaphylaxis. LAT-deficient bone marrow-derived mast cells (BMMC) showed normal growth and development. Whereas tyrosine phosphorylation of Fc(epsilon)RI, Syk, and Vav was intact in LAT-deficient BMMCs following Fc(epsilon)RI engagement, tyrosine phosphorylation of SLP-76, PLC-gamma1, and PLC-gamma2 and calcium mobilization were dramatically reduced. LAT-deficient BMMCs also exhibited profound defects in activation of MAPK, degranulation, and cytokine production after Fc(epsilon)RI cross-linking. These results show that LAT plays a critical role in Fc(epsilon)RI-mediated signaling in mast cells.

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.

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.

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.

Involvement of the SHP-1 tyrosine phosphatase in regulation of T cell selection.

The selection events shaping T cell development in the thymus represent the outcome of TCR-driven intracellular signaling cascades evoked by Ag receptor interaction with cognate ligand. In view of data indicating TCR-evoked thymocyte proliferation to be negatively modulated by the SHP-1 tyrosine phosphatase, a potential role for SHP-1 in regulating selection processes was investigated by analysis of T cell development in H-Y TCR transgenic mice rendered SHP-1 deficient by introduction of the viable motheaten mutation or a dominant negative SHP-1-encoding transgene. Characterization of thymocyte and peripheral T cell populations in H-Y TCR-viable motheaten mice revealed TCR-evoked proliferation as well as the positive and negative selection of H-Y-specific thymocytes to be enhanced in these mice, thus implicating SHP-1 in the negative regulation of each of these processes. T cell selection processes were also augmented in H-Y TCR mice carrying a transgene driving lymphoid-restricted expression of a catalytically inert, dominant-negative form of SHP-1. SHP-1-negative effects on thymocyte TCR signaling were not influenced by co-cross-linking of the CD28 costimulatory and/or CTLA-4 inhibitory receptors and appear, accordingly, to be realized independently of these comodulators. These observations indicate that SHP-1 raises the signaling threshold required for both positive and negative selection and reveal the inhibitory effects of SHP-1 on TCR signaling to be cell autonomous. The demonstrated capacity for SHP-1 to inhibit TCR-evoked proliferation and selection indicate SHP-1 modulatory effects on the magnitude of TCR-generated signal to be a key factor in determining the cellular consequences of TCR-ligand interaction.

Essential role of LAT in T cell development.

The linker molecule LAT is a substrate of the tyrosine kinases activated following TCR engagement. Phosphorylated LAT binds many critical signaling molecules. The central role of this molecule in TCR-mediated signaling has been demonstrated by experiments in a LAT-deficient cell line. To probe the role of LAT in T cell development, the LAT gene was disrupted by targeting. LAT-deficient mice appeared healthy. Flow cytometric analysis revealed normal B cell populations but the absence of any mature peripheral T cells. Intrathymic development was blocked within the CD4- CD8- stage. No gross abnormality of NK or platelet function was observed. LAT is thus critical to both T cell activation and development.

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.

Specific requirement for CD3epsilon in T cell development.

T cell antigen receptor (TCR) and pre-TCR complexes are composed of clonotypic heterodimers in association with dimers of signal transducing invariant subunits (CD3gamma, -delta, -epsilon, and zeta). The role of individual invariant subunits in T cell development has been investigated by generating gene-specific mutations in mice. Mutation of CD3gamma, -delta, or zeta results in an incomplete block in development, characterized by reduced numbers of mature T cells that express low levels of TCR. In contrast, mature T cells are absent from CD3epsilon-/- mice, and thymocyte development is arrested at the early CD4(-)CD8(-) stage. Although these results suggest that CD3epsilon is essential for pre-TCR and TCR expression/function, their interpretation is complicated by the fact that expression of the CD3gamma and CD3delta genes also is reduced in CD3epsilon-/- mice. Thus, it is unclear whether the phenotype of CD3epsilon-/- mice reflects the collective effects of CD3gamma, -delta, and -epsilon deficiency. By removing the selectable marker (PGK-NEO) from the targeted CD3epsilon gene via Cre/loxP-mediated recombination, we generated mice that lack CD3epsilon yet retain normal expression of the closely linked CD3gamma and CD3delta genes. These (CD3epsilonDelta/Delta) mice exhibited an early arrest in T cell development, similar to that of CD3epsilon-/- mice. Moreover, the developmental defect could be rescued by expression of a CD3epsilon transgene. These results identify an essential role for CD3epsilon in T cell development not shared by the CD3gamma, CD3delta, or zeta-family proteins and provide further evidence that PGK-NEO can influence the expression of genes in its proximity.

Induction of antigen-specific hyporesponsiveness by transplantation of hemopoietic cells containing an MHC class I transgene regulated by a lymphocyte-specific promoter.

We explored a novel approach to tolerance induction by the transplantation of bone marrow (BM) cells (BMCs) that themselves do not express a foreign histocompatibility Ag, but which give rise to mature lymphocytes that do so. Lines of transgenic (FVB) mice were generated that contained an MHC class I Dd cDNA regulated by a CD2 promoter. Because the CD2 promoter is lymphocyte-specific and activated relatively late in lymphocyte ontogeny, Dd is expressed on most mature lymphocytes in the periphery but only on developing B cells in the BM of transgenic mice. Transgenic BMCs are tolerogenic and reproducibly engraft in nontransgenic mice using a conditioning regimen that is nonpermissive for the engraftment of conventional (MHC promoter) Dd-transgenic BMCs. Engrafted BMCs generate transgene-expressing lymphocytes and confer a state of Ag-specific hyporesponsiveness on the host that is primarily attributable to a peripheral mechanism. The strategies by which tolerance can be optimized in this system are discussed.

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.

Characterization of an associated 16-kDa tyrosine phosphoprotein required for Ly-49D signal transduction.

Ly-49D is an activating receptor on NK cells that does not become tyrosine phosphorylated upon activation. This report demonstrates that immunoprecipitation of Ly-49D, following pervanadate treatment or specific Ab cross-linking, coprecipitates a 16-kDa tyrosine-phosphorylated protein (pp16). Immunoblotting experiments and data from TCR-zeta/Fc epsilonRIgamma double knockout mice confirm that pp16 is not TCR-zeta, TCR-eta, or Fc epsilonRIgamma. Association of pp16 with Ly-49D involves a transmembrane arginine since mutation to leucine (Ly-49D[R54L]) abolishes association with pp16 in transfected P815 cells. In addition, Ly-49D(R54L) transfectants fail to mediate Ca2+ mobilization following Ab cross-linking. Therefore, signaling through Ly49D on NK cells depends on association with a distinct tyrosine phosphoprotein (pp16) in a manner analogous to that of TCR and FcR. Expression of this novel signaling peptide in both the NK and myeloid lineages indicates that pp16 is likely involved in the signal transduction cascade of additional receptor families.

Regulation of TCR signal transduction in murine thymocytes by multiple TCR zeta-chain signaling motifs.

The alphabeta TCR is a multimeric protein complex comprising ligand-binding and signal-transducing subunits. The signal transduction processes are mediated by the immunoreceptor tyrosine-based activation motifs (ITAMs), and up to 10 ITAMs are present within a single TCR complex. This multiplicity may allow for signal amplification and/or the formation of qualitatively distinct intracellular signals. Notably, the TCR-zeta subunit contains three ITAMs, and exists as a disulfide-linked homodimer in the TCR complex. In normal murine thymocytes and peripheral T cells, a proportion of TCR-zeta molecules is constitutively tyrosine phosphorylated and associated with the ZAP-70 protein tyrosine kinase. We examined the contribution of the different TCR-zeta ITAMs in regulating the constitutive phosphorylation of the TCR-zeta subunit in thymocytes by analyzing TCR-zeta-deficient mice that had been reconstituted with either full-length or single ITAM-containing TCR-zeta subunits. We report in this work that in the absence of a full-length TCR-zeta subunit, there is no apparent constitutive phosphorylation of the remaining TCR/CD3 ITAMs. Following TCR ligation, all of the CD3 ITAMs become inducibly phosphorylated and associate with the ZAP-70 protein tyrosine kinase. Regardless of the number of TCR-zeta ITAMs present in the TCR complex, we report that a number of molecules involved in downstream signaling events, such as ZAP-70, SLP-76, and pp36, are all inducibly tyrosine phosphorylated following TCR ligation. These results support the notion that the different TCR ITAMs function in a quantitative rather than qualitative manner.

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.

Fc epsilonRI gamma can support T cell development and function in mice lacking endogenous TCR zeta-chain.

Fc epsilonRI gamma (Fc gamma) is a member of the zeta family of signal transducing molecules that function as components of both the TCR and Fc receptors (FcR). While the majority of thymocytes and T cells express TCRs containing zeta-chain homodimers, certain unique populations of T cells express TCRs that contain both zeta and Fc gamma. To examine the ability of Fc gamma to substitute for zeta-chain in T cell development and function, we introduced a transgene encoding Fc gamma into mice made genetically deficient for zeta-chain (zeta(e)-/-). Analysis of thymocyte development in zeta(e)-/-;Fc gamma Tg mice demonstrated that Fc gamma was able to support the maturation of both gammadelta TCR+ and alphabeta TCR+ T cells. However, positive selection of alphabeta TCR+ thymocytes was less efficient in zeta(e)-/-;Fc gamma Tg mice than in zeta(e)-/- mice reconstituted with zeta-chain. This difference may be due to the fact that Fc gamma contains a single immunoreceptor tyrosine-based activation motif (ITAM) whereas zeta-chain contains three ITAMs. Interestingly, the peripheral T cells that develop in zeta(e)-/- mice reconstituted with Fc gamma are functional and respond to TCR-specific stimuli. These data suggest that Fc gamma and zeta are interchangeable in their ability to mediate T cell development and function, however zeta-chain is more efficient at promoting positive selection and T cell maturation. The difference in efficiency between zeta and Fc gamma may be responsible in part for the unusual developmental and functional properties of T cells that constitutively express Fc gamma as a signaling component of their TCRs.

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.