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.     

A shift from negative to positive selection of autoreactive T cells by the reduced level of TCR signal in TCR-transgenic CD3 zeta-deficient mice

T cell selection is thought to be determined through the interaction between TCR and Ag/MHC. However, the contribution of the level of TCR signal to thymic selection remains unclear. To address this issue, we analyzed T cell selection of male Ag (HY)-specific TCR transgenic (HYTg) mice crossed with CD3 zeta-deficient (zeta KO) mice (HYTg/zeta KO), which have impaired signaling through TCR. In male HYTg/zeta KO mice, the number of thymocytes was comparable to that in normal mice, and almost all the peripheral T cells were HY specific, although these positively selected cells were anergic to male Ag. From these observations, the decrease in TCR signaling by CD3 zeta deficiency resulted in both the avoidance of negative selection and the acquisition of positive selection of autoreactive T cells in male HYTg/zeta KO mice. There was a shift of T cell selection from positive to no selection of HY-specific T cells in female HYTg/zeta KO mice also. Collectively, these findings suggest that the level of TCR signal directly regulates T cell selection; furthermore, the findings have integrated the models of T cell selection into a concept based on the quantity of TCR signal.     

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.     

Association of phosphatidylinositol 3-kinase with a specific sequence of the T cell receptor zeta chain is dependent on T cell activation

The T cell antigen receptor (TCR).CD3 complex contains several distinct but related signal transduction modules termed "Reth motifs": one each in the cytoplasmic domains of CD3-gamma, -delta, and -epsilon chains and three in the CD3-zeta polypeptide (zeta A, zeta B, and zeta C). Cross-linking of individual motifs expressed in chimeric molecules leads to early and late T cell activation events. Although the activated T cell receptor associates with nonreceptor tyrosine kinases, the sites of interaction with kinases and other potential effector molecules have not been fully mapped. Here we show that phosphatidylinositol 3-kinase (PI 3-kinase) preferentially associated with the zeta chain membrane proximal motif zeta A. Maximal PI 3-kinase/zeta A association occurred following TCR.CD3 activation and was dependent upon phosphorylation of both tyrosine residues in zeta A. The association of PI 3-kinase was specific for zeta A and could be ranked zeta A > zeta C > zeta B. Phosphorylation of the zeta A motif on tyrosine residues occurred in response to TCR.CD3 cross-linking in vivo. These results indicate that T cell activation leads to assembly of an intracellular signaling complex: recruitment of a tyrosine kinase, phosphorylation of zeta A, and association of PI 3-kinase. These data also support a model in which different Reth motifs of the TCR.CD3 complex recruit distinct signal transduction molecules. Thus, the subdomains of the T cell antigen receptor zeta chain may serve different roles during T cell maturation and antigen-driven activation.     

Development and function of T cells in T cell antigen receptor/CD3 zeta knockout mice reconstituted with Fc epsilon RI gamma

Engagement of alpha-beta T cell receptors (TCRs) induces many events in the T cells bearing them. The proteins that transduce these signals to the inside of cells are the TCR-associated CD3 polypeptides and zeta-zeta or zeta-eta dimers. Previous experiments using knockout (KO) mice that lacked zeta (zeta KO) showed that zeta is required for good surface expression of TCRs on almost all T cells and for normal T cell development. Surprisingly, however, in zeta KO mice, a subset of T cells in the gut of both zeta KO and normal mice bore nearly normal levels of TCR on its surface. This was because zeta was replaced by the Fc epsilon RI gamma (FcR gamma). These cells were relatively nonreactive to stimuli via their TCRs. In addition, a previous report showed that zeta replacement by the FcR gamma chain also might occur on T cells in mice bearing tumors long term. Again, these T cells were nonreactive. To understand the consequences of zeta substitution by FcR gamma for T cell development and function in vivo, we produced zeta KO mice expressing FcR gamma in all of their T cells (FcR gamma TG zeta KO mice). In these mice, TCR expression on immature thymocytes was only slightly reduced compared with controls, and thymocyte selection occurred normally and gave rise to functional, mature T cells. Therefore, the nonreactivity of the FcR gamma + lymphocytes in the gut or in tumor-bearing mice must be caused by some other phenomenon. Unexpectedly, the TCR levels of mature T cells in FcR gamma TG zeta KO mice were lower than those of controls. This was particularly true for the CD4+ T cells. We conclude that FcR gamma can replace the functions of zeta in T cell development in vivo but that TCR/CD3 complexes associated with FcR gamma rather than zeta are less well expressed on cells. Also, these results revealed a difference in the regulation of expression of the TCR/CD3 complex on CD4+ and CD8+ T cells.     

Calnexin associates with monomeric and oligomeric (disulfide-linked) CD3delta proteins in murine T lymphocytes

The antigen-binding receptor expressed on most T lymphocytes consists of disulfide-linked clonotypic alphabeta heterodimers noncovalently associated with monomeric CD3gamma,delta,epsilon proteins and disulfide-linked zeta zeta homodimers, collectively referred to as the T cell antigen receptor (TCR) complex. Here, we examined and compared the disulfide linkage status of newly synthesized TCR proteins in murine CD4(+)CD8(+) thymocytes and splenic T cells. These studies demonstrate that CD3delta proteins exist as both monomeric and oligomeric (disulfide-linked) species that differentially assemble with CD3epsilon subunits in CD4(+)CD8(+) thymocytes and splenic T cells. Interestingly, unlike previous results on glucose trimming and TCR assembly of CD3delta proteins in splenic T cells (Van Leeuwen, J. E. M., and K. P. Kearse (1996) J. Biol. Chem. 271, 9660-9665), we found that glucose residues were not invariably removed from CD3delta glycoproteins prior to their assembly with CD3epsilon subunits in CD4(+)CD8(+) thymocytes. Finally, these studies show that calnexin associates with both monomeric and disulfide-linked CD3delta proteins in murine T cells. The data in the current report demonstrate that CD3delta proteins exist as both monomeric and disulfide-linked molecules in murine T cells that differentially associate with partner TCR chains in CD4(+)CD8(+) thymocytes and splenic T cells. These results are consistent with the concept that folding and assembly of CD3delta proteins is a function of their oxidation state.     

Expression of a CD3 epsilon transgene in CD3 epsilon(null) mice does not restore CD3 gamma and delta expression but efficiently rescues T cell development from a subpopulation of prothymocytes

The TCR-associated CD3 complex consists of four subunits, i.e. CD3 gamma, delta, epsilon and zeta, which are expressed very early in T cell development prior to the expression of the TCR and the pre-TCR alpha chain. It is unclear whether the expression of each CD3 protein is independent of, or is influenced by, other CD3 subunits. To study whether CD3 epsilon regulates expression of CD3 gamma and delta genes, we generated a strain of CD3 epsilon-deficient mice termed CD3 epsilon(delta P/delta P) (epsilon(delta P)), in which the promoter of CD3E was disrupted, and subsequently reconstituted these mice with a CD3 epsilon transgene. In the epsilon(delta P) mice, T cell development is arrested at the double-negative stage and targeting the CD3 epsilon gene caused severe inhibition of CD3 gamma and delta gene expression. Introduction of the CD3 epsilon transgene did not restore CD3 gamma and delta expression. However, a very small fraction of prothymocytes that expressed CD3 gamma and delta was rescued upon reconstitution of the CD3 epsilon transgene. Remarkably, this rescue led to a very efficient differentiation and maturation of thymocytes, resulting in a significant T cell population in the periphery. These results demonstrate that CD3 epsilon does not regulate expression of CD3 gamma and delta genes, and underscore the capacity of each prothymocyte to give rise to a large number of mature peripheral T cells.     

Separately expressed T cell receptor alpha and beta chain transgenes exert opposite effects on T cell differentiation and neoplastic transformation

Two aspects of T cell differentiation in T cell receptor (TCR)-transgenic mice, the generation of an unusual population of CD4-CD8-TCR+ thymocytes and the absence of gamma delta cells, have been the focus of extensive investigation. To examine the basis for these phenomena, we investigated the effects of separate expression of a transgenic TCR alpha chain and a transgenic TCR beta chain on thymocyte differentiation. Our data indicate that expression of a transgenic TCR alpha chain causes thymocytes to differentiate into a CD4-CD8-TCR+ lineage at an early developmental stage, depleting the number of thymocytes that differentiate into the alpha beta lineage. Surprisingly, expression of the TCR alpha chain transgene is also associated with the development of T cell lymphosarcoma. In contrast, expression of the transgenic TCR beta chain causes immature T cells to accelerate differentiation into the alpha beta lineage and thus inhibits the generation of gamma delta cells. Our observations provide a model for understanding T cell differentiation in TCR-transgenic mice.     

CD16-expressing CD8alpha alpha+ T lymphocytes in the intestinal epithelium: possible precursors of Fc gammaR-CD8alpha alpha+ T cells

T lymphocytes normally express their Ag receptors in association with the CD3 proteins, which include CD3zeta. In CD3zeta eta(null) mice thymic and peripheral T lymphocytes do not express the TCR/CD3 complex on their surface due to retention in the endoplasmic reticulum of the remaining polypeptide chains. However, intestinal intraepithelial lymphocytes (iIEL) of CD3zeta eta(null) mice do express surface TCR, because the Fc epsilonRI gamma chain replaced the CD3zeta chain in the TCR/CD3 complex. Here we report that in a subset of CD8alpha alpha+ iIEL the presence of the Fc epsilonRI gamma chain could be accounted for by the surface expression of the Fc gammaRIII(CD16) complex. Because in wild-type (wt) mice only CD16+ iIEL coexpressed Fc epsilonRI gamma and CD3zeta, we concluded that the presence of Fc epsilonRI gamma was dictated by its required participation of CD16 complex. CD8alpha alpha+ iIEL bearing CD16 and B220 were also detected in the intestinal mucosa of RAG-2(null) mice from 12 days after birth onward. Two independent experimental settings were used in an attempt to demonstrate that CD16+ iIEL matured into CD16- T cells. First, in the RAG-2(null) mice, iIEL responded to in vivo administration of an anti-CD3epsilon mAb by progression to a more mature stage of development, characterized by a loss of CD16 and B220. Secondly, a conversion to CD16- iIEL occurred upon transfer of wt CD16+ iIEL into RAG-2(null) mice. We conclude from these experiments that in both RAG-2(null) and wt mice, a precursor/progeny relationship may exists between CD16+ B220+ CD8alpha alpha+ and CD16- B220- CD8alpha alpha+ iIEL.     

Immature thymocyte antigen-1: a novel thymocyte marker discriminating pre- and post-selected thymocytes

Previously, we described a mAb (1-23) reacting with a novel cell surface antigen expressed on thymocytes at late CD4-CD8- [(double negative (DN)] to early CD4+CD8+ [(double positive (DP)] differentiation stage. Since the expression of this molecule was restricted to immature thymocytes, we designated it as immature thymocyte antigen-1 (IMT-1). In this study, we have investigated the relevance of IMT-1 expression to thymocyte selection using TCR transgenic mice, scid mice or RAG-2-/- mice. The IMT-1+ population in DP thymocytes was decreased in the thymuses of MHC class I-restricted or class II-restricted TCR transgenic mice with a positively selecting MHC background when compared with that of the mice with a non-selecting MHC background. IMT-1+ DP thymocytes were also decreased in TCR transgenic mice in which negative selection occurs. When DP thymocytes in H-Y TCR transgenic mice were stimulated with CD3epsilon mAb in vitro as well as in vivo, the expression of IMT-1 on DP thymocytes was decreased. Furthermore, the expression of IMT-1 on DN thymocytes from RAG-2-/- mice was drastically reduced when CD3epsilon mAb was challenged in vivo. These results suggest that the expression of IMT-1 on DP or DN thymocytes is down-regulated by stimulation through TCR as well as pre-TCR. Taken together, these results show that IMT-1 is a unique surface marker which exquisitely separates pre-selected thymocytes from post-selected thymocytes.     

Requirements for differentiation of an immature CD4+8+ T-cell line

The CD3 epsilon and zeta chains of the TCR have been shown to possess independent signaling capabilities. Studies with chimeric molecules containing the cytoplasmic domains of either zeta or epsilon have suggested that these two structurally distinct members of the TCR-CD3 complex are able to function autonomously and have redundant features in the context of TCR-signal transduction in mature T cells. Expression of a chimeric human IL-2-receptor-zeta-chain molecule in the CD4+8+ T-cell line, DPK, has enabled us to directly analyze responses initiated by the zeta-chain-signaling module alone within the context of immature T-cell differentiation. In this paper, we show that antibody crosslinking of the chimeric zeta chain delivers only a limited activation signal as measured by Ca[2+] flux, induction of low-level CD5 expression, and minimal differentiation as assessed by loss of cell-surface CD8 expression. TCR-induced activation through antibody crosslinking of the endogenous CD3 epsilon receptor in the absence of costimulation was also relatively inefficient in initiating activation and differentiation. However, co-crosslinking of the CD4 coreceptor with CD3 resulted in a synergistic response, where as there was little effect of co-crosslinking of CD4 and the zeta-chain chimera. Striking differences were also observed in the substrate pattern of tyrosine phosphorylation, as well as lymphokine secretion following triggering through the intact TCR versus the zeta chain alone. These results indicate that although the zeta-chain may possess some signaling capacities similar to that of the intact TCR, it appears to have limited function as an autonomous subunit in initiating CD4+8+ T-cell differentiation.     

CD3 ligation on immature thymocytes generates antagonist-like signals appropriate for CD8 lineage commitment, independently of T cell receptor specificity

The signals that direct differentiation of T cells to the CD4 or CD8 lineages in the thymus remain poorly understood. Although it has been relatively easy to direct differentiation of CD4 single positive (CD4+) cells using combinations of antibodies and pharmacological agents that mimic receptor engagements, equivalent stimuli do not induce efficient maturation of CD8+ cells. Here we report that, irrespective of the MHC-restriction specificity of the TCR, differentiation of mature CD8+ thymocytes can be induced by ligation of CD3 polypeptides on immature thymocytes with a F(ab')2 reagent (CD3fos-F(ab')2). The tyrosine phosphorylation patterns stimulated by CD3fos-F(ab')2 have been shown to resemble those delivered to mature T cells by antagonist peptides, which are known to direct positive selection of CD8+ cells, and we can show that this reagent exhibits potent antagonistic-like activity for primary T cell responses. Our results suggest a distinction in the signals that specify lineage commitment in the thymus. We present a model of thymocyte differentiation that proposes that the relative balance of signals delivered by TCR engagement and by p56lck activation is responsible for directing commitment to the CD8 or CD4 lineages.     

The JNK pathway regulates the In vivo deletion of immature CD4(+)CD8(+) thymocytes

The extracellular signal-regulated kinase (ERK), the c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase pathways are triggered upon ligation of the antigen-specific T cell receptor (TCR). During the development of T cells in the thymus, the ERK pathway is required for differentiation of CD4(-)CD8(-) into CD4(+)CD8(+) double positive (DP) thymocytes, positive selection of DP cells, and their maturation into CD4(+) cells. However, the ERK pathway is not required for negative selection. Here, we show that JNK is activated in DP thymocytes in vivo in response to signals that initiate negative selection. The activation of JNK in these cells appears to be mediated by the MAP kinase kinase MKK7 since high levels of MKK7 and low levels of Sek-1/MKK4 gene expression were detected in thymocytes. Using dominant negative JNK transgenic mice, we show that inhibition of the JNK pathway reduces the in vivo deletion of DP thymocytes. In addition, the increased resistance of DP thymocytes to cell death in these mice produces an accelerated reconstitution of normal thymic populations upon in vivo DP elimination. Together, these data indicate that the JNK pathway contributes to the deletion of DP thymocytes by apoptosis in response to TCR-derived and other thymic environment- mediated signals.     

Requirement for p56lck tyrosine kinase activation in T cell receptor-mediated thymic selection

The nonreceptor protein tyrosine kinase p56lck (Lck) serves as a fundamental regulator of thymocyte development by delivering signals from the pre-T cell receptor (pre-TCR) that permit subsequent maturation. However, considerable evidence supports the view that Lck also participates in signal transduction from the mature TCR. We have tested this conjecture by expressing a dominant-negative form of Lck under the control of a promoter element (the distal lck promoter) that directs high expression in CD4+CD8+ thymocytes, mature thymocytes, and peripheral T cells, thereby avoiding, complications that result from the well-documented ability of dominant-negative Lck to block very early events in thymocyte maturation. Here we report that expression of the catalytically inactive Lck protein at twice normal concentrations inhibits thymocyte positive selection by as much as 80%, while leaving other aspects of cell maturation intact. This effect was studied in more detail in mice simultaneously bearing the male-specific H-Y alpha/beta TCR transgene and ovalbumin-specific DO10 alpha/beta TCR transgene, where even equimolar expression of the dominant-negative Lck protein substantially vitiated the positive selection process. Although deletion of H-Y alpha/beta thymocytes proceeded normally in male mice despite the presence of catalytically inactive Lck, modest inhibition of superantigen-mediated deletion was in some cases observed. These data further implicate Lck in the propagation of all TCR-derived signals, and indicate that even very modest deficiencies in the representation of functional Lck molecules could in humans, profoundly alter the character of the peripheral TCR repertoire.     

Rapid deletion of rearranged T cell antigen receptor (TCR) Valpha-Jalpha segment by secondary rearrangement in the thymus: role of continuous rearrangement of TCR alpha chain gene and positive selection in the T cell repertoire formation

A rearranged T cell receptor (TCR) Valpha and Jalpha gene from a cytochrome c-specific T cell hybridoma was introduced into the genomic Jalpha region. The introduced TCR alpha chain gene is expressed in a majority of CD3 positive and CD4 CD8 double-negative immature thymocytes. However, only a few percent of the double-positive and single-positive thymocytes express this TCR alpha chain. This decrease is caused by a rearrangement of TCR alpha chain locus, which deletes the introduced TCR gene. Analysis of the mice carrying the introduced TCR alpha chain and the transgenic TCR beta chain from the original cytochrome c-specific T cell hybridoma revealed that positive selection efficiently rescues double-positive thymocytes from the loss of the introduced TCR alpha chain gene. In the mice with negatively selecting conditions, T cells expressing the introduced TCR alphabeta chains were deleted at the double-positive stage. However, a large number of thymocytes escape negative selection by using an endogenous TCR alpha chain created by secondary rearrangement maintaining normal thymocyte development. These results suggest that secondary rearrangements of the TCR alpha chain gene play an important role in the formation of the T cell repertoire.     

The CD3gamma chain is essential for development of both the TCRalphabeta and TCRgammadelta lineages

CD3gamma and CD3delta are the most closely related CD3 components, both of which participate in the TCRalphabeta-CD3 complex expressed on mature T cells. Interestingly, however, CD3delta does not appear to participate functionally in the pre-T-cell receptor (TCR) complex that is expressed on immature T cells: disruption of CD3delta gene expression has no effect on the developmental steps controlled by the pre-TCR. Here we report that in contrast with CD3delta, CD3gamma is an essential component of the pre-TCR. We generated mice selectively lacking expression of CD3gamma, in which expression of CD3delta, CD3epsilon, CD3zeta, pTalpha and TCRbeta remained undisturbed. Thus, all components for composing a pre-TCR are available, with the exception of CD3gamma. Nevertheless, T-cell development is severely inhibited in CD3gamma-deficient mice. The number of cells in the thymus is reduced to <1% of that in normal mice, and the large majority of thymocytes lack CD4 and CD8 and are arrested at the CD44-CD25+ double negative (DN) stage of development. Peripheral lymphoid organs are also practically devoid of T cells, with absolute numbers of peripheral T cells reduced to only 2-5% of those in normal mice. Both TCRalphabeta and TCRgammadelta lineages fail to develop effectively in CD3gamma-deficient mice, although absence of CD3gamma has no effect on gene rearrangements of the TCRbeta, delta and gamma loci. Furthermore, absence of CD3gamma results in a severe reduction in the level of TCR and CD3epsilon expression at the cell surface of thymocytes and peripheral T cells. The defect in the DN to double positive transition in mice lacking CD3gamma can be overcome by anti-CD3epsilon-mediated cross-linking. CD3gamma is thus essential for pre-TCR function.     

Signaling pathways and their role in maturation and function of T lymphocytes

Signals delivered through the beta/gp33 (pre-TCR) and T-cell receptor alpha beta control proliferation and differentiation of thymocytes at two distinct control points of T cell maturation. Interaction between T-cell receptor (TCR) and peptide/MHC complex induce signaling pathways leading to activation of T cell. Signal transduction involves CD3 zeta phosphorylation by Lck tyrosine kinase and activation of ZAP-70 which regulates signaling pathways through PKC, Ca++ and Ras/Raf kinase cascade. Appropriate response of cell is preceded by integration of different signals in the nucleus.