JUN is a key transcriptional regulator of the unfolded protein response in acute myeloid leukemia.

The transcription factor JUN is frequently overexpressed in multiple genetic subtypes of acute myeloid leukemia (AML); however, the functional role of JUN in AML is not well defined. Here we report that short hairpin RNA (shRNA)-mediated inhibition of JUN decreases AML cell survival and propagation in vivo. By performing RNA sequencing analysis, we discovered that JUN inhibition reduces the transcriptional output of the unfolded protein response (UPR), an intracellular signaling transduction network activated by endoplasmic reticulum (ER) stress. Specifically, we found that JUN is activated by MEK signaling in response to ER stress, and that JUN binds to the promoters of several key UPR effectors, such as XBP1 and ATF4, to activate their transcription and allow AML cells to properly negotiate ER stress. In addition, we observed that shRNA-mediated inhibition of XBP1 or ATF4 induces AML cell apoptosis and significantly extends disease latency in vivo tying the reduced survival mediated by JUN inhibition to the loss of pro-survival UPR signaling. These data uncover a previously unrecognized role of JUN as a regulator of the UPR as well as provide key new insights into the how ER stress responses contribute to AML and identify JUN and the UPR as promising therapeutic targets in this disease.

Transcription factors NFAT2 and Egr1 cooperatively regulate the maturation of T-lymphoma in vitro.

We have demonstrated that transcription factors Egr1 and NFAT2 cooperate in regulation of the early stages of T-lymphocyte development, whereas the related factors Egr2 and Egr3 do not cooperate with NFAT2. Egr1 and NFAT2 are shown to cooperatively control gene expression of the regulatory factor Id3 and recombinase Rag2, whose functions are critical for T-lymphocyte differentiation. Thus, the concerted action of the transcription factors Egr1 and NFAT2 can play a crucial role in regulation of the T cell differentiation in vitro due to the cooperative regulation of Id3 and Rag2 gene expression.

On the dynamics of TCR:CD3 complex cell surface expression and downmodulation.

TCR downmodulation following ligation by MHC:peptide complexes is considered to be a pivotal event in T cell activation. Here, we analyzed the dynamics of TCR:CD3 cell surface expression on resting and antigen-activated T cells. We show that the TCR:CD3 complex is very stable and is rapidly internalized and recycled in resting T cells. Surprisingly, the internalization rate is not increased following TCR ligation by MHC:peptide complexes, despite significant TCR downmodulation, suggesting that constitutive internalization rather than ligation-induced downmodulation serves as the force that drives serial ligation. Furthermore, TCR downmodulation is mediated by the intracellular retention of ligated complexes and degradation by lysosomes and proteasomes. Thus, our data demonstrate that ligation induces TCR downmodulation by preventing recycling rather than inducing internalization.

Branching out to gain control: how the pre-TCR is linked to multiple functions.

How is signaling specificity achieved by the pre-TCR during selection of T-cell fate? Like the TCR, this receptor controls many functions, and recent studies define which pathways couple the pre-TCR to the molecular events controlling survival, proliferation, allelic exclusion at the TCRbeta locus, and further differentiation.

Competitive displacement of pT alpha by TCR-alpha during TCR assembly prevents surface coexpression of pre-TCR and alpha beta TCR.

During alphabeta T cell development, CD4(-)CD8(-) thymocytes first express pre-TCR (pTalpha/TCR-beta) before their differentiation to the CD4(+)CD8(+) stage. Positive selection of self-tolerant T cells is then determined by the alphabeta TCR expressed on CD4(+)CD8(+) thymocytes. Conceivably, an overlap in surface expression of these two receptors would interfere with the delicate balance of thymic selection. Therefore, a mechanism ensuring the sequential expression of pre-TCR and TCR must function during thymocyte development. In support of this notion, we demonstrate that expression of TCR-alpha by immature thymocytes terminates the surface expression of pre-TCR. Our results reveal that expression of TCR-alpha precludes the formation of pTalpha/TCR-beta dimers within the endoplasmic reticulum, leading to the displacement of pre-TCR from the cell surface. These findings illustrate a novel posttranslational mechanism for the regulation of pre-TCR expression, which may ensure that alphabeta TCR expression on thymocytes undergoing selection is not compromised by the expression of pre-TCR.

Identification of a novel pre-TCR isoform in which the accessibility of the TCR beta subunit is determined by occupancy of the 'missing' V domain of pre-T alpha.

We have identified a novel pre-TCR isoform that is structurally distinct from conventional pre-TCR complexes and whose TCR beta chains are inaccessible to anti-TCR beta antibodies. We term this pre-TCR isoform the MB (masked beta)-pre-TCR. Pre-T alpha (pT alpha) subunits of MB-pre-TCR complexes have a larger apparent mol. wt due to extensive modification with O:-linked carbohydrates; however, preventing addition of O-glycans does not restore antibody recognition of the TCR beta subunits of MB-pre-TCR complexes. Importantly, accessibility of TCR beta chains in MB-pre-TCR complexes is restored by filling in the 'missing' variable (V) domain of pT alpha with a V domain from TCR alpha. Moreover, the proportion of pre-TCR complexes in which the TCR beta subunits are accessible to anti-TCR beta antibody varies with the cellular context, suggesting that TCR beta accessibility is controlled by a trans-acting factor. The way in which this factor might control TCR beta accessibility as well as the physiologic relevance of TCR beta masking for pre-TCR function are discussed.

Extracellular signal-regulated kinase (ERK) activation by the pre-T cell receptor in developing thymocytes in vivo.

The first checkpoint in T cell development occurs between the CD4(-)CD8(-) and CD4(+)CD8(+) stages and is associated with formation of the pre-T cell receptor (TCR). The signaling mechanisms that drive this progression remain largely unknown. Here, we show that extracellular signal-regulated kinases (ERKs)-1/2 are activated upon engagement of the pre-TCR. Using a novel experimental system, we demonstrate that expression of the pre-TCR by developing thymocytes induces ERK-1/2 activation within the thymus. In addition, the activation of this pre-TCR signaling cascade is mediated through Lck. These findings directly link pre-TCR complex formation with specific downstream signaling components in vivo.

Peripheral CD4+ T cell maturation recognized by increased expression of Thy-1/CD90 bearing the 6C10 carbohydrate epitope.

The SM6C10 IgM autoantibody recognizes a surface determinant, 6C10, that is highly expressed on all immature thymocytes. In contrast, its expression on peripheral T cells appears developmentally regulated, i.e., absent from most naive T cells in spleen of neonatal mice, but expressed on 40-80% of naive CD4+ T cells in adult. In this paper, we demonstrate that SM6C10 recognizes a carbohydrate epitope on the Thy-1 glycoprotein using immunoprecipitation analysis, by binding to affinity-purified Thy-1 in an ELISA, and by sensitivity to N-glycosidase-F treatment. Retroviral Thy-1 gene transduction experiments into Thy-1- variant T cell lines and a pro-B cell line provide evidence that 6C10 glycosylated Thy-1 expression is not restricted to T cells but depends on the recipient cell. Therefore, differences in 6C10 levels among Thy-1+ T cells in mice likely reflect developmental regulation of posttranslational modification of the Thy-1 glycoprotein. The ability of naive CD4+ T cells to respond to anti-Thy-1 stimulation increases from neonate to adult, and 6C10- naive cells from adult mice respond poorly compared with 6C10+ cells, similar to the cells in neonatal mice. These results suggest that there is functional maturation by peripheral CD4+ T cells that coincides with 6C10 glycosylated Thy-1 up-regulation, and natural autoantibody recognizes this 6C10 carbohydrate epitope.

Control of early thymocyte development by the pre-T cell receptor complex: A receptor without a ligand?

Beta-selection refers to a developmental checkpoint linking thymocyte survival to the outcome of antigen receptor gene rearrangement. Immature thymocytes that productively rear-range the gene segments of the TCRbeta locus undergo proliferative expansion and mature to the CD4(+)CD8(+)stage; those failing to do so die by apoptosis. How are these precursor cells alerted that TCRbeta rearrangement has been productive? While it is clear that this process involves signals transduced by a surrogate form of the TCR termed the pre-TCR, it remains unclear how pre-TCR signals are triggered. In this review, we will discuss the implications of recent experimental attempts to address this issue, as well as how pre-TCR activation is linked to the changes in gene expression that underlie thymocyte development.

Signals transduced by CD3epsilon, but not by surface pre-TCR complexes, are able to induce maturation of an early thymic lymphoma in vitro.

Development of immature CD4-CD8- (double-negative) thymocytes to the CD4+CD8+ (double-positive) stage is linked to productive rearrangement of the TCRbeta locus by signals transduced through the pre-TCR. However, the mechanism whereby pre-TCR signaling is initiated remains unclear, in part due to the lack of an in vitro model system amenable to both biochemical and genetic analysis. In this study, we establish the thymic lymphoma Scid.adh as such a model system. Scid.adh responds to Ab engagement of surface IL-2Ra (TAC):CD3epsilon molecules (a signaling chimera that mimics pre-TCR signaling in vivo) by undergoing changes in gene expression observed following pre-TCR activation in normal thymocytes. These changes include down-regulation of CD25, recombinase-activating gene (RAG)-1, RAG-2, and pTalpha; and the up-regulation of TCRalpha germline transcripts. We term this complete set of changes in gene expression, in vitro maturation. Interestingly, Scid.adh undergoes only a subset of these changes in gene expression following Ab engagement of the pre-TCR. Our findings make two important points. First, because TAC:CD3epsilon stimulation of Scid.adh induces physiologically relevant changes in gene expression, Scid.adh is an excellent cellular system for investigating the molecular requirements for pre-TCR signaling. Second, Ab engagement of CD3epsilon signaling domains in isolation (TAC:CD3epsilon) promotes in vitro maturation of Scid.adh, whereas engagement of CD3epsilon molecules contained within the complete pre-TCR fails to do so. Our current working hypothesis is that CD3epsilon fails to promote in vitro maturation when in the context of an Ab-engaged pre-TCR because another pre-TCR subunit(s), possibly TCRzeta, qualitatively alters the CD3epsilon signal.

The connecting peptide domain of pT alpha dictates weak association of the pre-T cell receptor with the TCR zeta subunit.

Signals delivered through the pre-TCR, a heterodimer of pT alpha and TCR beta chains, are crucial for the maturation and proliferation of immature alphabeta lineage thymocytes from the CD4- CD8- to the CD4+ CD8+ stage. To gain insight into the structural and functional properties of the pre-TCR, chimeric TCR alpha chains were generated by replacing domains of the alpha chain cytoplasmic, transmembrane and constant regions with homologous domains from the pT alpha chain. All chimeric TCR could be expressed stably at the cell surface and induce Ca2+ mobilization as well as phosphorylation of several protein substrates on tyrosine residues. However, chimeras wherein the connecting peptide of TCR alpha chain was substituted by the one from pT alpha, were weakly associated with the TCR zeta chain, showing that functional but not physical interactions were preserved in such chimeras. In contrast, introduction of the connecting peptide of TCR alpha in the pT alpha chain was insufficient to confer stable association with the TCR zeta chain. These results demonstrate that the inability of the pre-TCR to interact strongly with TCR zeta is attributable to amino acid residues present throughout the region comprised between the intrachain Cys and the transmembrane domain. It remains to be determined whether the weak physical interaction between the pre-TCR alphand the zeta2 homodimer prevents the activation of specific TCR zeta-dependent signaling pathways, and thus confers unique signaling properties upon the pre-TCR. In addition, this structural difference between the pT alpha/beta and alphabeta TCR might constitute a means to regulate the expression of these receptors at the surface of thymocytes, at different stages of their maturation.

Limiting TCR expression leads to quantitative but not qualitative changes in thymic selection.

Thymic selection is controlled in part by the avidity of the interaction between thymocytes and APCs. In agreement, the selective outcome can be modulated by altering the expression levels of selecting ligands on APCs. Here we test the converse proposition, i. e., whether changing TCR levels on thymocytes can alter the selective outcome. To this end, we have generated mice in which all thymocytes express two transgenic TCRs simultaneously (dual TCR-expressing (DTE) mice), the class I-restricted HY TCR and the class II-restricted AND TCR. Due to mutual dilution, surface expression levels of the two individual transgenic TCRs are diminished in DTE relative to single TCR-expressing mice. We find that thymic selection is highly sensitive to these reductions in TCR surface expression. Positive selection mediated by the AND and HY TCRs is severely impaired or abolished, respectively. Negative selection of the HY TCR in male DTE mice is also partly blocked, leading to the appearance of significant numbers of double positive thymocytes. Also, in the periphery of male, but not female, DTE mice, substantial numbers of single positive CD8bright cells accumulate, which are positively selected in the thymus but by a highly inefficient hemopoietic cell-dependent process. Overall our results favor the interpretation that the outcome of thymic selection is not determined solely by avidity and the resulting signal intensity, but is also constrained by other factors such as the nature of the ligand and/or its presentation by different subsets of APCs.

ZAP-70 protein promotes tyrosine phosphorylation of T cell receptor signaling motifs (ITAMs) in immature CD4(+)8(+) thymocytes with limiting p56(lck).

As a result of interaction with epithelial cells in the thymic cortex, immature CD4(+)8(+) (double positive, DP) thymocytes express relatively few T cell receptors (TCRs) and contain diminished numbers of coreceptor-associated p56(lck) (lck) PTK molecules. As a result, TCR signal transduction in DP thymocytes is significantly impaired, despite its importance for repertoire selection. We report here that, in DP thymocytes, tyrosine phosphorylation of TCR signaling motifs (ITAMs) by lck, an early event in TCR signal transduction, is dependent upon ZAP-70 protein independent of ZAP-70's kinase activity. Furthermore, the dependence on ZAP-70 protein for ITAM phosphorylation diminishes as available lck increases. Importantly, ZAP-70's role in ITAM phosphorylation in DP thymocytes is not limited to protecting phosphorylated ITAMs from dephosphorylation. Rather, this study indicates that ZAP-70 protein augments ITAM phosphorylation in DP thymocytes and so compensates in part for the relative deficiency of coreceptor-associated lck.

Altered functional responsiveness of thymocyte subsets from CD3delta-deficient mice to TCR-CD3 engagement.

CD3delta-deficient (delta degrees) mice are defective in alphabeta T cell development. Here we explore the capacity of TCR-CD3 signaling complexes expressed on delta degrees thymocytes to mediate the following functional outcomes in response to antibody cross-linking: (i) the transition from the CD4-CD8- to CD4+CD8+ stage, (ii) the transition from the CD4+CD8+ to CD4+CD8- or CD4-CD8+ stages and (iii) the induction of apoptosis. We provide evidence that CD3deltaepsilon complexes are dispensable for mediating the anti-CD3-mediated CD4-CD8- to CD4+CD8+ transition. On the other hand, CD3delta is critical at the CD4+CD8+ stage. We demonstrate that CD4+CD8+ thymocytes from delta degrees mice, unlike delta degrees CD4-CD8- thymocytes and wild-type CD4+CD8+ thymocytes, require prolonged or consecutive stimuli to elicit functional responses. Depending on the nature of the secondary stimulus, delta degrees thymocytes can be induced to undergo apoptosis or preferential maturation to the CD4-CD8+ stage. Taken together these results indicate that the signaling capacity of the TCR-CD3 complex is noticeably altered in the absence of CD3delta. The essential role of CD3delta at the CD4+CD8+ stage of development correlates with the onset of TCRalpha rearrangement, consistent with a critical structural and/or functional relationship between CD3delta and TCRalpha.

Transmembrane polar residues of TCR beta chain are required for signal transduction.

Mutagenic analyses have identified structural motifs important for TCR-mediated signaling in the antigen-binding chains, CD3 and zeta subunits of the TCR complex. In this study, we altered selected residues in the transmembrane and extracellular constant regions of the TCR beta chain and expressed the mutants in a T hybridoma line bearing endogenous receptor. We measured cytokine production and apoptosis in response to antigen or antibody. We found that mutation of one or both of the transmembrane tyrosine residues in the TCR beta chain caused a marked reduction in responsiveness. Mutation of the transmembrane serine to alanine also reduced responses, although less markedly. Immunoprecipitation analyses showed that the TCR beta mutations did not alter association with zeta. These experiments identify a signaling role for the transmembrane domain of the TCR beta chain.

A novel mechanism for B cell repertoire maturation based on response by B cell precursors to pre-B receptor assembly.

The expression of different sets of immunoglobulin specificities by fetal and adult B lymphocytes is a long-standing puzzle in immunology. Recently it has become clear that production of immunoglobulin mu heavy chain and subsequent assembly with a surrogate light chain to form the pre-B cell receptor complex is critical for development of B cells. Here we show that instead of promoting pre-B cell progression as in adult bone marrow, this complex inhibits pre-B cell growth in fetal liver. Curiously, we identify a fetal-associated VH11 mu heavy chain that allows continued pre-B proliferation in fetal liver. Interestingly, this heavy chain does not associate efficiently with a surrogate light chain, providing a previously unrecognized mechanism for skewing the expression of distinctive VH genes toward fetal through early neonatal life.

Subunit composition of pre-T cell receptor complexes expressed by primary thymocytes: CD3 delta is physically associated but not functionally required.

Maturation of immature CD4-CD8- (DN) thymocytes to the CD4+CD8+ (DP) stage of development is driven by signals transduced through a pre-T cell receptor (TCR) complex, whose hallmark is a novel subunit termed pre-T alpha (pT alpha). However, the precise role of pre-TCRs in mediating the DN to DP transition remains unclear. Moreover, progress in understanding pre-TCR function has been hampered thus far because previous attempts to demonstrate expression of pT alpha-containing pre-TCRs on the surface of normal thymocytes have been unsuccessful. In this report, we demonstrate for the first time that pT alpha-containing pre-TCR complexes are expressed at low levels on the surface of primary thymocytes and that these pre-TCR complexes comprise a disulfide-linked pT alpha-TCR-beta heterodimer associated not only with CD3-gamma and -epsilon, as previously reported, but also with zeta and delta. Interestingly, while CD3-delta is associated with the pre-TCR complex, it is not required for pre-TCR function, as evidenced by the generation of normal numbers of DP thymocytes in CD3-delta-deficient mice. The fact that any of the signaling components of the pre-TCR are dispensable for pre-TCR function is indeed surprising, given that few pre-TCR complexes are actually expressed on the surface of primary thymocytes in vivo. Thus, pre-TCRs do not require the full array of TCR-associated signaling subunits (gamma, delta, epsilon, and zeta), possibly because pT alpha itself possesses signaling capabilities.

A spontaneously arising mutation in the DLAARN motif of murine ZAP-70 abrogates kinase activity and arrests thymocyte development.

Development of immature CD4+ CD8+ thymocytes into functionally mature CD4+ and CD8+ T cells is driven by selection events that require signals transduced through the T cell antigen receptor (TCR). Transduction of TCR signals in the thymus involves tyrosine phosphorylation of the protein tyrosine kinase ZAP-70 by p56(lck) and results in induction of ZAP-70 enzymatic activity. We have identified a novel, spontaneously arising point mutation within a highly conserved motif (DLAARN) in the kinase domain of murine ZAP-70 that uncouples tyrosine phosphorylation of ZAP-70 from induction of ZAP-70 kinase activity. Mice homozygous for this mutation are devoid of mature T cells because thymocyte development is arrested at the CD4+ CD8+ stage of differentiation. The developmental arrest is due to the inability of CD4+ CD8+ thymocytes to propagate TCR signals in the absence of ZAP-70 kinase activity despite tyrosine phosphorylation of TCR-associated ZAP-70 molecules.

Incomplete endoplasmic reticulum (ER) retention in immature thymocytes as revealed by surface expression of "ER-resident" molecular chaperones.

The folding and assembly of nascent proteins in the endoplasmic reticulum (ER) is assisted by molecular chaperones that are themselves retained within the ER. We now report that a number of different ER proteins, including molecular chaperones, are selectively expressed on the surface of immature thymocytes, but their surface expression is extinguished upon further differentiation. Escape from the ER is only possible for newly synthesized ER proteins before they become permanently retained. Thus, the cellular process of ER retention is incomplete in immature thymocytes and provides an explanation for surface expression of partial receptor complexes that transduce differentiative signals during thymic development.

TCR activation of ZAP70 is impaired in CD4+CD8+ thymocytes as a consequence of intrathymic interactions that diminish available p56lck.

The fate of developing CD4+CD8+ thymocytes is determined by signals transduced through surface TCR complexes. Here, we report that cross-linking of TCR on CD4+ CD8+ thymocytes fails to activate ZAP70 protein tyrosine kinase and fails to initiate downstream signaling events, unless the TCR are coaggregated with surface coreceptor molecules. TCR signaling in CD4+CD8+ thymocytes is impaired because the number of available p56lck molecules is diminished by intrathymic CD4-Ia interactions that initially activate p56lck molecules, which are subsequently degraded. As a consequence of intrathymic CD4-Ia interactions, TCR zeta chains are initially phosphorylated to recruit ZAP70 molecules, but the recruited ZAP70 molecules are not subsequently phosphorylated, resulting in TCR complexes that are stably associated with inactive ZAP70 molecules. Thus, intrathymic interactions that diminish p56lck regulate TCR signaling thresholds and affect TCR structure in developing CD4+CD8+ thymocytes.