CD3ζ ITAMs enable ligand discrimination and antagonism by inhibiting TCR signaling in response to low-affinity peptides.

The T cell antigen receptor (TCR) contains ten immunoreceptor tyrosine-based activation motif (ITAM) signaling sequences distributed within six CD3 subunits; however, the reason for such structural complexity and multiplicity is unclear. Here we evaluated the effect of inactivating the three CD3ζ chain ITAMs on TCR signaling and T cell effector responses using a conditional 'switch' mouse model. Unexpectedly, we found that T cells expressing TCRs containing inactivated (non-signaling) CD3ζ ITAMs (6F-CD3ζ) exhibited reduced ability to discriminate between low- and high-affinity ligands, resulting in enhanced signaling and cytokine responses to low-affinity ligands because of a previously undetected inhibitory function of CD3ζ ITAMs. Also, 6F-CD3ζ TCRs were refractory to antagonism, as predicted by a new in silico adaptive kinetic proofreading model that revises the role of ITAM multiplicity in TCR signaling. Finally, T cells expressing 6F-CD3ζ displayed enhanced cytolytic activity against solid tumors expressing low-affinity ligands, identifying a new counterintuitive approach to TCR-mediated cancer immunotherapy.

THEMIS increases TCR signaling in CD4+CD8+ thymocytes by inhibiting the activity of the tyrosine phosphatase SHP1.

The T cell lineage-restricted protein THEMIS plays a critical role in T cell development at the positive selection stage. In the SHP1 activation model, THEMIS is proposed to enhance the activity of the tyrosine phosphatase SHP1 (encoded by Ptpn6), thereby dampening T cell antigen receptor (TCR) signaling and preventing the inappropriate negative selection of CD4+CD8+ thymocytes by positively selecting ligands. In contrast, in the SHP1 inhibition model, THEMIS is proposed to suppress SHP1 activity, rendering CD4+CD8+ thymocytes more sensitive to TCR signaling initiated by low-affinity ligands to promote positive selection. We sought to resolve the controversy regarding the molecular function of THEMIS. We found that the defect in positive selection in Themis-/- thymocytes was ameliorated by pharmacologic inhibition of SHP1 or by deletion of Ptpn6 and was exacerbated by SHP1 overexpression. Moreover, overexpression of SHP1 phenocopied the Themis-/- developmental defect, whereas deletion of Ptpn6, Ptpn11 (encoding SHP2), or both did not result in a phenotype resembling that of Themis deficiency. Last, we found that thymocyte negative selection was not enhanced but was instead impaired in the absence of THEMIS. Together, these results provide evidence favoring the SHP1 inhibition model, supporting a mechanism whereby THEMIS functions to enhance the sensitivity of CD4+CD8+ thymocytes to TCR signaling, enabling positive selection by low-affinity, self-ligand-TCR interactions.

GRB2 promotes thymocyte positive selection by facilitating THEMIS-mediated inactivation of SHP1.

The T-lineage restricted protein THEMIS has been shown to play a critical role in T cell development. THEMIS, via its distinctive CABIT domains, inhibits the catalytic activity of the tyrosine phosphatase SHP1 (PTPN6). SHP1 and THEMIS bind to the ubiquitous cytosolic adapter GRB2, and the purported formation of a tri-molecular THEMIS-GRB2-SHP1 complex facilitates inactivation of SHP1 by THEMIS. The importance of this function of GRB2 among its numerous documented activities is unclear as GRB2 binds to multiple proteins and participates in several signaling responses in thymocytes. Here, we show that similar to Themis-/- thymocytes, the primary molecular defect in GRB2-deficient thymocytes is increased catalytically active SHP1 and the developmental block in GRB2-deficient thymocytes is alleviated by deletion or inhibition of SHP1 and is exacerbated by SHP1 overexpression. Thus, the principal role of GRB2 during T cell development is to promote THEMIS-mediated inactivation of SHP1 thereby enhancing the sensitivity of TCR signaling in CD4+CD8+ thymocytes to low affinity positively selecting self-ligands.

CD5 dynamically calibrates basal NF-κB signaling in T cells during thymic development and peripheral activation.

Immature T cells undergo a process of positive selection in the thymus when their new T cell receptor (TCR) engages and signals in response to self-peptides. As the T cell matures, a slew of negative regulatory molecules, including the inhibitory surface glycoprotein CD5, are up-regulated in proportion to the strength of the self-peptide signal. Together these regulators dampen TCR-proximal signaling and help avoid any subsequent peripheral activation of T cells by self-peptides. Paradoxically, antigen-specific T cells initially expressing more CD5 (CD5hi) have been found to better persist as effector/memory cells after a peripheral challenge. The molecular mechanisms underlying such a duality in CD5 function is not clear. We found that CD5 alters the basal activity of the NF-κB signaling in resting peripheral T cells. When CD5 was conditionally ablated, T cells were unable to maintain higher expression of the cytoplasmic NF-κB inhibitor IκBα. Consistent with this, resting CD5hi T cells expressed more of the NF-κB p65 protein than CD5lo cells, without significant increases in transcript levels, in the absence of TCR signals. This posttranslationally stabilized cellular NF-κB depot potentially confers a survival advantage to CD5hi T cells over CD5lo ones. Taken together, these data suggest a two-step model whereby the strength of self-peptide-induced TCR signal lead to the up-regulation of CD5, which subsequently maintains a proportional reserve of NF-κB in peripheral T cells poised for responding to agonistic antigen-driven T cell activation.

Ldb1 is required for Lmo2 oncogene-induced thymocyte self-renewal and T-cell acute lymphoblastic leukemia.

Prolonged or enhanced expression of the proto-oncogene Lmo2 is associated with a severe form of T-cell acute lymphoblastic leukemia (T-ALL), designated early T-cell precursor ALL, which is characterized by the aberrant self-renewal and subsequent oncogenic transformation of immature thymocytes. It has been suggested that Lmo2 exerts these effects by functioning as component of a multi-subunit transcription complex that includes the ubiquitous adapter Ldb1 along with b-HLH and/or GATA family transcription factors; however, direct experimental evidence for this mechanism is lacking. In this study, we investigated the importance of Ldb1 for Lmo2-induced T-ALL by conditional deletion of Ldb1 in thymocytes in an Lmo2 transgenic mouse model of T-ALL. Our results identify a critical requirement for Ldb1 in Lmo2-induced thymocyte self-renewal and thymocyte radiation resistance and for the transition of preleukemic thymocytes to overt T-ALL. Moreover, Ldb1 was also required for acquisition of the aberrant preleukemic ETP gene expression signature in immature Lmo2 transgenic thymocytes. Co-binding of Ldb1 and Lmo2 was detected at the promoters of key upregulated T-ALL driver genes (Hhex, Lyl1, and Nfe2) in preleukemic Lmo2 transgenic thymocytes, and binding of both Ldb1 and Lmo2 at these sites was reduced following Cre-mediated deletion of Ldb1. Together, these results identify a key role for Ldb1, a nonproto-oncogene, in T-ALL and support a model in which Lmo2-induced T-ALL results from failure to downregulate Ldb1/Lmo2-nucleated transcription complexes which normally function to enforce self-renewal in bone marrow hematopoietic progenitors.

Detection of Intracellular Reduced (Catalytically Active) SHP-1 and Analyses of Catalytically Inactive SHP-1 after Oxidation by Pervanadate or H2O2.

Oxidative inactivation of cysteine-dependent Protein Tyrosine Phosphatases (PTPs) by cellular reactive oxygen species (ROS) plays a critical role in regulating signal transduction in multiple cell types. The phosphatase activity of most PTPs depends upon a 'signature' cysteine residue within the catalytic domain that is maintained in the de-protonated state at physiological pH rendering it susceptible to ROS-mediated oxidation. Direct and indirect techniques for detection of PTP oxidation have been developed (Karisch and Neel, 2013). To detect catalytically active PTPs, cell lysates are treated with iodoacetyl-polyethylene glycol-biotin (IAP-biotin), which irreversibly binds to reduced (S-) cysteine thiols. Irreversible oxidation of SHP-1 after treatment of cells with pervanadate or H2O2 is detected with antibodies specific for the sulfonic acid (SO3H) form of the conserved active site cysteine of PTPs. In this protocol, we describe a method for the detection of the reduced (S-; active) or irreversibly oxidized (SO3H; inactive) form of the hematopoietic PTP SHP-1 in thymocytes, although this method is applicable to any cysteine-dependent PTP in any cell type.

THEMIS: Two Models, Different Thresholds.

THEMIS, a recently identified T-lineage-restricted protein, is the founding member of a large metazoan protein family. Gene inactivation studies have revealed a critical requirement for THEMIS during thymocyte positive selection, implicating THEMIS in signaling downstream of the T cell antigen receptor (TCR), but the mechanistic underpinnings of THEMIS function have remained elusive. A previous model posited that THEMIS prevents thymocytes from inappropriately crossing the positive/negative selection threshold by dampening TCR signaling. However, new data suggest an alternative model where THEMIS enhances TCR signaling enabling thymocytes to reach the threshold for positive selection, avoiding death by neglect. We review the data supporting each model and conclude that the preponderance of evidence favors an enhancing function for THEMIS in TCR signaling.

THEMIS enhances TCR signaling and enables positive selection by selective inhibition of the phosphatase SHP-1.

THEMIS, a T cell-specific protein with high expression in CD4+CD8+ thymocytes, has a crucial role in positive selection and T cell development. THEMIS lacks defined catalytic domains but contains two tandem repeats of a distinctive module of unknown function (CABIT). Here we found that THEMIS directly regulated the catalytic activity of the tyrosine phosphatase SHP-1. This action was mediated by the CABIT modules, which bound to the phosphatase domain of SHP-1 and promoted or stabilized oxidation of SHP-1's catalytic cysteine residue, which inhibited the tyrosine-phosphatase activity of SHP-1. Deletion of SHP-1 alleviated the developmental block in Themis-/- thymocytes. Thus, THEMIS facilitates thymocyte positive selection by enhancing the T cell antigen receptor signaling response to low-affinity ligands.

Themis1 enhances T cell receptor signaling during thymocyte development by promoting Vav1 activity and Grb2 stability.

The T cell signaling protein Themis1 is essential for the positive and negative selection of thymocytes in the thymus. Although the developmental defect that results from the loss of Themis1 suggests that it enhances T cell receptor (TCR) signaling, Themis1 also recruits Src homology 2 domain-containing phosphatase-1 (SHP-1) to the vicinity of TCR signaling complexes, suggesting that it has an inhibitory role in TCR signaling. We used TCR signaling reporter mice and quantitative proteomics to explore the role of Themis1 in developing T cells. We found that Themis1 acted mostly as a positive regulator of TCR signaling in vivo when receptors were activated by positively selecting ligands. Proteomic analysis of the Themis1 interactome identified SHP-1, the TCR-associated adaptor protein Grb2, and the guanine nucleotide exchange factor Vav1 as the principal interacting partners of Themis1 in isolated mouse thymocytes. Analysis of TCR signaling in Themis1-deficient and Themis1-overexpressing mouse thymocytes demonstrated that Themis1 promoted Vav1 activity both in vitro and in vivo. The reduced activity of Vav1 and the impaired T cell development in Themis1(-/-) mice were due in part to increased degradation of Grb2, which suggests that Themis1 is required to maintain the steady-state abundance of Grb2 in thymocytes. Together, these data suggest that Themis1 acts as a positive regulator of TCR signaling in developing T cells, and identify a mechanism by which Themis1 regulates thymic selection.

Impairment of immunological synapse formation in adaptively tolerant T cells.

Adaptive tolerance is a hyporesponsive state in which lymphocyte Ag receptor signaling becomes desensitized after prolonged in vivo encounter with Ag. The molecular mechanisms underlying this hyporesponsive state in T cells are not fully understood, although a major signaling block has been shown to be present at the level of ZAP70 phosphorylation of linker for activation of T cells (LAT). In this study, we investigated the ability of adaptively tolerant mouse T cells to form conjugates with Ag-bearing APCs and to translocate signaling molecules into the interface between the T cells and APCs. Compared with naive or preactivated T cells, adaptively tolerant T cells showed no dramatic impairment in their formation of conjugates with APCs. In contrast, there was a large impairment in immunological synapse formation. Adaptively tolerant T cells were defective in their translocation of signaling molecules, such as ZAP70, LAT, and phospholipase C γ1, into the T cell-APC contact sites. Although Ag-induced activation of VAV1 was normal, VAV's recruitment into the synapse was also impaired. Interestingly, expressions of both IL-2-inducible T cell kinase and growth factor receptor-bound protein 2-related adaptor downstream of SHC were decreased by 60-80% in adaptively tolerant T cells. These decreases, in addition to the impairment in LAT phosphorylation by ZAP70, appear to be the major impediments to the phosphorylation of SLP76 (SRC homology 2 domain-containing leukocyte protein of 76 kDa) and the recruitment of VAV1, which are important for stable immunological synapse formation.

Themis is a member of a new metazoan gene family and is required for the completion of thymocyte positive selection.

T cell antigen receptor (TCR) signaling in CD4(+)CD8(+) double-positive thymocytes determines cell survival and lineage commitment, but the genetic and molecular basis of this process is poorly defined. To address this issue, we used ethylnitrosourea mutagenesis to identify a previously unknown T lineage-specific gene, Themis, which is critical for the completion of positive selection. Themis contains a tandem repeat of a unique globular domain (called 'CABIT' here) that includes a cysteine motif that defines a family of five uncharacterized vertebrate proteins with orthologs in most animal species. Themis-deficient thymocytes showed no substantial impairment in early TCR signaling but did show altered expression of genes involved in the cell cycle and survival before and during positive selection. Our data suggest a unique function for Themis in sustaining positive selection.

The NS5A protein of hepatitis C virus represses gene expression of hRPB10alpha, a common subunit of host RNA polymerases, through interferon regulatory factor-1 binding site.

The nonstructural (NS) 5A protein of hepatitis C virus (HCV) plays important roles in both viral RNA replication and modulation of the physiology of the host cell. Here we report that NS5A repressed gene expression of hRPB10alpha, a common subunit of host RNA polymerases (Pol), in hepatoma cell lines and Huh-7 cells harboring HCV replicon. Analysis of the hRPB10alpha promoter region revealed that interferon regulatory factor-1 binding element (IRF-E) was essential for its transcription. The IRF-E was responsible for the NS5A-mediated repression of the hRPB10alpha transcription and its induction by IRF-1 that is known to be induced by interferon-alpha. Electrophoretic mobility shift assay showed that IRF-1 bound to the IRF-E and the binding reduced when NS5A was expressed. NS5A appeared to negatively regulate IRF-1 expression, which might be partly responsible for the decrease of hRPB10alpha expression. NS5A expression moderately decreased promoter-independent Pol activity in vitro. Transcription of adenoviral genes that are dependent on Pol II or III and propagation of adenoviral genome were impaired in HeLa cells with stable NS5A expression. The results suggest that NS5A may partly modulate host cell transcription by the down-regulation of hRPB10alpha.

Adeno-associated virus-mediated gene transfer of a secreted form of TRAIL inhibits tumor growth and occurrence in an experimental tumor model.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cell death in various tumor cells, but relatively spares normal cells. Recombinant adeno-associated virus (rAAV) vectors have a number of advantages including in vivo long-term gene expression. Here, we assessed the biological activity of a novel, secreted form of TRAIL (sTRAIL) for cancer gene therapy using a rAAV2 vector. METHODS: A plasmid and rAAV2 vectors were constructed encoding sTRAIL composed of a leader sequence, the isoleucine zipper, and the active domain of TRAIL (aa 95-281). The functionality of sTRAIL was validated by cell viability, FACS analysis, caspase-3 activity, and TUNEL staining. rAAV-sTRAIL was injected intratumorally to nude mice bearing human A549 lung tumor cells. Nude mice received A549 tumor cells after intravenous delivery of rAAV-sTRAIL. The antitumor effect was then evaluated by measuring tumor regression and occurrence in the experimental animal. RESULTS: sTRAIL was released from cells transfected with the sTRAIL expression construct or transduced with rAAV-sTRAIL, and induced apoptosis in cancer cells, but spared normal fibroblast cells. Secreted sTRAIL formed oligomers including trimers with intersubunit disulfide. Purified sTRAIL exerted much lower cytotoxicity on primary human hepatocytes compared to recombinant TRAIL. Intratumoral delivery of rAAV-sTRAIL significantly inhibited growth of A549 tumors established in nude mice. A number of apoptotic tumor cells were detected by TUNEL staining in mice treated with rAAV-sTRAIL. Systemic pretreatment with rAAV-sTRAIL significantly inhibited tumor formation in nude mice. CONCLUSION: The results suggest that rAAV-sTRAIL may be useful for local or systemic cancer gene therapy for treating TRAIL-sensitive tumors.

Identifying subpopulations of thymic epithelial cells by flow cytometry using a new specific thymic epithelial marker, Ly110.

We generated monoclonal antibodies reacting to a mouse thymic epithelial cell specific membrane protein, Thymic Stromal Co-transporter (TSCOT)/Ly110. These antibodies showed specificity to the peptide sequences derived from TSCOT/Ly110 determined by specific peptide inhibition in flow cytometric analyses with cells expressing the protein on the surface. TSCOT/Ly110 expressing subpopulation can be identified among the CDR1(+) or 6C3(+) cortical epithelial cells. Furthermore, CDR1 positive cortical thymic epithelial cells can be separated into further distinguishable populations; CDR1(+)6C3(+)Ly110(+), CDR1(+)6C3(-)/(low)Ly110(+), CDR1(+)Ly110(-). Some of TSCOT/Ly110 expressing cells negative for both CDR1 and 6C3 markers were found at the earlier stages of development, while most of the cells are positive for both at 1-week-old stage. After then, downregulation in 6C3 and/or CDR1 expression was noticed until 16 weeks of age. These results suggest that TSCOT/Ly110 is a new marker for the subpopulation of CDR1(+) or 6C3(+) epithelial cells in the neonatal and adult thymus and is useful for the studies on the epithelial cell differentiation process.

Oncolytic effects of adenovirus mutant capable of replicating in hypoxic and normoxic regions of solid tumor.

Solid tumors contain normoxic and hypoxic regions depending on the distance from the capillary. Normal cells may also be exposed to hypoxia under certain physiological conditions. Tumor hypoxia has been shown to associate strongly with tumor propagation and malignant progression. Hypoxia-inducible factor (HIF)-1alpha is stable under hypoxia and induces transcription of target genes by binding to the hypoxia-response element (HRE). Here we investigated the oncolytic effects of a novel adenovirus mutant with a deleted E1B55 gene (Ad.Delta55.HRE), in which the expression of E1A, which is essential for adenoviral replication, is regulated under the control of an HRE-expression system. Ad.Delta55.HRE expressed E1A under normoxia and more E1A under hypoxia and exhibited oncolytic effects on various cultured tumor cells, but its cytotoxic effect is relatively attenuated in normal fibroblast cells under normoxic and hypoxic conditions. Ad.Delta55.HRE lysed Huh-7 hepatoma cells stably expressing HIF-1alpha more effectively compared to parental cells. Ad.Delta55.HRE treatment exhibited significant antitumor activity in PC-3 prostate- and MDA-MB-435 breast tumor-bearing nude mice in which HIF-1alpha protein was immunohistochemically detected. The E1A and hexon proteins of adenovirus were immunostained in MDA-MB-435 xenografts after Ad.Delta55.HRE treatment, suggestive of viral replication. Our results suggest that Ad.Delta55.HRE may be useful for the treatment of solid tumors.