Signaling In Thymocytes Research Articles

Bisphenol A induces oxidative stress and apoptosis signaling in thymocytes and alters blood immune cells profiles in rats: the protection role of catechins

Bisphenol A induces oxidative stress and apoptosis signaling in thymocytes and alters blood immune cells profiles in rats: the protection role of catechins

Lack of Herpes Virus Entry Mediator Signals in Thymocytes Impairs Conventional CD8 T Cell Selection and Promotes Memory-like CD8 T Cell Development.

Thymocytes having diverse Ag specificities are selected in response to self-MHC-peptide expressed in thymic epithelial cells, which contributes to the formation of a T cell repertoire. However, it is not well understood whether additional signals from epithelial cells are required to drive positive selection. In this study, we found that one of the TNFR superfamily members, herpes virus entry mediator (HVEM), when expressed on thymocytes provides signals for positive selection. HVEM deficiency in double-positive (DP) thymocytes impaired positive selection of CD8 thymocytes. HVEM-deficient thymocytes in OT-1 TCR transgenic mice exhibited significant defects in positive selection and impaired CD69 upregulation of selected thymocytes. HVEM ligands (lymphotoxin-like, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes, and B and T lymphocyte attenuator) were expressed in cortical thymic epithelial cells. Weak TCR ligation combined with HVEM signals enhanced ERK activation in DP thymocytes developed invitro. Insufficient signals for positive selection in HVEM-deficient DP thymocytes led to the development of innate memory-like CD8 T cells expressing high levels of CD122, along with the increased development of PLZF+ NKT cells. These results suggest that thymocytes receive activation signals through HVEM during positive selection. Thus, our findings provide evidence that the threshold of thymocyte positive selection is set by signals from TCR in association with HVEM.

In Vitro Analysis of Thymocyte Signaling.

When a developing thymocyte expresses a TCR, it is subjected to numerous interactions with self-peptide/MHC complexes that determine its fate. These include death by neglect, negative selection (apoptosis and lineage deviation), positive selection, and lineage commitment. Identifying signals that govern these unique cell fates requires the ability to assess the activity, level of expression, subcellular location, and molecular associations between numerous proteins within the developing T cell. Given the unique, temporal, and developmental changes that occur during development, isolating and analyzing small populations of thymocytes are necessary to get a complete picture of the development process. Thus, this chapter describes methods designed to analyze thymocyte signaling under various types of peptide-based stimulation in vitro.

Effects of sex steroids on thymic epithelium and thymocyte development.

Sex steroid hormones have major effects on the thymus. Age-related increases in androgens and estrogens and pregnancy-induced increases in progestins all cause dramatic thymic atrophy. Atrophy can also be induced by treatment with exogenous sex steroids and reversed by ablation of endogenous sex steroids. Although these observations are frequently touted as evidence of steroid lymphotoxicity, they are often driven by steroid signaling in thymic epithelial cells (TEC), which are highly steroid responsive. Here, we outline the effects of sex steroids on the thymus and T cell development. We focus on studies that have examined steroid signaling in vivo, aiming to emphasize the actions of endogenous steroids which, via TEC, have remarkable programming effects on the TCR repertoire. Due to the dramatic effects of steroids on TEC, especially thymic involution, the direct effects of sex steroid signaling in thymocytes are less well understood. We outline studies that could be important in addressing these possibilities, and highlight suggestive findings of sex steroid generation within the thymus itself.

The phosphatidinositol-transfer protein Nir3 is a modulator of T cell development

Abstract Phosphatidylinositol (PI) 4,5-bisphosphate (PIP2) functions as a critical source of second messengers implicated in T cell activation. In T cells, hydrolysis of PIP2 by the lipid phosphatase PLCγ1 generates the second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), resulting in PKC and Ras/MAPK activation as well as calcium increases that contribute to T cell responses. A secondary consequence of PLCγ1 activation is a transient reduction in PIP2 levels. The PI transfer proteins Nir2 and Nir3 have been shown to replenish PIP2 levels at the PM through delivery of the precursor PI from the endoplasmic reticulum (ER) via non-vesicular transport. In particular, Nir3 has been shown to replenish PI levels during basal signaling and in response to weak signaling. Given the importance of weak signaling in thymocyte positive selection, as well as the elevated levels of Nir3 mRNA during the double positive (DP) stage of thymocytes development (Immgen), we hypothesize that Nir3 is a regulator of PI homeostasis during thymocyte development. Using CRISPR-Cas9 technology, we have generated a Nir3 knockout mouse. DP thymocytes from Nir3 knockout mice have reduced basal calcium levels, as well as reduced TCR-induced calcium flux. Furthermore, introduction of the OT1 transgenic TCR into Nir3 knockout mice resulted in elevated numbers of DP thymocytes and reduced numbers of CD8SP thymocytes, suggesting a block in positive selection. Analysis of TCR signaling in Nir3 knockout OT1 thymocytes revealed attenuated calcium flux as well as defective induction of pERK, pAkt, and pS6. These findings are the first to characterize the function of PI transfer proteins and their importance specifically in T cell development.

Elucidating roles of the Rag1 N-terminus and RAG DSBs in shaping the cellular response to TCRa recombination

Abstract The RAG1/RAG2 (RAG) endonuclease generates lymphocyte antigen receptor gene diversity via V(D)J recombination. The large numbers of V, D, and J segments and inherent imprecision in repair of RAG DNA double strand breaks (DSBs) together establish a vast diversity of antigen receptor specificities, including self-reactive receptors. Mechanisms have evolved to negatively select self-reactive cells and inhibit autoimmunity. In humans, deletion of the RAG1 N-terminus causes Omenn Syndrome, a fatal immunodeficiency with ab T cell-based autoimmunity. We discovered impaired negative selection in a spontaneous homozygous Rag1 mutant mouse with loss of the Rag1 N-terminus. This Rag1 region, which is not required for V(D)J recombination, has intrinsic ubiquitin ligase activity and interacts with another ubiquitin ligase and a kinase. We hypothesize the RAG1 N-terminus signals from RAG DSBs induced during TCRa recombination to shape the thymocyte proteome and enhance negative selection. To test our hypothesis, we have made mice whose thymocytes lack RAG DSBs or harbor RAG DSBs induced by wild-type Rag1 or mutant Rag1 lacking the N-terminus or intrinsic ubiquitin ligase activity. Our preliminary data suggest that RAG DSBs and distinct RAG1 N-terminus activities up-regulate expression of Zap70, an intracellular protein that transmits TCR signals, and CD80, a transmembrane protein that co-stimulates T cells engaging antigen. These data are consistent with a model wherein the RAG1 N-terminus facilitates negative selection through stimulating TCR signaling in thymocytes that bind self-antigens presented by thymocytes, dendritic cells, or thymic epithelial cells.

Excessive fluoride exposure induces thymocyte apoptosis and impairs cell division: Roles of the PERK and IRE1 pathways

• Excessive fluoride inhibited division and induced apoptosis of thymocytes. • Excessive fluoride induced activation of PERK and IRE1 signaling in thymocytes. • PERK and IRE1 signaling induced by fluoride plays a key role in immunosuppression.

Transforming Growth Factor-beta signaling in \u03b1\u03b2 thymocytes promotes negative selection

In the thymus, the T lymphocyte repertoire is purged of a substantial portion of highly self-reactive cells. This negative selection process relies on the strength of TCR-signaling in response to self-peptide-MHC complexes, both in the cortex and medulla regions. However, whether cytokine-signaling contributes to negative selection remains unclear. Here, we report that, in the absence of Transforming Growth Factor beta (TGF-β) signaling in thymocytes, negative selection is significantly impaired. Highly autoreactive thymocytes first escape cortical negative selection and acquire a Th1-like-phenotype. They express high levels of CXCR3, aberrantly accumulate at the cortico-medullary junction and subsequently fail to sustain AIRE expression in the medulla, escaping medullary negative selection. Highly autoreactive thymocytes undergo an atypical maturation program, substantially accumulate in the periphery and induce multiple organ-autoimmune-lesions. Thus, these findings reveal TGF-β in thymocytes as crucial for negative selection with implications for understanding T cell self-tolerance mechanisms.

Glucocorticoids Oppose Thymocyte Negative Selection by Inhibiting Helios and Nur77.

Glucocorticoid (GC) signaling in thymocytes shapes the TCR repertoire by antagonizing thymocyte negative selection. The transcription factors Nur77 and Helios, which are upregulated in TCR-signaled thymocytes, have been implicated in negative selection. In this study, we found that GCs inhibited Helios and, to a lesser extent, Nur77 upregulation in TCR-stimulated mouse thymocytes. Inhibition was increased by GC preincubation, and reductions in mRNA were prevented by a protein synthesis inhibitor, suggesting that GCs suppress indirectly via an intermediary factor. Upregulation of Helios in TCR-stimulated thymocytes was unaffected by deletion of Nur77, indicating Nur77 and Helios are regulated independently. Whereas CD4+ thymocytes are positively selected in wild-type AND TCR-transgenic B6 mice, loss of GC receptor expression resulted in increased negative selection. Correspondingly, Helios and Nur77 levels were elevated in TCRhiCD4+CD8+ (TCR-signaled) thymocytes. Notably, deletion of Helios fully reversed this negative selection, whereas deletion of Nur77 had no effect on CD4+CD8+ cell numbers but reversed the loss of mature CD4+ thymocytes. Thus, Nur77 and Helios are GC targets that play nonredundant roles in setting the signaling threshold for thymocyte negative selection.

Erythroid differentiation regulator 1 strengthens TCR signaling in thymocytes by modulating calcium flux

Erythroid differentiation regulator 1 (Erdr1) has been identified as a stromal survival factor released under stressful conditions. Previously, Erdr1 was reported to be expressed highly in thymus, but roles of Erdr1 in thymus were not known. Here, the effects of Erdr1 on T cell development were investigated. The expression of Erdr1 was higher in thymus than bone marrow and Erdr1 was detected in both the cortex and medulla of thymus. Erdr1 treatment significantly induced the expression of activation marker, CD69, from thymocytes in the presence of TCR stimuli in vitro and the induction was dependent on increased Ca2+ influx. In addition, in vivo administration of Erdr1 resulted in significant increase of total and positive selected thymocyte numbers, particularly in the number of CD3TCRhiCD69+ DP thymocytes. Taken together, our results show that Erdr1 enhances the strength of TCR signaling and cellularity of thymocytes by amplifying Ca2+ influx in thymocytes receiving TCR signals.

Cutting Edge: De Novo Glucocorticoid Synthesis by Thymic Epithelial Cells Regulates Antigen-Specific Thymocyte Selection

Glucocorticoid (GC) signaling in thymocytes counters negative selection and promotes the generation of a self-tolerant yet Ag-responsive T cell repertoire. Whereas circulating GC are derived from the adrenals, GC are also synthesized de novo in the thymus. The significance of this local production is unknown. In this study we deleted 11β-hydroxylase, the enzyme that catalyzes the last step of GC biosynthesis, in thymic epithelial cells (TEC) or thymocytes. Like GC receptor-deficient T cells, T cells from mice lacking TEC-derived but not thymocyte-derived GC proliferated poorly to alloantigen, had a reduced antiviral response, and exhibited enhanced negative selection. Strikingly, basal expression of GC-responsive genes in thymocytes from mice lacking TEC-derived GC was reduced to the same degree as in GC receptor-deficient thymocytes, indicating that at steady-state the majority of biologically active GC are paracrine in origin. These findings demonstrate the importance of extra-adrenal GC even in the presence of circulating adrenal-derived GC.

Caught in a Wnt storm: Complexities of Wnt signaling in hematopoiesis.

The Wnt signaling pathway is an evolutionary conserved pathway that is involved in the development of almost every organ system in the body and provides self-renewal signals for most, if not all, adult stem cell systems. In recent years, this pathway has been studied by various research groups working on hematopoietic stem cells, resulting in contradicting conclusions. Here, we discuss and interpret the results of these studies and propose that Wnt dosage, the source of hematopoietic stem cells, and interactions with other pathways explain these disparate results.

In Vitro Analysis of Thymocyte Signaling.

From the moment a developing thymocyte expresses a TCR, it is subjected to numerous interactions with self-peptide/MHC complexes that determine its ultimate fate. These include death by neglect, negative selection (apoptosis and lineage deviation), positive selection, and lineage commitment. The identification of signals that govern these unique cell fates requires the ability to assess the activity, level of expression, subcellular location, and the molecular associations of numerous proteins within the developing T cell. Thus, this chapter describes methods designed to analyze thymocyte signaling under various types of peptide-based stimulation in vitro.

Modulation of autoimmunity associated with blocked nuclear export of histone Deacetylase 7 in thymocytes by CCR9 and the NOD genetic background. (P4088)

Abstract Histone Deacetylase 7 (HDAC7) is a chromatin-modifier that, like other Class IIa HDACs, regulates cell differentiation through extracellular signal-dependent nucleo-cytoplasmic shuttling. We recently showed that nuclear export of HDAC7 in response to TCR signaling in thymocytes is required for negative thymic selection, and that thymic expression of a constitutively nuclear mutant of HDAC7 (HDAC7-ΔP) consequently causes lethal autoimmunity. Although the block in negative selection appears broad, in C57bl/6 mice autoimmunity is largely restricted to the abdominal viscera, with lethality due to exocrine pancreatitis. Investigating the cause of this restricted autoimmunity, we noted abnormally high expression of CCR9, a receptor associated with abdominal homing, in the CD8 T cells of HDAC7-ΔP mice. While this suggested that the type of disease caused by HDAC7-ΔP was due to its effects on both negative selection and chemokine receptor expression, we found that expression of HDAC7-ΔP in a CCR9-deficient background lengthened survival but did not alter the tissue distribution of autoimmune attack. In contrast to this result, we found that expression of HDAC7-ΔP in the NOD genetic background extensively altered the tissue distribution of autoimmunity and also greatly reduced mean survival time. These results shed significant new light on how a specific molecular lesion affecting thymic selection can interact with different genetic backgrounds to produce distinct disease outcomes.

The carboxy-terminal region of CD5 is required for c-CBL mediated TCR signaling downmodulation in thymocytes

CD5 functions as a negative regulator of TCR signaling during thymocyte development, however, the molecular mechanisms involved in this process remain elusive. A key molecule involved in the down modulation of TCR signaling is c-Cbl, an ubiquitin ligase that physically associates with CD5. Crosslinking of TCR in thymocytes leads to ubiquitylation and lysosomal/proteasomal degradation of TCR downstream signaling effectors and CD5 itself. The present report shows that co-engagement of CD3 with CD5 enhanced c-Cbl phosphorylation, which was not affected by the deletion of the pseudo-ITAM domain of CD5, the putative binding site for c-Cbl. However, amino acids present in the carboxy-terminal region of CD5, were necessary for this effect, indicating that ITAM-independent sites were involved in the interaction of c-Cbl with CD5. The carboxy-terminal region of CD5 was also required for Vav degradation, a well-known target for c-Cbl-dependent ubiquitylation. These results support the notion that the distal cytoplasmic domain of CD5, including Y463, plays a relevant role in the downmodulation of TCR signals in thymocytes via c-Cbl.

CD81 interacts with the T cell receptor to suppress signaling.

CD81 (TAPA-1) is a ubiquitously expressed tetraspanin protein identified as a component of the B lymphocyte receptor (BCR) and as a receptor for the Hepatitis C Virus. In an effort to identify trans-membrane proteins that interact with the T-cell antigen receptor (TCR), we performed a membrane yeast two hybrid screen and identified CD81 as an interactor of the CD3delta subunit of the TCR. We found that in the absence of CD81, in thymocytes from knockout mice, TCR engagement resulted in stronger signals. These results were recapitulated in T cell lines that express low levels of CD81 through shRNA mediated silencing. Increased signaling did not result from alterations in the levels of TCR on the surface of T lymphocytes. Although CD81 is not essential for normal T lymphocyte development, it plays an important role in regulating TCR and possibly pre-TCR signal transduction by controlling the strength of signaling. CD81 dependent alterations in thymocyte signaling are evident in increased CD5 expression on CD81 deficient double positive (DP) thymocytes. We conclude that CD81 interacts with the T cell receptor to suppress signaling.

SLAP deficiency increases TCR avidity leading to altered repertoire and negative selection of cognate antigen-specific CD8+ T cells

How T cell receptor (TCR) avidity influences CD8(+) T cell development and repertoire selection is not yet fully understood. To fill this gap, we utilized Src-like adaptor protein (SLAP)-deficient mice as a tool to increase TCR avidity on double positive (DP) thymocytes. We generated SLAP(-/-) mice with the transgenic MHC class I-restricted TCR (OT-1) and SLAP(-/-) Vβ5 mice, expressing only the β-chain of the TCR OT-1 transgene, to examine the effects of increased TCR surface levels on CD8(+) T cell development and repertoire selection. In comparing SLAP(-/-) OT-1 and Vβ5 mice with wild-type controls, we performed compositional analysis and assessed thymocyte signaling by measuring CD5 levels. In addition, we performed tetramer and compositional staining to measure affinity for the cognate antigen, ovalbumin (OVA) peptide, presented by MHC. Furthermore, we quantified differences in α-chain repertoire in SLAP(-/-) Vβ5 mice. We have found that SLAP(-/-) OT-1 mice have fewer CD8(+) thymocytes but have increased CD5 expression. SLAP(-/-) OT-1 mice have fewer DP thymocytes expressing Vα2, signifying increased endogenous α-chain rearrangement, and more non-OVA-specific CD8(+) splenocytes upon tetramer staining. Our data demonstrate that SLAP(-/-) Vβ5 mice also have fewer OVA-specific cells and increased Vα2 usage in the peripheral Vβ5 CD8(+) T cells that were non-OVA-specific, demonstrating differences in α-chain repertoire. These studies provide direct evidence that increased TCR avidity in DP thymocytes enhances CD8(+) T cell negative selection deleting thymocytes with specificity for cognate antigen, an antigen the mature T cells may never encounter. Collectively, these studies provide new insights into how TCR avidity during CD8(+) T cell development influences repertoire selection.

Negative Selection Assay Based on Stimulation of T Cell Receptor Transgenic Thymocytes with Peptide-MHC Tetramers

Thymocyte negative selection is a requirement for the development of self tolerance. Although it is possible to assay the induction of cell death in thymocytes in vitro using antibody cross-linking, this stimulus is much stronger than the normal range of T cell receptor ligands that could be encountered during normal development. Signaling in thymocytes is finely balanced between positive and negative selection stimuli, where a negative selecting ligand can be only marginally higher affinity than a positive selecting ligand. We have therefore developed an assay for the induction of negative selection that can distinguish such cases, and that is amenable to high-throughput analysis. The assay is based on the induction of activated caspase 3 in thymocytes expressing a defined T cell receptor, after stimulation with MHC-peptide tetramers in vitro for 24 hours or less.

Tespa1: another gatekeeper for positive selection

A previously unknown protein, Tespa1, that regulates the thymocyte positive-selection checkpoint has now been identified. The phenotype of Tespa1-deficient mice and the role of Tespa1 in thymocyte signaling are very similar to those of Themis-deficient mice and Themis itself, another recently described but unrelated protein.

The Long-Term Survival Potential of Mature T Lymphocytes Is Programmed During Development in the Thymus

The homeostatic maintenance of normal numbers of mature T lymphocytes in the immune system depends on signaling from the T cell antigen receptor (TCR) and the interleukin-7 receptor (IL-7R); however, it is unclear whether there is crosstalk between these two receptors. Here, we have identified a central role for TCR signaling during the development of T lymphocytes in the thymus in the determination of IL-7 function in mature T lymphocytes. We showed that Il7r expression in mature T cells was modulated by developmental TCR-dependent signals elicited during the process of positive selection in the thymus and that this mechanism was common to both CD4(+) and CD8(+) T cells. Control of Il7r expression by the TCR was limited to thymocytes because neither the abundance nor the function of IL-7Rα was affected by TCR signaling in peripheral T cells. Finally, we showed that thymocytes without optimal IL-7Rα abundance failed to form part of the pool of mature T lymphocytes that patrol the periphery of normal hosts, highlighting the importance of this mechanism in shaping the repertoire of lymphocytes that make up this population.