Tcra Locus Research Articles

Retention of duplicated ITAM-containing transmembrane signaling subunits in the tetraploid amphibian species Xenopus laevis

The ITAM-bearing transmembrane signaling subunits (TSS) are indispensable components of activating leukocyte receptor complexes. The TSS-encoding genes map to paralogous chromosomal regions, which are thought to arise from ancient genome tetraploidization(s). To assess a possible role of tetraploidization in the TSS evolution, we studied TSS and other functionally linked genes in the amphibian species Xenopus laevis whose genome was duplicated about 40 MYR ago. We found that X. laevis has retained a duplicated set of sixteen TSS genes, all except one being transcribed. Furthermore, duplicated TCRα loci and genes encoding TSS-coupling protein kinases have also been retained. No clear evidence for functional divergence of the TSS paralogs was obtained from gene expression and sequence analyses. We suggest that the main factor of maintenance of duplicated TSS genes in X. laevis was a protein dosage effect and that this effect might have facilitated the TSS set expansion in early vertebrates.

567. Enhanced Efficiency of CRISPR-Mediated Gene Knock-Out in Primary Human T-Cells

RNA-guided endonuclease (RGEN) technology has great promise for enabling efficient editing of essentially any target genomic locus. Here, we have evaluated application of S. pyogenes Cas9 for gene editing in primary human T-cells using mRNA-mediated delivery of a first generation spCas9 and adeno-associated virus (AAV) to drive guide RNA expression. We find that spCas9-mediated editing using this approach can achieve targeted gene disruption rates at the TCRa locus of up to 30%. Furthermore, evaluation of the dose response of editing efficiency at different Cas9 mRNA doses and over a range of AAV MOI suggests that editing efficiency is limited primarily by AAV-driven guide RNA expression. Evaluation of several approaches to achieve higher editing efficiencies led to the development of a novel method capable of achieving up to 90% TCRa disruption at reduced AAV MOI in selected cell populations. These results suggest that a Cas9-mRNA/AAV-guide approach can be applied to effectively disrupt multiple individual genes in primary human T-cells, and highlight an innovate method through which CRISPR/Cas9 technology may be applied with enhanced efficiency.

T-cell receptor α enhancer is inactivated in αβ T lymphocytes

The Tcra enhancer (Eα) is essential for Tcra locus germ-line transcription and primary Vα-to-Jα recombination during thymocyte development. We found that Eα is inhibited late during thymocyte differentiation and in αβ T lymphocytes, indicating that it is not required to drive transcription of rearranged Tcra genes. Eα inactivation resulted in the disruption of functional long-range enhancer-promoter interactions and was associated with loss of Eα-dependent histone modifications at promoter and enhancer regions, and reduced expression and recruitment of E2A to the Eα enhanceosome in T cells. Enhancer activity could not be recovered by T-cell activation, by forced expression of E2A or by the up-regulation of this and other transcription factors in the context of T helper differentiation. Our results argue that the major function of Eα is to coordinate the formation of a chromatin hub that drives Vα and Jα germ-line transcription and primary rearrangements in thymocytes and imply the existence of an Eα-independent mechanism to activate transcription of the rearranged Tcra locus in αβ T cells.

The Ability To Rearrange Dual TCRs Enhances Positive Selection, Leading to Increased Allo- and Autoreactive T Cell Repertoires

Thymic selection is designed to ensure TCR reactivity to foreign Ags presented by self-MHC while minimizing reactivity to self-Ags. We hypothesized that the repertoire of T cells with unwanted specificities such as alloreactivity or autoreactivity are a consequence of simultaneous rearrangement of both TCRα loci. We hypothesized that this process helps maximize production of thymocytes capable of successfully completing thymic selection, but results in secondary TCRs that escape stringent selection. In T cells expressing two TCRs, one TCR can mediate positive selection and mask secondary TCR from negative selection. Examination of mice heterozygous for TRAC (TCRα(+/-)), capable of only one functional TCRα rearrangement, demonstrated a defect in generating mature T cells attributable to decreased positive selection. Elimination of secondary TCRs did not broadly alter the peripheral T cell compartment, though deep sequencing of TCRα repertoires of dual TCR T cells and TCRα(+/-) T cells demonstrated unique TCRs in the presence of secondary rearrangements. The functional impact of secondary TCRs on the naive peripheral repertoire was evidenced by reduced frequencies of T cells responding to autoantigen and alloantigen peptide-MHC tetramers in TCRα(+/-) mice. T cell populations with secondary TCRs had significantly increased ability to respond to altered peptide ligands related to their allogeneic ligand as compared with TCRα(+/-) cells, suggesting increased breadth in peptide recognition may be a mechanism for their reactivity. Our results imply that the role of secondary TCRs in forming the T cell repertoire is perhaps more significant than what has been assumed.

Somatic hypermutation and use of immunoglobulin variable regions at shark T cell receptors (VET2P.1038)

Abstract The nurse shark (Ginglymostoma cirratum) is part of the earliest clade found to possess an adaptive immune system employing immunoglobulin superfamily lymphocyte antigen receptors. The hallmarks of adaptive immunity, memory and specificity, are accomplished through B or T cells employing immunoglobulin (Ig) or T-cell receptors (TCR) respectively. Both lineages undergo somatic gene rearrangement for receptor diversification, a process mediated by RAG proteins, in which V, D, and J gene segments are joined together. Surprisingly, we found somatic hypermutation (SHM, normally a B cell process) to occur extensively at shark TCRα loci. Unlike B cell SHM that follows antigen exposure in sharks and most other vertebrates, SHM at TCRα loci occurs during thymic development. TCRα SHM is supported with isolation of clusters of mutated clones having the same CDR3 rearrangement.We suggest that such SHM, in addition to TCRα receptor editing, permits developing T cells with evolving receptors to scan cortical epithelial cells for positive selection on self-peptide/self MHC. Additionally, we have found that shark TCRδ can employ variable gene segments of IgM and IgW in rearrangements, a situation that may be similar to the IgHV use by TCRδ noted in higher vertebrates. Future studies must further study the lineage commitment of shark B and T cells and the receptor plasticity of these lineages in all vertebrates.

Biallelic T cell receptor rearrangement enhances the efficiency of thymocyte development and positive selection. (HEM4P.234)

Abstract Biallelic rearrangement of TCR loci has been proposed to increase the likelihood of productive gene rearrangement, thereby promoting thymocyte survival. When allelic exclusion is incomplete, this process also results in dual TCR-expressing T cells, which may increase repertoire diversity, but may also increase autoreactive potential. We investigated the effects of biallelic TCR gene rearrangement by comparing wildtype (wt) T cells to those from mice hemizygous for the genes encoding TCR α, TCR β, or both. Using bone marrow chimera competition assays we found that Tcrb hemizygous thymocytes were impaired in the DN1 to DN2 transition relative to wt cells. In addition, Tcrb hemizygous mice exhibited a higher frequency of T cells committed to the γδ lineage. Bone marrow chimera competition assays revealed a dramatic increase in the ratio of wt thymocytes to Tcra hemizygous thymocytes between the pre-selection and post-selection DP compartments, demonstrating that biallelic Tcra rearrangement increases the rate of positive selection. Despite their impairments in thymocyte development, mice hemizygous for both Tcra and Tcrb generated polyclonal T cell repertoires similar to wt mice as assessed by numbers of antigen-specific naïve T cells, responses to multiple self and foreign antigens, and TCRβ sequence diversity. Thus, biallelic rearrangement of Tcra and Tcrb loci enhances the efficiency of thymocyte development, but is not necessary for normal TCR repertoire diversification.

Visualization and quantification of monoallelic TCRα gene rearrangement in αβ T cells

T-cell receptor α (TCRα) chain rearrangement is not constrained by allelic exclusion and thus αβ T cells frequently have rearranged both alleles of this locus. Thereby, stepwise secondary rearrangements of both TCRα loci further increase the odds for generation of an α-chain that can be positively selected in combination with a pre-existing TCRβ chain. Previous studies estimated that approximately 2-12% of murine and human αβ T cells still carry one TCRα locus in germline configuration, which must comprise a partially or even fully rearranged TCRδ locus. However, these estimates are based on a relatively small amount of individual αβ T-cell clones and αβ T-cell hybridomas analyzed to date. To address this issue more accurately, we made use of a mouse model, in which a fluorescent reporter protein is introduced into the constant region of the TCRδ locus. In this TcrdH2BeGFP system, fluorescence emanating from retained TCRδ loci enabled us to quantify monoallelically rearranged αβ T cells on a single-cell basis. Via fluorescence-activated cell sorting analysis, we determined the frequency of monoallelic TCRα rearrangements to be 1.7% in both peripheral CD4(+) and CD8(+) αβ T cells. Furthermore, we found a skewed 5' Jα gene utilization of the rearranged TCRα allele in T cells with monoallelic TCRα rearrangements. This is in line with previous descriptions of a tight interallelic positional coincidence of Jα gene segments used on both TCRα alleles. Finally, analysis of T cells from transgenic mice harboring only one functional TCRα locus implied the existence of very rare unusual translocation or episomal reintegration events of formerly excised TCRδ loci.

NKT Cell-TCR Expression Activates Conventional T Cells in Vivo, but Is Largely Dispensable for Mature NKT Cell Biology

Natural killer T (NKT) cell development depends on recognition of self-glycolipids via their semi-invariant Vα14i-TCR. However, to what extent TCR-mediated signals determine identity and function of mature NKT cells remains incompletely understood. To address this issue, we developed a mouse strain allowing conditional Vα14i-TCR expression from within the endogenous Tcrα locus. We demonstrate that naïve T cells are activated upon replacement of their endogenous TCR repertoire with Vα14i-restricted TCRs, but they do not differentiate into NKT cells. On the other hand, induced TCR ablation on mature NKT cells did not affect their lineage identity, homeostasis, or innate rapid cytokine secretion abilities. We therefore propose that peripheral NKT cells become unresponsive to and thus are independent of their autoreactive TCR.

Cell of origin in radiation‐induced premalignant thymocytes with differentiation capability in mice conditionally losing one Bcl11b allele

Bcl11b is a haploinsufficient tumor suppressor, mutations or deletion of which has been found in 10-16% of T-cell acute lymphoblastic leukemias. Bcl11b(KO) (/+) heterozygous mice are susceptible to thymic lymphomas, a model of T-cell acute lymphoblastic leukemia, when γ-irradiated, and irradiated Bcl11b(KO) (/+) mice generate clonally expanding or premalignant thymocytes before thymic lymphoma development. Cells with radiation-induced DNA damages are assumed to be the cells of origin in tumors; however, which thymocyte is the tumor cell origin remains obscure. In this study we generated Bcl11b(flox/+) ;Lck-Cre and Bcl11b(flox/+) ;CD4-Cre mice; in the former, loss of one Bcl11b allele occurs in thymocytes at the immature CD4(-) CD8(-) stage, whereas in the latter the loss occurs in the more differentiated CD4(+) CD8(+) double-positive stage. We examined clonal expansion and differentiation of thymocytes in mice 60days after 3Gy γ-irradiation. Half (9/18) of the thymuses in the Bcl11b(flox/+) ;Lck-Cre group showed limited rearrangement sites at the T-cell receptor-β (TCRβ) locus, indicating clonal cell expansion, but none in the Bcl11b(flox/+) ;CD4-Cre group did. This indicates that the origin of the premalignant thymocytes is not in double-positive cells but immature thymocytes. Interestingly, those premalignant thymocytes underwent rearrangement at various different sites of the TCRα locus and the majority showed a higher expression of TCRβ and CD8, and more differentiated phenotypes. This suggests the existence of a subpopulation of immature cells within the premalignant cells that is capable of proliferating and continuously producing differentiated thymocytes.

Generation of secondary TCRs is critical for T cell development and the formation of an alloreactive T cell repertoire (P2132)

Abstract A small number of peripheral T cells in humans and mice express two TCRs. Secondary TCRs arise from a lack of allelic exclusion at the TCRα locus. A functional role for secondary TCRs has been demonstrated in several immune responses, including a particularly important contribution to alloreactivity, the molecular basis for transplant rejection. Genetic elimination of secondary TCRs in mice resulted in a 50% reduction in the naive alloreactive T cell frequency. Recently, studies have elucidated a high degree of peptide specificity in alloreactivity, but the role of secondary TCRs is unknown. We hypothesize that secondary TCRs are important for the formation of the naive T cell repertoire capable of responding to individual allopeptides. To address this, we analyzed T cells in mice heterozygous for Trac (Cα+/-), which can only generate a single functional TCRα rearrangement, to look at T cell selection and alloantigen specificity. In TCR Cα+/- mice, there is a defect in the generation of mature T cells attributable to altered thymic selection. The effect on T cell development does not impact peripheral T cell numbers. Despite this, there are functional consequences for the inability to express secondary TCRs: binding to individual allopeptide-MHC tetramers was significantly diminished in naive T cells from TCR Cα+/- mice. Our work reveals a fundamental role for secondary TCRs in thymic selection and in the formation of complete allopeptide-binding T cell populations.

B cell immunogenetics in the T cell receptor repertoire generation of shark (P6069)

Abstract Rearranged immunoglobulin (Ig) variable (V) regions undergo somatic hypermutation (SHM) in activated B cells during a T-dependent humoral immune response, allowing affinity maturation. TCR loci were believed not to undergo SHM, until the demonstration of SHM at the TCRγ locus of sandbar sharks. Our study in the nurse shark corroborates this seminal finding, as we have found SHM of TCRγ loci in spleen; we further show that TCRγ V regions are highly mutated in clones isolated from the spiral valve (shark intestine), suggesting a role for T cell SHM in mucosal immunity. Surprisingly, we also found SHM to occur extensively at shark TCRα loci. Unlike B cell SHM that follows antigen exposure in sharks and most other vertebrates, SHM at TCRα loci occurs during thymic development. TCRα SHM is supported with isolation of clusters of mutated clones having the same CDR3 rearrangement.We suggest that such SHM, in addition to TCRα receptor editing, permits developing T cells with evolving receptors to scan cortical epithelial cells for positive selection on self-peptide/self MHC. Additionlly, we have found that shark TCRδ can employ variable gene segments of IgM and IgW in rearrangements, a situation that may be similar to the IgHV use by TCRδ noted in higher vertebrates. Future studies must further study the lineage commitment of shark B and T cells and the receptor plasticity of these lineages in all vertebrates.

Human peripheral Vδ1+ γδ T cells can develop into αβ T cells (P4460)

Abstract The T cell compartment can be divided in two large classes: αβ and γδ T cells. It is believed that they develop in the thymus from a common progenitor and that the choice between αβ and γδ T cell fate is the first lineage decision made by progenitors after they commit to the T-cell lineage. However, here we show that peripheral Vδ1+ γδ T cells in an inflammatory environment can transdifferentiate into αβ T cells. Upon their extrathymic route of differentiation, that resembles well-characterized molecular program of thymic αβ lineage development, Vδ1+ T cells upregulate CD4+ coreceptor, develop Vδ1+ CD4+CD8+ double positive cells, show heterodimeric CD8αβ, transcribe RAG, preTα and express a particular Vβchain on their surface. Simultaneously inflammation confers controlled initiation of rearrangement in the TCRα locus. Transdifferentiation of Vδ1+ T cells at the clonal and bulk-culture level into functional CD4+ or CD8+ αβ T cells via a Vδ1+TCR/αβ+TCR double positive stage suggests that, upon inflammatory stimuli, Vδ1+ T-cell conversion participates in the induction of adaptive immune responses. Identifying an innate T cell as an αβ T-cell progenitor and showing the developmental steps linking the progenitor to adaptive, thymus-independant αβ T-cell responses as inflammatory conditions is of utmost relevance and will deeply impact evaluation of immune responses in infection, malignancy and autoimmune processes.

Abstract 3598: Cell of origin in radiation-induced premalignant thymocytes with differentiation capability in mice conditionally losing one Bcl11b allele.

Abstract Mouse thymic lymphoma (TL) has been used for study of radiation carcinogenesis because of its high incidence. The lymphoma or leukemia is a mouse model of human T-cell acute lymphoblastic leukemias (T-ALL), which is a malignant clonal expansion of thymocytes that accounts for about 15% of ALL cases. Ionizing radiation can damage DNA, and the cells with damaged DNA are assumed to be the cells of origin in tumors. However, which cell in the thymus or the bone marrow leads to TL remains obscure. Bcl11b gene encodes zinc-finger transcription proteins, mutations or deletion of which has been found in 10-16% of T-ALL. Bcl11bKO/+ heterozygous mice are susceptible to thymic lymphomas when γ-irradiated, and irradiated Bcl11bKO/+ mice generate clonally expanding or premalignant thymocytes within atrophic thymus. Resembling clonal expansion of thymocytes was reported in the T-ALL model overexpressing LMO2 oncogene. Interestingly, LMO2-induced clonally expanding thymocytes persist long-term and comprise cells with the stem cell-like self-renewal property that function for the bone marrow stem cells before TL development. In this study, we developed Bcl11bflox/+;Lck-Cre and Bcl11bflox/+;CD4-Cre mice, in which loss of one Bcl11b allele occurs in thymocytes at the immature CD4−CD8− stage without expression of CD4 and CD8 cell surface markers and at the CD4+CD8+ double-positive (DP) stage, respectively. Those mice were subjected to γ-irradiation of 3Gy at 8 weeks of age and their thymocytes were characterized of clonal expansion, differentiation and cell number. The clonal expansion was observed in only the former mice, suggesting the origin of the premaligant thymocytes not in the DP cells, the cell type seen in a majority of TL. The clonally expanded thymocytes possessed common rearrangement sites at the TCRβ locus but they underwent rearrangement at various different sites at the TCRα locus, and their majority showed mature phenotypes, a higher expression of TCRβ and the CD8 expression. This suggests the presence of a subpopulation of immature thymocytes that is capable to rearrange DNA at the TCRα locus in DP cells to continuously produce further differentiated thymocytes. We also found the decrease in thymocyte number in irradiated Bcl11bflox/+;Lck-Cre mice. This probably reflects the reduced proliferation of bone marrow-derived progenitors, and this reduction may weaken the competition in niche with premaligant thymocytes and help for them to survive and proliferate. Citation Format: Ryo Kominami, Rieka Go, Satoshi Hirose, Yukio Mishima. Cell of origin in radiation-induced premalignant thymocytes with differentiation capability in mice conditionally losing one Bcl11b allele. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3598. doi:10.1158/1538-7445.AM2013-3598

Genetic association, seasonal infections and autoimmune basis of narcolepsy

In recent years, a growing number of potential autoimmune disorders affecting neurons in the central nervous system have been identified, including narcolepsy. Narcolepsy is a lifelong sleep disorder characterized by excessive daytime sleepiness with irresistible sleep attacks, cataplexy (sudden bilateral loss of muscle tone), hypnagogic hallucinations, and abnormalities of Rapid Eye Movement sleep. Narcolepsy is generally a sporadic disorder and is caused by the loss of hypocretin (orexin)-producing neurons in the hypothalamus region of the brain. Studies have established that more than 90% of patients have a genetic association with HLA DQB1*06:02. Genome-wide association analysis shows a strong association between narcolepsy and polymorphisms in the TCRα locus and weaker associations within TNFSF4 (also called OX40L), Cathepsin H and the P2RY11-DNMT1 (purinergic receptor subtype P2Y11 to DNMT1, a DNA methytransferase) loci, suggesting an autoimmune basis. Mutations in DNMT1 have also been reported to cause narcolepsy in association with a complex neurological syndrome, suggesting the importance of DNA methylation in the pathology. More recently, narcolepsy was identified in association with seasonal streptococcus, H1N1 infections and following AS03-adjuvanted pH1N1 influenza vaccination in Northern Europe. Potential immunological pathways responsible for the loss of hypocretin producing neurons in these cases may be molecular mimicry or bystander activation. Specific autoantibodies or T cells cross-reactive with hypocretin neurons have not yet been identified, however, thus narcolepsy does not meet Witebsky's criteria for an autoimmune disease. As the brain is not an easily accessible organ, mechanisms of disease initiation and progression remain a challenge to researchers.

Higher-Order Looping and Nuclear Organization of Tcra Facilitate Targeted RAG Cleavage and Regulated Rearrangement in Recombination Centers

V(D)J recombination is essential for generating a diverse array of B and Tcell receptors that can recognize and combat foreign antigens. As with any recombination event, tight control is essential to prevent the occurrence of genetic anomalies that drive cellular transformation. One important aspect of regulation is directed targeting of the RAG recombinase. Indeed, RAG accumulates at the 3' end of individual antigen receptor loci poised for rearrangement; however, it is not known whether focal binding is involved in regulating cleavage, and what mechanisms lead to enrichment of RAG in this region. Here, we show that monoallelic looping out of the 3' end of the Tcell receptor α (Tcra) locus, coupled with transcription and increased chromatin/nuclear accessibility, is linked to focal RAG binding and ATM-mediated regulation of monoallelic cleavage on looped-out 3' regions. Our data identify higher-order loop formation as a key determinant of directed RAG targeting and the maintenance of genome stability.

Human peripheral Vd1+ ?d T cells can develop into a� T cells

Event Abstract Back to Event Human peripheral Vδ1+ γδ T cells can develop into αβ T cells Karin Schilbach1*, Hendrik Ziegler1, Marko Sterk1, Jan Haarer1, Hans-Georg Rammensee2 and Rupert Handgretinger1 1 Childrens Hospital University Tübingen, Germany 2 Interfaculty Institute for Cell Biology University of Tübingen, Dept. of Immunology, Germany The T cell compartment can be divided in two large classes: αβ and γδ T cells. It is believed that they develop in the thymus from a common progenitor and that the choice between αβ and γδ T cell fate is the first lineage decision made by progenitors after they commit to the T-cell lineage. However, here we show that peripheral Vδ1+ γδ T cells in an inflammatory environment can transdifferentiate into αβ T cells. Upon their extrathymic route of differentiation, that resembles well-characterized molecular program of thymic αβ lineage development, Vδ1+ T cells upregulate CD4+ coreceptor, develop Vδ1+ CD4+CD8+ double positive cells, show heterodimeric CD8αβ, transcribe RAG, preTα and express a particular Vβchain on their surface. Simultaneously inflammation confers controlled initiation of rearrangement in the TCRα locus. Transdifferentiation of Vδ1+ T cells at the clonal and bulk-culture level into functional CD4+ or CD8+ αβ T cells via a Vδ1+/αβ+TCR double positive stage suggests that, upon inflammatory stimuli, Vδ1+ T-cell conversion participates in the induction of adaptive immune responses. Identifying an innate T cell as an αβ T-cell progenitor and showing the developmental steps linking the progenitor to adaptive, thymus-independant αβ T-cell responses as inflammatory conditions is of utmost relevance and will deeply impact evaluation of immune responses in infection, malignancy and autoimmune processes. Keywords: γδ T cells, T cell progenitor, T cell development, Inflammation, extrathymic, Autoimmunity Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Schilbach K, Ziegler H, Sterk M, Haarer J, Rammensee H and Handgretinger R (2013). Human peripheral Vδ1+ γδ T cells can develop into αβ T cells. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00361 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 22 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Karin Schilbach, Childrens Hospital University Tübingen, Tuebingen, 72076, Germany, schilbachk@web.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Karin Schilbach Hendrik Ziegler Marko Sterk Jan Haarer Hans-Georg Rammensee Rupert Handgretinger Google Karin Schilbach Hendrik Ziegler Marko Sterk Jan Haarer Hans-Georg Rammensee Rupert Handgretinger Google Scholar Karin Schilbach Hendrik Ziegler Marko Sterk Jan Haarer Hans-Georg Rammensee Rupert Handgretinger PubMed Karin Schilbach Hendrik Ziegler Marko Sterk Jan Haarer Hans-Georg Rammensee Rupert Handgretinger Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

TRIM28 mediates chromatin modifications at the TCRα enhancer and regulates the development of T and natural killer T cells

T-cell receptor-α (TCRα) rearrangement in CD4(+)CD8(+) double-positive immature thymocytes is a prerequisite for production of αβ T cells and invariant natural killer T cells. This developmental event is regulated by the TCRα enhancer (Eα), which induces chromatin modification and recruitment of the recombination-activating proteins Rag1 and Rag2. However, the molecular mechanism underlying the activation and long-range action of Eα remains incompletely understood. We show here that the chromatin-modifying factor TRIM28 is highly expressed in double-positive thymocytes and persistently phosphorylated at serine 473. TRIM28 binds to Eα and induces histone 3 lysine 4 trimethylation in the Eα and distant regions of the TCRα locus, coupled with recruitment of Rag proteins. T-cell-conditional ablation of TRIM28 impaired TCRα gene rearrangement and compromised the development of αβ T cells and invariant natural killer T cells. These findings establish TRIM28 as a unique regulator of thymocyte development and highlight an epigenetic mechanism involving TRIM28-mediated active chromatin modification in the TCRα locus.

Human Peripheral CD34+ γδ T Cells Can Transdifferentiate into αβ T Cells.

AbstractAbstract 2312Human peripheral CD34+ γδ T cells can transdifferentiate into αβ T cellsHaploidentical transplantation of peripheral mobilized T-cell depleted stem cells is associated with a delayed immune recovery resulting in severe and often lethal infectious complications after transplantation. For T-cell depletion, either positive selection of CD34+ stem cells as well as negative depletion of CD3+ T lymphocytes is used. However, delayed reconstitution of the adaptive T-cell immune system is seen with both approaches. Since adaptive immune cells are the progeny of haematopoietic precursors, one reason for the delayed immune reconstitution might be the depletion of a progenitor for the adaptive immune system with CD34+ positive selection and CD3 negative depletion. Therefore, we hypothesized the existence of a peripheral CD34+ CD3+ lymphoid progenitor cell. And indeed, we could identify a subset of peripheral circulating cells with a weaker expression of the CD34 antigen compared to CD34+ myeloid progenitor cells which coexpressed CD3, γδ TCR(Vδ1), and CD4lo and additionally express the hematopoietic progenitor markers CD105, CD117, CD135 and CXCR4. In an inflammatory environment, this CD34+ subset can transdifferentiate in vitro into αβ T cells. Upon its extrathymic route of differentiation, that resembles thymic development, CD34+ Vd1+ CD4+ cells increase CD4+ coreceptor expression, develop Vδ1+ CD4+CD8+ double positive cells, show heterodimeric CD8αβ, transcribe RAG and preTα and express a particular Vβ chain on their surface. Simultaneously inflammation confers controlled initiation of rearrangement in the TCRα locus. Transdifferentiation of Vδ1+ T cells at the clonal and bulk-culture level into functional CD4+ or CD8+ αβ T cells upon inflammatory stimuli, is in line with the findings that HSCs participate directly in the primary response to both acute and chronic infections.The identification of CD34+ Vδ1+ T-cells as precursor for adaptive immune cells under inflammatory conditions is of utmost clinical importance, since (i) this subset is selectively lost with the clinically used CliniMACS method of CD34 positive selection, which preferentially enriches for stem cells with a stronger expression of the CD34 antigen due to the binding of more magnetic particles and better retention of the CD34++ cells in the magnetic field and (ii) this subset is also lost using CD3 negative depletion due the coexpression of the CD3 antigen on the CD34+ Vd1+ cells.Our results might be an explanation why a more rapid T-cell recovery is seen after the transplantation of peripheral stem cells depleted of TcRαβ T-cells, which retains the CD34+ Vδ1+ cells in the graft. Furthermore, the assignment of this fundamental role for γδ T cells as a reservoir of an as-yet unappreciated lineage-committed extrathymic αβ T-lymphoid progenitor opens a new vista in immunology and necessitates reevaluation of adaptive immune responses in infection, autoimmunity and cancer. Disclosures:No relevant conflicts of interest to declare.

The DNA Damage- and Transcription-Associated Protein Paxip1 Controls Thymocyte Development and Emigration

Histone 3 lysine 4 trimethylation (H3K4me3) is associated with promoters of active genes and found at hot spots for DNA recombination. Here we have shown that PAXIP1 (also known as PTIP), a protein associated with MLL3 and MLL4 methyltransferase and the DNA damage response, regulates RAG-mediated cleavage and repair during V(D)J recombination in CD4(+) CD8(+) DP thymocytes. Loss of PAXIP1 in developing thymocytes diminished Jα H3K4me3 and germline transcription, suppressed double strand break formation at 3' Jα segments, but resulted in accumulation of unresolved Tcell receptor α-chain gene (Tcra) breaks. Moreover, PAXIP1 was essential for release of mature single positive (SP) αβ Tcells from the thymus through transcriptional activation of sphingosine-1-phosphate receptor S1pr1 as well as for natural killer Tcell development. Thus, in addition to maintaining genome integrity during Tcra rearrangements, PAXIP1 controls distinct transcriptional programs during DP differentiation necessary for Tcra locus accessibility, licensing mature thymocytes for trafficking and natural killer Tcell development.

Incomplete TCR‐β allelic exclusion accelerates spontaneous autoimmune arthritis in K/BxN TCR transgenic mice

Allelic exclusion of antigen receptor loci is a fundamental mechanism of immunological self-tolerance. Incomplete allelic exclusion leads to dual T-cell receptor (TCR) expression and can allow developing autoreactive αβ T lymphocytes to escape clonal deletion. Because allelic exclusion at the TCR-β locus is more stringent than at the TCR-α locus, dual TCR-β expression has not been considered a likely contributor to autoimmunity. We show here that incomplete TCR-β allelic exclusion permits developing thymocytes bearing the autoreactive, transgene-encoded KRN TCR to be positively selected more efficiently, thereby accelerating the onset of spontaneous autoimmune arthritis. Our findings highlight dual TCR-β expression as a mechanism that can enhance the maturation of autoreactive pathogenic T cells and lead to more rapid development of autoimmune disease.