TCRβ Locus Research Articles

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.

Immunoglobulin and T Cell Receptor Gene High-Throughput Sequencing Quantifies Minimal Residual Disease in Acute Lymphoblastic Leukemia and Predicts Post-Transplantation Relapse and Survival

Minimal residual disease (MRD) quantification is an important predictor of outcome after treatment for acute lymphoblastic leukemia (ALL). Bone marrow ALL burden ≥ 10−4 after induction predicts subsequent relapse. Likewise, MRD ≥ 10−4 in bone marrow before initiation of conditioning for allogeneic (allo) hematopoietic cell transplantation (HCT) predicts transplantation failure. Current methods for MRD quantification in ALL are not sufficiently sensitive for use with peripheral blood specimens and have not been broadly implemented in the management of adults with ALL. Consensus-primed immunoglobulin (Ig), T cell receptor (TCR) amplification and high-throughput sequencing (HTS) permit use of a standardized algorithm for all patients and can detect leukemia at 10−6 or lower. We applied the LymphoSIGHT HTS platform (Sequenta Inc., South San Francisco, CA) to quantification of MRD in 237 samples from 29 adult B cell ALL patients before and after allo-HCT. Using primers for the IGH-VDJ, IGH-DJ, IGK, TCRB, TCRD, and TCRG loci, MRD could be quantified in 93% of patients. Leukemia-associated clonotypes at these loci were identified in 52%, 28%, 10%, 35%, 28%, and 41% of patients, respectively. MRD ≥ 10−4 before HCT conditioning predicted post-HCT relapse (hazard ratio [HR], 7.7; 95% confidence interval [CI], 2.0 to 30; P = .003). In post-HCT blood samples, MRD ≥10−6 had 100% positive predictive value for relapse with median lead time of 89 days (HR, 14; 95% CI, 4.7 to 44, P < .0001). The use of HTS-based MRD quantification in adults with ALL offers a standardized approach with sufficient sensitivity to quantify leukemia MRD in peripheral blood. Use of this approach may identify a window for clinical intervention before overt relapse.

Noncore RAG1 Regions Promote Vβ Rearrangements and αβ T Cell Development by Overcoming Inherent Inefficiency of Vβ Recombination Signal Sequences

The RAG proteins are comprised of core endonuclease domains and noncore regions that modulate endonuclease activity. Mutation or deletion of noncore RAG regions in humans causes immunodeficiency and altered TCR repertoire, and mice expressing core but not full-length Rag1 (Rag1(C/C)) or Rag2 (Rag2(C/C)) exhibit lymphopenia, reflecting impaired V(D)J recombination and lymphocyte development. Rag1(C/C) mice display reduced D-to-J and V-to-DJ rearrangements of TCRβ and IgH loci, whereas Rag2(C/C) mice show decreased V-to-DJ rearrangements and altered Vβ/VH repertoire. Because Vβs/VHs only recombine to DJ complexes, the Rag1(C/C) phenotype could reflect roles for noncore RAG1 regions in promoting recombination during only the D-to-J step or during both steps. In this study, we demonstrate that a preassembled TCRβ gene, but not a preassembled DβJβ complex or the prosurvival BCL2 protein, completely rescues αβ T cell development in Rag1(C/C) mice. We find that Rag1(C/C) mice exhibit altered Vβ utilization in Vβ-to-DJβ rearrangements, increased usage of 3'Jα gene segments in Vα-to-Jα rearrangements, and abnormal changes in Vβ repertoire during αβ TCR selection. Inefficient Vβ/VH recombination signal sequences (RSSs) have been hypothesized to cause impaired V-to-DJ recombination on the background of a defective recombinase as in core-Rag mice. We show that replacement of the Vβ14 RSS with a more efficient RSS increases Vβ14 recombination and rescues αβ T cell development in Rag1(C/C) mice. Our data indicate that noncore RAG1 regions establish a diverse TCR repertoire by overcoming Vβ RSS inefficiency to promote Vβ recombination and αβ T cell development, and by modulating TCRβ and TCRα gene segment utilization.

Illegitimate V(D)J recombination-mediated deletions in Notch1 and Bcl11b are not sufficient for extensive clonal expansion and show minimal age or sex bias in frequency or junctional processing

Illegitimate V(D)J recombination at oncogenes and tumor suppressor genes is implicated in formation of several T cell malignancies. Notch1 and Bcl11b, genes involved in developing T cell specification, selection, proliferation, and survival, were previously shown to contain hotspots for deletional illegitimate V(D)J recombination associated with radiation-induced thymic lymphoma. Interestingly, these deletions were also observed in wild-type animals. In this study, we conducted frequency, clonality, and junctional processing analyses of Notch1 and Bcl11b deletions during mouse development and compared results to published analyses of authentic V(D)J rearrangements at the T cell receptor beta (TCRβ) locus and illegitimate V(D)J deletions observed at the human, nonimmune HPRT1 locus not involved in T cell malignancies. We detect deletions in Notch1 and Bcl11b in thymic and splenic T cell populations, consistent with cells bearing deletions in the circulating lymphocyte pool. Deletions in thymus can occur in utero, increase in frequency between fetal and postnatal stages, are detected at all ages examined between fetal and 7 months, exhibit only limited clonality (contrasting with previous results in radiation-sensitive mouse strains), and consistent with previous reports are more frequent in Bcl11b, partially explained by relatively high Recombination Signal Information Content (RIC) scores. Deletion junctions in Bcl11b exhibit greater germline nucleotide loss, while in Notch1 palindromic (P) nucleotides are more abundant, although average P nucleotide length is similar for both genes and consistent with results at the TCRβ locus. Non-templated (N) nucleotide insertions appear to increase between fetal and postnatal stages for Notch1, consistent with normal terminal deoxynucleotidyl transferase (TdT) activity; however, neonatal Bcl11b junctions contain elevated levels of N insertions. Finally, contrasting with results at the HPRT1 locus, we find no obvious age or gender bias in junctional processing, and inverted repeats at recessed coding ends (Pr nucleotides) correspond mostly to single-base additions consistent with normal TdT activity.

Peripheral subnuclear positioning suppresses Tcrb recombination and segregates Tcrb alleles from RAG2

Allelic exclusion requires that the two alleles at antigen-receptor loci attempt to recombine variable (V), diversity (D), and joining (J) gene segments [V(D)J recombination] asynchronously in nuclei of developing lymphocytes. It previously was shown that T-cell receptor β (Tcrb) alleles frequently and stochastically associate with the nuclear lamina and pericentromeric heterochromatin in CD4(-)CD8(-) thymocytes. Moreover, rearranged alleles were underrepresented at these locations. Here we used 3D immunofluorescence in situ hybridization to identify recently rearranged Tcrb alleles based on the accumulation of the DNA-repair protein 53BP1. We found that Tcrb alleles recombine asynchronously in double-negative thymocytes and that V(D)J recombination is suppressed on peripheral as compared with central Tcrb alleles. Moreover, the recombination events that did take place at the nuclear periphery preferentially occurred on Tcrb alleles that were partially dissociated from the nuclear lamina. To understand better the mechanism by which V(D)J recombination is suppressed at the nuclear periphery, we evaluated the subnuclear distribution of recombination-activating gene 2 (RAG2) protein. We found that RAG2 abundance was reduced at the nuclear periphery. Moreover, RAG2 was distributed differently from RNA polymerase II and histone H3K4 trimethylation. Our data suggest that the nuclear periphery suppresses V(D)J recombination, at least in part, by segregating Tcrb alleles from RAG proteins.

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.

T cell receptor repertoire plasticity in peripheral CD8 T cells (P1446)

Abstract Using a technique for the paired amplification of TCR α and β chains from single cells, we have discovered a surprising plasticity in the peripheral TCR repertoire, particularly under conditions of inflammation. This plasticity includes multiple instances of TCR revision, where changes are made in the TCR coding sequence by de novo rearrangement. Here we describe the induction of this process in murine CD8+ T cells derived from influenza-infected lungs and human T cells derived from CMV-infected individuals ex vivo or stimulated in single-cell cultures in vitro. Taking advantage of the co-expression of productive and non-productive Tcra alleles, we demonstrated secondary rearrangement in the TCRβ locus ex vivo, while the in vitro cultures demonstrated a remarkably high rate of revision following strong stimulation. Not surprisingly, revision was found to be RAG-dependent. Utilizing a model of peripheral deletion of RAG2, we showed that mice deficient in revision showed delayed onset and severity of disease in an experimental autoimmune encephalomyelitis (EAE) model, but also produced a less functional T cell response following infectious challenge. Thus, peripheral TCR revision appears to be a robust process that contributes to optimal T cell functionality during infection but may also contribute to the development of autoimmunity.

ATM Influences the Efficiency of TCRβ Rearrangement, Subsequent TCRβ-Dependent T Cell Development, and Generation of the Pre-Selection TCRβ CDR3 Repertoire

Generation and resolution of DNA double-strand breaks is required to assemble antigen-specific receptors from the genes encoding V, D, and J gene segments during recombination. The present report investigates the requirement for ataxia telangiectasia-mutated (ATM) kinase, a component of DNA double-strand break repair, during TCRβ recombination and in subsequent TCRβ-dependent repertoire generation and thymocyte development. CD4−CD8− double negative stage 2/3 thymocytes from ATM-deficient mice have both an increased frequency of cells with DNA break foci at TCRβ loci and reduced Vβ-DJβ rearrangement. Sequencing of TCRβ complementarity-determining region 3 demonstrates that ATM-deficient CD4+CD8+ double positive thymocytes and peripheral T cells have altered processing of coding ends for both in-frame and out-of-frame TCRβ rearrangements, providing the unique demonstration that ATM deficiency alters the expressed TCRβ repertoire by a selection-independent mechanism. ATMKO thymi exhibit a partial developmental block in DN cells as they negotiate the β-selection checkpoint to become double negative stage 4 and CD4+CD8+ thymocytes, resulting in reduced numbers of CD4+CD8+ cells. Importantly, expression of a rearranged TCRβ transgene substantially reverses this defect in CD4+CD8+ cells, directly linking a requirement for ATM during endogenous TCRβ rearrangement to subsequent TCRβ-dependent stages of development. These results demonstrate that ATM plays an important role in TCRβ rearrangement, generation of the TCRβ CDR3 repertoire, and efficient TCRβ-dependent T cell development.

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

Requirement for Dicer in Survival of Proliferating Thymocytes Experiencing DNA Double-Strand Breaks

The Dicer nuclease generates small RNAs that regulate diverse biological processes through posttranscriptional gene repression and epigenetic silencing of transcription and recombination. Dicer-deficient cells exhibit impaired differentiation, activity, proliferation, and survival. Dicer inactivation in developing mouse lymphocytes impairs their proliferation and survival and alters Ag receptor gene repertoires for largely undefined reasons. To elucidate functions of Dicer in lymphocyte development and Ag receptor locus transcription and recombination, we analyzed mice with conditional Dicer deletion in thymocytes containing unrearranged or prerearranged TCRβ loci. Expression of either a preassembled functional TCRβ gene (Vβ1(NT)) or the prosurvival BCL2 protein inhibited death and partially rescued proliferative expansion of Dicer-deficient thymocytes. Notably, combined expression of Vβ1(NT) and BCL2 completely rescued proliferative expansion of Dicer-deficient thymocytes and revealed that Dicer promotes survival of cells attempting TCRβ recombination. Finally, inclusion of an endogenous preassembled DJβ complex that enhances Vβ recombination increased death and impaired proliferative expansion of Dicer-deficient thymocytes. These data demonstrate a critical role for Dicer in promoting survival of thymocytes experiencing DNA double-strand breaks (DSBs) during TCRβ recombination. Because DSBs are common and ubiquitous in cells, our findings indicate that impaired cellular survival in response to DSBs should be considered when interpreting Dicer-deficient phenotypes.

Control of antigen receptor gene assembly by cis-element mediated looping

The adaptive immune system endows mammals with a sophisticated mechanism to survive in a world filled with pathogens. This dynamic defense is generated by V(D)J recombination, which enables lymphocytes to uniquely interact with an enormous range of epitopes on foreign antigens. V(D)J recombination is a set of sequentially controlled DNA cleavage and repair events, which brings variable (V), diversity (D) and joining (J) gene segments together to form functional antigen receptor genes. However, the recombination process must be stringently regulated to prevent formation of chromosomal translocations, which can lead to tumors. The process of V(D)J recombination is regulated at the level of tissue, stage and allele specificity by genetic and epigenetic mechanisms. Our lab has characterized several genetic elements that regulate chromatin accessibility and recombination at the T cell receptor beta (Tcrb) locus. These include transcriptional promoters and enhancers, which interact with each other in conformational space to form a promoter-enhancer holocomplex. The holocomplex subsequently recruits the SWI/SNF chromatin remodeling complex, leading to locus activation. However, the factors that mediate conformation and epigenetic changes required for efficient Tcrb gene assembly are unknown. We hypothesized that active transcription may stabilize the holocomplex. In order to test this, we chemically induced promoter-proximal-pausing in proT cells. We discovered that in the absence of elongating RNA polymerase II, the promoter-enhancer holocomplex is disrupted without any robust changes in transcription factor binding, structural protein recruitment or histone acetylation across the locus. Ongoing work seeks to decipher whether the transcriptional machinery is required at all for holocomplex stability. Meanwhile, in order to dissect the spatial and epigenetic mechanisms that lead to Tcrb activation, our lab has generated mutated versions of Tcrb cis-elements in a proT cell line model using Zinc Finger Nucleases. Preliminary studies on these mutants have shown that the holocomplex formation requires the enhancer (or enhancer bound elements) and also CTCF sites flanking the locus. Ongoing studies seek to identify which CTCF sites are necessary and sufficient for holocomplex stability. Using these and additional mutants, we are testing the role played by cis-elements in maintaining the chromatin and looping of Tcrb, which are essential for recombination.

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.

An ectopic CTCF-dependent transcriptional insulator influences the choice of Vβ gene segments for VDJ recombination at TCRβ locus

Insulators regulate transcription as they modulate the interactions between enhancers and promoters by organizing the chromatin into distinct domains. To gain better understanding of the nature of chromatin domains defined by insulators, we analyzed the ability of an insulator to interfere in VDJ recombination, a process that is critically dependent on long-range interactions between diverse types of cis-acting DNA elements. A well-established CTCF-dependent transcriptional insulator, H19 imprint control region (H19-ICR), was inserted in the mouse TCRβ locus by genetic manipulation. Analysis of the mutant mice demonstrated that the insulator retains its CTCF and position-dependent enhancer-blocking potential in this heterologous context in vivo. Remarkably, the inserted H19-ICR appears to have the ability to modulate cis-DNA interactions between recombination signal sequence elements of the TCRβ locus leading to a dramatically altered usage of Vβ segments for Vβ-to-DβJβ recombination in the mutant mice. This reveals a novel ability of CTCF to govern long range cis-DNA interactions other than enhancer–promoter interactions and suggests that CTCF-dependent insulators may play a diverse and complex role in genome organization beyond transcriptional control. Our functional analysis of mutated TCRβ locus supports the emerging role of CTCF in governing VDJ recombination.

DNA Double-Strand Breaks Relieve USF-Mediated Repression of Dβ2 Germline Transcription in Developing Thymocytes

Activation of germline promoters is central to V(D)J recombinational accessibility, driving chromatin remodeling, nucleosome repositioning, and transcriptional read-through of associated DNA. We have previously shown that of the two TCRβ locus (Tcrb) D segments, Dβ1 is flanked by an upstream promoter that directs its transcription and recombinational accessibility. In contrast, transcription within the DJβ2 segment cluster is initially restricted to the J segments and only redirected upstream of Dβ2 after D-to-J joining. The repression of upstream promoter activity prior to Tcrb assembly correlates with evidence that suggests DJβ2 recombination is less efficient than that of DJβ1. Because inefficient DJβ2 assembly offers the potential for V-to-DJβ2 recombination to rescue frameshifted V-to-DJβ1 joints, we wished to determine how Dβ2 promoter activity is modulated upon Tcrb recombination. In this study, we show that repression of the otherwise transcriptionally primed 5'Dβ2 promoter requires binding of upstream stimulatory factor (USF)-1 to a noncanonical E-box within the Dβ2 12-recombination signal sequence spacer prior to Tcrb recombination. USF binding is lost from both rearranged and germline Dβ2 sites in DNA-dependent protein kinase, catalytic subunit-competent thymocytes. Finally, genotoxic dsDNA breaks lead to rapid loss of USF binding and gain of transcriptionally primed 5'Dβ2 promoter activity in a DNA-dependent protein kinase, catalytic subunit-dependent manner. Together, these data suggest a mechanism by which V(D)J recombination may feed back to regulate local Dβ2 recombinational accessibility during thymocyte development.

Multiple clonal Ig/TCR products: implications for interpretation of clonality findings

Clonality assessment via analysis of immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements has become an important and valuable adjunct in the diagnostic process of suspect lymphoproliferations. One of the major issues in correct interpretation of clonality testing results appears to be the occurrence of multiple clonal PCR products. The presence of two different PCR products could reflect biclonality but can often more easily be attributed to the occurrence of biallelic rearrangements in a single clone. Furthermore, due to the specific configuration of the IGK and TCRB loci, multiple rearrangements can occur on one allele, resulting in the possibility that up to four PCR products could be compatible with a single clone. Finally, >2 clonal Ig/TCR products (>4 for IGK and TCRB) could reflect biclonality, oligoclonality, or even pseudoclonality. It is not always easy to distinguish between those options, but the use of duplicates will be helpful to determine reproducibility and relevance of the detected clonal PCR products. In conclusion, straightforward interpretation of clonality testing results can be hampered by the occurrence of multiple clonal products. Only careful consideration of immunobiological and technical explanations will prevent incorrect interpretation in such cases.

Downregulation of the transcription factor KLF4 is required for the lineage commitment of T cells

The roles of the reprogramming factors Oct4, Sox2, c-Myc and Klf4 in early T cell development are incompletely defined. Here, we show that Klf4 is the only reprogramming factor whose expression is downregulated when early thymic progenitors (ETPs) differentiate into T cells. Enforced expression of Klf4 in uncommitted progenitors severely impaired T cell development mainly at the DN2-to-DN3 transition when T cell lineage commitment occurs and affected the transcription of a variety of genes with crucial functions in early T cell development, including genes involved in microenvironmental signaling (IL-7Rα), Notch target genes (Deltex1), and essential T cell lineage regulatory or inhibitory genes (Bcl11a, SpiB, and Id1). The survival of thymocytes and the rearrangement at the Tcrb locus were impaired in the presence of enforced Klf4 expression. The defects in the DN1-to-DN2 and DN2-to-DN3 transitions in Klf4 transgenic mice could not be rescued by the introduction of a TCR transgene, but was partially rescued by restoring the expression of IL-7Rα. Thus, our data indicate that the downregulation of Klf4 is a prerequisite for T cell lineage commitment.

Interaction of Ras with P110γ Is Required for Thymic β-Selection in the Mouse

Thymocytes are tested for productive rearrangement of the tcrb locus by expression of a pre-TCR in a process termed β-selection, which requires both Notch1 and CXCR4 signaling. It has been shown that activation of the GTPase Ras allows thymocytes to proliferate and differentiate in the absence of a Pre-TCR; the direct targets of Ras at this checkpoint have not been identified, however. Mice with a mutant allele of p110γ unable to bind active Ras revealed that CXCR4-mediated PI3K activation is Ras dependent. The Ras-p110γ interaction was necessary for efficient β-selection-promoted proliferation but was dispensable for the survival or differentiation of thymocytes. Uncoupling Ras from p110γ provides unambiguous identification of a Ras interaction required for thymic β-selection.

Out-of-Frame T Cell Receptor Beta Transcripts Are Eliminated by Multiple Pathways In Vivo

Non-productive antigen receptor genes with frame shifts generated during the assembly of these genes are found in many mature lymphocytes. Transcripts from these genes have premature termination codons (PTCs) and could encode truncated proteins if they are not either inactivated or destroyed by nonsense-mediated decay (NMD). In mammalian cells, NMD can be activated by pathways that rely on the presence of an intron downstream of the PTC; however, NMD can also be activated by pathways that do not rely on these downstream introns, and pathways independent of NMD can inactivate PTC-containing transcripts. Here, through the generation and analysis of mice with gene-targeted modifications of the endogenous T cell receptor beta (Tcrb) locus, we demonstrate that in T cells in vivo, optimal clearance of PTC-containing Tcrb transcripts depends on the presence of an intron downstream of the PTC.

Absence of TCR loci chromosomal translocations in cutaneous T-cell lymphomas

Chromosomal aberrations involving T-cell receptor (TCR) gene loci have been described in several T-cell malignancies. In primary cutaneous T-cell lymphomas (CTCL), the frequency of these aberrations has not yet been well established. We analyzed TCR gene loci (TCRAD, TCRB, and TCRG) status in CTCLs by fluorescence in situ hybridization (FISH). Twenty-five patients with CTCLs were included in the study: 13 Sézary syndromes (SS), six tumoral stage mycosis fungoides (MFt), and six primary cutaneous anaplastic large cell lymphomas CD30(+) (cALCL-CD30(+)). FISH was performed with three break-apart probes flanking TCRAD (14q11), TCRB (7q34), and TCRG (7p14) loci in each case. TCR gene chromosomal rearrangements were not detected in any of the analyzed cases. Gains of TCRB and TCRG genes were observed in 23% (3 of 13) of SS and 50% (3 of 6) of MFt, reflecting the presence of trisomy and/or tetrasomy of chromosome 7 already detected by conventional cytogenetics and array comparative genetic hybridization techniques. TCR loci rearrangements are not frequent in CTCLs; however, we cannot exclude a pathogenic role in these malignancies.

A Barrier-Type Insulator Forms a Boundary between Active and Inactive Chromatin at the Murine TCRβ Locus

In CD4(-)CD8(-) double-negative thymocytes, the murine Tcrb locus is composed of alternating blocks of active and inactive chromatin containing Tcrb gene segments and trypsinogen genes, respectively. Although chromatin structure is appreciated to be critical for regulated recombination and expression of Tcrb gene segments, the molecular mechanisms that maintain the integrity of these differentially regulated Tcrb locus chromatin domains are not understood. We localized a boundary between active and inactive chromatin by mapping chromatin modifications across the interval extending from Prss2 (the most 3' trypsinogen gene) to D(β)1. This boundary, located 6 kb upstream of D(β)1, is characterized by a transition from repressive (histone H3 lysine 9 dimethylation [H3K9me2]) to active (histone H3 acetylation [H3ac]) chromatin and is marked by a peak of histone H3 lysine 4 dimethylation (H3K4me2) that colocalizes with a retroviral long terminal repeat (LTR). Histone H3 lysine 4 dimethylation is retained and histone H3 lysine 9 dimethylation fails to spread past the LTR even on alleles lacking the Tcrb enhancer (E(β)) suggesting that these features may be determined by the local DNA sequence. Notably, we found that LTR-containing DNA functions as a barrier-type insulator that can protect a transgene from negative chromosomal position effects. We propose that, in vivo, the LTR blocks the spread of heterochromatin, and thereby helps to maintain the integrity of the E(β)-regulated chromatin domain. We also identified low-abundance, E(β)-dependent transcripts that initiate at the border of the LTR and an adjacent long interspersed element. We speculate that this transcription, which extends across D(β), J(β) and C(β) gene segments, may play an additional role promoting initial opening of the E(β)-regulated chromatin domain.