Inefficient V(D)J recombination underlies monogenic T cell receptor ß expression.

The assembly of T cell receptor (TCR) and immunoglobulin (Ig) genes by V(D)J recombination generates the antigen receptor (AgR) diversity that is vital for adaptive immunity. At most AgR loci, V(D)J recombination is regulated so that only one allele assembles a functional gene, ensuring that nearly every T and B cell expresses a single type, or specificity, of AgR. The genomic organizations of some AgR loci permit the assembly and expression of two distinct genes on each allele; however, this is prevented by undetermined mechanisms. We show that the poor qualities of recombination signal sequences (RSSs) flanking Vß gene segments suppress the assembly and expression of two distinct TCRß genes from a single allele. Our data demonstrate that an intrinsic genetic mechanism that stochastically limits Vß recombination efficiency governs monogenic TCRß expression, thereby restraining the expression of multiple AgRs on αß T cells.

Colitogenic effector T cells: roles of gut-homing integrin, gut antigen specificity and γδ T cells.

Disturbance of T-cell homeostasis could lead to intestinal inflammation. Naive CD4 T cells undergoing spontaneous proliferation, a robust proliferative response that occurs under severe lymphopenic conditions, differentiate into effector cells producing Th1- and/or Th17-type cytokines and induce a chronic inflammation in the intestine that resembles human inflammatory bowel disease. In this study, we investigated the key properties of CD4 T cells necessary to induce experimental colitis. α4ß7 upregulation was primarily induced by mesenteric lymph node (mLN) resident CD11b(+) dendritic cell subsets via transforming growth factor beta (TGFß)/retinoic acid-dependent mechanism. Interestingly, α4ß7 expression was essential but not sufficient to induce inflammation. In addition to gut-homing specificity, expression of gut Ag specificity was also crucial. T-cell acquisition of the specificity was dramatically enhanced by the presence of γδ T cells, a population previously shown to exacerbate T-cell-mediated colitis. Importantly, interleukin (IL)-23-mediated γδ T cell stimulation was necessary to enhance colitogenicity but not gut antigen reactivity of proliferating CD4 T cells. These findings demonstrate that T-cell colitogenicity is achieved through multiple processes, offering a therapeutic rationale by intervening these pathways.

PTH expands short-term murine hemopoietic stem cells through T cells.

Intermittent parathyroid hormone (iPTH) treatment expands hemopoietic stem and progenitor cells (HSPCs), but the involved mechanisms and the affected HSPC populations are mostly unknown. Here we show that T cells are required for iPTH to expand short-term HSPCs (ST-HSPCs) and improve blood cell engraftment and host survival after BM transplantation. Silencing of PTH/PTH-related protein receptor (PPR) in T cells abrogates the effects of iPTH, thus demonstrating a requirement for direct PPR signaling in T cells. Mechanistically, iPTH expands ST-HSPCs by activating Wnt signaling in HSPCs and stromal cells (SCs) through T-cell production of the Wnt ligand Wnt10b. Attesting to the relevance of Wnt10b, iPTH fails to expand ST-HSPCs in mice with Wnt10b(-/-) T cells. Moreover, iPTH fails to promote engraftment and survival after BM transplantation in Wnt10b null mice. In summary, direct PPR signaling in T cells and the resulting production of Wnt10b play a pivotal role in the mechanism by which iPTH expands ST-HSPCs. The data suggest that T cells may provide pharmacologic targets for HSPC expansion.

Emergence of T cells that recognize nonpolymorphic antigens during graft-versus- host disease.

Chronic GVHD is a major cause of morbidity and mortality in allogeneic stem cell transplantation recipients and typically develops from antecedent acute GVHD. In contrast to acute GVHD, chronic GVHD has much broader tissue involvement and clinical manifestations that bear striking similarity to what is observed in autoimmune diseases. How autoimmunity arises out of alloimmunity has been a longstanding unresolved issue. To address this question, in the present study, we performed a comprehensive analysis of the clonotypic T-cell response using complementary murine models that simulate what occurs during the transition from acute to chronic GVHD. These studies revealed repertoire skewing and the presence of high-frequency clonotypes that had undergone significant in vivo expansion, indicating that GVHD-associated autoimmunity was characterized by antigen-driven expansion of a limited number of T-cell clones. Furthermore, we observed that T cells with identical TCRß CDR3 nucleotide sequences were capable of recognizing donor and host antigens, providing evidence that the loss of self-tolerance during acute GVHD leads to the emergence of self-reactive donor T cells that are capable of recognizing nonpolymorphic tissue or commensally derived antigens. These data provide a mechanistic framework for how autoimmunity develops within the context of preexisting GVHD and provide additional insight into the pathophysiology of chronic GVHD.

Screening of differentially expressed genes and predominant expression of ß variable region of T cell receptor in peripheral T cells of psoriatic patients.

UNLABELLED: Psoriasis is a skin disease featuring epithelial cell hyper-proliferation and T cell infiltration. Abnormal T cell immune responses play an important role in psoriatic pathogenesis. To screen differentially expressed genes in T cells of patients with plaque psoriasis, analyze the predominant expression of the ß variable region of T cell receptors and discuss the role of T cells in the pathogenesis of psoriasis. High throughput RNA sequencing and Real-time PCR were used. RESULTS: A total of 907 genes were differentially expressed in peripheral T cells of patients with psoriasis. Among them, 695 genes were mapped to the Gene Ontology database, 14 gene terms were significantly enriched, and 418 genes were involved in signaling pathways such as apoptosis, B cell receptor signaling and T cell receptor signaling. TRBV2, TRBV5-7, TRBV6-6/6-9, TRBV12, TRBV24 and TRBV29 were significantly up-regulated in psoriatic patients compared to healthy subjects, among which, TRBV6-6/6-9, TRBV12 and TRBV29 are predominantly expressed in psoriatic patients. Many genes were differentially expressed in T cells of psoriatic patients, especially the TRBV gene family, which were predominantly expressed in T cells and might play an important role in abnormal immune responses of T cells in psoriatic patients.

The microbiota mediates pathogen clearance from the gut lumen after non-typhoidal Salmonella diarrhea.

Many enteropathogenic bacteria target the mammalian gut. The mechanisms protecting the host from infection are poorly understood. We have studied the protective functions of secretory antibodies (sIgA) and the microbiota, using a mouse model for S. typhimurium diarrhea. This pathogen is a common cause of diarrhea in humans world-wide. S. typhimurium (S. tm(att), sseD) causes a self-limiting gut infection in streptomycin-treated mice. After 40 days, all animals had overcome the disease, developed a sIgA response, and most had cleared the pathogen from the gut lumen. sIgA limited pathogen access to the mucosal surface and protected from gut inflammation in challenge infections. This protection was O-antigen specific, as demonstrated with pathogens lacking the S. typhimurium O-antigen (wbaP, S. enteritidis) and sIgA-deficient mice (TCRß(-/-)δ(-/-), J(H) (-/-), IgA(-/-), pIgR(-/-)). Surprisingly, sIgA-deficiency did not affect the kinetics of pathogen clearance from the gut lumen. Instead, this was mediated by the microbiota. This was confirmed using 'L-mice' which harbor a low complexity gut flora, lack colonization resistance and develop a normal sIgA response, but fail to clear S. tm(att) from the gut lumen. In these mice, pathogen clearance was achieved by transferring a normal complex microbiota. Thus, besides colonization resistance ( = pathogen blockage by an intact microbiota), the microbiota mediates a second, novel protective function, i.e. pathogen clearance. Here, the normal microbiota re-grows from a state of depletion and disturbed composition and gradually clears even very high pathogen loads from the gut lumen, a site inaccessible to most "classical" immune effector mechanisms. In conclusion, sIgA and microbiota serve complementary protective functions. The microbiota confers colonization resistance and mediates pathogen clearance in primary infections, while sIgA protects from disease if the host re-encounters the same pathogen. This has implications for curing S. typhimurium diarrhea and for preventing transmission.

Position-dependent silencing of germline Vß segments on TCRß alleles containing preassembled VßDJßCß1 genes.

The genomic organization of TCRbeta loci enables Vbeta-to-DJbeta2 rearrangements on alleles with assembled VbetaDJbetaCbeta1 genes, which could have deleterious physiologic consequences. To determine whether such Vbeta rearrangements occur and, if so, how they might be regulated, we analyzed mice with TCRbeta alleles containing preassembled functional VbetaDJbetaCbeta1 genes. Vbeta10 segments were transcribed, rearranged, and expressed in thymocytes when located immediately upstream of a Vbeta1DJbetaCbeta1 gene, but not on alleles with a Vbeta14DJbetaCbeta1 gene. Germline Vbeta10 transcription was silenced in mature alphabeta T cells. This allele-dependent and developmental stage-specific silencing of Vbeta10 correlated with increased CpG methylation and decreased histone acetylation over the Vbeta10 promoter and coding region. Transcription, rearrangement, and expression of the Vbeta4 and Vbeta16 segments located upstream of Vbeta10 were silenced on alleles containing either VbetaDJbetaCbeta1 gene; sequences within Vbeta4, Vbeta16, and the Vbeta4/Vbeta16-Vbeta10 intergenic region exhibited constitutive high CpG methylation and low histone acetylation. Collectively, our data indicate that the position of Vbeta segments relative to assembled VbetaDJbetaCbeta1 genes influences their rearrangement and suggest that DNA sequences between Vbeta segments may form boundaries between active and inactive Vbeta chromatin domains upstream of VbetaDJbetaCbeta genes.

Pre-TCR signaling inactivates Notch1 transcription by antagonizing E2A.

Precise control of the timing and magnitude of Notch signaling is essential for the normal development of many tissues, but the feedback loops that regulate Notch are poorly understood. Developing T cells provide an excellent context to address this issue. Notch1 signals initiate T-cell development and increase in intensity during maturation of early T-cell progenitors (ETP) to the DN3 stage. As DN3 cells undergo beta-selection, during which cells expressing functionally rearranged TCRbeta proliferate and differentiate into CD4(+)CD8(+) progeny, Notch1 signaling is abruptly down-regulated. In this report, we investigate the mechanisms that control Notch1 expression during thymopoiesis. We show that Notch1 and E2A directly regulate Notch1 transcription in pre-beta-selected thymocytes. Following successful beta-selection, pre-TCR signaling rapidly inhibits Notch1 transcription via signals that up-regulate Id3, an E2A inhibitor. Consistent with a regulatory role for Id3 in Notch1 down-regulation, post-beta-selected Id3-deficient thymocytes maintain Notch1 transcription, whereas enforced Id3 expression decreases Notch1 expression and abrogates Notch1-dependent T-cell survival. These data provide new insights into Notch1 regulation in T-cell progenitors and reveal a direct link between pre-TCR signaling and Notch1 expression during thymocyte development. Our findings also suggest new strategies for inhibiting Notch1 signaling in pathologic conditions.

Notch signaling is required for proliferation but not for differentiation at a well-defined beta-selection checkpoint during human T-cell development.

Notch signaling is absolutely required for beta-selection during mouse T-cell development, both for differentiation and proliferation. In this report, we investigated whether Notch has an equally important role during human T-cell development. We show that human CD34(+) thymocytes can differentiate into CD4(+)CD8beta(+) double positive (DP) thymocytes in the absence of Notch signaling. While these DP cells phenotypically resemble human beta-selected cells, they lack a T-cell receptor (TCR)-beta chain. Therefore, we characterized the beta-selection checkpoint in human T-cell development, using CD28 as a differential marker at the immature single positive CD4(+)CD3(-)CD8alpha(-) stage. Through intracellular TCR-beta staining and gene expression analysis, we show that CD4(+)CD3(-)CD8alpha(-)CD28(+) thymocytes have passed the beta-selection checkpoint, in contrast to CD4(+)CD3(-)CD8alpha(-)CD28(-) cells. These CD4(+)CD3(-)CD8alpha(-)CD28(+) thymocytes can efficiently differentiate into CD3(+)TCRalphabeta(+) human T cells in the absence of Notch signaling. Importantly, preselection CD4(+)CD3(-)CD8alpha(-)CD28(-) thymocytes can also differentiate into CD3(+)TCRalphabeta(+) human T cells without Notch activation when provided with a rearranged TCR-beta chain. Proliferation of human thymocytes, however, is clearly Notch-dependent. Thus, we have characterized the beta-selection checkpoint during human T-cell development and show that human thymocytes require Notch signaling for proliferation but not for differentiation at this stage of development.

Use of a Pan-Vbeta primer permits the amplification and sequencing of TCRbeta chains expressed by bovine T-cell clones following a single semi-nested PCR reaction.

We report in this study the design and validation of a Pan-Vbeta primer that in combination with Cbeta-specific primers enables the amplification, in a single semi-nested PCR, of TCRbeta chains expressed by bovine T-cell clones irrespective of the expressed Vbeta sequence. Using the Pan-Vbeta primer we examined the TCRbeta chains expressed by 16 Theileria parva-specific clones that had not been previously analysed. TCRbeta chain sequence was obtained from 15 of the clones following direct sequencing of the PCR product, whilst the other clone appeared to express 2 different TCRbeta chains which were characterised following sub-cloning of the PCR product. We have also successfully used the Pan-Vbeta primer to amplify the TCRbeta chains expressed by 19 T-cell clones, on which previous analysis using Vbeta-subfamily-specific primers had failed to do. Sequencing of these TCRbeta chains has identified members of 2 novel bovine Vbeta subfamilies-Vbeta5 and VbetaX. This method offers a simple and rapid method of analyzing the TCRbeta chains of bovine T-cell clones that has many potential applications in the investigation of bovine T-cell responses.

CD4 T cell-independent antibody response promotes resolution of primary influenza infection and helps to prevent reinfection.

It is generally believed that the production of influenza-specific IgG in response to viral infection is dependent on CD4 T cells. However, we previously observed that CD40-deficient mice generate influenza-specific IgG during a primary infection, suggesting that influenza infection may elicit IgG responses independently of CD4 T cell help. In the present study, we tested this hypothesis and show that mice lacking CD40 or CD4 T cells produce detectable titers of influenza-specific IgG and recover from influenza infection in a manner similar to that of normal mice. In contrast, mice completely lacking B cells succumb to influenza infection, despite the presence of large numbers of functional influenza-specific CD8 effector cells in the lungs. Consistent with the characteristics of a T-independent Ab response, long-lived influenza-specific plasma cells are not found in the bone marrow of CD40-/- and class II-/- mice, and influenza-specific IgG titers wane within 60 days postinfection. However, despite the short-lived IgG response, CD40-/- and class II-/- mice are completely protected from challenge infection with the same virus administered within 30 days. This protection is mediated primarily by B cells and Ab, as influenza-immune CD40-/- and class II-/- mice were still resistant to challenge infection when T cells were depleted. These data demonstrate that T cell-independent influenza-specific Ab promotes the resolution of primary influenza infection and helps to prevent reinfection.

Changes to peptide structure, not concentration, contribute to expansion of the lowest avidity cytotoxic T lymphocytes.

The efficient in vitro expansion of antigen-specific CD8+ cytotoxic T lymphocytes (CTL) for use in adoptive immunotherapy represents an important clinical goal. Furthermore, the avidity of expanded CTL populations often correlates closely with clinical outcome. In our study, high-avidity CTL lines could be expanded ex vivo from an antigen-primed animal using low peptide concentration, and intermediate peptide concentrations favored the generation of lower avidity CTL. Further increases in peptide concentration during culture inhibited the expansion of all peptide-specific CD8+ cells. In contrast, a single amino acid variant peptide efficiently generated functional CTL populations at high or low peptide concentration, which responded to wild-type epitope with the lowest average avidity seen in this study. We propose that for some peptides, the efficient generation of low-avidity CTL responses will be favored by stimulation with altered peptide rather than high concentrations of wild-type epitope. In addition, some variant peptides designed to have improved binding to major histocompatibility complex class I may reduce rather than enhance the functional avidity for the wild-type peptide of ex vivo-expanded CTL. These observations are relevant to in vitro expansion of CTL for immunotherapy and strategies to elicit regulatory or therapeutic immunity to neo-self-antigen when central tolerance has eliminated high-avidity, cognate T cells.

Obligatory role for cooperative signaling by pre-TCR and Notch during thymocyte differentiation.

The first checkpoint during T cell development, known as beta selection, requires the successful rearrangement of the TCR-beta gene locus. Notch signaling has been implicated in various stages during T lymphopoiesis. However, it is unclear whether Notch receptor-ligand interactions are necessary during beta selection. Here, we show that pre-TCR signaling concurrent with Notch receptor and Delta-like-1 ligand interactions are required for the survival, proliferation, and differentiation of mouse CD4(-)CD8(-) thymocytes to the CD4(+)CD8(+) stage. Furthermore, we address the minimal signaling requirements underlying beta selection and show a hierarchical positioning of key proximal signaling molecules. Collectively, our results demonstrate an essential role for Notch receptor-ligand interactions in enabling the autonomous signaling capacity of the pre-TCR complex.

2-deoxycoformycin in the treatment of T-large granular lymphocyte leukemia.

A 52-year-old woman presented to our clinic for investigation of agranulocytosis and mild lymphocytosis. A diagnosis of T-large granular lymphocyte leukemia was made, based on immunophenotyping findings of the peripheral blood lymphocytes (CD3, CD8, CD16, CD57). Flow cytometric analysis of TCR-Vbeta repertoire showed single Vbeta9 expression on peripheral T-cells. Clonality was also demonstrated with PCR analysis which revealed clonal rearrangement of TCRgamma-chain gene. Granulocyte-macrophage colony-stimulating factor (G-CSF) (G-CSF), cyclosporine, methylprednisolone and oral methotrexate failed to correct the neutropenia. Finally, treatment with 2-deoxycoformycin (DCF) was successful and resulted in complete correction of the neutrophil count. Flow cytometric analysis of TCR-Vbeta repertoire proved to be an effective method to assess the therapeutic response to various treatments and to evaluate residual disease.

Intrahepatic and circulating HLA class II-restricted, hepatitis C virus-specific T cells: functional characterization in patients with chronic hepatitis C.

To compare the functional features of circulating and intrahepatic hepatitis C virus (HCV)-specific CD4+ T cells in chronic HCV infection, peripheral blood and liver-infiltrating lymphocytes from 29 patients with chronic hepatitis C were stimulated with structural and nonstructural HCV proteins to produce antigen-specific T-cell lines and clones. Antigen specificity, fine specificity, phenotype, cytokine production, and T-cell receptor (TCR)-vbeta chain expression were analyzed. The results indicate a hierarchy of stimulatory capacity by the different HCV proteins, core being the antigen most frequently recognized by CD4+ intrahepatic lymphocytes, followed by NS4 and NS5. The CD4 response was directed simultaneously against different HCV proteins in individual patients, but fine-specificity analysis indicated that the response was generally focused on a limited number of immunodominant epitopes. Although the narrowly focused nature of this response may favor the emergence of escape mutations, this event was not observed by following-up over time the sequence of 2 epitopes strongly immunodominant for intrahepatic CD4 cells of a patient with chronic HCV infection. In conclusion, simultaneous analysis of peripheral blood and intrahepatic CD4 cells in the same patients indicated a predominant Th1 profile of HCV-specific CD4 cells and suggests a specific compartmentalization of virus-specific T cells into the liver.

Allelic exclusion at the TCRbeta locus.

Assembly of TCRbeta chain variable-region genes is regulated in the context of allelic exclusion. Differential epigenetic modifications of the two TCRbeta alleles established early in embryonic development may be important for permitting allelic exclusion by ordering rearrangement of the two alleles in double-negative thymocytes. Expression of a TCRbeta chain, as part of the pre-TCR complex, activates signaling pathways that enforce allelic exclusion in double-positive thymocytes. These signaling pathways, which utilize p56(lck) and SLP-76, may be distinct from those used to promote other processes initiated by pre-TCR expression. In double-positive thymocytes allelic exclusion is enforced, in part, by changes in Vbeta gene segment accessibility promoted by cis-acting elements that may be distinct from those regulating accessibility of D/Jbeta gene segments.

Novel system for generating cytotoxic effector lymphocytes using carcinoembryonic antigen (CEA) peptide and cultured dendritic cells.

We have established a practical system for generating antitumor effector lymphocytes using the tumor antigen peptide CEA and cultured dendritic cells (DCs), and have also characterized effector cells. DCs were induced from the adherent cell population of autologous peripheral blood mononuclear cells (PBMCs) obtained from HLA-A0201 normal or tumor-bearing donors using IL-4 and GM-CSF. The cultured DCs were shown to express class I, class II, CD80 and CD86 molecules. The PBMCs were stimulated for 7 days with the DCs pulsed with the HLA-A0201-restricted CEA peptide CEA9 671 and then expanded in an anti-CD3 antibody (1 microgram/ml)-coated flask in the presence of a 80 U/ml IL-2 (IL-2/CD3 system). The effector cells, which were designated as CEA peptide-pulsed dendritic cell-activated killer (CEA-PDAK) cells, were preferentially CD3+CD8+, and capable of killing T2 cells pulsed with CEA peptide but not T2 cells alone. The CA-PDAK cells also lysed the gastric cancer cell line KATO III (HLA-A0201, CEA (+)), but not the WiDr (HLA-A2402, CEA(+)) cells. The cytotoxicity was abrogated when the CEA-PDAK cells were treated with anti-TCR alpha beta antibody or when the target cells were treated with the anti-class I antibody prior to the cytotoxicity assay. The CEA-PDAK cells exerted their cytotoxic activity even in the presence of a high amount of CEA protein at the effector phase, which mimicked the clinical setting. The CEA-PDAK cells showed approximately a hundred-fold expansion in total cell numbers yielded without any loss of the specific lysis, when stimulated with the IL-2/CD3 system compared to those stimulated with IL-2 alone. The TCR V beta gene analysis for the CEA-PDAK cells, conducted by means of RT-PCR-Southern blotting, demonstrated oligoclonal expression of TCR beta 7 and 12, and the latter was shown to be responsible for the killing activity. SSCP analysis indicated the clonotype of the TCR V beta 12 gene, indicating a selective expansion of lymphocytes bearing a limited TCR variable region by the stimulation with CEA peptide-pulsed DCs. Taken together, the effector lymphocytes reactive with the CEA antigen can be generated from PBMCs with the antigenic CEA peptide and cultured DCs. The IL-2/CD3 system is effective and practical in activating the effector cells for the clinical use of CEA-PDAK cells. Adoptive immunotherapy using this system may be promising for treating CEA-expressing tumors.

Premature TCR alpha beta expression and signaling in early thymocytes impair thymocyte expansion and partially block their development.

In thymocyte ontogeny, Tcr-a genes rearrange after Tcr-b genes. TCR alpha beta transgenic (Tg) mice have no such delay, consequently expressing rearranged TCR alpha beta proteins early in the ontogeny. Such mice exhibit reduced thymic cellularity and accumulate mature, nonprecursor TCR(+)CD8(-)4(-) thymocytes, believed to be caused by premature Tg TCR alpha beta expression via unknown mechanism(s). Here, we show that premature expression of TCR alpha beta on early thymocytes curtails thymocyte expansion and impairs the CD8(-)4(-) --> CD8(+)4(+) transition. This effect is accomplished by two distinct mechanisms. First, the early formation of TCR alpha beta appears to impair the formation and function of pre-TCR, consistent with recently published results. Second, the premature TCR alpha beta contact with intrathymic MHC molecules further pronounces the block in proliferation and differentiation. These results suggest that the benefit of asynchronous Tcr-a and Tcr-b rearrangement is not only to minimize waste during thymopoiesis, but also to simultaneously allow proper expression/function of the pre-TCR and to shield CD8(-)4(-) thymocytes from TCR alpha beta signals that impair thymocyte proliferation and CD8(-)4(-) --> CD8(+)4(+) transition.

Cytotoxic effector T cells elicited by the killed tumor vaccine differ significantly from the effectors generated during active growth of a murine B-cell lymphoma.

Active specific immunotherapy of neoplastic diseases is an elusive goal. Using a murine B lymphoma 2C3, we showed that vaccination with the killed tumor cells effectively induces protective immunity and a cytotoxic T cell (CTL) response. Similar protection, however, is rarely observed in mice bearing live tumor cells. These animals usually succumb to the progressively growing tumor. In this study, we inquired whether the splenic CTL induced during tumor progression in mice differ from those evoked by the killed tumor cells. Here we demonstrate that the CTL generated following vaccination are significantly different from those induced in the tumor-bearing hosts. Adding to the complexity, the CTL from the early tumor bearers also differ significantly from those induced at the late stages. These differences are based on their cytotoxic activity, MHC allele specificity, mitogen responsiveness, cytokine secretion profile and T cell receptor Vbeta gene expression. The results clearly indicate that passive immunization with killed tumor is most effective, possibly because the CTL induced are not subject to the same regulatory pressure as those induced during active tumor growth. This decreasing effectiveness of CTL could be due to greater variability in antigenic stimulus, less involvement of innate immunity, changes in cytokine milieu and/or costimulatory factors.

Conserved cAMP responsive element and core promoter complex are critical for specificity of the distal T-cell receptor beta chain enhancer for its native promoter.

The Vbeta 8.1 promoter is regulated by T-cell-specific and ubiquitous transcription factors, which bind immediately upstream of and inside the core promoter region. The various Vbeta promoters contain two conserved elements, a cAMP responsive element (CRE) located upstream of the core promoter and a basal initiator flanked by two regulatory motifs. Here we have studied the interplay between the distal enhancer and its native promoter. We show that the remote enhancer acts specifically through its native promoter. Specific enhancer-promoter interplay is mediated through the conserved regions of the Vbeta promoters. Importantly, the conserved CRE serves as a functional recognition element for the enhancer whereas it barely contributes to promoter activity. The other conserved regions surrounding the initiation site are critical for activators that bind at and function through the core promoter region and thereby regulate both promoter and enhancer activity. The enhancer is highly sensitive to E1A-12S, which represses both general and specific enhancer activities. Enhancer activity and promoter-enhancer specificity is, at least in part, mediated by the coactivators CBP/p300.