Lymphocyte antigen receptor gene assembly

Lymphocyte antigen receptor genes are assembled through the cutting and joining of segments of DNA in developing lymphocytes. The basic features of the biochemical steps of this assembly process, referred to as the V(D)J recombination, are similar for the assembly of all lymphocyte antigen receptor genes, yet this assembly is precisely regulated in several important contexts during lymphocyte development. It has long been appreciated that this occurs through modulation of accessibility of antigen receptor loci to the enzymatic complex that assembles antigen receptor genes. However, recent studies have suggested that some regulatory constraints may be enforced at the level of the V(D)J recombination reaction itself. This review focuses on recent advances in the understanding of the regulation of antigen receptor gene assembly, with particular attention paid to the assembly of T-cell receptor β-chain genes during T-cell development.     

Regulation of T cell antigen receptor alpha chain gene expression]

A T cell antigen receptor (TcR) gene is considered to be a model system for studies of developmentally-regulated, lineage-specific gene expression. For the TcR alpha gene expression, it has been shown that the enhancer region located at 3.0 kb in mouse or 4.5 kb in human downstream of TcR C alpha gene is essential. The enchancer is demonstrated to contain at least four nuclear protein binding sites called T alpha 1-T alpha 4. In this report, we analysed the molecular requirement for murine TcR alpha gene enhancer function by using in vitro mutagenesis system and CAT assay, and obtained the following results: 1. The T alpha 2 element, especially ACATCC sequence, is important for enhancer activity, because the mutation in the sequence completely abolished the activity. 2. The sequence TCTGG near by the ACATCC sequence seems also important for the enhancer function, since the mutation lost the half of the activity. 3. The ets1, which is known to bind the ACATCC site, upregulates the enhancer activity of the reporter gene containing the concatemers of T alpha 2 region. The results indicate that ets1 has a trans-activating activity to the T alpha 2 region of TcR alpha chain enhancer.     

Negative regulation of antigen receptor signaling in lymphocytes

The negative regulation of antigen receptor signal transduction is essential for the maintenance of thresholds for activation in lymphocytes. CD45 and SHP-1 are tyrosine phosphatases that are important in maintaining the proper level of tyrosine phosphorylation. Regulation of the src family of tyrosine kinases is mediated by the coordinated action of the tyrosine kinase Csk and the tyrosine phosphatase CD45. B cell receptor signaling is negatively regulated by the recruitment of SHP-1 to bind the B cell transmembrane proteins CD22 and FcγRIIb1. SHP-1 also functions to negatively regulate T cell receptor signaling by dephosphorylating and inactivating tyrosine kinases. Received: 13 October 1997 / Accepted: 19 December 1997     

Regulation of immunoglobulin gene expression in normal lymphocytes. II. Mechanisms of down-regulation of immunoglobulin secretion after engagement of the B cell antigen receptor.

When B cells are stimulated with lipopolysaccharide (LPS) they start to proliferate and mature into immunoglobulin (Ig)-secreting cells. Co-stimulation with F(ab')2 fragments of antibodies directed against the B cell antigen receptor leads to an inhibition of Ig secretion but not of proliferation. This effect can be mimicked by phorbol esters alone or by a combination of phorbol esters and the Ca2+ ionophore ionomycin, which activate protein kinase C. Here we report that co-stimulation with phorbol esters and ionomycin differentially affects a group of genes normally up-regulated during the course of LPS-dependent B cell activation. Thus, the mRNA coding for the membrane-bound form of IgM and the interleukin 2 receptor (55-kDa protein) continue to be expressed at the levels typical of LPS-stimulated cells, while the mRNA coding for the secreted form of IgM (mu S) and for the J chain are reduced. The loss of mu S mRNA is attributable to an altered processing behavior with respect to the mu precursor and/or a decreased stability of the mRNA itself.     

Regulation of T cell receptor alpha gene assembly by a complex hierarchy of germline Jalpha promoters

Assembly of the gene encoding T cell receptor alpha (Tcra) is characterized by an orderly progression of primary and secondary V(alpha)-to-J(alpha) recombination events across the J(alpha) array, but the targeting mechanisms responsible for this progression are mostly unknown. Studies have shown that the T early-alpha promoter is important in targeting primary Tcra rearrangements. We found that T early-alpha and a previously unknown promoter associated with J(alpha)49 targeted primary recombination to discrete sets of constant alpha region (C(alpha))-distal J(alpha) segments and together directed nearly all normal primary recombination events. Furthermore, deletion of the T early-alpha promoter activated previously suppressed downstream promoters and stimulated primary rearrangement to centrally located J(alpha) segments. Central promoter derepression also occurred after primary rearrangement, thereby providing a mechanism to target secondary recombination events.     

The antigen receptor complex on cord B lymphocytes.

The neonatal immune system responds to a restricted range of antigens, producing largely IgM antibody of low affinity. Comparison of the components of the B-cell antigen receptor complex shows significantly elevated membrane levels of IgM in neonatal B cells, compared with adult cells. CD79, which acts as the signal transducer for membrane immunoglobulin, is elevated in parallel with IgM, while IgD is elevated to a lesser degree. CD19, CD21, CD22 and CD81, which are all involved in transmitting activation signals when immunoglobulin is engaged, are not elevated. CD32, which is involved in negative regulation of activation, is present at reduced levels on cord B cells. The elevation of B-cell membrane IgM persists during infancy. Neonatal B cells respond in vitro to interleukin-4 (IL-4) by further elevation of membrane IgM levels. The elevated level of membrane IgM may make neonatal B cells easier to trigger by low concentrations of antigen, but in vitro activation and immunoglobulin modulation experiments did not show significant differences between cord and adult B-cell responses to anti-IgM.     

Mitogenic lectins bind to the antigen receptor on human lymphocytes

The specificity of interactions between mitogenic and non-mitogenic lectins and disulfide-linked cell surface receptors on human lymphocytes was explored. Lysates (Nonidet-P40) of surface-radioiodinated tonsil lymphocytes and T lymphoblastoid cells (HPB-ALL) were absorbed with lectin-agarose derivatives (or bovine serum albumin, BSA-agarose) or immunoprecipitated with appropriate monoclonal antibodies (mAb). Lectin eluates and solubilized immunoprecipitates were analyzed by two-dimensional (nonreduced/reduced) sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiolabeled polypeptides were visualized by autoradiography. Among the various lectin-binding polypeptides, two disulfide-linked heterodimers (II and III) and two apparent homodimers (I and IV) are bound by pea lectin, concanavalin A and lentil lectin on tonsil lymphocytes; II, III and IV are bound both leukoagglutinating (L)- and erythroagglutinating (E)-phytohemagglutinins from Phaseolus vulgaris (PHA). Pokeweed mitogen recognizes only II and III. These molecules are weakly bound by peanut agglutinin, soybean agglutinin, Ulex europaeus agglutinin-I, Dolichos biflorus agglutinin, Vicia villosa agglutinin and Sophora japonica agglutinin, but are not bound by Helix pomatia agglutinin or BSA-agarose. Heterodimer II (82-88 kDa), comprised of 50-55-kDa and 40-43-kDa subunits, probably represents the alpha/beta T cell antigen receptor (TcR alpha/beta). Heterodimer III (64-72 kDa), comprised of 41-kDa and 37-kDa subunits, may represent TcR gamma. The homodimers, I (120-130 kDa) and IV (55-61 kDa), comprised of 55-60-kDa and 30-kDa polypeptides, respectively, have apparently not been previously described. Evidence that H1-2D4, a mAb directed against the antigen receptor on HPB-ALL cells, and E-PHA interact with a common molecule includes: (a) immunoprecipitation of TcR with H1-2D4 from the glycopeptide fraction specifically eluted from insolubilized lectin with N-acetylgalactosamine; and (b) adsorption of TcR from a solubilized H1-2D4 immunoprecipitate by E-PHA-agarose. Recognition of CD3 by E-PHA is indicated by immunoprecipitation of CD3 protein by UCHT1 from the glycopeptide fraction specifically eluted from E-PHA. The results are consistent with the view that mitogenic lectins interact with certain disulfide-linked molecules on human lymphocytes, including the TcR alpha/beta and perhaps TcR gamma; while some nonmitogenic lectins also recognize these receptors, the interaction is of low affinity.     

丁酸钠及其G蛋白偶联受体对T淋巴细胞的调节作用

短链脂肪酸通常由肠道微生物对食物纤维的发酵而产生,已有报道其在免疫和炎症中起重要作用。本文研究丁酸钠通过其受体GPR43对T淋巴细胞的调节作用,探讨丁酸钠在T细胞水平的抗炎作用和机制。以Hut78和Jurkat细胞株为模型,用RT-PCR检测T细胞中GPR41和GPR43的表达情况。用实时荧光定量PCR检测PMA/Ionomycin诱导的T淋巴细胞经丁酸钠处理后IL-2和IL-4水平变化,并以ELISA等方法验证。通过RT-PCR以及胞内钙离子检测探究丁酸钠对T细胞的具体作用机制及作用靶标。结果显示,GPR41和GPR43在T细胞中存在表达且在PMA刺激后表达显著上升。丁酸钠能刺激胞内钙离子的释放,抑制IL-2、促进IL-4的产生,且这种调节作用不受Gαi抑制剂PTX影响。另外,丁酸钠可以显著抑制MOG诱导EAE小鼠脾脏淋巴细胞的体外增殖。这些结果说明,在T淋巴细胞中丁酸钠主要通过GPR43受体调节其细胞因子的产生,抑制炎症性T淋巴细胞的增殖从而发挥其抗炎功能。     

Epigenetic mechanisms that regulate antigen receptor gene expression

Functional immunoglobulin and T-cell receptor genes are generated from germline V, D and J gene segments by a series of site-specific recombination events. This process is regulated by the availability of recombination machinery and by the ordered accessibility of appropriate target gene segments. Recent studies highlight the importance of chromatin remodelling and locus positioning for controlling antigen receptor gene expression and recombination.     

Regulation of dihydropyridine receptor and ryanodine receptor gene expression in regenerating skeletal muscle

 One of the the major properties of mature skeletal muscle is its ability to regenerate after injury. The purpose of the present study was to determine whether the expression of genes encoding the dihydropyridine receptor calcium channel (DHPR) and the ryanodine receptor (RyR), which play a critical role in excitation–contraction coupling, is regulated by skeletal muscle regeneration. The process of regeneration was induced by bupivacaine injection in surgically exposed rat extensor digitorum longus (EDL) muscle. After total RNA isolation from the injected and the contralateral control EDL muscles performed 3, 7, 15 and 30 days following injection, Northern blot and RNase protection assays were carried out with four cDNA probes specific for the skeletal and cardiac muscle isoforms of both the DHPR α1-subunit and the RyR. After 3 days, an initial precipitous decrease in the expression of the genes encoding the skeletal muscle isoforms of the DHPR and RyR was observed, followed by an increase. Moreover, regenerating skeletal muscle transiently expressed mRNA for the DHPR cardiac isoform, mainly at the beginning of regeneration. No expression of mRNA for the cardiac RyR was observed. Contraction experiments, performed using EDL muscle at the same times after bupivacaine injection, showed that twitch amplitude was markedly decreased in the absence of external calcium, but only during the early stages of regeneration. Similar findings in relation to expression of skeletal and cardiac muscle DHPR message were previously reported from experiments conducted during early developmental stages using fetal skeletal muscle and muscle cell cultures [Chaudhari N, Beam KG (1993) Dev Biol 155:507–515]. These results suggest that expression of the DHPR cardiac isoform in skeletal muscle could explain certain cardiac-like aspects of excitation–contraction coupling of regenerating skeletal muscle and developing skeletal muscle as well. Received: 2 July 1996 / Received after revision: 18 September 1996 / Accepted: 20 September 1996     

Regulation of T-cell receptor β-chain gene assembly by recombination signals: the beyond 12/23 restriction

Summary:  Assembly of antigen receptor genes is regulated in several important contexts during lymphocyte development. This regulation occurs through modulation of gene segment accessibility to the V(D)J recombinase and/or at the level of the recombination reaction due, in part, to constraints imposed by recombination signal (RS) sequences. RSs are composed of conserved heptamer and nonamer sequences that flank relatively non-conserved spacer sequences of either 12 or 23 base pairs. Recombination occurs only between RSs of dissimilar spacer lengths, a restriction known as the 12/23 rule. Recently, we have shown that RSs can impose significant constraints on antigen receptor gene assembly beyond enforcing the 12/23 rule. This restriction, termed B12/23, was revealed by analysis of T-cell receptor β (TCRβ) locus rearrangements, where Dβ 12RSs and not Jβ 12RSs are capable of efficiently targeting Vβ 23RSs' rearrangement. The B12/23 restriction occurs at or prior to the DNA-cleavage step of the V(D)J recombination reaction, relies on features of the Dβ 12RSs and Vβ 23RSs, and is not absolutely dependent on lymphoid-specific factors other than the recombinase-activating gene-1 (RAG-1) and RAG-2 proteins. By preserving Dβ gene segment utilization, the B12/23 restriction is required, at a minimum, for the generation of a diverse repertoire of TCRβ chains.     

Structure, assembly and intracellular transport of the T cell receptor for antigen.

The T cell receptor for antigen (TCR) is responsible for the recognition of antigen associated with the major histocompatibility complex (MHC). The TCR expressed on the surface of T cells is associated with an invariant structure, CD3. CD3 is assumed to be responsible for intracellular signaling following occupancy of the TCR by ligand. The TCR/CD3 complex consists of six different polypeptides, and represents a uniquely complex multisubunit assembly problem for the cell. The cell copes with this problem by regulating the intracellular assembly of the complex. Within the endoplasmic reticulum, the newly-synthesised chains assemble into the complete structure prior to transport to the cell surface. There are a series of different isoforms of the receptor involving differential use of the TCR heterodimer (alpha-beta or gamma-delta), zeta-family member, and CD3 gamma or delta chains. These are presumably linked to different TCR functions. Assembly of the TCR/CD3 complex competes with specific degradation of unassembled polypeptides. The fate of the receptor depends on the presence of subtle signals on individual chains which determine pairing and assembly or degradation. The T cell is thus able to select a completely assembled fully functional series of distinct TCR/CD3 complexes for expression at the cell surface.     

B cell antigen receptor assembly and Syk activation in the S2 cell reconstitution system

Signal transduction from the B cell antigen receptor (BCR) involves a multitude of signaling molecules often organized in dynamic protein complexes. The molecular mechanisms operating during signaling are difficult to study solely by loss-of-function analysis. For a better understanding of the transient interaction of signaling molecules and their regulation by feedback loops, as well as their dynamic behavior in living cells, new techniques are required. We have developed a method allowing the reconstitution of the BCR complex and several of its key signaling elements in the evolutionary distant environment of the Drosophila S2 Schneider cell line. With this gain-of-function approach, we study here the assembly of the BCR complex and the control of its transport to the cell surface of S2 cells. We find that without binding to a light chain, the membrane-bound microm heavy chain (micromHC) homodimer, together with the Ig-alpha/Ig-beta heterodimer, can come to the cell surface where it is signaling competent. This finding could have implications for potential signaling functions of such a receptor molecule during pro-/pre-B cell development. We also studied the activation of the BCR-proximal kinase Syk. We found that a truncated Syk mutant lacking the first (N-terminal) SH2 domain and the linker regions, is still regulated by autoinhibition and can only become activated in the presence of the BCR. This indicates that the C-terminal SH2 domain of Syk is the dominant regulatory subunit of this kinase.     

Regulation and chance in the ontogeny of B and T cell antigen receptor repertoires

The adaptive immune system has to economically generate a large array of T and B cell antigen receptors (T cell receptors [TCRs], B cell receptors [BCRs]) that eliminate both longstanding and novel antigens from the host while preventing the production of deleterious (e.g., autoreactive) antigen receptors. Our studies focus on the mechanisms that shape the development of these antigen receptor repertoies during human ontogeny. The key to BCR and TCR diversity is the third complementarity determining region (CDR3) of the variable domain, which in the immunoglobulin heavy chain and TCR β chain, is created by the junction between the variable, diversity, and joining gene segments. The CDR3 diversity is constrained by overrepresentation of gene segments and lack of N regions during the first trimester of gestation and then increases exponentially during ontogeny until it reaches adult levels months after birth. This process parallels, and may contribute to, the stepwise acquisition of the ability to respond to specific antigens. Recent studies indicate that maturation of the CDR 3 repertoire is not accelerated by premature exposition to extrauterine antigen and thus appears to follow a strictly developmentally regulated program whose pacemaker(s) is still unknown.     

Functional responses of human neonatal B lymphocytes to antigen receptor cross-linking and CpG DNA

Human neonates are immunologically immature and consequently are highly susceptible to infection. The cellular basis for the dysfunctional immune responses of neonates is not clear, but is likely to reflect the immaturity of both B and T cell populations. Here we have examined the ability of human cord blood B cells to respond to antigen receptor cross-linking and also to CpG containing oligodeoxynucleotides (ODN), and compared their responses with those of adult peripheral blood B cells. Antigen receptor cross-linking with soluble F(ab')2 anti-IgM antibodies, induced HLA-DR and CD86 up-regulation and proliferation to a similar extent in adult and cord blood B cells. Both interleukin (IL)-2 and IL-4 co-stimulated anti-IgM-induced proliferation, but cord blood B cells were less sensitive than adult B cells to the co-stimulatory effects of IL-2. Antigen receptor cross-linking induced secretion of the chemokines macrophage inflammatory protein-1 alpha (MIP-1 alpha) and MIP-1 beta in adult and cord blood B cells, and secretion was enhanced by IL-2 or IL-4. CpG-ODN induced up-regulation of HLA-DR and CD86 expression and proliferation of adult and cord blood B cells, and anti-IgM and CPG-ODN synergized in the induction of proliferation. CpG-ODN also induced MIP-1 alpha and MIP-1 alpha secretion in adult and cord blood B cells. In addition to functional studies we examined the expression of CD62L (l-selectin), CCR7 and CXCR5. Our data show that surface expression of CD62L and CCR7 is lower on cord blood B cells than on adult B cells, suggesting that human cord blood B cells may exhibit homing defects.