Antigen Receptor Variable Region Genes Research Articles

Functional redundancy between the XLF and DNA-PKcs DNA repair factors in V(D)J recombination and nonhomologous DNA end joining

Classical nonhomologous end joining (C-NHEJ) is a major mammalian DNA double-strand break (DSB) repair pathway that is required for assembly of antigen receptor variable region gene segments by V(D)J recombination. Recombination activating gene endonuclease initiates V(D)J recombination by generating DSBs between two V(D)J coding gene segments and flanking recombination signal sequences (RS), with the two coding ends and two RS ends joined by C-NHEJ to form coding joins and signal joins, respectively. During C-NHEJ, recombination activating gene factor generates two coding ends as covalently sealed hairpins and RS ends as blunt 5'-phosphorylated DSBs. Opening and processing of coding end hairpins before joining by C-NHEJ requires the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). However, C-NHEJ of RS ends, which do not require processing, occurs relatively normally in the absence of DNA-PKcs. The XRCC4-like factor (XLF) is a C-NHEJ component that is not required for C-NHEJ of chromosomal signal joins or coding joins because of functional redundancy with ataxia telangiectasia mutated kinase, a protein that also has some functional overlap with DNA-PKcs in this process. Here, we show that XLF has dramatic functional redundancy with DNA-PKcs in the V(D)J SJ joining process, which is nearly abrogated in their combined absence. Moreover, we show that XLF functionally overlaps with DNA-PKcs in normal mouse development, promotion of genomic stability in mouse fibroblasts, and in IgH class switch recombination in mature B cells. Our findings suggest that DNA-PKcs has fundamental roles in C-NHEJ processes beyond end processing that have been masked by functional overlaps with XLF.

MECHANISM AND CONTROL OF V(D)J RECOMBINATION AT THE IMMUNOGLOBULIN HEAVY CHAIN LOCUS

V(D)J recombination assembles antigen receptor variable region genes from component germline variable (V), diversity (D), and joining (J) gene segments. For B cells, such rearrangements lead to the production of immunoglobulin (Ig) proteins composed of heavy and light chains. V(D)J is tightly controlled at the Ig heavy chain locus (IgH) at several different levels, including cell-type specificity, intra- and interlocus ordering, and allelic exclusion. Such controls are mediated at the level of gene segment accessibility to V(D)J recombinase activity. Although much has been learned, many long-standing questions regarding the regulation of IgH locus rearrangements remain to be elucidated. In this review, we summarize advances that have been made in understanding how V(D)J recombination at the IgH locus is controlled and discuss important areas for future investigation.

Immune system learning and memory quantified by graphical analysis of B-lymphocyte phylogenetic trees

The immune system learns from its encounters with pathogens and memorizes its experiences. One of the mechanisms it uses for this purpose is the intra-individual evolution of antigen receptors on B lymphocytes, achieved via hypermutation and selection of antigen receptor variable region genes during an immune response. We have developed a novel method for analyzing the graphical properties of phylogenetic trees of receptor genes which have been mutated and selected during an immune response. In the study presented here, we address the artifacts introduced by experimental methods of cell collection for DNA analysis, the meaning of each parameter measured on the tree graphs, and the differences between the dynamics of the humoral immune response in different lymphoid tissues.

Receptor revision in T cells: an open question?

During lymphocyte development, both B and T cells assemble antigen receptor variable region genes from germline gene segments, allowing the expression of unique receptors in each clonally derived lymphocyte. Previously, it was shown that in certain cases, progenitor and immature B cells are capable of editing their receptors to a new specificity on encounter with self-antigens. Although the existence of such a process in T cells remains controversial, recent studies suggest that mature T cells are able to similarly revise their receptors in the periphery.

Accessibility control of antigen-receptor variable-region gene assembly: role of cis-acting elements.

Antigen receptor variable region genes are assembled from germline variable (V), diversity (D), and joining (J) gene segments. This process requires expression of V(D)J recombinase activity, and "accessibility" of variable gene segments to this recombinase. The exact mechanism by which variable gene segments become accessible during development is not known. However, several studies have shown that cis-acting elements that regulate transcription may also function to regulate accessibility. Here we review the evidence that transcriptional promoters, enhancers, and silencers are involved in regulation of accessibility. The manner in which these elements may combine to regulate accessibility is addressed. In addition, current and potential strategies for identifying and analyzing cis-acting elements that mediate locus accessibility are discussed.

Analysis of T-Cell Antigen Receptor Variable Region Gene Usage in Patients with Thyroid-Related Pretibial Dermopathy

It is unknown whether T cells infiltrating the pretibial skin of patients with thyroid-related pretibial dermopathy represent a primary immune response or participate in a nonspecific inflammatory process. To characterize these T cells at the molecular level, we examined the T-cell antigen receptor variable region gene usage in pretibial skin biopsy specimens obtained from patients with early and late stages of pretibial dermopathy and from individuals with unrelated inflammatory conditions of the pretibial skin. RNA extracted from pretibial biopsy specimens and peripheral blood lymphocytes was reverse transcribed and amplified with the polymerase chain reaction and 22 V alpha and 23 V beta gene-specific oligonucleotide primers. The resulting T-cell receptor (TcR) V alpha and V beta transcripts were verified by Southern hybridization analysis using TcR C-region-specific, digoxigenin-labeled oligonucleotide probes. Compared with matched peripheral blood lymphocytes, the pretibial TcR V alpha and V beta gene repertoire expressed was heterogeneous but revealed marked restriction of V alpha and V beta gene usage in samples derived from patients with active inflammatory pretibial dermopathy of recent onset. In contrast, greater diversity of the TcR V alpha gene repertoire and loss of TcR V beta gene restriction were noted in patients with long-standing, clinically inactive pretibial dermopathy. TcR V gene usage in pretibial tissue and peripheral blood lymphocyte samples obtained from control subjects was unrestricted. Limited variability of TcR V gene usage in early pretibial dermopathy may reflect a primary immune response of antigen-specific T lymphocytes infiltrating the pretibial skin in thyroid-related pretibial dermopathy.

Potential targets for autosomal SCID mutations

Significant advances have been recently made in the molecular characterization of genes that encode proteins with activities that are directly, or indirectly, involved in the assembly of antigen receptor variable region genes. Such genes are candidate targets for human autosomal mutations that lead to severe combined immune deficiencies characterized by a lack of both T and B cells.

Analysis of retroorbital T cell antigen receptor variable region gene usage in patients with Graves' ophthalmopathy

To date, it has remained unclear whether orbit-infiltrating T cells in patients with Graves' ophthalmopathy (GO) represent a primary immune response in which a limited number of T cell clones driving the disease are activated against specific antigens, or whether they participate in a non-specific inflammatory process. To characterize these T cells at the molecular level, we examined the T cell antigen receptor (TcR) V gene repertoire in situ in retroorbital tissue specimens obtained from patients with early and late stages of clinically severe GO and from patients with non-GO orbital conditions. Ribonucleic acid extracted from orbital tissue and peripheral blood lymphocytes (PBL) was reverse transcribed and amplified using the polymerase chain reaction and 22 V alpha and 24 V beta gene-specific oligonucleotide primers. The resulting TcR V alpha and V beta transcripts were verified by Southern hybridization analysis using TcR C region-specific, digoxigenin-labeled oligonucleotide probes. Compared with matched PBL, the retroorbital TcR V alpha and V beta gene repertoire expressed was heterogeneous, but revealed marked restriction of V gene usage in samples derived from retroorbital connective tissue and extraocular muscle of all eight patients with severe GO of short duration studied. In contrast, greater diversity of the TcR V beta gene repertoire and loss of TcR V alpha gene restriction was noted in four patients with late GO undergoing reconstructive eye muscle surgery. Unrestricted TcR V gene usage was demonstrated in orbital tissue and PBL samples obtained from control subjects. These results suggest that retroorbital TcR V gene usage is variable but markedly restricted during the earlier stages of GO. With increasing disease duration, greater diversity of the TcR V gene repertoire appears to develop, and oligoclonality of the T cell response may be lost. Selection of patients with early stages of GO will be important when further dissecting TcR usage and antigen specificity of orbit-infiltrating T lymphocytes in GO.

Mechanisms that control antigen receptor variable region gene assembly

The somatic assembly of antigen receptor genes—known as V(D)J recombination—is a highly regulated process at several different levels. V(D)J rearrangement is limited to stages of early B and T cell development by the lymphoid specific expression of recombinase activity. A common recombinase is apparently responsible for the rearrangement of all antigen receptor loci. Thus, the tissue specific and ordered rearrangement of each antigen receptor locus must depend on controlling their 'accessibility' to the recombinase. Here, we outline recent work that has contributed to the current understanding of mechanisms that control V(D)J rearrangement.

Erratum: Lack of N Regions in Antigen Receptor Variable Region Genes of TdT-Deficient Lymphocytes

Note 29 on page 1175 of the report "Lack of N regions in antigen receptor variable region genes of TdT-deficient lymphocytes" by T. Komori et al . (27 Aug., p. 1171) should have included the information that the murine TdT clone used in that study was obtained from N. R. Landau, as described in a 1987 paper by N. R. Landau, D. G. Schatz, M. Rosa, and D. Baltimore [ Mol. Cell. Biol. 7, 3237 (1987)] (reference 13 of the report by Komori et al .).

Lack of N Regions in Antigen Receptor Variable Region Genes of TdT-Deficient Lymphocytes

During the assembly of immunoglobulin and T cell receptor variable region genes from variable (V), diversity (D), and joining (J) segments, the germline-encoded repertoire is further diversified by processes that include the template-independent addition of nucleotides (N regions) at gene segment junctions. Terminal deoxynucleotidyl transferase (TdT)-deficient lymphocytes had no N regions in their variable region genes, which shows that TdT is responsible for N region addition. In addition, certain variable region genes appeared at increased frequency in TdT-deficient thymocytes, which indicates that N region addition also influences repertoire development by alleviating sequence-specific constraints imposed on the joining of particular V, D, and J segments.