Robert Monroe

RAG2 :GFP KNOCKIN MICE REVEAL NOVEL ASPECTS OF RAG2 EXPRESSION IN PRIMARY AND PERIPHERAL LYMPHOID TISSUES

We generated mice in which a functional RAG2:GFP fusion gene is knocked in to the endogenous RAG2 locus. In bone marrow and thymus, RAG2:GFP expression occurs in appropriate stages of developing B and T cells as well as in immature bone marrow IgM+ B cells. RAG2:GFP also is expressed in IgD+ B cells following cross-linking of IgM on immature IgM+ IgD+ B cells generated in vitro. RAG2:GFP expression is undetectable in most immature splenic B cells; however, in young RAG2:GFP mice, there are substantial numbers of splenic RAG2:GFP+ cells that mostly resemble pre-B cells. The latter population decreases in size with age but reappears following immunization of older RAG2:GFP mice. We discuss the implications of these findings for current models of receptor assembly and diversification.

The Igκ 3′ Enhancer Influences the Ratio of Igκ versus Igλ B Lymphocytes

Abstract We generated mice harboring germline mutations in which the enhancer element located 9 kb 3′ of the immunoglobulin κ light chain gene (3′E κ ) was replaced either by a single loxP site (3′E κ Δ) or by a neomycin resistance gene (3′E κ N). Mice homozygous for the 3′E κ Δ mutation had substantially reduced numbers of κ-expressing B cells and increased numbers of λ-expressing B cells accompanied by decreased κ versus λ gene rearrangement. In these mutant mice, κ expression was reduced in resting B cells, but was normal in activated B cells. The homozygous 3′E κ N mutation resulted in a similar but more pronounced phenotype. Both mutations acted in cis . These studies show that the 3′E κ is critical for establishing the normal κ/λ ratio, but is not absolutely essential for κ gene rearrangement or, surprisingly, for normal κ expression in activated B cells. These studies also imply the existence of additional regulatory elements that have overlapping function with the 3′E κ element.

Immature Thymocytes Employ Distinct Signaling Pathways for Allelic Exclusion versus Differentiation and Expansion

T cell receptor (TCR) beta chain allelic exclusion occurs at the thymocyte CD4- 8- (double-negative, or DN) to CD4+ 8+ (double-positive, or DP) transition, concurrently with differentiation and cellular expansion, and is imposed by a negative feedback loop in which a product of the first rearranged TCRbeta allele arrests further recombination in the TCRbeta locus. All of the major events associated with the development of DP cells can be induced by the introduction of TCRbeta or activated Lck transgenes. Here, we present evidence that the signaling pathways that promote thymocyte differentiation and expansion of RAG-deficient DN cells but not those that suppress rearrangements of endogenous TCRbeta genes in normal DN cells are engaged by activated Ras. We propose that TCRbeta allelic exclusion is mediated by effector pathways downstream of Lck but independent of Ras.

Deletion of the RAG2 C terminus leads to impaired lymphoid development in mice.

The recombination-activating gene (RAG)1 and RAG2 proteins comprise the lymphocyte-specific components of the V(D)J recombinase and are required for the assembly of antigen-receptor variable-region genes. A mutant truncated RAG2 protein (“core” RAG2) lacking the C-terminal 144 amino acids, together with core RAG1, is able to mediate the basic biochemical steps required for V(D)J recombination in vitro and in transfected cell lines. Here we examine the effect of replacing the endogenous RAG2 locus in mice with core RAG2. These mice generate substantial numbers of B and T cells, demonstrating that the core RAG2 protein retains significant in vivo function. However, core RAG2 mice display a reduction in the total number of B and T cells, reflecting impaired lymphocyte development at the progenitor stage associated with reduced chromosomal V(D)J recombination. We discuss potential roles of the RAG2 C terminus in mediating rearrangement of endogenous antigen-receptor loci.

Impaired V(D)J Recombination and Lymphocyte Development in Core RAG1-expressing Mice

RAG1 and RAG2 are the lymphocyte-specific components of the V(D)J recombinase. In vitro analyses of RAG function have relied on soluble, highly truncated “core” RAG proteins. To identify potential functions for noncore regions and assess functionality of core RAG1 in vivo, we generated core RAG1 knockin (RAG1c/c) mice. Significant B and T cell numbers are generated in RAG1c/c mice, showing that core RAG1, despite missing ∼40% of the RAG1 sequence, retains significant in vivo function. However, lymphocyte development and the overall level of V(D)J recombination are impaired at the progenitor stage in RAG1c/c mice. Correspondingly, there are reduced numbers of peripheral RAG1c/c B and T lymphocytes. Whereas normal B lymphocytes undergo rearrangement of both JH loci, substantial levels of germline JH loci persist in mature B cells of RAG1c/c mice, demonstrating that DJH rearrangement on both IgH alleles is not required for developmental progression to the stage of VH to DJH recombination. Whereas VH to DJH rearrangements occur, albeit at reduced levels, on the nonselected alleles of RAG1c/c B cells that have undergone D to JH rearrangements, we do not detect VH to DH rearrangements in RAG1c/c B cells that retain germline JH alleles. We discuss the potential implications of these findings for noncore RAG1 functions and for the ordered assembly of VH, DH, and JH segments.

Developmental Regulation of TCRδ Locus Accessibility and Expression by the TCRδ Enhancer

Abstract We have used gene-targeted mutation to assess the role of the T cell receptor δ (TCRδ) enhancer (Eδ) in αβ and γδ T cell development. Mice lacking Eδ exhibited no defects in αβ T cell development but had a severe reduction in thymic and peripheral γδ T cells and decreased VDJδ rearrangements. Simultaneous deletion of both Eδ and the TCRα enhancer (Eα) demonstrated that residual TCRδ rearrangements were not driven by Eα, implicating additional elements in TCRδ locus accessibility. Surprisingly, while deletion of Eδ severely impaired germline TCRδ expression in double-negative thymocytes, absence of Eδ did not affect expression of mature δ transcripts in γδ T cells. We conclude that Eδ has an important role in TCRδ locus regulation at early, but not late, stages of γδ T cell development.

Accessibility control of variable region gene assembly during T‐cell development

Summary: T‐cell development is a complex and ordered process that is regulated in part by the progressive assembly and expression of antigen receptor genes. T cells can be divided into two lineages based on expression of either an αβ or γδ T‐cell antigen receptor (TCR), The genes that encode the TCR β and y chains lie in distinct loci, whereas the genes that encode the TCR a and S chains he in a single locos (TCR α/δ locus). Assembly of TCR variable region genes is mediated by a site‐specific recombination process that is common among all lymphocytes. Despite the common nature of this process, recombination of TCR genes is tightly regulated within the context of the developing T cell. TCR β, γ and δ variable region genes are assembled prior to TCR α variable region genes. Furthermore, assembly of TCR β variable region genes is regulated within the context of allelic exclusion. The regulation of rearrangement arid expression of genes within the TCR α/δ locus presents a complicated problem. TCR α and δ variable region genes are assembled at different stages of T‐cell development, and fully assembled TCR α and δ variable region genes must be expressed in distinct hneages of T cells, αβ and γδ. respectively We have developed several experimental approaches lo assess the role of cis‐acting elements in regulating recombination and expression of TCR genes. Here we describe these approaches and discuss our analyses of the regulation of accessibility of the TCR β and TCR α/δ foci during T‐cell development.

T cell receptor (TCR) α/δ locus enhancer identity and position are critical for the assembly of TCR δ and α variable region genes

T cell receptor (TCR) δ and α variable region genes are assembled from germ-line gene segments located in a single chromosomal locus in which TCRδ segments are situated between TCRα segments. The TCRα enhancer (Eα) located at the 3′ end of the TCRα/δ locus functions over a long chromosomal distance to promote TCRα rearrangement and maximal TCRδ expression; whereas the TCRδ enhancer (Eδ) is located among the TCRδ segments and functions with additional element(s) to mediate TCRδ rearrangement. We used gene-targeted mutation to evaluate whether the identity of Eα and the position of Eδ are critical for the developmental stage-specific assembly of TCR δ and α variable region genes. Specific replacement of Eα with Eδ, the core Eα element (EαC), or the Ig heavy chain intronic enhancer (iEμ), all of which promote accessibility in the context of transgenic V(D)J recombination substrates, did not promote a significant level of TCRα rearrangement beyond that observed in the absence of Eα. Therefore, the identity and full complement of Eα-binding sites are critical for promoting accessibility within the TCRα locus. In the absence of the endogenous Eδ element, specific replacement of Eα with Eδ also did not promote TCRδ rearrangement. However, deletion of intervening TCRα/δ locus sequences to restore the inserted Eδ to its normal chromosomal position relative to 5′ sequences rescued TCRδ rearrangement. Therefore, unlike Eα, Eδ lacks ability to function over the large intervening TCRα locus and/or Eδ function requires proximity to additional upstream element(s) to promote TCRδ accessibility.