Barry P. Sleckman

Function of the TCRα Enhancer in αβ and γδ T Cells

Abstract We have used gene targeted mutational approaches to assess the role of the T cell receptor α (TCRα) enhancer (Eα) in the control of TCRα and TCRδ gene rearrangement and expression. We show that Eα functions in cis to promote Vα to Jα rearrangement across the entire Jα locus, a distance of greater than 70 kb. We also show that Eα is required for normal αβ T cell development; in this lineage, Eα is required for germline Jα expression, for normal expression levels of rearranged VαJα genes, and for expression of a diverse Vα repertoire. In γδ T cells, Eα is not required for VδDJδ rearrangement, but, surprisingly, is required for normal expression levels of mature VδDJδ transcripts and for expression of germline Jα transcripts. Our findings imply that Eα function is not limited to the TCRα components of the TCRα/δ locus or to the αβ lineage; rather, Eα function is important in both αβ and γδ lineage T cells.

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.

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.

Host-parasite relationships between Echinostoma caproni and RAG-2-deficient mice

Abstract The RAG-2-deficient mouse, a strain of genetically altered mice lacking B- and T-lymphocytes, was used as a host for Echinostoma caproni. In all, 12 male RAG mice were exposed to 25 cysts each, and 12 served as uninfected controls. Mice were necropsied at 2 and 3 weeks postinfection (p.i.). The mean number u2009±SE (9.7u2009±u20092.4) of worms recovered from infected mice at 2 weeks p.i. was not significantly different from that recovered at 3 weeks p.i. (6.5u2009±u20092.2). The intestinal circumference of infected RAG mice was significantly greater than that of the controls at 2 and 3 weeks p.i. A significant goblet cell hyperplasia occurred at 2 weeks p.i., but the response was not effective in eliminating worms from the RAG mice. The effect of a high cyst burden was examined by exposure of 8 RAG and 8 ICR mice to 100 cysts each. The body length and area and the oral sucker area of worms grown in RAG mice were significantly greater than those of worms grown in ICR mice. Worm recovery at up to 3 months p.i. was examined in RAG mice exposed to 25 cysts and necropsied every 2 weeks p.i. The mean worm recovery recorded at 2 weeks p.i. was significantly greater than that noted at 12 weeks p.i., at which time worm rejection from the RAG mouse host first occurred. The RAG mouse is a useful host for studies on E. caproni in a murine host that lacks B- and T-lymphocytes.

Analysis of the complex genomic structure of Bcl-x and its relationship to Bcl-xγ expression after CD28-dependent costimulation

The Bcl-x(gamma) cytosolic protein is essential for costimulatory activity after CD3/CD28 coligation. Here we delineate the Bcl-x(gamma)/Bcl-x genomic organization and the molecular mechanism that allows expression. We show that exon 4 of the Bcl-x gene encodes the unique C-terminal end of the Bcl-x(gamma) molecule while exons 5, 6, 7 and 8 are differentially transcribed to yield three alternative Bcl-x(gamma) 3 untranslated regions (UTR). CD28-dependent signals may increase levels of Bcl-x(gamma) protein through induction of an alternatively-spliced Bcl-x(gamma) 3 UTR that contains stem loop structures that stabilize Bcl-x(gamma) RNA. The ability receptor-induced signals to regulate the splicing pattern of the complex Bcl-x gene may allow T-cells to respond appropriately to antigenic stimuli.

Functional Analysis of Cd2, cd4, and cd8 in t‐Cell Activationa

T cells may be activated by the antigen-specific T-cell receptor (TCR)-CD3 complex upon interaction with allogeneic major histocompatibility complex (MHC) antigens or foreign antigens in association with syngeneic MHC molecules. In addition t o the TCR. several other cell-surface molecules are important for T-cell adhesion and activation. These molecules include lymphocyte-function associated antigen (LFA)-I. CD2 (TII, Leu 5, LFA-2)CD4 (T4, Leu 3 in the human, L3T4 in the mouse), and CD8 (T8, Leu 2 in the human, Lyt-2 in the mouse) on the T cell, and LFA-3 on the target or stimulator cell (TABLE 1) . LFA-1 is a heterodimer involved in antigen-independent conjugate formation between the T cell and target or stimulator cell, and may play a wider role in cell adhesion of lymphoid cells.’.? CD2 is a receptor on T cells whose natural ligand on the stimulator cell is LFA-3, a broadly distributed glycoprotein.1-4 Although the CD2/ LFA-3 interaction clearly functions in cell-cell adhesion, recent data suggest that it may play a role in T-cell activation. Functional studies and monoclonal antibody (mAb)-inhibition data indicate that the CD4 and CD8 glycoproteins appear to interact with nonpolymorphic regions of MHC class I1 and class I molecules. respectively. and may serve to enhance antigen-specific recognition.’ Much of our understanding about the cell-surface antigens CD2, CD4, and CD8 has been derived from the analysis of the functional effect of mAb directed against these molecules. In the last several years, it has become clear that the