Knock-ins and conditional knockouts: in vivo analysis of glucocorticoid receptor regulation and function.

To determine the cellular targets for glucocorticoid (GC) action, we have generated mice in which a green fluorescent protein-glucocorticoid receptor (GFP-GR) fusion gene is knocked into the endogenous GR locus. We found that GFP-GR function is indistinguishable from endogenous GR on both a cellular and systemic level. Furthermore, the green fluorescence intensity of the GFP-GR protein is proportional to its expression, allowing quantitation of GR expression in single living cells. We initiated our analysis of GR regulation in the thymus. Using multicolor flow cytometry, we found that GR expression is uniform among embryonic thymocyte subpopulations, but gradually "matures" over a three-week period after birth. In the adult, analysis of GFP-GR expression on RAG2-/- and HY T cell receptor (TCR) transgenic genetic backgrounds, showed that GR is induced to high levels in immature CD25+ CD4- CD8- thymocytes and down-regulated by activation of the pre-TCR during positive but not negative selection. Additionally, relative GR expression is dissociated from GC-induced apoptosis in vivo. These results implicate pre-TCR signaling as a mechanism for GR down-regulation and separate receptor abundance from susceptibility to apoptosis across thymocyte populations.

Assessing a role for enhancer-blocking activity in gene regulation within the murine T-cell receptor alpha/delta locus.

Although situated close together within the T-cell receptor (TCR) alpha/delta locus, TCR delta and TCR alpha gene segments are controlled by two developmental stage-specific enhancers and are activated according to distinct developmental programmes. We previously used a stable transfection colony assay to identify an enhancer-blocking element, blocking element alpha/delta-1 (BEAD-1), between the TCR delta and alpha gene segments of the human TCR alpha/delta locus. We hypothesized that enhancer-blocking by BEAD-1 might be required to prevent the TCR delta enhancer from activating TCR alpha gene segment transcription and rearrangement at the double negative stage of thymocyte development. Here, we used a transfection approach to define partial enhancer-blocking activity in an analogous position of the murine TCR alpha/delta locus. To test the functional significance of this activity in vivo, we used gene targeting to delete the region from the endogenous locus. We found no perturbation of TCR delta and TCR alpha gene expression and rearrangement on targeted alleles, indicating that enhancer-blocking activity in this region is not required to maintain the developmentally distinct activation profiles of the two genes. We suggest that appropriate regulation may be achieved as a result of intrinsic biases in enhancer-promoter interactions or a developmental stage specificity to promoter function that is distinct from any additional specificity imposed by the enhancers themselves.

Recombination signal sequences restrict chromosomal V(D)J recombination beyond the 12/23 rule.

The genes encoding the variable regions of lymphocyte antigen receptors are assembled from variable (V), diversity (D) and joining (J) gene segments. V(D)J recombination is initiated by the recombinase activating gene (RAG)-1 and -2 proteins, which introduce DNA double-strand breaks between the V, D and J segments and their flanking recombination signal sequences (RSSs). Generally expressed DNA repair proteins then carry out the joining reaction. The conserved heptamer and nonamer sequences of the RSSs are separated by non-conserved spacers of 12 or 23 base pairs (forming 12-RSSs and 23-RSSs). The 12/23 rule, which is mediated at the level of RAG-1/2 recognition and cutting, specifies that V(D)J recombination occurs only between a gene segment flanked by a 12-RSS and one flanked by a 23-RSS. Vbeta segments are appended to DJbeta rearrangements, with little or no direct Vbeta to Jbeta joining, despite 12/23 compatibility of Vbeta 23-RSSs and Jbeta12-RSSs. Here we use embryonic stem cells and mice with a modified T-cell receptor (TCR)beta locus containing only one Dbeta (Dbeta1) gene segment and one Jbeta (Jbeta1) gene cluster to show that the 5' Dbeta1 12-RSS, but not the Jbeta1 12-RSSs, targets rearrangement of a diverse Vbeta repertoire. This targeting is precise and position-independent. This additional restriction on V(D)J recombination has important implications for the regulation of variable region gene assembly and repertoire development.

Mechanisms that direct ordered assembly of T cell receptor beta locus V, D, and J gene segments.

T cell receptor (TCR) beta variable region genes are assembled in progenitor T cells from germ-line Vbeta, Dbeta, and Jbeta segments via an ordered two-step process in which Dbeta to Jbeta rearrangements occur on both alleles before appendage of a Vbeta to a preexisting DJbeta complex. Direct joining of Vbeta segments to nonrearranged Dbeta or Jbeta segments, while compatible with known restrictions on the V(D)J recombination mechanism, are infrequent within the endogenous TCRbeta locus. We have analyzed mechanisms that mediate ordered Vbeta, Dbeta, and Jbeta assembly via an approach in which TCRbeta minilocus recombination substrates were introduced into embryonic stem cells and then analyzed for rearrangement in normal thymocytes by recombinase-activating gene 2-deficient blastocyst complementation. These analyses demonstrated that Vbeta segments are preferentially targeted for rearrangement to Dbeta as opposed to Jbeta segments. In addition, we further demonstrated that Vbeta segments can be appended to nonrearranged endogenous Dbeta segments in which we have eliminated the ability of Dbeta segments to join to Jbeta segments. Our findings are discussed in the context of the mechanisms that regulate the ordered assembly and utilization of V, D, and J segments.

Cloning and functional characterization of the early-lymphocyte-specific Pb99 gene.

The Pb99 gene is specifically expressed in pre-B cells and thymocytes and not in mature B and T cells or nonlymphoid tissues, implying that it may function in early lymphoid development. We have previously described the cloning of an incomplete cDNA for Pb99. Here we report the isolation of full-length cDNAs and genomic clones for the murine Pb99 gene and the mapping of its location to mouse chromosome 8. Sequence analyses of different Pb99 cDNA clones suggest that there may be at least three forms of the Pb99 protein generated by differential processing of the Pb99 transcript. The cDNA with the longest open reading frame encodes a putative protein that has seven hydrophobic domains similar to those of seven membrane-spanning proteins, such as the classical G protein-coupled receptors. To directly address the role of the Pb99 protein in lymphoid development, Pb99-deficient mice were generated by gene targeting, and lymphocyte development in these mice was analyzed.

Requirement for B cell linker protein (BLNK) in B cell development.

Linker proteins function as molecular scaffolds to localize enzymes with substrates. In B cells, B cell linker protein (BLNK) links the B cell receptor (BCR)-activated Syk kinase to the phosphoinositide and mitogen-activated kinase pathways. To examine the in vivo role of BLNK, mice deficient in BLNK were generated. B cell development in BLNK-/- mice was blocked at the transition from B220+CD43+ progenitor B to B220+CD43- precursor B cells. Only a small percentage of immunoglobulin M++ (IgM++), but not mature IgMloIgDhi, B cells were detected in the periphery. Hence, BLNK is an essential component of BCR signaling pathways and is required to promote B cell development.

A developmental switch from TCR delta enhancer to TCR alpha enhancer function during thymocyte maturation.

V(D)J recombination and transcription within the TCR alpha/delta locus are regulated by three characterized cis-acting elements: the TCR delta enhancer (Edelta), TCR alpha enhancer (Ealpha), and T early alpha (TEA) promoter. Analysis of enhancer and promoter occupancy and function in developing thymocytes in vivo indicates Edelta and Ealpha to be developmental-stage-specific enhancers, with Edelta "on" and Ealpha "off" in double-negative III thymocytes and Edelta "off" and Ealpha "on" in double-positive thymocytes. Edelta downregulation reflects a loss of occupancy. Surprisingly, Ealpha and TEA are extensively occupied even prior to activation. TCR delta downregulation in double-positive thymocytes depends on two events, Edelta inactivation and removal of TCR delta from the influence of Ealpha by chromosomal excision.

Developmental regulation of TCR delta locus accessibility and expression by the TCR delta enhancer.

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

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.

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

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+/-SE (9.7+/-2.4) of worms recovered from infected mice at 2 weeks p.i. was not significantly different from that recovered at 3 weeks p.i. (6.5+/-2.2). 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.

Recombination and transcription of the endogenous Ig heavy chain locus is effected by the Ig heavy chain intronic enhancer core region in the absence of the matrix attachment regions.

The intronic Ig heavy chain (IgH) enhancer, which consists of the core enhancer flanked by 5' and 3' matrix attachment regions, has been implicated in control of IgH locus recombination and transcription. To elucidate the regulatory functions of the core enhancer and its associated matrix attachment regions in the endogenous IgH locus, we have introduced targeted deletions of these elements, both individually and in combination, into an IgHa/b-heterozygous embryonic stem cell line. These embryonic stem cells were used to generate chimeric mice by recombination activating gene-2 (Rag-2)-deficient blastocyst complementation, and the effects of the introduced mutations were assayed in mutant B cells. We find that the core enhancer is necessary and sufficient to promote normal variable (V), diversity (D), and joining (J) segment recombination in developing B lineage cells and IgH locus transcription in mature B cells. Surprisingly, the 5' and 3' matrix attachment regions were dispensable for these processes.

Accessibility control of variable region gene assembly during T-cell development.

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 alpha beta or gamma delta T-cell antigen receptor (TCR). The genes that encode the TCR beta and gamma chains lie in distinct loci, whereas the genes that encode the TCR alpha and delta chains lie in a single locus (TCR alpha/delta 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 beta, gamma and delta variable region genes are assembled prior to TCR alpha variable region genes. Furthermore, assembly of TCR beta variable region genes is regulated within the context of allelic exclusion. The regulation of rearrangement and expression of genes within the TCR alpha/delta locus presents a complicated problem. TCR alpha and delta variable region genes are assembled at different stages of T-cell development, and fully assembled TCR alpha and delta variable region genes must be expressed in distinct lineages of T cells, alpha beta and gamma delta, respectively. We have developed several experimental approaches to 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 beta and TCR alpha/delta loci during T-cell development.

Epstein-Barr virus in Hodgkin's disease: correlation of risk factors and disease characteristics with molecular evidence of viral infection.

Risk factors suggestive of relatively late exposure to EBV have been consistently associated with Hodgkin's disease (HD) in younger adults. In addition, evidence of EBV infection has been found in the Reed-Sternberg cells themselves in about one-third to one-half of all HD cases. However, no study yet published has correlated these childhood social environment risk factors with the presence of EBV in Hodgkin's tumor cells. We examined whether EBV-positive HD occurs in those patients whose childhood environment would predispose them to relatively late exposure to EBV. The study population consisted of 102 cases of mixed cellularity (MC; n = 25) or nodular sclerosing (n = 77) HD. Samples that tested positive for either EBV-encoded RNA or latent membrane protein or both were considered EBV-positive. Of the 102 cases, 83 completed a questionnaire regarding childhood social environment. The association with EBV-positivity was estimated by the odds ratio (OR) with 95% confidence intervals (CI). Twenty-two percent of the cases were EBV-positive. These cases were more likely to be MC (OR, 6.2; CI, 2.3-16.3) and male (OR, 3.4; CI, 1.3-9.0). History of infectious mononucleosis (IM) was not predictive of EBV-positivity, with only 3 of 14 such patients being EBV-positive (P = 0.82). Contrary to our hypothesis, no association between EBV and childhood environment risk factors was identified. The association of EBV with MC histology and male gender agrees with previous reports. The most intriguing finding was the dissociation between IM history and EBV-positivity, in that almost all of the cases with a history of IM were EBV-negative.

Assembly of productive T cell receptor delta variable region genes exhibits allelic inclusion.

The generation of a productive "in-frame" T cell receptor beta (TCR beta), immunoglobulin (Ig) heavy (H) or Ig light (L) chain variable region gene can result in the cessation of rearrangement of the alternate allele, a process referred to as allelic exclusion. This process ensures that most alphabeta T cells express a single TCR beta chain and most B cells express single IgH and IgL chains. Assembly of TCR alpha and TCR gamma chain variable region genes exhibit allelic inclusion and alphabeta and gammadelta T cells can express two TCR alpha or TCR gamma chains, respectively. However, it was not known whether assembly of TCR delta variable regions genes is regulated in the context of allelic exclusion. To address this issue, we have analyzed TCR delta rearrangements in a panel of mouse splenic gammadelta T cell hybridomas. We find that, similar to TCR alpha and gamma variable region genes, assembly of TCR delta variable region genes exhibits properties of allelic inclusion. These findings are discussed in the context of gammadelta T cell development and regulation of rearrangement of TCR delta genes.

Function of the TCR alpha enhancer in alphabeta and gammadelta T cells.

We have used gene targeted mutational approaches to assess the role of the T cell receptor alpha (TCR alpha) enhancer (E alpha) in the control of TCR alpha and TCR delta gene rearrangement and expression. We show that E alpha functions in cis to promote V alpha to J alpha rearrangement across the entire J alpha locus, a distance of greater than 70 kb. We also show that E alpha is required for normal alphabeta T cell development; in this lineage, E alpha is required for germline J alpha expression, for normal expression levels of rearranged V alpha J alpha genes, and for expression of a diverse V alpha repertoire. In gamma delta T cells, E alpha is not required for VdeltaDJdelta rearrangement, but, surprisingly, is required for normal expression levels of mature VdeltaDJdelta transcripts and for expression of germline J alpha transcripts. Our findings imply that E alpha function is not limited to the TCR alpha components of the TCRalpha/delta locus or to the alpha beta lineage; rather, E alpha function is important in both alphabeta and gammadelta lineage T cells.

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.

Disruption of the CD4-p56lck complex is required for rapid internalization of CD4.

CD4 is a cell surface glycoprotein expressed by a subset of T lymphocytes and functions to enhance T-cell activation. CD4 is noncovalently associated via the cytoplasmic domain with the protein-tyrosine kinase p56lck, a member of the src protein-tyrosine kinase family. Upon activation of protein kinase C by phorbol ester, CD4 is phosphorylated on cytoplasmic serine residues and internalized from the cell surface, and disruption of the CD4-p56lck complex occurs. The exact relationship between these events is likely to be functionally significant, as cytoplasmic-domain serine phosphorylation and internalization have been shown to regulate the function of receptors that possess intrinsic protein-tyrosine kinase activity. Here we demonstrate that p56lck slows the rate of phorbol 12-myristate 13-acetate-induced internalization of CD4 in a manner that depends on a physical association between p56lck and CD4. This decreased rate is due at least in part to a requirement for disruption of the CD4-p56lck complex prior to internalization of CD4. Furthermore, disruption of the CD4-p56lck complex appears to depend on the integrity of the cytoplasmic-domain serine at position 408, probably due to a requirement for phosphorylation.

Glycolipid-anchored form of CD4 increases intercellular adhesion but is unable to enhance T cell activation.

CD4 functions to enhance T cell activation by increasing intercellular adhesion and/or by transduction of an intracellular signal. To study the role of human CD4 in T cell activation we have used a murine T cell hybridoma, By 155.16, which produces IL-2 when stimulated by HLA-DR-bearing cells. Previously, we have shown that expression of human CD4 by this hybridoma enhances its ability to produce IL-2 in response to HLA-DR-bearing cells. Furthermore, deletion of the majority of the cytoplasmic domain renders CD4 less efficient at enhancing IL-2 production. We describe studies of a glycolipid-anchored mutant of the CD4 molecule, CD4PI. This mutant is composed of the entire extracellular domain of CD4 anchored to the outlet leaflet of the membrane via a covalent bond to glycosylphosphatidylinositol and, therefore, has no transmembrane or cytoplasmic domains. When expressed in By155.16, CD4PI shows no defect in its ability to increase intercellular adhesion but is unable to augment IL-2 production. These results clearly demonstrate that CD4 enhances T cell activation by mechanisms other than increasing intercellular adhesion.

Human immunodeficiency virus infection is efficiently mediated by a glycolipid-anchored form of CD4.

Two broad roles have been revealed for the CD4 molecule. It serves as a receptor for both class II major histocompatibility complex molecules and human immunodeficiency virus (HIV). Upon binding class II major histocompatibility molecules, CD4 functions to enhance T-cell activation. By binding to CD4, HIV gains entry into the cell. We have used a chimeric molecule of CD4 and lymphocyte function-associated antigen 3 (LFA-3), CD4PI, which lacks a membrane-spanning domain and is instead anchored in the membrane by linkage to glycosyl-phosphatidylinositol. To further define the structural attributes of viral receptors, and specifically those of CD4 required for HIV infection, we have expressed CD4PI and CD4 in a human T-cell line, HSB-2. We find that CD4PI is able to mediate infection of these cells by HIV with similar, if not greater efficiency, compared with wild-type CD4. Thus the membrane-spanning region of CD4 is not required for HIV infection, and a lipid-anchored protein can serve as a viral receptor.

The cytoplasmic domain of CD4 is required for stable association with the lymphocyte-specific tyrosine protein kinase p56lck.

The CD4 T cell surface molecule binds MHC class II determinants expressed on antigen-presenting cells. CD4 is thought to enhance T cell activation by serving as an adhesion molecule as well as possibly by transducing an independent intracellular signal during the process of antigen stimulation. The recent observation that CD4 is physically associated with the Src-related tyrosine protein kinase p56lck suggests that tyrosine phosphorylation might be involved in these CD4 "signaling" events. The results presented in this report demonstrate that deletion of the cytoplasmic domain of CD4 significantly diminishes its ability to stably associate with p56lck. This observation provides a biochemical basis for the decreased ability of this mutant CD4 molecule to enhance T cell activation during suboptimal antigen stimulation.