Dennis Y. Loh

Mice Lacking p27Kip1 Display Increased Body Size, Multiple Organ Hyperplasia, Retinal Dysplasia, and Pituitary Tumors

SUMMARY Mice lacking p27(Kip1) have been created by gene targeting in embryonic stem cells. These mice are larger than the control animals, with thymus, pituitary, and adrenal glands and gonadal organs exhibiting striking enlargement. CDK2 activity is elevated about 10-fold in p27(-/-) thymocytes. Development of ovarian follicles seems to be impaired, resulting in female sterility. Similar to mice with the Rb mutation, the p27(-/-) mice often develop pituitary tumors spontaneously. The retinas of the mutant mice show a disturbed organization of the normal cellular layer pattern. These findings indicate that p27(Kip1) acts to regulate the growth of a variety of cells. Unexpectedly, the cell cycle arrest mediated by TGFbeta, rapamycin, or contact inhibition remained intact in p27(-/-) cells, suggesting that p27(Kip1) is not required in these pathways.

Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo

An adoptive transfer system was used to monitor physically the behavior of a trace population of TCR transgenic T cells in vivo. After subcutaneous injection of antigen in adjuvant, the antigen-specific cells accumulated first in the paracortical region of the draining lymph nodes, proliferated there for several days, and then moved into lymph node follicles, where they accounted for most of the T cells. They then disappeared slowly from the draining nodes, and the remaining cells were hypersensitive to antigenic stimulation in vitro. In contrast, when the antigen was introduced into the blood, the antigen-specific cells rapidly accumulated in the paracortical regions of all lymph nodes, proliferated there for a short time, but never entered follicles. Most of the cells then rapidly disappeared, leaving behind a population that was hyporesponsive to antigenic stimulation. These results provide a physical basis for the classical finding that antigen-specific memory and tolerance can be influenced by the form of antigen administration.

Expression of the p56 lck Y505F Mutation in CD45-Deficient Mice Rescues Thymocyte Development

ABSTRACT Mice deficient in the transmembrane protein tyrosine phosphatase CD45 exhibit a block in thymocyte development. To determine whether the block in thymocyte development was due to the inability to dephosphorylate the inhibitory phosphorylation site (Y505) in p56 lck (Lck), we generated CD45-deficient mice that express transgenes for the Lck Y505F mutation and the DO11.10 T-cell antigen receptor (TCR). CD4 single-positive T cells developed and accumulated in the periphery. Treatment with antigen resulted in thymocyte apoptosis and the loss of transgenic-TCR-bearing cells. Peripheral CD45-deficient T cells from the mice expressing both transgenes responded to antigen by increasing CD69 expression, interleukin-2 production, and proliferation. These results indicate that thymocyte development requires the dephosphorylation of the inhibitory site in Lck by CD45.

Functional analysis of the murine T-cell receptor beta enhancer and characteristics of its DNA-binding proteins.

The minimal T-cell receptor (TCR) beta-chain (TCR beta) enhancer has been identified by transfection into lymphoid cells. The minimal enhancer was active in T cells and in some B-lineage cells. When a larger fragment containing the minimal enhancer was used, its activity was apparent only in T cells. Studies with phytohemagglutinin and 4 beta-phorbol-12,13-dibutyrate revealed that the enhancer activity was increased by these agents. By a combination of DNase I footprinting, gel mobility shift assay, and methylation interference analysis, seven different motifs were identified within the minimal enhancer. Furthermore, competition experiments showed that some of these elements bound identical or similar factors that are known to bind to the TCR V beta promoter decamer or to the immunoglobulin enhancer kappa E2 or muEBP-E motif. These shared motifs may be important in the differential gene activity among the different lymphoid subsets.

Identification of T-cell receptor Vβ deletion mutant mouse strain AU/ssJ (H-2q) which is resistant to collagen-induced arthritis

Our laboratory is involved in investigating the role of T-cell receptor (Tcr) in collagen-induced arthritis (CIA). During these studies we found AU/ssJ (H-2q) mice to be resistant to CIA like SWR (H-2q), as compared with other H-2q strains with wild-type Tcr like DBA/1 and B 10. Q. Upon screening with monoclonal antibodies F23.1 and KJ23a, AU/ssJ was found to be F23.1 negative (Vβ8 Tcr negative) and KJ23a positive (Vβ17a Tcr positive). Southern blot analysis on liver DNA using specific Tcr-Vβ probes confirmed the deletion of Vβ8 gene family and also showed that AU/ssJ mice have deletions of Vβ9, Vβ13, Vβ12, and Vβ11 genes of Tcr. Further, these mice show a restriction fragment length polymorphism pattern with Vβ10, Vβ6, and Vβ17 probes similar to SWR mice as compared with 1310 mice. Since SWR and AU/ssJ are from different backgrounds, these studies indicate that specific variable region β chain genes of Tcr are crucial for susceptibility to CIA in mice. Furthermore, these studies identify an additional inbred strain which has also deleted 50% of its Tcr-Vβ genes.

Transcription from a murine T-cell receptor V beta promoter depends on a conserved decamer motif similar to the cyclic AMP response element.

We identified a regulatory region of the murine V beta promoter by both in vivo and in vitro analyses. The results of transient transfection assays indicated that the dominant transcription-activating element within the V beta 8.3 promoter is the palindromic motif identified previously as the conserved V beta decamer. Elimination of this element, by linear deletion or specific mutation, reduced transcriptional activity from this promoter by 10-fold. DNase I footprinting, gel mobility shift, and methylation interference assays confirmed that the palindrome acts as the binding site of a specific nuclear factor. In particular, the V beta promoter motif functioned in vitro as a high-affinity site for a previously characterized transcription activator, ATF. A consensus cyclic AMP response element (CRE) but not a consensus AP-1 site, can substitute for the decamer in vivo. These data suggest that cyclic AMP response element-binding protein (ATF/CREB) or related proteins activate V beta transcription.

T-cell receptor VTβ genes in natural populations of mice

The composition of 15 VTβ gene subfamilies has been examined by Southern hybridization among a broad spectrum of colony bred rat and mouse species extending phylogenetically from Rattus to Mus musculus domesticus. Most mouse species contain a similar content of VTβ genes as determined by the number of hybridizing restriction fragment (RF) bands. Furthermore, the extent of restriction fragment length polymorphism (RFLP) appears to be limited. Some VTβ gene families, however, are missing from Rattus (VTβ7, VTβ12) and M. shortridgei (VTβ9, VTβ16). Extension of the VTβ survey to a panel of 38 wild-caught mice reveals that nearly a third lack specific hybridization to the VTβ5 probe. Previous reports have established that the mouse inbred strains SJL, C57BR, C57L, and SWR lack 50% of their VTβ repertoire, including VTβ5 (Behlke et al. 1985). This study demonstrates that natural populations of mice also carry a significantly reduced VTβ gene repertoire.

Transgenic Mouse Model of Lymphocyte Development

Publisher Summary This chapter discusses lymphocyte development by the transgenic mouse model. The fundamental assumption underlying the positive selection model is that there is a requirement for a specific interaction between the TCR and the self-MHC molecules displayed in the thymus. By using intra-H-2 recombinant mice in an experiment described in the chapter, the positive-selecting element was mapped for the 2C TCR as H-2K b . The origin of alloreactive cells can be any self-MHC molecule, not necessarily the allelic counterpart of the recognized allomolecule. The origin of the alloreactive T cells is nothing but self-MHC positively selected cells that happen to cross-react with allo-MHC. It was found in the experiment that there is a direct interaction between the 2C TCR and the K b molecule that leads to positive selection. The interaction of these two molecules must transduce appropriate signals in the developing thymocytes that eventually lead to the differentiation that accompanies positive selection.

Life and Death of a T Cell

Thymus-derived lymphocytes or T cells recognize nominal antigens in the context of the products of the self major histocompatibility locus (MHC)--a phenomenon called MHC restriction. In addition, T cells must be self-tolerant to antigens that are endogenous to the organism in order to avoid an autoimmune condition. How the developing T cells acquire these two characteristics is the main theme of my laboratory.

T-Cell Receptor Genes

To defend against disease, the immune system must be able to recognize a wide variety of foreign antigens with a specificity fine enough to distinguish these foreign antigens from self molecules. For T cells, the T-cell receptor (TcR), a heterodimer composed of an α and a β chain is responsible for the recognition of antigen. Each of the TcR chains is composed of two domains: a constant domain, which is membrane-proximal, and a membrane-distal variable domain, which is responsible for antigen—MHC recognition. The variable domain is encoded by up to three different types of gene segments, which are noncontiguous in the germline; variable (V), joining (J), and at least in the β chain, diversity (D) segments. During T-cell maturation, one of each type is randomly selected and brought together by somatic recombination to form a mature T-cell receptor gene. Thus, the diversity of the T-cell receptor depends on two factors: the number of different gene segments in the germline and the combinatorial diversity generated during the rearrangement process. The murine TcR Vβ gene family consists of ~16 Vβ subfamilies encoding a total of 20 Vβ gene segments (Barth et al., 1985; Behlke et al., 1985). During T-cell ontogeny, these V gene segments undergo somatic DNA rearrangements to Dβ—Jβ or -Jα gene segments, resulting in a complete V-(D)-J assembly in a fashion similar to the rearrangement process undergone by immunoglobulin genes (Kronenberg et al., 1986).