Faculty Bibliography

Immunocompetent mice are nonpermissive for the development and maturation of the human filarial parasite, Brugia malayi. We and others have shown that the absence of T-lymphocytes, alone or in combination with B-lymphocytes, renders mice permissive to infection. In a previous study, we showed that mice lacking CD8+ T-lymphocytes are also completely nonpermissive for B. malayi, indicating that CD8+ T-lymphocytes are not an obligate requirement for resistance. In the present study, we have examined the role of CD4+ T-lymphocytes in resistance to filarial infection using two experimental systems. In the first, we used an anti-CD4 monoclonal antibody to deplete CD4+ T-cells in vivo in immunocompetent BALB/c mice. In the second system, we used mutant mice in which the gene encoding the CD4 antigen had been disrupted by homologous recombination, resulting in a lack of CD4+ T-cells. Challenge of either the anti-CD4 antibody depleted BALB/c mice or CD4 knockout mice with B. malayi infective-stage larvae demonstrated that mice lacking CD4+ T-lymphocytes were resistant to infection. These data indicate that CD4+ T-cells are not an obligate requirement for murine resistance to B. malayi

Thymocytes co-expressing the CD4 and CD8 co-receptors differentiate into mature T cells that express either CD4 or CD8 and have helper or cytotoxic functions, respectively. Recent studies indicate that commitment to the CD4+ or CD8+ lineages occurs stochastically, but retention of the appropriate co-receptor is required to complete development

To complete their maturation, most immature thymocytes depend on the simultaneous engagement of their antigen receptor [alpha beta T cell receptor (TCR)] and their CD4 or CD8 coreceptors with major histocompatibility complex class II or I ligands, respectively. However, a normal subset of mature alpha beta TCR+ thymocytes did not follow these rules. These thymocytes expressed NK1.1 and a restricted set of alpha beta TCRs that are intrinsically class I-reactive because their positive selection was class I-dependent but CD8-independent. These cells were CD4+ and CD4-8- but never CD8+, because the presence of CD8 caused negative selection. Thus, neither CD4 nor CD8 contributes signals that direct their maturation into the CD4+ and CD4-8- lineages

Despite the differences in the antigens that they recognize and in the effector functions they carry out, B and T lymphocytes utilize remarkably similar signal transduction components to initiate responses. They both use oligomeric receptors that contain distinct recognition and signal transduction subunits. Antigen receptors on both cells interact with at least two distinct families of PTKs via common sequence motifs, ARAMs, in the cytoplasmic tails of their invariant chains, which have likely evolved from a common evolutionary precursor. Coreceptors appear to serve to increase the sensitivity of both of these receptor systems through events that influence ligand binding and signal transduction. The critical role of tyrosine phosphorylation of downstream signaling components, such as phospholipase C, is the net result of changes in the balance of the action of antigen receptor-regulated PTKs and PTPases. The identification of downstream effectors, including calcineurin and Ras, that regulate cellular responses, such as lymphokine gene expression, promises the future possibility of connecting the complex pathway from the plasma membrane to the nucleus in lymphocytes. Insight gained from studies of the signaling pathways downstream of TCR and BCR stimulation is likely to contribute significantly to future understanding of mechanisms responsible for lymphocyte differentiation and for the discrimination of self from nonself in developing and mature cells

Several domains of CD4 have been suggested to play a critical role in events that follow its binding to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120-gp41). It has been reported previously that cells expressing a chimeric molecule consisting of the first 177 residues of human CD4 attached to residues from the hinge, transmembrane, and cytoplasmic domains of human CD8 did not form syncytia with HIV-1-infected cells (L. Poulin, L.A. Evans, S. Tang, A. Barboza, H. Legg, D.R. Littman, and J.A. Levy, J. Virol. 65: 4893-4901, 1991). In contrast, we found that the hybrid CD4.CD8 molecule expressed in human cells did render them susceptible to fusion with cells expressing HIV-1IIIB or HIV-1RF envelope glycoproteins encoded by vaccinia virus recombinants, but only after long lag times. The lag time of membrane fusion mediated by the hybrid CD4.CD8 molecule was fivefold longer than that for the wild-type CD4 molecule. However, the rate of binding to and the affinity of soluble gp120 for membrane-associated CD4.CD8 were the same as for CD4. Both molecules were laterally mobile, as determined by patching experiments. Coexpression of the CD4.CD8 chimera with wild-type CD4 did not lead to interference in fusion but had an additive effect. Therefore, the proximal membrane domains of CD4 play an important role in determining the kinetics of postbinding events leading to membrane fusion. We hypothesize that the long lag time is due to the inability of the CD4.CD8-gp120-gp41 complex to undergo the rapid conformational changes which occur during the fusion mediated by wild-type CD4

The ectodomains of the T cell surface glycoproteins CD4 and CD8 bind to membrane-proximal domains of MHC class II and class I molecules, respectively, while both cytoplasmic domains interact with the protein tyrosine kinase (PTK) p56lck (lck) through a shared cysteine-containing motif. Function of CD4 and CD8 requires their binding to the same MHC molecule as that recognized by the T cell antigen receptor (TCR). In vitro studies indicate that CD4-associated lck functions even in the absence of kinase activity. In vivo experiments show that, whereas helper T cell development is impaired in CD4-deficient mice, high level expression of a transgenic CD4 that cannot bind lck rescues development of this T cell subset. These studies suggest that CD4 is an adhesion molecule whose localization is regulated through protein-protein interactions of the associated PTK and whose function is to increase the stability of the TCR signalling complex by binding to the relevant MHC. The function of CD4 in development has been further studied in the context of how double positive (CD4+CD8+) thymocytes mature into either CD4+ T cells with helper function and TCR specificity for class II or into CD8+ T cells with cytotoxic function and specificity for class I. Studies using CD4-transgenic mice indicate that development of single positive T cells involves stochastic downregulation of either CD4 or CD8, coupled to activation of a cytotoxic or helper program, respectively, and subsequent selection based on the ability of the TCR and remaining co-receptor to engage the same MHC molecule

Expression of either the CD4 or CD8 glycoproteins discriminates two functionally distinct lineages of T lymphocytes. A null mutation in the gene encoding CD4 impairs the development of the helper cell lineage that is normally defined by CD4 expression. Infection of CD4-null mice with Leishmania has revealed a population of functional helper T cells that develops despite the absence of CD4. These CD8- alpha beta T cell receptor+ T cells are major histocompatibility complex class II-restricted and produce interferon-gamma when challenged with parasite antigens. These results indicate that T lymphocyte lineage commitment and peripheral function need not depend on the function of CD4

A T-lymphocyte-specific enhancer located 13 kb upstream of the murine CD4 gene was recently shown to be required for the developmentally regulated expression of CD4. We have previously identified three nuclear protein binding sites in this enhancer; one of these sites, CD4-3, is essential for expression and contains two E-box core motifs (CANNTG) adjacent to each other in the sequence TAACAGGTGTCAGCTGGT. In electrophoretic mobility shift assays using the CD4-3 oligonucleotide as a probe, three nuclear protein complexes, termed CD4-3A, -B, and -C, were detected with nuclear extracts from T-cell lines. CD4-3A, which involves nuclear protein binding to the 5' E-box, was detected only with nuclear extracts from lymphoid cells. Specific antisera were used to show that the CD4-3A complex contains a heterodimer or heterooligomer of basic helix-loop-helix transcriptional factors, E12 or a related factor and HEB, which is expressed predominantly in thymus. Consistent with this finding, in vitro-translated E12 and HEB proteins, as homodimers or heterodimers, bound preferentially to the 5' E-box. Point mutations in the 5' E-box, but not in the 3' E-box, abolished CD4 enhancer activity. Furthermore, overexpression of Id, a protein that forms inactive heterodimers with E12/E47, blocked CD4 enhancer activity in T cells. These results suggest that a heterodimer composed of HEB and E12 or a closely related protein plays a critical role in CD4 enhancer function by interacting with the 5' E-box motif of the CD4-3 site in vivo