Craig B. Davis

Pyk2 regulates multiple signaling events crucial for macrophage morphology and migration

The biological role of the protein tyrosine kinase, Pyk2, was explored by targeting the Pyk2 gene by homologous recombination. Pyk2–/– mice are viable and fertile, without overt impairment in development or behavior. However, the morphology and behavior of Pyk2–/– macrophages were impaired. Macrophages isolated from mutant mice failed to become polarized, to undergo membrane ruffling, and to migrate in response to chemokine stimulation. Moreover, the contractile activity in the lamellipodia of Pyk2–/– macrophages was impaired, as revealed by measuring the rearward movement toward the nucleus of fibronectin-coated beads on the lamellipodia in opposition to an immobilizing force generated by optical tweezers. Consistently, the infiltration of macrophages into a carageenan-induced inflammatory region was strongly inhibited in Pyk2–/– mice. In addition, chemokine stimulation of inositol (1, 4, 5) triphosphate production and Ca2+ release, as well as integrin-induced activation of Rho and phosphatidyl inositol 3 kinase, were compromised in Pyk2–/– macrophages. These experiments reveal a role for Pyk2 in cell signaling in macrophages essential for cell migration and function.

Evidence for a stochastic mechanism in the differentiation of mature subsets of T lymphocytes

Thymocytes that coexpress the CD4 and CD8 glycoproteins differentiate into mature CD4+ helper or CD8+ cytotoxic cells depending on whether their antigen receptors are specific for MHC class II or class I molecules, respectively. The mechanism of this decision process was investigated in mice whose T cell development was biased toward the class II-specific lineage. We found that constitutive expression of CD4 allows a developmentally arrested population of thymocytes that have mismatched class II-specific TCRs and the CD8 coreceptor to be rescued and to acquire a cytotoxic phenotype. This result is consistent with a two-step process of thymocyte maturation, in which there is stochastic down-regulation of either CD4 or CD8 and subsequent selection based on the ability of the TCR and remaining coreceptor to engage the same MHC molecule.

Thymocyte lineage commitment: is it instructed to stochastic?

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.

Signal Transduction During T Cell Development

The CD4 molecule has important roles in signaling during both T cell development and peripheral helper T cell activation1,2. The ectodomain of CD4 binds to a membrane-proximal domain of MHC class II molecules on antigen presenting cells3, while its cytoplasmic tail interacts with the cytoplasmic protein tyrosine kinase (PTK) p56 lck (lck), a member of the src family of protein tyrosine kinases (reviewed in reference 4). These properties of CD4 contribute to the simultaneous interaction of the T cell antigen receptor (TCR) and CD4 with the same MHC class II molecule and to the localization of lck to the TCR complex. It has been thought that the function of CD4 (and of the class I-specific coreceptor CD8) in signal transduction is to facilitate localization of lck within the TCR complex, thus initiating a PTK signaling cascade. However, recent studies suggest that the primary function of CD4 may be to physically stabilize the interaction of the TCR with MHC, and that a direct signaling role of the CD4-associated lck molecule may not be required4,5. In this chapter, we discuss a non-catalytic role of the CD4-associated lck molecule in facilitating T cell activation and developmental signaling.

Differential Involvement of Protein Tyrosine Kinases p56lck and p59fyn in T Cell Development

Mature CD4+ and CD8+ T cells recognize and respond to processed antigens associated with major histocompatibility complex-encoded molecules (MHC). It is during thymic development that selection processes act on immature CD4+CD8+ thymocytes to ensure the fonnation of a repertoire of functional T cells1,2. Thus, immature T cells expressing an ∝β TCR with specificity for self-peptides plus MHC class I or class II molecules are positively selected, differentiating into CD8+ or CD4+ T cells, respectively3,4,5,6. Immature thymocytes lacking or expressing a non-selectable TCR undergo programmed cell death1. Additionally, those CD4+CD8+ T cells expressing an autospecific TCR may undergo programmed cell death 7,8 (negative selection). The CD4 and CD8 molecules are thought to actively participate in these T cell repertoire selection events through their coreceptor functions9. In a coreceptor model, the TCR and appropriate coreceptor molecule recognize and interact with the same MHC molecule, with CD4 or CD8 potentiating TCR-derived intracellular signals10,1l.12. Recent reports have demonstrated that mutations affecting CD8/class I MHC interactions disrupt both positive and negative selection in the thymus13,14.

Role of CD4 and CD8 in Determining Cell Fate in the Thymus

Publisher Summary CD8 and CD4 are cell surface receptors that are expressed on non-overlapping sets of mature T cells. CD8 binds to class I major histocompatibility complex (MHC) molecules and is found on T cells that have an antigen receptor (TCR) that is restricted to class I MHC molecules. CD4 binds to class II MHC molecules and is found on class II-specific T cells. CD8 and CD4 expression correlates with T cell effector function: CD4 + class II-specific T cells are predominantly helper T cells, whereas CD8 + class I-specific T cells have cytotoxic activity. CD8 and CD4 are required for efficient recognition of peptide-MHC complexes by mature T cells. Although several T cell surface molecules are required for efficient T cell responses, CD8 and CD4 are unique because they function in concert with the TCR, by binding to the same MHC molecules. This property has led to the designation of CD8 and CD4 as coreceptors. In addition to their role in mature T cell responses to foreign peptide and MHC molecules, CD4 and CD8 are involved in the recognition of self MHC molecules, which occurs during thymic development. To survive and mature, thymocytes must recognize self MHC molecules as they develop in a process known as “positive selection.”