Role of chemokines in thymocyte development.

As they mature, thymocytes migrate to specific regions of the thymus, interact with different types of stromal cells, and thereby receive signals for survival, differentiation, or death. Despite its importance, the molecular control of thymocyte trafficking remains poorly understood. Chemokines and their receptors probably control the homing of T cell progenitors to the thymus, their intrathymic migration, and exit to the periphery. Certain chemokines are abundant in the thymus, and their receptors are expressed during distinct developmental stages. Below, we discuss recent studies of chemokines and their receptors in the thymus, speculating on their function in the frame work of thymocyte trafficking.

Murine CCR9, a chemokine receptor for thymus-expressed chemokine that is up-regulated following pre-TCR signaling.

Chemokines are likely to play an important role in regulating the trafficking of developing T cells within the thymus. By using anti-CD3varepsilon treatment of recombinase-activating gene 2 (Rag2-/-) mice to mimic pre-TCR signaling and drive thymocyte development to the double positive stage, we have identified murine GPR-9-6 as a chemokine receptor whose expression is strongly induced following pre-TCR signaling. GPR-9-6 mRNA is present at high levels in the thymus, and by RT-PCR analysis its expression is induced as normal thymocytes undergo the double negative to double positive transition. Furthermore we show that TECK (thymus-expressed chemokine), a chemokine produced by thymic medullary dendritic cells, is a functional ligand for GPR-9-6. TECK specifically induces a calcium flux and chemotaxis of GPR-9-6-transfected cells. In addition, TECK stimulates the migration of normal double positive thymocytes, as well as Rag2-/- thymocytes following anti-CD3varepsilon treatment. Hence, GPR-9-6 has been designated as CC chemokine receptor 9 (CCR9). Our results suggest that TECK delivers signals through CCR9 important for the navigation of developing thymocytes.

Replacement of pre-T cell receptor signaling functions by the CD4 coreceptor.

An important checkpoint in early thymocyte development ensures that only thymocytes with an in-frame T cell receptor for antigen beta (TCR-beta) gene rearrangement will continue to mature. Proper assembly of the TCR-beta chain into the pre-TCR complex delivers signals through the src-family protein tyrosine kinase p56lck that stimulate thymocyte proliferation and differentiation to the CD4+CD8+ stage. However, the biochemical mechanisms governing p56lck activation remain poorly understood. In more mature thymocytes, p56lck is associated with the cytoplasmic domain of the TCR coreceptors CD4 and CD8, and cross-linking of CD4 leads to p56lck activation. To study the effect of synchronously inducing p56lck activation in immature CD4-CD8- thymocytes, we generated mice expressing a CD4 transgene in Rag2-/- thymocytes. Remarkably, without further experimental manipulation, the CD4 transgene drives maturation of Rag2-/- thymocytes in vivo. We show that this process is dependent upon the ability of the CD4 transgene to bind Lck and on the expression of MHC class II molecules. Together these results indicate that binding of MHC class II molecules to CD4 can deliver a biologically relevant, Lck-dependent activation signal to thymocytes in the absence of the TCR-alpha or -beta chain.

A binding site for the T-cell co-receptor CD8 on the alpha 3 domain of HLA-A2.

Adhesion measurements between CD8 and 48 point mutants of HLA-A2.1 show that the CD8 alpha-chain binds to the alpha 3 domain of HLA-A2.1. Three clusters of alpha 3 residues contribute to the binding, with an exposed, negatively charged loop (residues 223-229) playing a dominant role. CD8 binding correlates with cytotoxic T-cell recognition and sensitivity to inhibition by anti-CD8 antibodies. Impaired alloreactive T-cell recognition of an HLA-A2.1 mutant with reduced affinity for CD8 is not restored by functional CD8 binding sites on an antigenically irrelevant class I molecule. Therefore, complexes of CD8 and the T-cell receptor bound to the same class I major histocompatibility complex molecule seem to be necessary for T-cell activation.

Alternatively spliced mRNA encodes a secreted form of human CD8 alpha. Characterization of the human CD8 alpha gene.

We have determined the organization and nucleotide sequence of the gene encoding the human T cell surface glycoprotein CD8 alpha. This gene spans approximately 8 kb and is organized into six exons which encode separate functional domains of the protein. Exon 1 encodes the 5' untranslated region and leader peptide, exon II the Ig V-like region, exon III the hinge-like region, exon IV the transmembrane domain, and exons V and VI the cytoplasmic tail. Alternative splicing that excludes nucleotide sequences from exon IV results in a transcript which encodes a secreted form of the protein. This transcript accounts for approximately 15% of the total CD8 alpha mRNA in human T cell leukemia lines and in normal human tissues. Secreted CD8 alpha protein can be detected in culture supernatants of T cell leukemia lines and PHA-stimulated PBMC by immunoprecipitation with the anti-CD8 alpha mAb OKT8 or with a polyclonal rabbit antiserum specific for the 28 amino acid cytoplasmic domain of CD8 alpha. The secreted CD8 alpha protein forms homodimers; when analyzed by SDS-PAGE, the protein migrates with an apparent molecular mass of 27 or 54 kDa under reducing or non-reducing conditions, respectively. Human secreted CD8 alpha may serve an immunoregulatory role for the interactions of T cells with their targets in vivo.

Polymorphism in the alpha 3 domain of HLA-A molecules affects binding to CD8.

Cytotoxic T lymphocytes (CTL) expressing the CD8 glycoprotein recognize peptide antigens presented by class I major histocompatibility complex (MHC) molecules. This correlation and the absence of CD8 polymorphism led to the hypothesis that CD8 binds to a conserved site of class I MHC molecules. Using a cell-cell binding assay we previously demonstrated specific interaction between human class I MHC (HLA-A,B,C) molecules and CD8. Subsequent analysis of the products of 17 HLA-A,B alleles revealed a natural polymorphism for CD8 binding in the human population. Two molecules, HLA-Aw68.1 and HLA-Aw68.2, which do not bind CD8, have a valine residue at position 245 whereas all other HLA-A,B,C molecules have alanine. Site-directed mutagenesis shows that this single substitution in the alpha 3 domain is responsible for the CD8 binding phenotype and also affects recognition by alloreactive and influenza-specific CTL. Our results indicate that CD8 binds to the alpha 3 domain of class I MHC molecules.

Cell-cell adhesion mediated by CD8 and MHC class I molecules.

CD4 and CD8 are cell-surface glycoproteins expressed on mutually exclusive subsets of peripheral T cells. T cells that express CD4 have T-cell antigen receptors that are specific for antigens presented by major histocompatibility complex class II molecules, whereas T cells that express CD8 have receptors specific for antigens presented by MHC class I molecules (reviewed in ref. 1). Based on this correlation and on the observation that anti-CD4 and anti-CD8 antibodies inhibit T-cell function, it has been suggested that CD4 and CD8 increase the avidity of T cells for their targets by binding to MHC class II or MHC class I molecules respectively. Also, CD4 and CD8 may become physically associated with the T-cell antigen receptor, forming a higher-affinity complex for antigen and MHC molecules, and could be involved in signal transduction. Cell-cell adhesion dependent CD4 and MHC II molecules has recently been demonstrated. To determine whether CD8 can interact with MHC class I molecules in the absence of the T-cell antigen receptor, we have developed a cell-cell binding assay that measures adhesion of human B-cell lines expressing MHC class I molecules to transfected cells expressing high levels of human CD8. In this system, CD8 and class I molecules mediate cell-cell adhesion, showing that CD8 directly binds to MHC class I molecules.

A second subunit of CD8 is expressed in human T cells.

The CD8 glycoprotein plays important functions in T cell development and in T cell activation. In rodents, CD8 is a heterodimer, consisting of an alpha-chain (Lyt2) and a beta-chain (Lyt3). In humans, only the alpha-chain has been detected, and it has been thought that CD8 consists of homodimers of this protein. We have isolated functional cDNA clones encoding human CD8 beta, and show that the CD8 beta protein is expressed on the surface of CD8+ human T cells. cDNA clones encoding multiple forms of the human CD8 beta-chain have been isolated and characterized. These structural variants, which are likely to arise by alternative splicing, differ in the sequences encoding the cytoplasmic domain, which can consist of 19, 30, or 52 amino acids. One of the cDNAs lacks nucleotide sequences corresponding to a hydrophobic transmembrane domain, and may encode a secreted CD8 beta protein. The protein product of the human CD8 beta gene can be detected by a recently described anti-CD8 monoclonal antibody, 597. Expression of the epitope recognized by this antibody requires co-expression of the CD8 alpha and CD8 beta gene products. About 90% of human CD8 alpha positive thymocytes and peripheral blood lymphocytes express CD8 beta at the cell surface. Expression of the CD8 beta chain is thus conserved between human and rodents, and the variant CD8 beta polypeptides may have distinct roles in T cell function and development.