Michael A. Bowen

CD5 Negatively Regulates the T-Cell Antigen Receptor Signal Transduction Pathway: Involvement of SH2-Containing Phosphotyrosine Phosphatase SHP-1

ABSTRACT The negative regulation of T- or B-cell antigen receptor signaling by CD5 was proposed based on studies of thymocytes and peritoneal B-1a cells from CD5-deficient mice. Here, we show that CD5 is constitutively associated with phosphotyrosine phosphatase activity in Jurkat T cells. CD5 was found associated with the Src homology 2 (SH2) domain containing hematopoietic phosphotyrosine phosphatase SHP-1 in both Jurkat cells and normal phytohemagglutinin-expanded T lymphoblasts. This interaction was increased upon T-cell receptor (TCR)-CD3 cell stimulation. CD5 co-cross-linking with the TCR-CD3 complex down-regulated the TCR-CD3-increased Ca2+ mobilization in Jurkat cells. In addition, stimulation of Jurkat cells or normal phytohemagglutinin-expanded T lymphoblasts through TCR-CD3 induced rapid tyrosine phosphorylation of several protein substrates, which was substantially diminished after CD5 cross-linking. The CD5-regulated substrates included CD3ζ, ZAP-70, Syk, and phospholipase Cγl but not the Src family tyrosine kinase p56 lck . By mutation of all four CD5 intracellular tyrosine residues to phenylalanine, we found the membrane-proximal tyrosine at position 378, which is located in an immunoreceptor tyrosine-based inhibitory (ITIM)-like motif, crucial for SHP-1 association. The F378 point mutation ablated both SHP-1 binding and the down-regulating activity of CD5 during TCR-CD3 stimulation. These results suggest a critical role of the CD5 ITIM-like motif, which by binding to SHP-1 mediates the down-regulatory activity of this receptor.

CD6—ligand interactions: a paradigm for SRCR domain function?

The scavenger receptor cysteine-rich (SRCR) superfamily, which includes proteins expressed by leukocytes, can be subdivided into groups A and B. Group B contains the lymphocyte cell-surface receptor CD6. This article reviews recent progress in understanding the interaction between CD6 and its ligand, activated leukocyte cell adhesion molecule (ALCAM). Analysis of the CD6-ALCAM interaction may help to understand how other SRCR domains bind to their ligands.

The Amino-terminal Immunoglobulin-like Domain of Activated Leukocyte Cell Adhesion Molecule Binds Specifically to the Membrane-proximal Scavenger Receptor Cysteine-rich Domain of CD6 with a 1:1 Stoichiometry

Activated leukocyte cell adhesion molecule (ALCAM) was recently identified as a ligand for CD6, a signaling receptor expressed on T cells, a subset of B cells, and some cells in the brain. Receptor-ligand binding assays, antibody blocking experiments, and examination of the tissue distribution of these two cell surface proteins suggest that CD6-ALCAM interactions play an important role in mediating the binding of thymocytes to thymic epithelial cells and of T cells to activated leukocytes. Presently, the details of CD6-ALCAM interactions and of signaling through CD6 are unknown. A series of truncated human ALCAM and CD6 immunoglobulin fusion proteins were produced and tested in different binding assays to analyze ALCAM-CD6 interactions in more detail. In this study, we report that the amino-terminal Ig-like domain of human ALCAM specifically binds to the third membrane-proximal scavenger receptor cysteine-rich (SRCR) domain of human CD6. Using thrombin-cleaved Ig fusion proteins containing single or multiple ALCAM or CD6 domains, we were able to determine that the stoichiometry of the interaction between the amino-terminal ALCAM domains and the membrane-proximal CD6 SRCR domain is 1:1. These results provide the first example of an Ig-like domain mediating an interaction with an SRCR domain.

Cell surface receptors and their ligands: in vitro analysis of CD6-CD166 interactions.

CD6 is a cell surface receptor belonging to the scavenger receptor cysteine‐rich (SRCR) protein superfamily (SRCRSF). It specifically binds activated leukocyte cell adhesion molecule (ALCAM, CD166), a member of the immunoglobulin (Ig) superfamily (IgSF). CD166 was among the first molecules identified as a ligand for an SRCRSF receptor, and the CD6‐CD166 interaction was the first interaction characterized involving SRCRSF and IgSF proteins. We focus here on what has been learned about the specifics of the CD6‐CD166 interaction from in vitro analysis. The studies are thought to provide an instructive example for the analysis of interactions between single‐path transmembrane cell surface proteins. Using soluble recombinant forms, the extracellular binding domains of receptor and ligand have been identified and characterized in a variety of assay systems. Both CD6 and CD166 have been subjected to intense mutagenesis and monoclonal antibody (mAb) binding studies and residues critical for their interaction have been identified. The availability of structural prototypes of both superfamilies has made it possible to map the binding site in CD166 and, more recently, in CD6 and compare these regions to epitopes of mAbs that block, or do not block, the interaction. In addition, the molecular basis of observed cross‐species receptor‐ligand interactions could be rationalized. These studies illustrate the value of structural templates for the interpretation of sequence and mutagenesis analyses. Proteins 2000;40:420–428.

Interaction of recombinant and natural soluble CD5 forms with an alternative cell surface ligand

CD5, a member of the scavenger receptor cysteine‐rich (SRCR) receptor family, plays a role in the thymocyte maturation, T cell activation and T cell‐antigen‐presenting cell interactions. To date only CD5 ligands (CD5L) compatible with a T‐B co‐stimulatory role have been described (CD72, gp40‐80 and IgVH framework region) so the existence of alternative CD5L involved in other aspects of T cell biology warrants further exploration. Here we characterize the cell binding properties of a recombinant soluble human CD5 extracellular domain glycoprotein (rsCD5). In contrast to previously characterized ligands, this molecule binds to a broadly distributed cell surface receptor expressed on monocytes, lymphocytes and various cell lines of lymphoid, myelomonocytic and epithelial origin. The cell binding of rsCD5 is divalent cation independent and inhibited by high molar concentrations of certain monosaccharides. Both human CD5 Ig fusion proteins and a natural soluble CD5 form (present in human serum and resulting from proteolytic cleavage following lymphocyte activation) reproduce the cell binding pattern of rsCD5 and block its binding in a competitive form. The involvement of the most N‐terminal CD5 SRCR domains (D1 and D2) in binding is deduced from competition cell binding assays with CD5 Ig fusion proteins. These results imply a novel CD5/CD5L interaction model recalling some aspects of the interaction of CD6 with activated leukocyte cell adhesion molecule (ALCAM).

CD5 Is A Potential Selecting Ligand for B-Cell Surface Immunoglobulin: A Possible Role in Maintenance and Selective Expansion of Normal and Malignant B Cells

Although the function of CD5 on B cells is unknown, previous studies suggested that CD5 interaction with VH framework regions of surface immunoglobulins (Igs) may contribute to survival and expansion of B cells. Here we used B-chronic lymphocytic leukemia (B-CLL) cells and transformed B-cell lines from normal and B-CLL patients to study CD5-Ig interactions. Immobilized Ig binds and permits isolation of CD5 from lysates of CD5-expressing cell lines. Immunoglobulins or Fab fragments of different VH families varied in their effectiveness as inhibitors of anti-CD5 staining of CLL cells, appendix and tonsil tissue sections. Human Ig also binds to purified recombinant CD5. We show here for the first time that the unconventional Ig-CD5 interaction maps to the extracellular CD5-D2 domain whereas conventional epitopes recognized by anti-CD5 antibodies are localized in the Dl domain of CD5. We propose that interactions of VH framework regions with CD5 as a ligand may maintain, select or expand normal, autoimmune or transformed B cells and also contribute to skewing of the normal VH repertoire.

CD6 as a potential target for treating multiple sclerosis

Significance Multiple sclerosis (MS), an autoimmune disease that affects the central nervous system, is driven by activated T lymphocytes that invade the brain and spinal cord, leading to damage to the myelin sheaths that surround nerve axons. Current treatments are only partly successful, especially for the later stages of chronic forms of MS. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, suitable for testing new approaches to treatment. By genetically altering mice to eliminate a T lymphocyte surface protein known as CD6, we show that the CD6 molecule is essential for the development of EAE. Furthermore, an anti-CD6 monoclonal antibody is effective in treating EAE in mice that express human CD6 but not mouse CD6 on their T lymphocytes. CD6 was established as a marker of T cells more than three decades ago, and recent studies have identified CD6 as a risk gene for multiple sclerosis (MS), a disease in which autoreactive T cells are integrally involved. Nevertheless, the precise role of CD6 in regulating T-cell responses is controversial and its significance in the pathogenesis of various diseases remains elusive, partly due to the lack of animals engineered to alter expression of the CD6 gene. In this report, we found that CD6 KO mice showed decreased pathogenic T-cell responses, reduced spinal cord T-cell infiltration, and attenuated disease severity in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. CD6-deficient T cells exhibited augmented activation, but also significantly reduced survival and proliferation after activation, leading to overall decreased Th1 and Th17 polarization. Activated CD6-deficient T cells also showed impaired infiltration through brain microvascular endothelial cell monolayers. Furthermore, by developing CD6 humanized mice, we identified a mouse anti-human CD6 monoclonal antibody that is highly effective in treating established EAE without depleting T cells. These results suggest that (i) CD6 is a negative regulator of T-cell activation, (ii) at the same time, CD6 is a positive regulator of activated T-cell survival/proliferation and infiltration; and (iii) CD6 is a potential new target for treating MS and potentially other T-cell–driven autoimmune conditions.

CD166 Expression, Characterization, and Localization in Salivary Epithelium: Implications for Function During Sialoadenitis

CD166 is an Ig superfamily molecule that binds homotypically to itself and heterotypically to CD6. Interactions between CD6 and CD166 are important during immune development and in alloreactivity. CD166 is expressed at increased levels in selected cancers and in rheumatoid arthritis synovium. Knowledge that CD166 was expressed in normal human salivary epithelium led to these studies of CD166 and CD6 in diseased mouse salivary glands, that resemble pathology seen in the human disease, Sjögrens syndrome. We showed that in mouse salivary epithelium CD166 was expressed but that expression of CD166 did not necessarily predict its function. Recombinant soluble CD6-Ig bound to CD6 ligands (CD6L) on transformed and freshly isolated salivary epithelial cells. Cross-blocking studies showed that binding of CD6-Ig to salivary epithelium was in part dependent on CD166, but that CD6-Ig binding may also involve additional CD6L. Binding of CD6-Ig was sensitive to trypsin digestion but resistant to digestion by collagenase and sialidase. Anti-CD166 ab precipitated CD166 from salivary epithelium pre- and post-treatment with the pro-inflammatory cytokine IFN-γ. In contrast CD6-Ig only precipitated CD166 from IFN-γ treated cells. More extensive colocalization between CD166 and the actin cytoskeleton was observed in sialoadenitis epithelium compared to control. We conclude that during sialoadenitis, CD166 undergoes a gain of function, resulting in closer association with the actin cytoskeleton and increased capacity to bind CD6. We suggest that altered CD166 function may contribute to the pro-inflammatory milieu during sialoadenitis seen in Sjögrens syndrome.