Talitha R. Bakker

The nature of molecular recognition by T cells.

Considerable progress has been made in characterizing four key sets of interactions controlling antigen responsiveness in T cells, involving the following: the T cell antigen receptor, its coreceptors CD4 and CD8, the costimulatory receptors CD28 and CTLA-4, and the accessory molecule CD2. Complementary work has defined the general biophysical properties of interactions between cell surface molecules. Among the major conclusions are that these interactions are structurally heterogeneous, often reflecting clear-cut functional constraints, and that, although they all interact relatively weakly, hierarchical differences in the stabilities of the signaling complexes formed by these molecules may influence the sequence of steps leading to T cell activation. Here we review these developments and highlight the major challenges remaining as the field moves toward formulating quantitative models of T cell recognition.

CD45 ectodomain controls interaction with GEMs and Lck activity for optimal TCR signaling

The transmembrane phosphatase CD45 regulates both Lck activity and T cell receptor (TCR) signaling. Here we have tested whether the large ectodomain of CD45 has a role in this regulation. A CD45 chimera containing the large ectodomain of CD43 efficiently rescues TCR signaling in CD45-null T cells, whereas CD45 chimeras containing small ectodomains from other phosphatases do not. Both basal Lck activity in unstimulated cells and the TCR-induced increase in tyrosine phosphorylation of the TCR ζ-chain and in Lck activity depend on the expression of CD45 with a large ectodomain. Unlike CD45 chimeras containing small ectodomains, both the CD45 chimera with a large ectodomain and wild-type CD45 itself are partially localized to glycosphingolipid-enriched membranes (GEMs). Taken together, these data show that the large CD45 ectodomain is required for optimal TCR signaling.

Glycosylation Influences the Lectin Activities of the Macrophage Mannose Receptor

The mannose receptor (MR) is a heavily glycosylated endocytic receptor that recognizes both mannosylated and sulfated ligands through its C-type lectin domains and cysteine-rich (CR) domain, respectively. Differential binding properties have been described for MR isolated from different sources, and we hypothesized that this could be due to altered glycosylation. Using MR transductants and purified MR, we demonstrate that glycosylation differentially affects both MR lectin activities. MR transductants generated in glycosylation mutant cell lines lacked most mannose internalization activity, but could internalize sulfated glycans. Accordingly, purified MR bearing truncated Man5-GlcNAc2 glycans (Man5 -MR) or non-sialylated complex glycans (SA0-MR) did not bind mannosylated glycans, but could recognize SO4-3-Gal in vitro. Additional studies showed that, although mannose recognition was largely independent of the oligomerization state of the protein, recognition of sulfated carbohydrates was mostly mediated by self-associated MR and that, in SA0-MR, there was a higher proportion of oligomeric MR. These results suggest that self-association could lead to multiple presentation of CR domains and enhanced avidity for sulfated sugars and that non-sialylated MR is predisposed to oligomerize. Therefore, the glycosylation of MR, terminal sialylation in particular, could influence its binding properties at two levels. (i) It is required for mannose recognition; and (ii) it modulates the tendency of MR to self-associate, effectively regulating the avidity of the CR domain for sulfated sugar ligands.

Immunology: How Do T Cells Recognize Antigen?

T cells recognize small fragments of microorganisms (antigens) on the surface of other cells using T cell antigen receptors. The mechanism by which these receptors signal into T cells is controversial, but two recent studies provide important new clues.

Self-help in T cell recognition?

Activation of T cells requires TCR engagement of foreign peptide complexed with MHC. New evidence suggests that TCR engagement of self-peptide–MHC complexes may enhance recognition of foreign antigen.

Glycosylation Influences the Ligand Binding Activities of Mannose Receptor

Murine mannose receptor (MR) contains seven N-linked and three O-linked oligosaccharides and differential binding properties have been described for MR isolated from the liver and the lung. We hypothesised that these different binding activities could be controlled by glycosylation. In this study the relationship between MR glycosylation and its function has been investigated using MR transductants generated in both wild type CHO cells and glycosylation-deficient LEC cells. The investigation shows that glycosylation does not affect the subcellular distribution, proteolytic processing and endocytic capacity of the receptor, but has a major effect in its binding capacity. Cells bearing MR modified with 52 sugars (Man-5 MR) completely lost its mannose-internalisation activity, which is associated with CRD4–5 of MR. In agreement with this observation purified soluble Man-5 MR lost the capability to bind mannan in vitro. The desialylation modification of MR also results in a 70% reduction of cellular internalisation activity and a low efficient mannan binding activity in vitro. However, cells bearing MR modified with 52 sugars or desialylated glycans do retain their sulphated sugar internalisation activity, which is associated to the cysteine-rich (CR) domain. Interestingly, in vitro 4 binding study indicated desialylated MR has better affinity than wild-type MR. Subsequent gel filtration and BIAcore studies showed that desialylated MR tend to form self-associated structure and multiple presentation of CR domain could enhance its affinity to sulphated sugars dramatically. These results, for the first time, suggest a role for glycosylation, especially terminal sialylation of MR, in manipulating its dual ligand binding activities in vivo.