Hyaluronan is a glycosaminoglycan present in the extracellular matrix. When hyaluronan is degraded during infection and injury, low m.w. forms are generated whose interactions influence inflammation and angiogenesis. Intact high m.w. hyaluronan, conversely, conveys anti-inflammatory signals. We demonstrate that high m.w. hyaluronan enhances human CD4+CD25+ regulatory T cell functional suppression of responder cell proliferation, whereas low m.w. hyaluronan does not. High m.w. hyaluronan also up-regulates the transcription factor FOXP3 on CD4+CD25+ regulatory T cells. These effects are only seen with activated CD4+CD25+ regulatory T cells and are associated with the expression of CD44 isomers that more highly bind high m.w. hyaluronan. At higher concentrations, high m.w. hyaluronan also has direct suppressive effects on T cells. We propose that the state of HA in the matrix environment provides contextual cues to CD4+CD25+ regulatory T cells and T cells, thereby providing a link between the innate inflammatory network and the regulation of adaptive immune responses.

T cell recognition of autoantigens is critical to progressive immunemediated destruction of islet cells, which leads to autoimmune diabetes. We identified a naturally presented autoantigen from the human islet antigen glutamic acid decarboxylase, 65-kDa isoform (GAD65), by using a combination of chromatography and mass spectrometry of peptides bound by the type I diabetes (insulindependent diabetes mellitus, IDDM)-associated HLA-DR4 molecule. Peptides encompassing this epitope-stimulated GAD65-specific T cells from diabetic patients and a DR4-positive individual at high risk for developing IDDM. T cell responses were antagonized by altered peptide ligands containing single amino acid modifications. This direct identification and manipulation of GAD65 epitope recognition provides an approach toward dissection of the complex CD4 ؉ T cell response in IDDM.T ype I diabetes (insulin-dependent diabetes mellitus, IDDM), like many autoimmune diseases, exhibits exquisite target organ specificity. IDDM is characterized by immune-mediated destruction of beta cells in the pancreatic islet, coincident with the sparing of neighboring alpha and delta cells. The precise target-cell specificity in this disease implies the existence of antigenic self proteins derived from beta cells that are recognized by autoimmune T lymphocytes. Extensive analysis of serum antibodies in patients with IDDM has documented several self proteins that are candidates for this role (1, 2). The 65-kDa isoform of human glutamic acid decarboxylase (hGAD65) is expressed in pancreatic beta cells at high levels, and antibodies to hGAD65 are present in up to 70% of newly diagnosed diabetics. These antibodies are also often present for several years before the development of clinical diabetes, providing a useful serum marker for prediction of disease onset (1, 3, 4). Recently, it has been shown that the suppression of GAD expression in nonobese diabetic (NOD) mice prevents autoimmune diabetes (5), directly implicating GAD as a likely participant in IDDM progression.Studies of T cell reactivity to autoantigens in diabetics have confirmed the immunogenicity of hGAD65 with reports of both CD4 ϩ and CD8 ϩ T cell responses (6)(7)(8)(9)(10)(11)(12)(13)(14). Approximately 70% of Caucasoid diabetics express the DRB1*0401, *0404, and *0405 MHC class II alleles. These DR4 ϩ alleles are in linkage disequilibrium with the DQB1*0302 gene, the HLA-DQ marker most highly associated with IDDM (15). T cell responses to the hGAD65 proteins that are restricted by HLA-DR molecules predominate in patients with these HLA disease-susceptibility haplotypes, and previous studies using overlapping synthetic peptides identified approximately 10 peptides that were capable of efficient binding to DR4 molecules. Indeed, three of these candidate autoantigen epitopes, corresponding to residues 115-127, 274-286, and 554-566 of hGAD65, were immunogenic when used to immunize mice transgenic for HLA-DR4 (16). A separate study, also using DR4-transgenic mice, found that these same three epitopes ...

Antigen-specific T cells acquire a distinctive phenotype during activation, with characteristic acquisition of surface markers and patterns of gene expression. Early after antigen stimulation, CD4(+) T lymphocytes increase their surface density of the CD4 marker, a trait which has been used to identify antigen-activated cells. The recent development of MHC tetramer technologies has greatly improved the ability to detect HLA class I-restricted T cells specific for known antigen epitopes. We have recently extended these studies to human class II-restricted CD4(+) T cell responses and now describe antigen-specific T cell responses from human peripheral blood in which elevated CD4 expression levels in human T cells following antigen stimulation identify the activated and proliferating subset of cells. The CD4(high) population is substantially enriched in epitope-specific cells identified by class II tetramer staining and almost all tetramer-positive cells are contained within the CD4(high) population. T cell clones derived from the tetramer-positive, CD4(high) population demonstrate antigen specificity and maintain tetramer staining, while the substantial number of CD4(high) cells which fail to stain with tetramer appear to proliferate as a result of bystander activation. Epitope-specific components of a polyclonal immune response are directly visualized and quantitated by tetramer detection, providing a direct measure of the heterogeneity of the human immune response.