Mary Rieck

Genetic Variation in PTPN22 Corresponds to Altered Function of T and B Lymphocytes

A variant of the PTPN22 gene, 1858C/T, is associated with an increased risk for the development of a wide array of autoimmune disorders. It is known that the protein tyrosine phosphatase Lyp encoded by this gene has an inhibitory effect on the proximal TCR signaling pathways. However, the consequences of carrying this variant and the mechanism by which it contributes to the development of autoimmunity are poorly understood. In this study, we demonstrate that homozygosity for this variant results in a profound deficit in T cell responsiveness to Ag stimulation. Heterozygosity for the variant allele is associated with reduced responsiveness of CD4+ memory T cells, characterized by diminished calcium mobilization, expression of CD25, and IL-10 production upon TCR stimulation. Additionally, the presence of the variant allele is associated with an increase in circulating memory T cells. We further demonstrate that these effects are not limited to the T cell compartment. Individuals with the variant allele have fewer memory B cells and these cells display a reduced response to stimulation via the BCR indicative of a B cell intrinsic defect. By identifying an immunologic phenotype in healthy subjects which correlates with the PTPN22 1858C/T genotype, we can now explore specific hypotheses regarding pathogenesis of diseases associated with the PTPN22 1858T variant.

Identification and functional characterization of T cells reactive to citrullinated-vimentin in HLA-DRB1*0401 humanized mice and RA patients

OBJECTIVEnAntibodies toward the citrullinated form of the synovial antigen vimentin are specific for rheumatoid arthritis (RA) and are associated with HLA-DRB1*0401. This suggests that T cells specific for peptides derived from citrullinated vimentin presented in the context of HLA-DRB1*0401 may contribute to the etiopathogenesis of RA. The aim of this study was to identify immunodominant epitopes from citrullinated vimentin presented by HLA-DRB1*0401 and to characterize the resulting T cell responses.nnnMETHODSnWe first predicted an HLA-binding T cell epitope from citrullinated vimentin based on the binding motif of HLA-DRB1*0401 and then confirmed its affinity. A class II major histocompatibility complex (MHC) tetramer loaded with the citrullinated form of vimentin aa 59-78 (cit-vimentin aa 59-78) was constructed and used to screen for specific T cells in HLA-DRB1*0401-transgenic mice, patients with RA, and healthy control subjects. Additionally, the cytokine output following cit-vimentin aa 59-78 challenge was analyzed in patients and healthy control subjects by multicolor flow cytometry and Luminex-based analysis.nnnRESULTSnThe citrullinated form of vimentin aa 59-78 bound to HLA-DRB1*0401, but the native form could not. Subsequently, cit-vimentin aa 59-78-specific T cells were detected in immunized mice and in the periphery of both HLA-DR*0401-positive healthy control subjects and HLA-DR*0401-positive patients with RA, using class II MHC tetramers, CD154 up-regulation, and intracellular cytokine measurements. As demonstrated in cell culture supernatants, the production of cytokines (predominantly interferon-γ) in response to cit-vimentin aa 59-78 was significantly higher in patients compared with controls.nnnCONCLUSIONnHere, we describe a posttranslational modification of an RA candidate autoantigen toward which HLA-DRB1*0401-restricted T cells can be detected in both patients with RA and healthy controls but for which a proinflammatory response is observed uniquely in patients with RA.

Functional and Structural Characterization of a Novel HLA-DRB1*04:01-Restricted α-Enolase T Cell Epitope in Rheumatoid Arthritis

Antibodies to citrullinated proteins, common in rheumatoid arthritis (RA) patients, are strongly associated to a specific set of HLA-DR alleles including HLA-DRB1*04:01, *04:04, and *01:01. Here, we first demonstrate that autoantibody levels toward the dominant citrullinated B cell epitope from α-enolase are significantly elevated in HLA-DRB1*04:01-positive RA patients. Furthermore, we identified α-enolase-derived T cell epitopes and demonstrated that native and citrullinated versions of several peptides bind with different affinities to HLA-DRB1*04:01, *04:04, and *01:01. The citrulline residues in the eight identified peptides are distributed throughout the entire length of the presented epitopes and more specifically, localized at peptide positions p-2, p2, p4, p6, p7, p10, and p11. Importantly, in contrast to its native version peptide 26 (TSKGLFRAAVPSGAS), the HLA-DRB1*04:01-restricted citrullinated peptide Cit26 (TSKGLFCitAAVPSGAS) elicited significant functional T cell responses in primary cells from RA patients. Comparative analysis of the crystal structures of HLA-DRB1*04:01 in complex with peptide 26 or Cit26 demonstrated that the posttranslational modification did not alter the conformation of the peptide. And since citrullination is the only structural difference between the two complexes, this indicates that the neo-antigen Cit26 is recognized by T cells with high specificity to the citrulline residue.

α-enolase specific T cells in rheumatoid arthritis – a MHC class II tetramer approach

Backgroundand objectives Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis (RA) and found in approximately 60% of patients and their presence is strongly associated with the human leucocyte antigen (HLA)-DRB1*0401 allele. α-enolase is one of these citrullinated antigens and antibodies against certain epitopes of this autoantigen are found in 40% of RA patients and are enriched in their synovial fluid. Here, the authors aimed to identify novel HLA-DRB1*0401 restricted T cell epitopes of α-enolase in order to construct and utilise major histocompatibility complex (MHC) class II tetramers to determine the frequency of cit-α-enolase-specific T cells in blood of DRB1*0401 RA patients. Material and methods Overlapping peptides, both native and citrullinated versions, of full length α-enolase were analysed for 0401 binding via the Proimmune Class II Reveal assay and 13 citrullinated peptides came out as candidates. Following our own validation of HLA-DRB1*0401 binding, the authors constructed class II MHC tetramers loaded with either the native or citrullinated form of one of the peptides. Peripheral blood mononuclear cells (PBMCs) from HLA-DRB1*0401 RA patients were then screened for α-enolase specific T cells. T cells recognising the α-enolase peptide were identified using direct ex vivo analysis and additional analysis of the T cell phenotypes was performed by multi-colour flow cytometry. PBMCs from these patients were also expanded in vitro for 14 days with haemagglutinin (HA) or either the native or the citrullinated version of the α-enolase peptide and analysed on a FACS calibur. Results In our binding assays the native and citrullinated form of our novel epitope bound to HLA-DRB1*0401 with a similar affinity. Tetramers were constructed and allowed for the detection of α-enolase specific T cells in the blood of DRB1*0401 RA patients. The frequency varied and was markedly lower than that of HA-specific T cells in the same individuals. When comparing the phenotype, our preliminary results suggest that T cells specific for the native α-enolase peptide are predominantly of a naïve phenotype, whereas the T cells specific for the citrullinated version are of a memory phenotype. Conclusions The authors have identified a novel α-enolase T cell epitope that binds HLA-DRB1*0401 in both its native and citrullinated form. Interestingly it appears that the cit-specific T cells are antigen-experienced and have previously been activated in the patients, while the native-specific T cells appeared naïve. In the future the authors plan to further enumerate and characterise cit-α-enolase specific T cells in RA and controls and to extend these studies to synovial fluid.

Memory T cells specific to citrullinated alpha-enolase are enriched in the rheumatic joint.

ACPA-positive rheumatoid arthritis (RA) is associated with distinct HLA-DR alleles and immune responses to many citrullinated self-antigens. Herein we investigated the T cell epitope confined within α-enolase326-340 in the context of HLA-DRB1*04:01 and assessed the corresponding CD4+ T cells in both the circulation and in the rheumatic joint. Comparative crystallographic analyses were performed for the native and citrullinated α-enolase326-340 peptides in complex with HLA-DRB1*04:01. HLA-tetramers assembled with either the native or citrullinated peptide were used for exxa0vivo and inxa0vitro assessment of α-enolase-specific T cells in peripheral blood, synovial fluid and synovial tissue by flow cytometry. The native and modified peptides take a completely conserved structural conformation within the peptide-binding cleft of HLA-DRB1*04:01. The citrulline residue-327 was located N-terminally, protruding towards TCRs. The frequencies of T cells recognizing native eno326-340 were similar in synovial fluid and peripheral blood, while in contrast, the frequency of T cells recognizing cit-eno326-340 was significantly elevated in synovial fluid compared to peripheral blood (3.6-fold, pu202f=u202f0.0150). Additionally, citrulline-specific T cells with a memory phenotype were also significantly increased (1.6-fold, pu202f=u202f0.0052) in synovial fluid compared to peripheral blood. The native T cell epitope confined within α-enolase326-340 does not appear to lead to complete negative selection of cognate CD4+ T cells. In RA patient samples, only T cells recognizing the citrullinated version of α-enolase326-340 were found at elevated frequencies implicating that neo-antigen formation is critical for breach of tolerance.

Identification and functional characterization of T cells reactive to citrullinated vimentin in HLA-DRB1*0401-positive humanized mice and rheumatoid arthritis patients: T Cell Reactivity to Citrullinated Antigens

OBJECTIVEnAntibodies toward the citrullinated form of the synovial antigen vimentin are specific for rheumatoid arthritis (RA) and are associated with HLA-DRB1*0401. This suggests that T cells specific for peptides derived from citrullinated vimentin presented in the context of HLA-DRB1*0401 may contribute to the etiopathogenesis of RA. The aim of this study was to identify immunodominant epitopes from citrullinated vimentin presented by HLA-DRB1*0401 and to characterize the resulting T cell responses.nnnMETHODSnWe first predicted an HLA-binding T cell epitope from citrullinated vimentin based on the binding motif of HLA-DRB1*0401 and then confirmed its affinity. A class II major histocompatibility complex (MHC) tetramer loaded with the citrullinated form of vimentin aa 59-78 (cit-vimentin aa 59-78) was constructed and used to screen for specific T cells in HLA-DRB1*0401-transgenic mice, patients with RA, and healthy control subjects. Additionally, the cytokine output following cit-vimentin aa 59-78 challenge was analyzed in patients and healthy control subjects by multicolor flow cytometry and Luminex-based analysis.nnnRESULTSnThe citrullinated form of vimentin aa 59-78 bound to HLA-DRB1*0401, but the native form could not. Subsequently, cit-vimentin aa 59-78-specific T cells were detected in immunized mice and in the periphery of both HLA-DR*0401-positive healthy control subjects and HLA-DR*0401-positive patients with RA, using class II MHC tetramers, CD154 up-regulation, and intracellular cytokine measurements. As demonstrated in cell culture supernatants, the production of cytokines (predominantly interferon-γ) in response to cit-vimentin aa 59-78 was significantly higher in patients compared with controls.nnnCONCLUSIONnHere, we describe a posttranslational modification of an RA candidate autoantigen toward which HLA-DRB1*0401-restricted T cells can be detected in both patients with RA and healthy controls but for which a proinflammatory response is observed uniquely in patients with RA.

Identification and function of T cells reactive with citrullinated vimentin in HLA-DRB1*0401 humanised mice and rheumatoid arthritis patients

The pathological mechanisms leading to a lost of immunological tolerance and inciting rheumatoid arthritis (RA) are not fully understood. However, autoimmunity towards several post-translationally modified (citrullinated) synovial antigens is thought to have a central role in disease pathogenesis. Of particular interest is vimentin, a synovial antigen to which antibodies towards its citrullinated form are commonly found in both RA sera and synovial fluid. …