| 1. |
|
|
| 2. |
- Chemin, Karine, et al.
(författare)
-
A Novel HLA-DRB1*10:01-Restricted T Cell Epitope From Citrullinated Type II Collagen Relevant to Rheumatoid Arthritis
- 2016
-
Ingår i: Arthritis & Rheumatology. - : Wiley. - 2326-5191 .- 2326-5205. ; 68:5, s. 1124-1135
-
Tidskriftsartikel (refereegranskat)abstract
- Objective. Antibodies against citrullinated type II collagen (Cit-CII) are common in the sera and synovial fluid of patients with rheumatoid arthritis (RA); however, the known T cell epitope of CII is not dependent on citrullination. The aim of this study was to identify and functionally characterize the Cit-CII-restricted T cell epitopes that are relevant to RA. Methods. Peripheral blood mononuclear cells (PBMCs) from HLA-DRB1*10:01-positive patients with RA and healthy donors were stimulated in vitro with candidate CII peptides. CD154 up-regulation was measured as a marker of antigen-specific activation, and anti-HLA-DR-blocking experiments confirmed HLA restriction. Cytokine production was measured using a Luminex technique. Direct peptide-binding assays using HLA-DRB1*10:01 and HLA-DRB1*04:01 monomeric proteins were performed. The T cell receptor (TCR) beta-chain of CD154-enriched antigen-specific T cells was analyzed using high-throughput sequencing. Results. A novel Cit-CII peptide was identified based on its ability to activate CD4+ T cells from HLA-DRB1*10:01-positive individuals. When stimulated in vitro, Cit-CII autoreactive T cells produced proinflammatory cytokines. Cit-CII311-325 bound (with low affinity) to HLA-DRB1*10:01 but not to HLA-DRB1*04:01, while the native form was unable to bind either protein. In addition, highly expanded clones were identified in the TCR beta repertoire of Cit-CII311-325-stimulated PBMCs. Conclusion. These results illustrate the ability of the citrullination process to create T cell epitopes from CII, a cartilage-restricted protein that is relevant to RA pathogenesis. The exclusive binding of Cit-CII311-325 to HLA-DRB1*10:01 suggests that recognition of citrullinated epitopes might vary between individuals carrying different RA-associated HLA-DR molecules.
|
|
| 3. |
- Gerstner, Christina
(författare)
-
Anti-citrulline immunity in rheumatoid arthritis : characterization of peptide-HLA interactions and CD4+ T cell responses
- 2018
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Rheumatoid Arthritis (RA) is a complex, systemic autoimmune disorder characterized by chronic inflammation in small joints in hands and feet. It is a common disease that if untreated leads to joint destruction, disability, comorbidities and a reduced lifespan. Both genetic and environmental factors contribute to disease pathogenesis that in the majority of patients is characterized by the occurrence of antibodies against citrullinated proteins (ACPA). The strong genetic association with certain MHC class II alleles led to autoreactive T cells being assigned a major role in the course of disease. Today, citrulline-specific CD4+ T cells are regarded the perfect target for antigen-specific immunotherapy and have so far been investigated in a handful of studies. The focus of this thesis was therefore to expand our knowledge of citrulline-reactive T cells with a specific emphasis on their phenotype as well as the interaction with their cognate peptide-HLA complexes on antigen-presenting cells. We have examined functional T cell responses to native and citrullinated epitopes derived from the candidate antigen α-enolase and compared their relevance in peripheral blood of patients with different RA-associated HLA-DR alleles. While HLA-DRB1*04:01 and *04:04 presented a similar set of peptides, a clear bias in functional responses towards citrullinated epitopes was observed in HLA-DRB1*04:01 patients. When analysing crystal structures of both native and citrullinated versions of two α-enolase epitopes in complex with HLADRB1* 04:01, we found the citrulline residues at peptide positions p-1 and p2, respectively. In both cases the citrulline was not involved in binding to the HLA molecule but instead pointed upwards readily available for interaction with the TCR. Particular recognition of these citrullinated epitopes by the T cells is thus based on the creation of neoantigens. Using HLA class II tetramer technology, we even detected cross-reactive T cells in some patients recognizing both native and citrullinated version of the epitope with citrulline at position p-1 implicating the existence of TCRs with different docking patterns towards these pHLA complexes. When comparing frequencies of T cells reactive to either of the two versions, we found memory T cells specific for the citrullinated version enriched in the synovial fluid compared to peripheral blood. Additionally, by successfully combining HLA class II tetramer sorting with single cell RNAsequencing, we could compare the transcriptional profile of citrulline- versus virus-specific CD4+ T cells in peripheral blood and synovial fluid of RA patients. Here, we repeatedly found genes associated with cytolytic and cytotoxic features upregulated in the citrulline-specific T cells both in blood and synovial fluid. Furthermore, we developed a multi-tetramer staining panel that allows the simultaneous assessment of multiple specificities making it applicable for longitudinal monitoring of T cells in clinical samples. Using this tool to examine the frequency and phenotype of T cells specific for eight citrullinated peptides from four RA candidate antigens, we could show that the frequencies of citrulline-specific CD4+ T cells in early RA patients decline upon disease improvement. In summary, our data demonstrate that autoreactive citrulline-specific T cells are present in RA patients both early and late in the disease course and that the breach of tolerance includes several non-related autoantigens.
|
|
| 4. |
- Gerstner, Christina, et al.
(författare)
-
Functional and Structural Characterization of a Novel HLA-DRB1*04:01-Restricted alpha-Enolase T Cell Epitope in Rheumatoid Arthritis
- 2016
-
Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 7
-
Tidskriftsartikel (refereegranskat)abstract
- 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 alpha-enolase are significantly elevated in HLA-DRB1*04:01-positive RA patients. Furthermore, we identified alpha-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.
|
|
| 5. |
- Gerstner, Christina, et al.
(författare)
-
Multi-HLA class II tetramer analyses of citrulline-reactive T cells and early treatment response in rheumatoid arthritis
- 2020
-
Ingår i: BMC Immunology. - : Springer Science and Business Media LLC. - 1471-2172. ; 21:1
-
Tidskriftsartikel (refereegranskat)abstract
- Background HLA class II tetramers can be used for ex vivo enumeration and phenotypic characterisation of antigen-specific CD4+ T cells. They are increasingly applied in settings like allergy, vaccination and autoimmune diseases. Rheumatoid arthritis (RA) is a chronic autoimmune disorder for which many autoantigens have been described. Results Using multi-parameter flow cytometry, we developed a multi-HLA class II tetramer approach to simultaneously study several antigen specificities in RA patient samples. We focused on previously described citrullinated HLA-DRB1*04:01-restricted T cell epitopes from alpha-enolase, fibrinogen-beta, vimentin as well as cartilage intermediate layer protein (CILP). First, we examined inter-assay variability and the sensitivity of the assay in peripheral blood from healthy donors (n = 7). Next, we confirmed the robustness and sensitivity in a cohort of RA patients with repeat blood draws (n = 14). We then applied our method in two different settings. We assessed lymphoid tissue from seropositive arthralgia (n = 5) and early RA patients (n = 5) and could demonstrate autoreactive T cells in individuals at risk of developing RA. Lastly, we studied peripheral blood from early RA patients (n = 10) and found that the group of patients achieving minimum disease activity (DAS28 < 2.6) at 6 months follow-up displayed a decrease in the frequency of citrulline-specific T cells. Conclusions Our study demonstrates the development of a sensitive tetramer panel allowing simultaneous characterisation of antigen-specific T cells in ex vivo patient samples including RA 'at risk' subjects. This multi-tetramer approach can be useful for longitudinal immune-monitoring in any disease with known HLA-restriction element and several candidate antigens.
|
|
| 6. |
- Hensvold, Aase, et al.
(författare)
-
The human bone marrow plasma cell compartment in rheumatoid arthritis-Clonal relationships and anti-citrulline autoantibody producing cells
- 2023
-
Ingår i: Journal of Autoimmunity. - : Elsevier BV. - 0896-8411 .- 1095-9157. ; 136
-
Tidskriftsartikel (refereegranskat)abstract
- A majority of circulating IgG is produced by plasma cells residing in the bone marrow (BM). Long-lived BM plasma cells constitute our humoral immune memory and are essential for infection-specific immunity. They may also provide a reservoir of potentially pathogenic autoantibodies, including rheumatoid arthritis (RA)-associated anti-citrullinated protein autoantibodies (ACPA). Here we investigated paired human BM plasma cell and peripheral blood (PB) B-cell repertoires in seropositive RA, four ACPA+ RA patients and one ACPA- using two different single-cell approaches, flow cytometry sorting, and transcriptomics, followed by recombinant antibody generation. Immunoglobulin (Ig) analysis of >900 paired heavy-light chains from BM plasma cells identified by either surface CD138 expression or transcriptome profiles (including gene expression of MZB1, JCHAIN and XBP1) demonstrated differences in IgG/A repertoires and N-linked glycosylation between patients. For three patients, we identified clonotypes shared between BM plasma cells and PB memory B cells. Notably, four individuals displayed plasma cells with identical heavy chains but different light chains, which may indicate receptor revision or clonal convergence. ACPA-producing BM plasma cells were identified in two ACPA+ patients. Three of 44 recombinantly expressed monoclonal antibodies from ACPA+ RA BM plasma cells were CCP2+, specifically binding to citrullinated peptides. Out of these, two clones reacted with citrullinated histone-4 and activated neutrophils. In conclusion, single-cell investigation of B-cell repertoires in RA bone marrow provided new understanding of human plasma cells clonal relationships and demonstrated pathogenically relevant disease-associated autoantibody expression in long-lived plasma cells.
|
|
| 7. |
- Pieper, Jennifer, et al.
(författare)
-
Memory T cells specific to citrullinated alpha-enolase are enriched in the rheumatic joint
- 2018
-
Ingår i: Journal of Autoimmunity. - : ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD. - 0896-8411 .- 1095-9157. ; 92, s. 47-56
-
Tidskriftsartikel (refereegranskat)abstract
- 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 alpha-enolase(326-340) 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 alpha-enolase(326-340) peptides in complex with HLA-DRB1*04:01. HLA-tetramers assembled with either the native or citrullinated peptide were used for ex vivo and in vitro assessment of a 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 eno(326-340) were similar in synovial fluid and peripheral blood, while in contrast, the frequency of T cells recognizing cit-eno(326-340) was significantly elevated in synovial fluid compared to peripheral blood (3.6-fold, p = 0.0150). Additionally, citrulline-specific T cells with a memory phenotype were also significantly increased (1.6-fold, p = 0.0052) in synovial fluid compared to peripheral blood. The native T cell epitope confined within alpha-enolase(326-340) 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 alpha-enolase(326-340) were found at elevated frequencies implicating that neo-antigen formation is critical for breach of tolerance. (C) 2018 Elsevier Ltd. All rights reserved.
|
|
