scholarly journals POS0008 IDENTIFICATION AND CHARACTERIZATION OF HISRS+ CD4+ T CELLS PATIENTS WITH IDIOPATHIC INFLAMMATORY MYOPATHIES

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 206.2-206
Author(s):  
B. Horuluoglu ◽  
A. S. Galindo-Feria ◽  
K. Chemin ◽  
G. Kozhukh ◽  
A. Dubnovitsky ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Class Ii ◽  
Hla Class Ii ◽  
Idiopathic Inflammatory Myopathies ◽  
Inflammatory Myopathies ◽  
Disease Pathogenesis ◽  
Stimulation Of

Background:Idiopathic inflammatory myopathies (IIM) also known as myositis, are rare chronic autoimmune disorders which are represented by muscle weakness and extra-muscular features such as skin rash, interstitial lung disease (ILD) and arthritis. One of the most common autoantibodies in myositis, with a prevalence of 25-35%, is the anti Jo-1 autoantibodies, targeting the histidyl-transfer RNA synthetase (HisRS). Although the exact mechanism of how these antibodies are developed is unknown, we have previously shown that upon stimulation of both peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage fluid cells (BALF) with HisRS protein, CD4+ T cells were activated and produced inflammatory cytokines. Hitherto the presence of antigen specific autoreactive T cells has not been established in myositis, however previous studies by our group showed a strong indication of their presence with a reactivity to a specific HisRS peptide.Objectives:The main aim of this project is to detect and characterize HisRS specific CD4+T cells using HLA Class II tetramers. HLA Class II tetramers allow the detection of rare antigen specific CD4+ T cells and are widely used in studies of immunity, vaccine development, allergy monitoring and in autoimmunity. These cells are of specific interest to understand autoimmunity and to develop new therapies in autoimmune diseasesMethods:HLA-DRB1*03:01 monomers with selected tetanus and HisRS peptides were in-house in E.coli system. The peptides of interest were attached to the N-terminus of the HLA b-chain via a flexible peptide linker. HLA-tetramers were assembled using a commercial fluorescently labeled streptavidin. The efficacy of the peptide-HLA tetramers was validated by stimulating PBMCs from HLA-matched healthy controls with tetanus peptide. The drequency of tetanus specific CD4+ T cells were detected at different time points (6,13 and 21 days) from the cultures using tetanus peptides bound HLA-DRB1*0301 tetramers. The presence of tetanus specific T cells was confirmed by the secretion of significantly higher IFNg levels upon re-stimulation of cells with tetanus peptide. The same protocol is applied for the HisRS-peptide tetramers. Peripheral blood cells are analysed from anti-Jo1+ and HLA-DRB1*0301 positive patients with IIM.Results:Applying this method, our preliminary findings demonstrate the presence of HisRS+CD4+ T cells in peripheral blood from Jo-1+ patients (n=3) using HisRS tetramers following stimulation with the respective peptide. We are now including more patient samples to confirm our findings, and further characterize their phenotype and functionalities by flow cytometry and ELISA/fluorospot assaysConclusion:Myositis is a rare and chronic autoimmune disorder, with no currently available cure. Previous studies indicate the importance of T cells in this disease. However, the phenotype, functionality and role of these cells in the disease pathogenesis has not been fully established. Characterization of this autoreactive T-cell population will help us enhance our understanding of the disease pathogenesis and thus to develop better treatment options.Acknowledgements:This work has been supported by grants from Karolinska Instiutet Resarch Foundation, Professor Nanna Svartz Stiftelse, Hjärt-Lung Fonden and Vetenskapsrådet in Sweden.Disclosure of Interests:Begum Horuluoglu: None declared, Angeles Shunashy Galindo-Feria: None declared, Karine Chemin: None declared, Genadiy Kozhukh: None declared, Anatoly Dubnovitsky: None declared, Vivianne Malmström: None declared, Ingrid E. Lundberg Consultant of: Consulting fees from Corbus Pharmaceuticals, Ind, Grant/research support from: Research grants from Bristol Myers Squibb and Astra Zeneca.

scholarly journals Multi-HLA class II tetramer analyses of citrulline-reactive T cells and early treatment response in rheumatoid arthritis

2020 ◽  
Author(s):  
Christina Gerstner ◽  
Sara Turcinov ◽  
Aase H Hensvold ◽  
Karine Chemin ◽  
Hannes Uchtenhagen ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
At Risk ◽  
T Cells ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Class Ii ◽  
Hla Class Ii ◽  
Phenotypic Characterization ◽  
Early Ra

Abstract Background: HLA class II tetramers can be used for ex vivo enumeration and phenotypic characterization 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 α-enolase, fibrinogen-b, 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 characterization 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.

scholarly journals Multi-HLA class II tetramer analyses of citrulline-reactive T cells and early treatment response in rheumatoid arthritis

2020 ◽  
Author(s):  
Christina Gerstner ◽  
Sara Turcinov ◽  
Aase H Hensvold ◽  
Karine Chemin ◽  
Hannes Uchtenhagen ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
At Risk ◽  
T Cells ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Class Ii ◽  
Hla Class Ii ◽  
Phenotypic Characterization ◽  
Early Ra

Abstract Background: HLA class II tetramers can be used for ex vivo enumeration and phenotypic characterization 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 α-enolase, fibrinogen-b, 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 characterization 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.

scholarly journals Multi-HLA class II tetramer analyses of citrulline-reactive T cells and early treatment response in rheumatoid arthritis

2019 ◽  
Author(s):  
Christina Gerstner ◽  
Sara Turcinov ◽  
Aase H Hensvold ◽  
Karine Chemin ◽  
Hannes Uchtenhagen ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
At Risk ◽  
T Cells ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Class Ii ◽  
Hla Class Ii ◽  
Phenotypic Characterization ◽  
Early Ra

Abstract Background: HLA class II tetramers can be used for ex vivo enumeration and phenotypic characterization 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 α-enolase, fibrinogen-b, 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 memory T cells. Conclusions: Our study demonstrates the development of a sensitive tetramer panel allowing simultaneous characterization 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.

Identification of 4 new HLA-DR–restricted minor histocompatibility antigens as hematopoietic targets in antitumor immunity

Blood ◽  
2009 ◽  
Vol 114 (17) ◽  
pp. 3684-3692 ◽  
Author(s):  
Anita N. Stumpf ◽  
Edith D. van der Meijden ◽  
Cornelis A. M. van Bergen ◽  
Roel Willemze ◽  
J. H. Frederik Falkenburg ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Hematopoietic Cells ◽  
Class Ii ◽  
Hla Class Ii ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Histocompatibility Antigens ◽  
Minor Histocompatibility Antigens ◽  
Hla Dr

Abstract Potent graft-versus-leukemia (GVL) effects can be mediated by donor-derived T cells recognizing minor histocompatibility antigens (mHags) in patients treated with donor lymphocyte infusion (DLI) for relapsed hematologic malignancies after HLA-matched allogeneic stem cell transplantation (alloSCT). Donor-derived T cells, however, may not only induce GVL, but also mediate detrimental graft-versus-host disease (GVHD). Because HLA-class II is under noninflammatory conditions predominantly expressed on hematopoietic cells, CD4+ T cells administered late after alloSCT may selectively confer GVL without GVHD. Although a broad range of different HLA-class I–restricted mHags have been identified, the first 2 autosomal HLA-class II–restricted mHags have only recently been characterized. By screening a recombinant bacteria cDNA expression library, we identified 4 new HLA-class II–restricted mHags recognized by CD4+ T cells induced in a patient with relapsed chronic myeloid leukemia who achieved long-term complete remission and experienced only mild GVHD of the skin after DLI. All CD4+ T cells were capable of recognizing the mHags presented by HLA-DR surface molecules on primary hematopoietic cells, but not on skin-derived (cytokine-treated) fibroblasts. The selective recognition of hematopoietic cells as well as the balanced population frequencies and common HLA-DR restriction elements make the novel mHags possible targets for development of immunotherapeutic strategies.

scholarly journals IL-6 down-regulates HLA class II expression and IL-12 production of human dendritic cells to impair activation of antigen-specific CD4+ T cells

2016 ◽  
Vol 65 (2) ◽  
pp. 193-204 ◽  
Author(s):  
Yosuke Ohno ◽  
Hidemitsu Kitamura ◽  
Norihiko Takahashi ◽  
Junya Ohtake ◽  
Shun Kaneumi ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Cd4 T Cells ◽  
Class Ii ◽  
Hla Class Ii ◽  
Human Dendritic Cells

scholarly journals Presentation of the Goodpasture Autoantigen to CD4 T Cells Is Influenced More by Processing Constraints Than by HLA Class II Peptide Binding Preferences

1998 ◽  
Vol 273 (19) ◽  
pp. 11440-11447 ◽  
Author(s):  
Richard G. Phelps ◽  
Victoria L. Jones ◽  
Mary Coughlan ◽  
A. Neil Turner ◽  
Andrew J. Rees
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Peptide Binding ◽  
Class Ii ◽  
Hla Class Ii

scholarly journals Integrative molecular profiling of autoreactive CD4 T cells in autoimmune hepatitis

2020 ◽  
Author(s):  
Amédée Renand ◽  
Iñaki Cervera-Marzal ◽  
Laurine Gil ◽  
Chuang Dong ◽  
Erwan Kervagoret ◽  
...  
Keyword(s):  
T Cells ◽  
Disease Activity ◽  
Autoimmune Hepatitis ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
B Cell Differentiation ◽  
Tcr Repertoire ◽  
First Time

AbstractBackground & AimsIn most autoimmune disorders, crosstalk of B cells and CD4 T cells results in the accumulation of autoantibodies. In autoimmune hepatitis (AIH), the presence of anti-Soluble Liver Antigen (SLA or SepSecs) autoantibodies is associated with significantly reduced overall survival, but the associated autoreactive CD4 T cells have not been characterized yet. Here we isolated and deeply characterized SLA-specific CD4 T cells in AIH patients.MethodsWe used brief ex vivo restimulation with overlapping SLA-derived peptides to isolate and phenotype circulating SLA-specific CD4 T cells, and integrative single-cell RNA-seq (scRNA-seq) to characterize their transcriptome and TCR repertoire in n=5 AIH patients. SLA-specific CD4 T cells were tracked in peripheral blood through TCR sequencing, to identify their phenotypic niche. We further characterized disease-associated peripheral blood T cells by high content flow cytometry in an additional cohort of n=46 AIH patients and n=18 non-alcoholic steatohepatitis (NASH) controls.ResultsAutoreactive SLA-specific CD4 T cells were only detected in patients with anti-SLA autoantibodies and had a memory PD-1+CXCR5−CCR6−CD27+ phenotype. ScRNA-seq revealed their pro-inflammatory/B-Helper profile (IL21, IFNG, TIGIT, CTLA4, NR3C1, CD109, KLRB1 and CLEC2D). Autoreactive TCR clonotypes were restricted to the memory PD-1+CXCR5− CD4 T cells. This subset was significantly increased in the blood of AIH patients and supported B cell differentiation through IL-21. Finally, we identified a specific phenotype (PD-1+CD38+CD27+CD127−CXCR5−) of CD4 T cells linked to disease activity and IgG response during AIH.ConclusionsThis work provides for the first time a deep characterization of rare circulating autoreactive CD4 T cells and the identification of their peripheral reservoir in AIH. We also propose a generic phenotype of pathogenic CD4 T cells related to AIH disease activity.

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