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.

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 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 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.

scholarly journals Phenotypic characterization of swine peripheral blood monocyte-derived macrophages and ex vivo infection with Salmonella enterica serovar Typhimurium

2019 ◽  
Vol 50 (2) ◽  
pp. 539-546 ◽  
Author(s):  
Carlos Eduardo Real Pereira ◽  
Márcio Sobreira Silva Araújo ◽  
Juliana Pinto da Silva Mol ◽  
José Paulo Hiroji Sato ◽  
Amanda Gabrielle de Souza Daniel ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Ex Vivo ◽  
Peripheral Blood Monocyte ◽  
Blood Monocyte ◽  
Phenotypic Characterization ◽  
Serovar Typhimurium

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

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Christina Gerstner ◽  
Sara Turcinov ◽  
Aase H. Hensvold ◽  
Karine Chemin ◽  
Hannes Uchtenhagen ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
Treatment Response ◽  
Early Treatment ◽  
Class Ii ◽  
Hla Class Ii ◽  
Early Treatment Response

scholarly journals THU0033 ALTERATIONS IN THE PHENOTYPIC LANDSCAPE AND SPECIFICITY OF CD4+ T CELLS IN CCP+ AT RISK SUBJECTS BEFORE THE ONSET OF RHEUMATOID ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 230.1-230
Author(s):  
C. Rims ◽  
V. Muir ◽  
K. Deane ◽  
S. Nagpal ◽  
N. Rao ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
At Risk ◽  
Flow Cytometry ◽  
T Cells ◽  
Immune Responses ◽  
Cd4 T Cells ◽  
Research Support ◽  
Phenotypic Analysis ◽  
Tetramer Staining ◽  
Early Ra

Background:The “Targeting Immune Responses for Prevention of RA” (TIP-RA) collaboration studies individuals at high risk for developing rheumatoid arthritis (RA) because of serum anti-citrullinated protein antibody (ACPA) positivity in absence of arthritis at baseline, and is focused on defining how they transition from at-risk to classifiable disease. One potential mechanism is the expansion of antigen specific T cells that recognize self-antigens and acquisition of disease associated T cell phenotypes. ACPA emerge years prior to clinically apparent disease and subsequently increase in their titer and breadth of specificity. However, few studies have characterized T cells during this transition.Objectives:To identify features associated with progression to RA by examining the specificity and surface phenotype of CD4+ T cells in individuals from the TIP-RA cohort by HLA class II tetramer staining and multi-parameter flow cytometry.Methods:Tetramer staining and flow cytometry were performed on peripheral blood samples from a baseline visit from CCP3- controls (n=34), CCP3+ at-risk (n=26), CCP3+ positive individuals who transitioned in the near-term to RA (called “RA converters”, n=4), and seropositive early-RA (n=21). Our staining panel allowed us to measure the frequencies of T cells specific for citrullinated alpha-enolase, aggrecan, cartilage intermediate layer protein (CILP), fibrinogen and vimentin. We then applied both supervised phenotyping and a cluster-based computational approach to compare the phenotypic landscape and specificity of antigen specific and total CD4+ T cells in each cohort.Results:We observed higher overall frequencies of T cells that recognize citrullinated epitopes in CCP3+ at-risk subjects than CCP- controls (p< 0.05). Among the individual specificities, elevated frequencies prior to disease onset were most prominent for CILP specific T cells. Supervised phenotypic analysis revealed an increase in CCR4+ CD4+ T cells in CCP3+ at risk subjects (p< 0.001) and a corresponding decrease in CXCR3+ CD4+ T cells that was most pronounced in RA converters and seropositive early-RA (p< 0.05). Cluster-based phenotypic analysis defined ten distinct phenotypic states present within all subjects. Each of these ten immunotypes contained T cells that recognize citrullinated epitopes. However, the predominant immunotype varied for different antigens. During progression, the frequencies of Ag specific T cells diminished when onset was imminent, but rebounded shortly after diagnosis. Concomitantly, Ag specific T cells with memory phenotypes were diminished, but subsequently reverted to TSCM, Th1, and Th1-17 like phenotypes.Conclusion:Our data show that disease associated changes in the antigen specificity of CD4+ T cells are present in CCP3+ at-risk subjects. Furthermore, the number of antigen specific T cells and their phenotype are perturbed before the onset of symptoms and development of classified RA. These findings support a continuum of immunologic changes that underlie risk and drive disease, motivating new approaches for early intervention.Acknowledgments:We gratefully acknowledge the Targeting Immune Responses for Prevention of Rheumatoid Arthritis (TIP-RA) for designing and executing this collaborative studyDisclosure of Interests:Cliff Rims: None declared, Virginia Muir: None declared, Kevin Deane Grant/research support from: Janssen, Consultant of: Inova, ThermoFisher, Janseen, BMS and Microdrop, Sunil Nagpal Shareholder of: Janssen Pharmaceuticals, Employee of: Janssen Pharmaceuticals, Navin Rao Shareholder of: Janssen Pharmaceuticals, Employee of: Janssen Pharmaceuticals, Frederic Baribaud Shareholder of: Janssen Research & Development, LLC, Employee of: Janssen Research & Development, LLC, George Vratsanos Shareholder of: Janssen Pharmaceuticals, Employee of: Janssen Pharmaceuticals, V. Michael Holers Grant/research support from: Janssen, Celgene, and BMS, Peter Linsley Consultant of: BMS, Eddie A. James Grant/research support from: Janssen, Pfizer, Sanofi, Novartis, Jane Buckner Grant/research support from: Bristol-Myers Squibb, Janssen

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