Dysbiosis in the oral microbiomes of anti-CCP positive individuals at risk of developing rheumatoid arthritis

ObjectivesAn increased prevalence of periodontitis and perturbation of the oral microbiome has been identified in patients with rheumatoid arthritis (RA). The periodontal pathogen Porphyromonas gingivalis may cause local citrullination of proteins, potentially triggering anti-citrullinated protein antibody production. However, it is not known if oral dysbiosis precedes the onset of clinical arthritis. This study comprehensively characterised the oral microbiome in anti-cyclic citrullinated peptide (anti-CCP) positive at-risk individuals without clinical synovitis (CCP+at risk).MethodsSubgingival plaque was collected from periodontally healthy and diseased sites in 48 CCP+at risk, 26 early RA and 32 asymptomatic healthy control (HC) individuals. DNA libraries were sequenced on the Illumina HiSeq 3000 platform. Taxonomic profile and functional capability of the subgingival microbiome were compared between groups.ResultsAt periodontally healthy sites, CCP+at risk individuals had significantly lower microbial richness compared with HC and early RA groups (p=0.004 and 0.021). Microbial community alterations were found at phylum, genus and species levels. A large proportion of the community differed significantly in membership (523 species; 35.6%) and structure (575 species; 39.1%) comparing CCP+at risk and HC groups. Certain core species, including P. gingivalis, had higher relative abundance in the CCP+at risk group. Seventeen clusters of orthologous gene functional units were significantly over-represented in the CCP+at risk group compared with HC (adjusted p value <0.05).ConclusionAnti-CCP positive at-risk individuals have dysbiotic subgingival microbiomes and increased abundance of P. gingivalis compared with controls. This supports the hypothesis that the oral microbiome and specifically P. gingivalis are important in RA initiation.

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

10.21203/rs.2.14731/v2 ◽

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.

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THU0111 T-Cell Subsets in Lymph Node Biopsies of Autoantibody Positive Subjects at Risk of Rheumatoid Arthritis (RA) and Early RA Patients

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2013-eular.639 ◽

2013 ◽

Vol 72 (Suppl 3) ◽

pp. A200.1-A200

Author(s):

T. H. Ramwadhdoebe ◽

J. Hähnlein ◽

K. I. Maijer ◽

J. Boorsma ◽

L. J. van Boven ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

At Risk ◽

Lymph Node ◽

T Cell ◽

T Cell Subsets ◽

Early Ra

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

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.1918 ◽

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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The oral microbiome in early rheumatoid arthritis patients and individuals at risk differs from healthy controls

Arthritis & Rheumatology ◽

10.1002/art.41780 ◽

2021 ◽

Author(s):

Johanna M. Kroese ◽

Bernd W. Brandt ◽

Mark J. Buijs ◽

Wim Crielaard ◽

Frank Lobbezoo ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

At Risk ◽

Early Rheumatoid Arthritis ◽

Oral Microbiome ◽

Healthy Controls

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

10.21203/rs.2.14731/v3 ◽

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.

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POS1441 SYMPTOMS CHARACTERISTICS OF SEROPOSITIVE INDIVIDUALS AT-RISK FOR DEVELOPING RHEUMATOID ARTHRITIS ARE VERSATILE AND COMPARABLE TO THOSE IN PEOPLE WITH EARLY RHEUMATOID ARTHRITIS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.3884 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 1005.3-1005

Author(s):

P. Studenic ◽

A. Circiumaru ◽

D. Aletaha ◽

K. Chatzidionysiou ◽

A. Hensvold ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

At Risk ◽

Symptom Burden ◽

Sensitivity Analyses ◽

Medical Attention ◽

Pain Patient ◽

Symptom Duration ◽

Early Ra ◽

Quality Register ◽

Patient Reported

Background:The symptom burden of typical rheumatoid arthritis (RA) symptoms in those at risk for developing RA - positive for anti-citrullinated peptide antibodies (ACPA) and musculoskeletal complaints - has not been explored.Objectives:To evaluate patient-reported symptoms in individuals at-risk to develop RA in comparison to people with newly diagnosed, early seropositive RA.Methods:Two datasources from the region of Stockholm were used: The RISK RA cohort follows up ACPA positive individuals with arthralgia, but without signs of joint inflammation in a structured program over 3 years. The baseline visit of this cohort was used. Patients with early seropositive (ACPA and/or rheumatoid factor positive) RA (symptom duration of 12 months maximum) reported in the Swedish Rheumatology Quality Register (SRQ) at their closest visit to their diagnosis date before receiving DMARD treatment have been identified. At-risk individuals were matched 1:3 by sex and age using the nearest neighbour method utilizing Mahalanobis distance, corrected for sample bias and exact matches on sex. Effect estimates of being at-risk compared to early RA for pain, patient global (GH), fatigue (all visual analogue scales), health assessment questionnaire (HAQ), TJC28 and the EuroQol-5D (EQ5D; range: 0-1) have been derived. Propensity score matching was used as sensitivity analyses.Results:A total of 223 individuals at risk for developing RA were compared to 820 matched early RA patients. The summary of distribution of variables and effect estimates of differences between at-risk and early RA individuals are shown in the Figure 1. At-risk individuals show 24mm of lower pain scores than early RA. This difference is even less (-17mm, 95%CI: -24 to -11) when the estimate is additionally matched for the number of tender joints. The TJC28 was on average 5.4 joints lower than in early RA patients. People at-risk show 22mm lower GH scores (rate themselves better) and 17mm lower fatigue scores than early RA patients. Fatigue was scored highest (mean:35mm, 95%CI: 30 to 29) among the three VAS. HAQ was on average lower by 0.6 points in at-risk individuals and EQ5D showed 0.24 higher index-values, outlining a better health status. Still the mean EQ5D in at-risk individuals was only 0.74 (95%CI: 0.71 to 0.77), which is lower than average values of an age-matched general population (0.92 to 0.96). Sensitivity analyses revealed similar results.Figure 1.Boxplots of pain, global health, fatigue, TJC28, EQ5D and HAQ, separately displayed for people with early seropositive RA and individuals at-risk. Population effect estimates (all p<0.001) comparing at-risk to early RA are provided for every pair, with 95% confidence interval in brackets.Conclusion:Not surprisingly, individuals at risk for RA report less symptom burden then early diagnosed RA patients. However, these differences only range around minimal clinically important differences and for fatigue even below, which stresses the need for medical attention and management strategies for symptomatic at-risk individuals.Acknowledgements:This study was supported through the New Horizon Fellowship and the FOREUM research fellowship grant and is part of the Innovative Medicines Initiative Joint Undertaking under grant agreement no 777357 (RTCure).Hensvold AH and Catrina IA contributed equally.Disclosure of Interests:None declared

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

10.21203/rs.2.14731/v1 ◽

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.

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