Compartmentalization and persistence of dominant (regulatory) T cell clones indicates antigen skewing in juvenile idiopathic arthritis

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    In this study, the authors performed mass cytometry (CyTOF) analysis and T cell receptor (TCR) sequencing to study immune cell composition and expansion of joint-derived Tregs and non-Tregs in Juvenile Idiopathic Arthritis (JIA). They studied different joints affected at the same time and found that the composition and functional characteristics of immune infiltrates are strikingly similar between joints within one patient. The research design of this study is appropriate and the methods used in this study are adequately described in the manuscript. The study may be potentially beneficial for the JIA treatment.

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Abstract

Autoimmune inflammation is characterized by tissue infiltration and expansion of antigen-specific T cells. Although this inflammation is often limited to specific target tissues, it remains yet to be explored whether distinct affected sites are infiltrated with the same, persistent T cell clones. Here we performed CyTOF analysis and T cell receptor (TCR) sequencing to study immune cell composition and (hyper-)expansion of circulating and joint-derived Tregs and non-Tregs in Juvenile Idiopathic Arthritis (JIA). We studied different joints affected at the same time, as well as over the course of relapsing-remitting disease. We found that the composition and functional characteristics of immune infiltrates are strikingly similar between joints within one patient, and observed a strong overlap between dominant T cell clones, especially Treg, of which some could also be detected in circulation and persisted over the course of relapsing remitting disease. Moreover, these T cell clones were characterized by a high degree of sequence similarity, indicating the presence of TCR clusters responding to the same antigens. These data suggest that in localized autoimmune disease there is auto-antigen driven expansion of both Teffector and Treg clones, that are highly persistent and are (re)circulating. These dominant clones might represent interesting therapeutic targets.

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  1. Author Response

    Reviewer #2 (Public Review):

    In the manuscript, Mijnheer et al mainly exploited CyTOF Helios mass cytometer and TCRβ repertoire sequencing to investigate the T cell composition and distribution in peripheral blood and synovial fluid, and further explored the temporal and spatial dynamics of regulatory T cells (Tregs) and non-Tregs in the inflamed joints of Juvenile Idiopathic Arthritis (JIA) patients. Their results indicate that the activated effector T cells and hyper-expanded Treg TCRβ clones found at the inflamed joints are highly persistent in the circulation, and the dominant of high degree of sequence similarity of Treg clones could serve as the novel therapeutic targets for the JIA treatment. Overall, the research design is appropriate, and the methods are adequately described in the study. However, several issues are required to be addressed.

    (1) The criteria for the JIA patient's recruitment should be clearly presented in the method section. For example, what is the specific included criteria and excluded criteria? Or did the patients take medicines for the treatment during the study?

    A total of 9 JIA patients were included in this study. Of these, n=2 were diagnosed with extended oligo JIA, n=2 with rheumatoid factor negative poly-articular JIA, and n=5 with oligo JIA, according to the revised criteria for JIA. The average age at the time of inclusion was 13,1 years (range 3,2 – 18,1 years) with a disease duration of 7,3 years (range 0.4 – 14.2 years). Due to limited sample availability, we did not have strict inclusion or exclusion criteria for JIA patient recruitment. For CyTOF analysis, patients were selected based on the criteria that the left and right knee joints should both be affected at the time of inclusion. For sequential TCR sequencing analysis, we included patients with a refractory disease course. At the time of first inclusion, patients were treated with non-steroidal anti-inflammatory drugs (NSAIDs) or methotrexate, but no biologicals. For the refractory time point samples, patients were treated with disease modifying anti-rheumatic drug (DMARDs) (leflunomide) and/or biologicals (Humira) after first sample inclusion due to the refractory nature of their disease.

    We have now updated the methods section (lines 455-463) of the revised manuscript with more information on patient recruitment, and included information on diagnosis, sex, age, disease duration and medication for every patient in Supplementary File 1.

    (2) As for the correlation analysis of the entire spectrum of node frequencies, the SFMCs and PBMCs isolated from 3 patients were conducted in the study. The sample size is too limited to obtain robust results and to make a convincing conclusion from the correlation analysis. And it is shown that a total of 9 JIA patients have been involved in the study. Could the author clarify it?

    In order to strengthen our observations, we now included single-cell transcriptomics data obtained from Zhang, et al. (https://doi.org/10.1038/s41590-019-0378-1). In this data, we identified a cluster of CD4+FOXP3+ Tregs (new Figure 2-figure supplement 2A and 3B) that showed increased frequency in RA patients (new Figure 2-figure supplement 2C), consistent with the high frequency of Tregs that we observed in our JIA SFMC samples. Additionally, the expression of markers of chronic TCR activation (PDCD1 (PD1), CTLA4 and ICOS), and cytokines (TNF, IFNG and GZMB) were significantly increased in RA compared to OA, in line with what we observed in JIA SFMC (new Figure 2-figure supplement 2D). These results demonstrate that, although the number of JIA patients included in our study is low, obtained results are robustly reproducible in an independent, comparable dataset.

    We do agree with the reviewer that the low number of patients included in our study warrants further validation. Therefore, we have now added the following line in the discussion to highlight this (lines 369-371): “Further validation of our observations in larger cohorts of JIA patients should help to substantiate these results and aid the identification of pathogenic Treg populations across patients.”.

    Regarding the number of patients included in our studies, we have now included Supplemental File 1, which clarifies which JIA patients have been used for each analysis in our study.

    (3) The results of the study indicate that the hyper-expanded T cell clones are shared between left and right knee joints. Since JIA may affect one or more joints, did the author check other joints to see if the same expanded T cell clones infiltrate multiple joints, such as hand or wrist?

    Indeed, it would be interesting to see whether hyper-expanded clones are shared between multiple inflamed joints other than knees. However, samples from other locations are very difficult to obtain and very little synovial fluid can be extracted from joints such as hands and wrists. Therefore, the number of cells obtained from these joints would be too limited to perform mass cytometry or TCR sequencing. Thus, we chose to focus on synovial fluid from knee joints in our studies. Moreover, for oligoarticular JIA patients, only the large joints are affected (of which the knees are most typical), so for these patients it would not be possible to include other joints.

    (4) For Fig.2B, the Treg CD25+FOXP3+ population was significantly enriched in synovial fluid (SF). Is it from the left knee joints or the right knee joints?

    Figure 2B shows data from both knee joints. We have now clarified this in the figure legend by adding “For SFMCs, data from the right and left knee joints for all patients is shown” (lines 179-180).

    And in the context of Line 144-148, it indicated the SF, however, the title of axis in Fig.2B indicated Synovial Fluid Mononuclear Cells (SFMCs). Please keep consistent.

    We thank the reviewer for bringing this to our attention. We have critically revised the manuscript and made the use of SF versus SFMCs more consistent.

    (5) For the longitudinal sampling timelines of JIA patients shown in Supplementary Fig.3, the interval of PB and SF sample collection is not consistent. And only 1 patient completed 4 visits and the sample collection. It is hard to make any conclusion from 1 patient.

    In our study, we had longitudinal samples available for 5 JIA patients for which we performed TCR sequencing of Tregs from SFMCs from different joints (right or left) at least two time points. In the manuscript we mainly focused on patient 1, as for this patient the largest amount of data was available. However, for all other longitudinal patient samples included, we also show that dominant clones persist over time (Figure 4A and 5A). To further highlight that our observations are not just applicable to one patient, but consistent for all patients included, we now included detailed analysis for all patients in Figure 4-figure supplement 3 and Figure 5-figure supplement 1. This analysis shows that frequencies of shared TCRβs are consistent over time in all patients.

  2. eLife assessment

    In this study, the authors performed mass cytometry (CyTOF) analysis and T cell receptor (TCR) sequencing to study immune cell composition and expansion of joint-derived Tregs and non-Tregs in Juvenile Idiopathic Arthritis (JIA). They studied different joints affected at the same time and found that the composition and functional characteristics of immune infiltrates are strikingly similar between joints within one patient. The research design of this study is appropriate and the methods used in this study are adequately described in the manuscript. The study may be potentially beneficial for the JIA treatment.

  3. Reviewer #1 (Public Review):

    The paper studied spatial-temporal characteristics of dominant T cell clones in juvenile idiopathic arthritis. The authors found that the composition and functional characteristics of immune infiltrates are strikingly similar between joints within one patient, and observed a strong overlap between dominant T cell clones, especially Treg. Moreover, in localized autoimmune disease there is auto-antigen driven expansion of both T effector and Treg clones, that are highly persistent and are (re)circulating.

  4. Reviewer #2 (Public Review):

    In the manuscript, Mijnheer et al mainly exploited CyTOF Helios mass cytometer and TCRβ repertoire sequencing to investigate the T cell composition and distribution in peripheral blood and synovial fluid, and further explored the temporal and spatial dynamics of regulatory T cells (Tregs) and non-Tregs in the inflamed joints of Juvenile Idiopathic Arthritis (JIA) patients. Their results indicate that the activated effector T cells and hyper-expanded Treg TCRβ clones found at the inflamed joints are highly persistent in the circulation, and the dominant of high degree of sequence similarity of Treg clones could serve as the novel therapeutic targets for the JIA treatment. Overall, the research design is appropriate, and the methods are adequately described in the study. However, several issues are required to be addressed.

    (1) The criteria for the JIA patient's recruitment should be clearly presented in the method section. For example, what is the specific included criteria and excluded criteria? Or did the patients take medicines for the treatment during the study?
    (2) As for the correlation analysis of the entire spectrum of node frequencies, the SFMCs and PBMCs isolated from 3 patients were conducted in the study. The sample size is too limited to obtain robust results and to make a convincing conclusion from the correlation analysis. And it is shown that a total of 9 JIA patients have been involved in the study. Could the author clarify it?
    (3) The results of the study indicate that the hyper-expanded T cell clones are shared between left and right knee joints. Since JIA may affect one or more joints, did the author check other joints to see if the same expanded T cell clones infiltrate multiple joints, such as hand or wrist?
    (4) For Fig.2B, the Treg CD25+FOXP3+ population was significantly enriched in synovial fluid (SF). Is it from the left knee joints or the right knee joints?
    And in the context of Line 144-148, it indicated the SF, however, the title of axis in Fig.2B indicated Synovial Fluid Mononuclear Cells (SFMCs). Please keep consistent.
    (5) For the longitudinal sampling timelines of JIA patients shown in Supplementary Fig.3, the interval of PB and SF sample collection is not consistent. And only 1 patient completed 4 visits and the sample collection. It is hard to make any conclusion from 1 patient.