There have been a lot of studies dealing with the iR of T cells in human RA, but only little studies have been dedicated to the iR of B cells in RA, especially in patients under bDMARD treatments. The present investigation might have demonstrated a consequential change in iR of BCR in RA patients for the first time. It is accepted that clonal diversity is generally getting low in disease state8, 23. We have shown that RA disease baseline condition is matching to what would be expected in CDR3 sequence diversity change.
The present investigation has also shown that RA disease activity was inversely correlated with D50 level, i.e., for the CD3 clonal diversity, patients with active RA demonstrated low D50. Both csDMARD and bDMARD treatment groups have shown steady growing trend of D50 values compared with their baseline samples along the treatment course, which is matching to what would be expected for disease improvement. The DAS28 values of two patients, No.13 and No.15 were decreasing accompanied with clonal expansion in the bDMARD group. The DAS28 values of Patient No. 13, which were 5.50, 5.43, 4.65, and 4.06 in sequence along the timeline, exhibited a decreasing trend in disease activity. The DAS28 values of Patient No. 15, which were 6.31, 5.59, 5.52, and 4.85 in sequence along the timeline, also exhibited a decreasing trend in disease activity. But in csDMARD group, one patient (No. 4) showed irrelevance of DAS28 to D50 level (Table 2 & Table 3), i.e., while improving D50 level, there was no improvement in terms of DAS28. Another patient (No.5) revealed the same trend as that in bDMARD group, which were 4.48, 4.71, 3.49 and 3.19 of D50 in sequence along the timeline, exhibiting a decreasing trend in disease activity. However, in contrary to increase in clonal diversity, the last DAS28 value on both cases was still above 3.2, indicating no significant improvement. Beside possible confounders such as undercurrent infection, this may conversely suggest D50 level is more sensitive than traditional DAS28 value to reflect disease activity. After all, pain or tenderness is so subjective that varies among patients, irrelevant to actual inflammatory status. Since tender joint count is one of the components of DAS28, the disease activity can also not be so reliably assessed by DAS28. Instead, D50 can be a better companion indicator of disease improvement trend, superior to DAS28 score. For a long time, lacking reliable biomarkers for prompt stratification of individual patients to fit the most appropriate and effective medications has rendered current treatment consensus for RA so challenging24. D50 level may potentially serve as an immediate biomarker to overcome this predicament.
Regarding the PCA, both “specific treatment” naïve samples did show a clustering effect, implying that specific clonal expansion pattern is present for RA. We have performed shared profile analysis on patient datasets of disease baseline to identify shared dominant CDR3 sequences. We identified a shared profile of 608 CDR3 clone sequences, i.e. clone expansion sequences which were present in at least two patients. Sequence “ARDLDY” (A=alanine R=arginine D=aspartic acid L=leucine Y=tyrosine) is the most significant CDR3 expansion sequence in these RA patients. Although currently, we still could not identify specific peptide that contains this sequence, it is expected that it could eventually be clarified when more data are accumulated.
We have also performed shared profile analysis on patient datasets of disease progression to identify shared dominant CDR3 sequences in patients with csDMARD/ bDMARD treatment. PCA analysis on CDR3 shared profile revealed a divergent tendency is some patients both in csDMARD/ bDMARD groups (Sample No. 5 in DMARD group, No.13, No. 15 in biologic group) indicating changes in immune diversity. Besides, the D50 values of Patient No. 5, No. 13 and No. 15 also exhibited an increasing trend in immune diversity, which were 9.7, 5.3, 12.8, 35.6; 5.3, 8.9, 14.2, 11.2; and 3.8, 8.0, 12.4, 5.6 in sequence along the timeline respectively, exhibiting an increasing trend in immune diversity. Taken together, PCA-measured divergent profiles can be a good indicator for an increase in immune diversity expressed as D50 values. On the other hand, serial detections during treatment course also exhibit clustering effect, supporting our hypothesis that specific clonal expansion pattern in individual patient may occur in response to some unique exogenous stimuli such as csDMARD/ bDMARD treatments within a same kind of underlying disease such as RA, regardless of disease improvement or deterioration.
Oral and gut microbiota have been thought to be one of the environmental factors that may enhance the development of RA25–28. For example, periodontitis caused by microorganisms might alter post-transcriptional regulation and citrullinated self-protein, leading to autoantibody production and local inflammation, and finally triggering the onset of RA. The change of healthy microbiota and pathogen-host immune system interaction may integrate the reduced immune diversity and BCR CD3 sequences clustering effect. Other environmental risk factor, especially cigarette smoking may also result in same clustering effect, eventually precipitating RA development29–31. On the contrary, treatments such as csDMARD/bDMARD break down the vicious cycle and lead to increased immune diversity. Clinically, autoantibodies such as RF and anti-CCP, though with very high specificity for disease entity, can be detected up to a decade before the development of RA32. Hence, no one can predict the disease onset of RA. CDR3 sharing profile analysis may potentially predict the development of RA and guide physicians to a timely and appropriate intervention. Clustering effect may represent inchoate of disease and serves as a predictor biomarker; however, more data are needed to confirm this hypothesis.
In spite of the present promising findings, our investigation holds some limitations. Firstly, this study was restrained by a small sample size, which may limit the statistical power for detecting the variation of the B-cell repertoire in clinical assessments. Yet, we still observed a trend toward a decrease of BCR iR diversity in RA patients prior to treatment and an increase in it after either csDMARDs or biologics, and a larger cohort study is required for solid validation. Secondly, our study only focuses on peripheral B cell repertoire in RA patients without B-cell and/ or plasma-cell clones in synovial tissue, and we will plan to extend our study in the future. Finally, we measured and analyzed the BCR IGH CD3 iR sequencing to monitor the disease activity, and further functional investigation will be conducted to elucidate the potential roles in the pathogenesis of RA.
In conclusion, we have successfully established a pipeline of experimental design, data acquisition, processing, and analysis to be used in monitoring the disease progression of RA. PCA is used to perform dimension reduction to make sense of CDR3 sequences variations, and the BCR CDR3 sequences do have a clustering pattern upon PCA representation which is an indication of specific clonal expansion in RA. We demonstrated that BCR CD3 iR sequencing can be a good tool to follow up on the disease progression of individual RA patients. Low BCR repertoire diversity was noted in treatment naïve RA patients. Conversely, immune diversity expands upon recovery or remission from active disease after csDMARDs and biologics treatment.