Th17 and CD24^hiCD27⁺ regulatory B lymphocytes are biomarkers of response to biologics in rheumatoid arthritis

The development of different targeted immunotherapies and treatment strategies (treat to target (T2T) and tight control) has allowed significant improvement in the prognosis of rheumatoid arthritis (RA), and long-lasting clinical remission may now be a consideration [1]. Approximately one third of patients, however, experience primary non-response or insufficient response to these different drugs [2, 3]. The search for biomarkers that could predict response to biologic drugs is therefore crucial, in order to ensure that the treatment chosen will be sufficiently effective to halt the progress of the disease, whilst avoiding prescribing patients ineffective, potentially deleterious (leading to infectious or neoplastic complications, etc.) and expensive treatment [4].

Different lymphocyte subpopulations play a major role in the physiopathology of RA, such as regulatory T cells (Treg), T helper 17 (Th17) cells and regulatory B lymphocytes (Breg). In the physiological condition, CD4 + CD25^hiFoxP3⁺ regulatory T cells constitute 5–10% of the population of CD4⁺ T lymphocytes in the blood [5]. A functional deficit in Treg with a reduction of their suppressive activity has been observed in RA [6]. A loss of Treg capacity to suppress effector CD4⁺CD25⁻ T cells has been observed, prior to anti-TNF treatment for active RA, and a recovery of this defect was observed in patients who respond clinically to anti-TNF drugs with a quantitative increase in Treg frequencies post treatment [6]. Moreover, the quantitative restoration of the Treg upon achieving remission with anti-TNF has been associated with greater probability of long-term remission [7]. An increase in T lymphocytes, and Treg in particular, was furthermore recorded after the use of abatacept, a biologic drug that reproduces the physiological inhibitor role of CTLA4 [8].

The identification of the Th17 lymphocytes, a subpopulation of CD4⁺ T-lymphocytes that produces the highly proinflammatory cytokine IL-17 [9, 10], opened the way for new understanding of the physiopathological and new treatment options (secukinumab or ixekizumab) for RA [11, 12]. Th17 cells are characterised by considerable plasticity (interconversion with Th1 cells), which themselves can also develop into Th17 under some pathophysiological circumstances [13‐16]. An inverse correlation between blood Th17 and anti-citrullinated peptide antibodies (ACPA) levels has been observed in patients with RA [17], illustrating the interrelationship between this subpopulation and humoral immunity in this autoimmune rheumatism. The role of Th17 appears to be particularly relevant during the onset of RA and later may be influenced by treatments, increasing under methotrexate (MTX) [18] and decreasing in patients that do not respond to infliximab [19].

B lymphocytes and humoral immunity also play a key role in RA, notably with ACPA and rheumatoid factor (RF) production [20]. Breg, by analogy to Treg, represent a subpopulation of B cells that have immune-regulating properties [21] and play a role in peripheral tolerance [22], mainly via the substantial secretion of IL-10 [22, 23]. Discrepancies in the phenotype of human Breg have been described [24‐28], although the most commonly accepted is the inclusion of putative Breg within the immature transitional T2 population (CD19+CD24^hiCD38^hi). Additional regulatory properties have been described in the memory B cells CD24^hiCD27⁺ phenotype (also producing IL-10) [29]. These cells are reported to be able to inhibit the proliferation of CD4⁺ T cells differentiating between Th1 and Th17 cells, producing different proinflammatory cytokines. Although they are reported to be quantitatively and functionally altered in patients with active RA (reduction of their capacity to induce both Treg from CD4+ and prevent the conversion of Treg into Th17 cells) [30, 31], a recent report observed that a high baseline level of Breg appeared to be predictive of a clinical European League Against Rheumatism (EULAR) response at 3 months upon anti-TNF treatment [25].

Thus, the main aim of our study was to describe the different lymphocyte subpopulation frequencies in RA (regulatory B and T cells and Th17 lymphocytes) before and under biologic drugs, in order to test the correlation with subsequent response to the drug received and to identify potential biomarker value that could be used to predict therapeutic response.

Our data describe the effects of biologic drugs on B cells (CD24^hiCD27⁺ Breg and T2/Breg), Treg and Th17 subsets, immuno-regulating lymphocyte subpopulations, involved in numerous autoimmune pathologic conditions such as RA. In our study, participants were representative of the patients commonly encountered in clinical practice (2/3 women, median age approximately 60 years). One third of the RA had developed within the previous 2 years. The median DAS28 was 4.24 and 3/4 of the subjects had immunopositive (RF and/or CCP+) RA and almost 50% of them had erosive RA.

In our study, T2/Breg cells were significantly reduced, both in percentage and in absolute numbers, in patients compared with controls. Other studies report a quantitative decrease in Breg in patients with RA compared to healthy control subjects and in patients with active RA compared with inactive disease [30, 31]. This lymphocyte subpopulation may therefore represent an objective biomarker of the activity of the disease. Moreover, we observed positive correlation in absolute numbers between T2/Breg and Treg cells in controls but not in patients, both at inclusion and during treatment, which may reflect dysfunction of the T2/Breg-Treg cell balance in RA. The T2/Bregs were also lower in patients with RF, as described in another study [30], in which negative correlation was observed between Bregs and RF and ACPA levels. There is no consensus as yet on the role of these antibodies in RA, but it has been proven that their presence is a marker of poor prognosis. Our data showing lower T2/Breg levels in patients with RA compared to controls, and in RF⁺ patients with RA, may therefore indicate a “protective” role.

DAS28 remission at M6 was associated with significantly higher CD24^hiCD27⁺Breg frequency at baseline. Likewise, higher CD24^hiCD27⁺ Breg frequency was initially observed in patients who had a good EULAR response at M6 (although not significant). The literature suggests that higher pre-treatment levels of CD24^hiCD27⁺ Breg and T2/Breg may be predictive of a good EULAR response at 3 months with anti-TNF treatment [25]. A retrospective study of patients with RA receiving abatacept, showed that the level of CD24^hiCD27⁺ Breg prior to treatment was not significantly different between patients who were EULAR responders and non-responder patients at M6 [34], whereas our results showed that a high initial level of CD24^hiCD27⁺ Breg is significantly associated with a good EULAR response at M6 under abatacept. Therefore, CD24^hiCD27⁺ Breg, that were higher at M0 in patients with DAS28 remission and in patients with good EULAR responses at M6, may represent a predictive biomarker for response to treatment, notably with abatacept. Finally, the activity of the disease (evaluated by DAS28) correlated negatively with the proportion of T2/Breg cells (and CD5⁺CD1d^hi [25] and IL-10 + CD5Cd1d^hi and IL-10⁺TIM-1⁺ [30, 31]) which also suggest a potential of this subset for disease monitoring.

Several studies reported more Treg in healthy subjects than in patients with early RA, but others reported similar levels [35‐37]. Our data showed no significant difference in Treg at baseline between the patients and the controls, most likely due to the small number of participants in our current work, as we previously identified significant differences with larger numbers of subjects [38]. Whereas the frequency of Treg cells was similar at baseline in patients treated with the different drugs (anti-TNF, tocilizumab and abatacept), we observed a decrease in Tregs under abatacept but an increase under anti-TNF drugs. As previously described [6, 7], this may be partly explained by the removal of the repression exerted by this major proinflammatory cytokine on these cells.

In our study, the pre-treatment level of Treg was not predictive of DAS28 remission or EULAR response at M6. However, we observed a significant evolution of Treg under biologic drugs, with a recovery of these cells in patients with DAS28 remission at M6. Their level is described in the literature as being higher in drug-naive patients with early disease, who will achieve remission with MTX [38] and recovery of these cells under anti-TNF drugs was associated with a greater probability of long-term remission on anti-TNF drugs [7]. Other authors also report an increase in Treg over the course of anti-TNF [6] and abatacept [8] treatment only in patients with a good response. Treg may therefore represent an efficient biomarker for monitoring patients but not for predicting their personal response.

Th17 cell levels were significantly lower in patients than in the control group. Another study, however, described elevated levels of Th17 cells in RA, but only in patients with recent onset of disease [18]. There is a large amount of literature on such quantification with major discrepancies due to the use of different techniques (with or without a polarization-inducing condition) and the use of different phenotypes to identify Th17 cells [39, 40]. We did not find any association between the level of Th17 cells and the presence or absence of ACPA, although inverse correlation between the frequency of Th17 cells in the blood and the ACPA level had been previously described [17]. In addition, there was positive correlation between the absolute numbers of CD24^hiCD27⁺ Breg and Th17 cells in patients with RA at baseline, but not in the controls. This was no longer observed after 3 and 6 months of treatment. This would therefore suggest initial pathologic correlation in RA, which disappeared over the course of a biology-modifying treatment such as a biologic drug.

Reduction in Th17 cells under MTX [18] and increase in non-response to infliximab [19] have been described In our study, patients with a good EULAR response at M6 had a significantly lower frequency of Th17 cells compared with moderate responders or non-responders, both at inclusion and at all the treatment points. A small proportion of Th17 cells at baseline was associated with a good EULAR response at M6 under anti-cytokine treatments but not under abatacept. The levels also did not evolve under treatment, whether the patients responded or not, which possibly reflects a lack of implication of this cell population in RA at late stages of the disease continuum (i.e. DMARD-resistant, biologic-drug-naive), which appears to contribute to the explanation of the disappointing results with treatments targeting IL-17 in clinical trials among patients with established RA [41]. In contrast, the positive correlation between the absolute numbers of Treg and Th17 cells, both in the controls and in the patient groups is likely to reinforce the hypothesis of an early role of Th17 cells in establishing chronicity [18]. It should finally be noted that low levels of Th17 cells may have predictive value for response to “anti-cytokine” treatments.

This work aimed to establish pilot data using routine immunology hospital services protocols towards describing the evolution of different lymphocyte subpopulations in patients with RA under biologic drugs with different mechanisms of action, and assessing whether there is correlation between their levels and the later therapeutic response. Whereas, to date, only RF and ACPA are used to attempt to stratify patients for personalised treatment, CD24^hiCD27⁺ B cells and Th17 cells may offer predictive value as biomarkers of therapeutic response under biologic drugs and may be able to guide clinicians in their choice of a tailored treatment. If validated, our data would suggest the prescribing of abatacept if the patient has high baseline levels of CD24^hiCD27⁺ B cells, whereas a low initial level of Th17 cells may guide the choice of an anti-cytokine. As this was a pilot study, it remains crucial that our results are confirmed on a wider scale before being used to guide our therapeutic strategies.