Τhere are several lines of experimental evidence that B cell depletion treatment may be related, directly or indirectly, with alterations of T cell subpopulations as well. Experimental animal studies have shown that B cell depletion in mice with collagen-induced arthritis, a T cell mediated disease, delayed arthritis [1]. In murine proteoglycan-induced arthritis, B cell-depleting therapy resulted in reduced CD4+ T cell reactivity [2], or in an increased number of Tregs and an enhanced suppressive capacity of Tregs [3]. In humans, B cell depleting therapies have also provoked alterations of T cells in patients with autoimmune diseases [4]. Patients with lupus nephritis receiving RTX displayed an enhanced expression of genes such as FoxP3 [5]. A study from our department suggested that patients with scleroderma developed reduced numbers of CD4+IL4+ T cells following B cell depletion [6].
In patients with RA, RTX reduced the number of circulating CD4+ T cells [7] particularly after multiple RTX cycles [8]. Lymph node resident T cells decreased in patients with RA following RTX administration [9]. Peripheral CD20+ T cells were also depleted in patients with RA receiving RTX [10]. RTX induced the expression of the previously decreased chemokine receptor CCR5 on the surface of CD4 + T cells in patients with RA [11].
Giollio et al studied 51 patients with RA treated with RTX for 4 years. The previously reduced numbers of peripheral natural killer cells in patients with RA were reportedly increased following RTX treatment during the 1st and even further during the 2nd year of treatment [12]. B cell depletion therapy did not alter the numbers of Tregs in the circulation of patients with RA [13]. RTX was reportedly capable of reducing Th17, but not Th1 and Treg T cells in the synovial tissue of patients with RA [14]. Our study analyzed peripheral Th17 cells and Tregs in patients with RA following RTX treatment.
We report herein that circulating Th17 cells were significantly decreased after RTX treatment. A potential downside of our study is the small number of patients enrolled as well as the relatively short follow-up period (16wk), that might not be entirely representative of the longer-lasting effects of RTX. The previously mentioned study of van de Veerdonk et al examined for potential changes of synovial but not circulating Th17 cells after RTX treatment in a small number of patients with RA (n = 12) after 12 weeks of treatment [14]. The authors reported that synovial Th17 cells (defined as CD3+IL17A+ T cells) were reduced following RTX administration and, at the functional level, the production of cytokines IL-17A, IL-21 and IL-22 was similarly reduced.
We also evaluated for potential changes in sTh17 cells in our patients with RA receiving RTX. Surface Th17 cells are a subset of “bona fide” Th17 cells expressing IL-17A on their surface membrane [37]. They represent a small subpopulation of the “classical phenotype” Th17 cells. Such sTh17 T cells express quantitatively less IL-17A on their surface compared to the IL-17A expressed intracellularly. Nevertheless, sTh17 cells are considered as potent effector cells because of the high expression of costimulatory, adhesion and activation molecules. In our study, evaluating a small number of patients, RTX significantly reduces this small but potentially “inflammatory” subset as well.
In order to address the question of specificity of our results assigned to RTX treatment, we chose to evaluate 3 different patient groups: apart from the RTX group, we included in our study 2 additional control populations. Biological disease-controls in our study were RA patients treated for the first time with the T cell-targeting agent ABA and disease-control group consisted of patients with RA already receiving standard treatment. Our data suggest that percentages of Th17 cells decrease in patients with RA treated with B cell depletion; nevertheless, we did not directly ask questions regarding their functional status or the production and/or secretion of relevant cytokines.
We further analyzed our results and report that responders had a more pronounced reduction of Th17 cells when compared to non-responders. In addition, our data suggest that ACPA (+) patients had a significantly greater reduction of Th17 cells, unlike ACPA (-) patients; such a differential response was also seen in RF (-) but not in RF (+) patients.
In contrast to our Th17 data, our experiments suggest that no changes in Treg numbers are seen following B cell depletion. Our Treg cells data are in agreement with previously published results. In their uncontrolled study, Feuchtenberger et al. measured Tregs at 6 and 12 months after treatment with RTX in 17 patients with RA [13]; CD4+CD25+FoxP3+T cells were analyzed and were reportedly unchanged in numbers after RTX treatment. It has been suggested in other studies that Tregs in patients with RA may be enriched either in the periphery [15] or in the synovial fluid [17]. In contrast, other studies have reported diminished Treg numbers in the periphery of patients with early RA [16] and diminished expression of CTLA4 and hence decreased Treg suppressive capacity in patients with RA [18]. In our study, the comparison of Treg cell numbers between patients with RA and healthy volunteers revealed no differences.
The experimental design of our study clearly suggests that RTX-induced reductions of peripheral Th17 are RTX-specific. In contrast to B cell depletion, treatment with other biologicals, such as ABA did not change the T cell subpopulations that we analyzed. In addition, the disease itself, being a process with remissions and relapses, is not responsible for the changes of Th17 cells reported herein, as suggested by the comparisons with our disease-control group of patients, because neither Th17 nor Tregs were different in numbers over time in patients with RA already receiving standard treatment. Our data depict that such T cell subset percentages remained similar at 2 different time points, despite changes of the DAS28 scores of our patients, i.e despite changes in their disease activity and therefore of their inflammatory status.
Potential changes in T lymphocyte subpopulations have been evaluated in patients with RA being treated with other biological agents. For instance, Nakayamada et al. studied 108 patients with RA receiving for the first time TNF-α blockers (n = 42), abatacept (n = 40) and tocilizumab (TOCI) (n = 22) [19]. Peripheral T cells were examined at 24 weeks post treatment; TNF-α inhibitors were associated with increased Th17 cells. ABA reportedly significantly reduced Th17 and Tregs. TOCI treatment had no significant impact on numbers of the T cell subpopulations examined in that study.
Treatment with ABA in a study of Alvarez-Quiroga et al in 30 patients with RA revealed a decline of Treg number but, in contrast, an enhancement of their suppressive capacity at week 12 compared to baseline [20]. Adding to the confusion, the exact opposite findings came from another study. In that, Bonelli et al enrolled 15 patients with RA to receive ABA [21]. The study concluded that ABA augmented Treg numbers and inhibited activity of Tregs in patients with RA at weeks 2 and 4 after administration of ABA. Picchianti et al studied 25 patients with RA that received ABA due to unresponsiveness to TNF-alpha blockers [22]. The results suggested an improvement of suppressive function of Treg T cells although there were no significant changes in their numbers following ABA treatment. In another study of Pesce et al., TOCI was infused monthly in a rather small number of patients with RA (n = 8) [23]. T cells were evaluated before and every 8 weeks after treatment. Th17 were not significantly altered by week 24. Tregs and “Th1/Th17 cells” (a subpopulation commonly found in inflamed tissues of RA) were increased after treatment with TOCI. Samson et al also concluded that TOCI corrects the disturbed balance of the ratio Tregs/Th17 in patients with RA [24]. Results obtained from 15 patients revealed that TOCI decreased Th17 and increased Tregs after 2 months compared to baseline. The small number of patients (n = 15) and the early evaluation of the T cell subpopulations (at 2 months) could potentially be considered as limitations of this study. Perhaps more importantly, one cannot aim towards the restoration of the imbalance between Tregs and Th17 (expressed as the Treg/Th17 ratio), since this a clearly arbitrary value.
Previous studies have implied the positive effect of TNF-alpha blockers on increasing and restoring Treg numbers and function in patients with RA [25]. Szalay et al studied 32 patients with early RA unresponsive to DMARDs that received TNF-α inhibitors [26]. Tregs were counted 4 and 8 weeks later and they were reportedly increased when compared to baseline values, whereas the number of peripheral Th17 cells was practically unchanged. However, the potential effects of TNF-α blockers on Treg and Th17 cells can be judged as controversial at best [27–32].
Our group of RTX-treated disease-control patients clearly displayed no changes in the number of Th17 cells. Tregs though were decreased after B-cell depletion. The effects of RTX on Tregs in patients with SLE have been previously reported. Tregs have been reportedly decreased in the periphery in patients with active SLE [33, 34]. Tregs were significantly increased after RTX treatment in patients with SLE [5, 35, 36]. These studies, in contrast to ours, included patients with lupus nephritis. In the study by Sfikakis et al, Tregs were evaluated by mRNA extraction and not by flow-cytometric phenotyping [5]. Other studies employed flow cytometry evaluations and analyzed Tregs either at months 1, 2 and 3 of treatment with B cell depletion [35] or at certain time points after treatment with a special emphasis on time of B-cell depletion and B cell repopulation [36]. In our study we enrolled in our RTX-treated patients only 5 patients with SLE; none had nephritis. The comparative analysis was performed at week 8 after RTX treatment. Therefore, previous studies of RTX effects on Tregs in other autoimmune diseases are not directly comparable with our study. A limitation of all studies mentioned above, including our own, is that studies are not longitudinal and analyze subgroups of patients who share certain characteristics.
To summarize all the above, it is evident that the numbers of patients with RA in each study, the time intervals the T cell subpopulations are evaluated after the administration of a biologic agent and even the very definition of the phenotype of the T cell subpopulations of interest, display a great heterogeneity among different studies resulting perhaps in heterogeneous and perhaps even contrasting results.