Periductal aggregation of lymphocytes in the lacrimal and salivary glands (SGs) is promoted by autoimmune epitheliitis (1) and leads to xerophthalmia, xerostomia, and the production of antibodies to self (2). This disorder may occur alone as primary Sjögren's syndrome (SS) or against a background of other connective tissue diseases as a secondary symptom. Contrary to the long-held notion that primary SS is a T cell–mediated disorder, B cells appear to be key to its pathogenesis (3).
The paradigm based on B cell involvement, coupled with the ever-growing variety of B cell subsets, raises the issue as to which of these subsets underpins the autoimmune features characteristic of the disease. As soon as B cell antigen receptors are functional, B cells migrate from the bone marrow to secondary lymphoid organs, where they mature further. The emerging transitional B cells have long been described in rodents (4) and have recently been identified in the peripheral blood (PB) of humans (5). As transitional type 1 (T1) B cells, they present as CD20+,CD5+, CD10+/−,CD21+/−,CD23+/−,IgM+,IgD+/−,CD38++. Once they have evolved to type 2 (T2) B cells, they become CD20+,CD5+/−,CD21++,CD23+/−,IgM++, IgD++,CD38+/−. The latter subset differentiates either to noncirculating cells (6) that populate the marginal zone or to circulating cells that constitute germinal centers within lymphoid follicles (7). Developmental stages are then identified on the basis of the relative expression levels of IgD and CD38 on mature B (Bm) lymphocytes (8). As a consequence of their selection outside germinal centers in the presence of short-lived plasma cells, CD38−,IgD+ naive Bm1 cells progress to become CD38+,IgD+ activated Bm2 cells, some of which are selected as CD38++,IgD+ Bm2′ germinal center founder cells. Then, they differentiate into CD38++,IgD− Bm3 centroblasts and Bm4 centrocytes. Two types of B cells arise from germinal centers: CD38−,IgD− Bm5 memory B cells and CD38++, IgD− plasmablasts, which were first described by Odendahl et al (9). These return to the bone marrow, where they differentiate into long-lived plasma cells.
Patients with primary SS exhibit an increase in Bm2/Bm2′ cells (10) and a decrease in memory Bm5 cells (11). Cells of the second subset accumulate in the SGs, germinal centers of which include T2 B cells and B cells resembling marginal zone B cells (12). That the B cell–activating factor BAFF participates in the development of such abnormalities is suggested by the correlation between its high rate of production in primary SS and the increased number of circulating Bm2/Bm2′ cells (13). This view is reinforced by the presence of receptors for BAFF on B cells sequestered within the SGs (14).
The importance of B cells in autoimmune processes was the rationale behind the use of the anti-CD20 monoclonal antibody (mAb) rituximab in the treatment of primary SS. Furthermore, the pioneering work by Edwards and Cambridge (15) in rheumatoid arthritis (RA) has spawned a number of trials in autoimmune diseases (16–18) such as idiopathic thrombocytopenic purpura (ITP). The kinetics of B cell reconstitution have been examined in patients with systemic lupus erythematosus (SLE), RA, and more recently, lymphoma (19–21).
Rituximab has been administered to patients with primary SS and has been claimed to lead to B cell depletion in the PB (22) and SGs (23). However, studies of the sequential repopulation of B cells following treatment of this disease have not been performed. There are several postulated mechanisms for the action of rituximab in B cell depletion. Should it turn out that antibody-dependent cell-mediated cytotoxicity (ADCC) predominates, its effects in primary SS would be dependent on the V and the F alleles of the Fcγ receptor IIIa (FcγRIIIa) –158 polymorphism, because the V and F alleles display a high affinity and a low affinity for IgG1, respectively (24). In addition, the baseline level of serum BAFF might influence the subsequent B cell regeneration but, in turn, depletion of B cells might affect the production of BAFF (25).
This study provides a unique opportunity to gain insight into the developmental stage at which autoreactive B cells escape tolerance, since data concerning whether rituximab depletion of B cells extends to tissue B cells are lacking. Our study was therefore conducted to address 3 issues. What are the dynamics of B cell subset redistribution following rituximab treatment in primary SS patients as compared with ITP patients? Do FcγRIIIa polymorphism and the preexisting BAFF level influence B cell depletion and repopulation? What are the kinetics of the reappearance of B cell subsets in the PB and SGs of the treated patients? Our findings are presented below.
- Top of page
- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
In this study, we show that 2 infusions of rituximab reduced B cells to <0.1% of circulating lymphocytes for >2 months in all but 1 of the primary SS patients. In addition, we provide evidence that all B cell subsets were equally sensitive to this treatment. Further B cell depletion was also achieved in solid tissues as early as 4 months after treatment, and this lasted for 12 months or more. Parenthetically, B cell reconstitution seems to begin earlier in primary SS than in SLE (19) and RA (20); however, differences in the treatment protocols mean that comparison of our results with previous reports should be viewed with caution. We also observed that a number of plasmablasts were sustained in the PB because of their protection from the effects of rituximab by their lack of CD20 expression as well as their BAFF-promoted survival (37). Similar plasma cell precursors have been detected after rituximab treatment in SLE (19) and RA (20).
The pattern of B cell repopulation was the same in all treated patients with primary SS. A high frequency of naive B cells carrying IgD and CD38 were up-regulated, and a few memory B cells were characteristically seen. The former B cells, IgD+,CD38++, are naive B cells exiting the bone marrow, and unlike germinal center founder cells, they express CD10, CD24, CD62L, and CD38. The latter cells distinguish T1 B cells, which are CD38++, from Bm1/Bm2 cells, which are CD38+/–. Thus, as hypothesized by Anolik et al (21), B cell repopulation after rituximab treatment recapitulates B cell ontogeny.
Interestingly, similar to immature B cells, mature Bm1/Bm2 cells carry CD5. Restricted progenitors of CD5+ cells, which have just been identified (38), could differentiate into circulating CD5+ B cells during adulthood. Thus, rituximab-induced B cell elimination appears to trigger a second round of ontogeny, with immature innate CD5+ B cells (39) and conventional B cells acquiring CD5 within the Bm2 subset (40).
The sole difference between the patients was in the timing of the reappearance of B cells. In this regard, we addressed the issue of which factors predict the duration of B cell depletion. Previous reports link the response to rituximab in SLE patients with FcγRIIIa polymorphism (35). Our limited data do not support a major role of FcγRIIIa polymorphism in patients with primary SS, at least in this group of study patients. Yet, whether or not ADCC is important in the therapeutic effectiveness of rituximab remains to be established. It is possible that FcγR polymorphisms other than FcγRIIIa-158 affect the killing function of macrophages and natural killer cells (41). Also related to dosing is the appearance in the recipient of an immune response to the infused rituximab (18). These were the findings that prompted us to launch a multicenter trial to compare a large number of primary SS patients. Correlation between rituximab levels and B cell depletion is currently being evaluated.
Relative to complement-mediated cellular cytotoxicity, caspase-dependent apoptosis, and sensitization to cytotoxic agents (18), the importance of ADCC as a mechanism for the activity of rituximab may vary from patient to patient. The efficacy of rituximab treatment is also diminished if the disease mechanism provides B cells with survival signals. In particular, repopulation of B cells might be modulated by BAFF through its effects on their lifespan (42). It is known, for example, that BAFF controls a steady-state number of circulating B cells that survive differentiation (43). This is consistent with equality in the length of time required for B cell repopulation in 3 of the 5 ITP patients and in the 6 group II primary SS patients, all of whom had low levels of BAFF. The influence of BAFF on B cell return is further supported by our previous finding that B cells infiltrating the SGs not only expressed BAFF receptors, but they also had the ability to synthesize this cytokine (14). As a result, rituximab-induced B cell depletion would decrease the synthesis of BAFF. Even so, BAFF could bind the newly formed B cells and thus be rendered undetectable in the ELISA.
Relevant to this issue is the suggestion that the beneficial effects of rituximab-based B cell depletion may be offset by a BAFF-mediated antiapoptotic effect on reemerging B cells (25). In this regard, the higher the level before treatment, the shorter the duration of the B cell lymphopenia. Similar bindings were reported in patients with B cell chronic lymphocytic leukemia (44). Thus, if rituximab is to be routinely applied as a treatment strategy for primary SS, advance knowledge of the level of BAFF may help in tailoring the doses of rituximab and the frequency of administration. Our results at 4 months posttreatment are consistent with the report that BAFF levels rose at 1–2 months (25) and decreased to pretreatment levels thereafter. Indeed, as stated by Cambridge et al (25), the relationship between the timing and speed of B cell repopulation and the levels of BAFF is likely to be complex.
One existing view is that treatment with rituximab may be less effective at purging B cells from sites of disease in affected tissues, although data on this topic are lacking. Hence, our study is novel in that sequential biopsies allowed us to compare the repopulating B cells in PB and SGs. The results indicate that at 24 months after rituximab treatment, B cells aggregating in SGs are T1 B cells. Since transfer experiments have established that T1 B cells give rise to T2 B cells, T2 B cells present in SGs at baseline should originate from T1 B cells. Accordingly, in primary SS patients, B cells should be initiating lymphoid neogenesis (45).
Memory B cells were detected early during repopulation, first in the PB and subsequently in SGs (compare Figure 5B with Figure 7). Later, they represented as few as 1% of circulating B cells. These findings are consistent with those reported by Anolik et al (19), Sanz and Anolik (46), and Rouzière et al (47). These are likely to be autoreactive memory B cells, and their depletion may have been less complete in solid tissues than in the PB. They might recirculate and home preferentially to afflicted organs. This sequence of events resembles the pathophysiology of memory B cell accumulation in the SGs, as proposed by Hansen and colleagues (11). Therefore, it is tempting to increase the number of infusions, rather than the dosage, of rituximab in an attempt to prolong the depletion of memory B cells and thereby delay their entrapment in SGs (48). Alternatively, due to the absence of resident memory B cells after treatment with rituximab (Weil JC: personal communication), new plasmablasts and plasma cells might have competed successfully in the bone marrow (49). Since the characteristic abnormalities in the proportions of PB and SG B cell subsets, and possibly in their functions (21), reappear 24 months after depletion, our data provide strong evidence that the microenvironment contributes to B cell abnormalities in this disease.
In summary, rituximab depletes B cells from the PB and SGs in patients with primary SS. A combination therapy that targets BAFF and CD20 can postpone B cell repopulation in patients with excessive BAFF because plasmablasts are protected from depletion by BAFF (30, 37). However, this picture is still far from complete (50). Rituximab should therefore be administered before irreversible atrophic changes occur in the SGs of patients with primary SS (51).
- Top of page
- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
Drs. Youinou, Pers, and Saraux had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
Study design. Pers, Youinou.
Acquisition of data. Daridon, Bendaoud, Le Berre, Bordron, Hutin, Renaudineau, Dueymes, Loisel, Berthou.
Analysis and interpretation of data. Pers, Devauchelle, Daridon, Youinou.
Manuscript preparation. Pers, Youinou.
Statistical analysis. Devauchelle, Saraux.