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Rituximab is widely used in autoimmune diseases including immune thrombocytopenia (ITP), although the mechanism of effect remains unclear. This study describes the effects of rituximab on platelet-associated antibodies (PA-APAs), B and T cell counts and clonality (IGHV and TRG@ gene rearrangements), FCGR3A (FcγRIIIa) and FCGR2A (FcγRIIa) polymorphisms and correlation to anti-CD40 ligand (CD40L) response. PA-APA levels fell more frequently in responders (6/8) than in non-responders (2/10: P = 0·08–0·15). Two responders had no PA-APAs. Two non-responders with a fall in PA-APAs had very high CD8 levels. One non-responder had a B cell clone, one responder and one non-responder had a T cell clone. 15/16 patients had the same responses to rituximab and antiCD40L. Patients with FCGR3A V/V polymorphisms were more likely to respond to rituximab (P = 0·03). In summary, the fall in PA-APAs in responders confirms the humoural effect of rituximab. Failure to respond in patients with very high CD8 levels, despite PA-APA fall indicates a role for T cell-mediated platelet/megakaryocyte destruction. Concordance of response to anti-CD40L suggests autoantibody-producing cells are under T cell control. Finally, the effect of FCGR polymorphisms on response confirms the importance of FCGR-mediated depletion of B cells in autoimmunity. This has implications on the pathology of ITP as well as the immunological effect of B cell depletion.
Immune thrombocytopenia (ITP) is an autoimmune disease in which the thrombocytopenia results from both accelerated destruction and sub-optimal production of platelets (Cines et al, 2009). Initial studies demonstrated a plasma-derived destructive effect, later identified as an anti-platelet antibody (APA) (Harrington et al, 1951). More recently, defects in cellular responses have been identified, including abnormalities in T-helper cells, defects in regulatory T cells, evidence that cytotoxic T cells can cause thrombocytopenia (Semple et al, 1996; Semple, 2002; Olsson et al, 2003, 2005; Yu et al, 2008).
The use of rituximab in autoimmunity developed because of its ability to deplete circulating B cells (Protheroe et al, 1999; Edwards et al, 2005). However, why B cell depletion is effective is not completely clear for a number of reasons. When rituximab is successful, resolution of autoimmunity occurs despite no change in immunoglobulin G (IgG) levels (Cambridge et al, 2006; Ferraro et al, 2008). Furthermore, changes in T cell number and function, seen in patients responding to rituximab (Stasi et al, 2007, 2008) and in mice models of B cell depletion (Bouaziz et al, 2007), imply that auto-reactive B cells contribute to the pathology either as an antigen-presenting cell (APC) or via cytokine effects stimulating auto-reactive, cytotoxic T cells.
This study takes the approach of extensive analysis in a small number of patients. Reported here are the laboratory findings from the original clinical cohort (Cooper et al, 2004) including changes in APA levels after rituximab and the influence of the T cell compartment and the FCGR3A V/F polymorphism on responses to treatment. In addition, in view of the impact on T cells on response to rituximab and the nature of CD40-CD40L in the T-B cell interaction, responses to rituximab were compared with results of treatment with anti-CD40 ligand (anti-CD40L).
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Despite almost universal B cell depletion, not all patients with ITP have a platelet response after rituximab. Differences in the occurrence, timing and quality of the response cause us to question our current understanding of how B cell depletion restores tolerance in patients with autoimmunity. Recent studies in patients with ITP imply that responses to rituximab are related to changes in T cells, in particular, resolution of TRBV skewing, changes in T-helper subsets, and improvement in regulatory T cell function (Stasi et al, 2007, 2008). Assessment of APAs after rituximab has not previously been reported. By intensively measuring both T cell numbers and anti-platelet antibodies in a small number of patients and their relationship to subsequent differences in responses to B cell depletion, we can speculate about the mechanism of effect of rituximab in ITP.
Despite well-known limitations (depending upon the assay) of the sensitivity and specificity of anti-platelet antibodies, reduction in PA-APA levels was associated with an increase in the platelet count in the majority of patients. Six of the eight patients with a fall in PA-APAs had an increase in their platelet counts. Furthermore, the timing of the PA-APA fall corresponded to that of the platelet increment; PA-APA fall was seen at 5 weeks in early responders compared to 26 weeks in late responders. Consistent with patients with systemic lupus erythematosus (SLE) (Cambridge et al, 2006; Ferraro et al, 2008), the autoantibody responses occurred despite no clinically significant fall in overall immunoglobulin levels, suggesting that rituximab targets autoantibody-producing B cells out of proportion to other B cells and long-lived plasma cells. This presumably reflects production of autoantibodies by B cells or short-lived plasma cells (Stasi, 2010).
In the two responding patients without detectable PA-APA, the antibody may not have been optimally measured. Alternatively, responses to rituximab in these patients may relate to removal of B cell activation of T cells. No obvious changes in T or NK cells were seen in these patients, although regulatory T cells and TRBV repertoire were not assessed.
In those patients who did not respond, the persistence of PA-APAs suggests that rituximab, despite peripheral blood B cell depletion, had not removed the antibody-producing cell. Further analysis also reveals the potential role of CD8 cell-mediated platelet or megakaryocyte destruction in non-responders. Two patients who did not respond to rituximab, despite a fall in PA-APAs, had very high CD8 levels, much higher than those of the remaining patients (Fig. 2). These patients presumably had both antibody and cellular elements contributing to platelet/megakaryocyte destruction. Removal of the antibody-mediated arm was not sufficient to cause a significant platelet increment. These patients may require dual therapy, attacking both the cytotoxic T cell and autoantibody production. This is consistent with a recent mouse model of ITP in which both cellular and antibody-mediated disease is described, and where responses to treatment are different (Chow et al, 2010). It is also consistent with data showing the direct effect of cytotoxic T cells in ITP (Olsson et al, 2003).
The similar responses to anti-CD40L and anti-CD20 (rituximab) implies that the autoantibody-producing cells in patients who respond to rituximab are driven by T cells, i.e., the interruption of the T-B cell interaction has the same effect as B cell depletion, suggesting the prime pathology remains in the autoreactive T cell. Alternatively, stimulation may be bidirectional, in which case, removal of the autoreactive B cell prevents further T cell activation. This would explain the responses seen in patients who did not have APAs before treatment and is consistent with earlier findings described above (Stasi et al, 2007, 2008). It is also consistent with the findings that only one patient (who did not respond) had a detectable B cell clone – implicating the importance of reducing overall B cell numbers – and that the presence of T cell clones did not predict response to rituximab. Descriptions of abnormal T cell responses in patients who are congenitally without B cells further highlight the importance of the B to T cell stimulation, a concept that is relatively under-examined (Martini et al, 2011).
The data also demonstrates that patients who responded to rituximab were more likely to have the FCGR3A V/V polymorphism. Although the numbers are small, the results are the same as previously reports in both lymphoma and SLE (Anolik et al, 2003). This is consistent with data from this patient population in which responders had a longer duration of B cell depletion (Cooper et al, 2004) and confirms an important role for FCGR3A in depleting B cells coated with antibody. Interestingly, using a double dose of rituximab in a trial for relapsed responders resulted in only a marginally longer B cell depletion and patients relapsed again in the same time window, suggesting a handling effect of antibody-coated B cells rather than a rituximab dose-effect (Hasan et al, 2009). The more efficient B cell depletion in V/V responders may also represent a more profound B cell depletion in the lymph nodes and bone marrow, interrupting antigen presentation and autoantibody production, hence the better platelet responses in these individuals. Of note, FCGR polymorphisms did not affect the timing (early versus late) of response.
Finally, thrombopoietin levels had no particular relevance to responses to rituximab. The lower neutrophil counts in non-responders may relate to complicated, poorly understood features of pathophysiology of patients with ITP.
The primary limitation of this study is the relatively the small numbers of ITP patients investigated in relation to treatment. However, by extensively studying these patients, we can begin to see patterns, which will help in the design of studies to further explore the nature of loss of tolerance in autoimmunity and its restoration with different therapies. Overall, the autoantibody data and associated T cell changes presented here, in limited numbers of patients, demonstrate four potential pathophysiological mechanisms of ITP: (i) Patients with predominantly B cell autoantibody-mediated disease, (ii) Patients with antibody-mediated disease and cytotoxic T cell disease, (iii) Patients with T cell-mediated diseases responding to B cell depletion; and (iv) Patients with plasma cell-producing autoantibodies, which are not destroyed by rituximab.
Another limitation is that these patients received other therapies, both before and during the study. All the therapies could potentially influence autoantibody measurement, such as IVIG therapy during the first 5 weeks given to both responders and non-responders, or the earlier CD40L therapy. The numbers in this study remain too small to evaluate all possibilities, but the almost significant difference between responders and non-responders, despite all these limitations and the time interval between these earlier therapies and rituximab, suggest that these are important preliminary findings.
The emerging data both from clinical trials of B cell depletion therapy and intriguing new mice and human data, including the in vivo data described here, show a prominent role for the B cell in modulating the immune system in autoimmunity. These data also re-validate the importance of measurement of PA-APA. Further investigation of T cell-B cell dynamics in ITP could promote development of novel therapeutic strategies or at least improve the ability to individualize therapy. It also suggests that ideal treatment of particularly difficult cases may require ameliorating both cytotoxic T cells and APA. Optimizing this approach would potentially involve serial testing of each pathway with modification of therapy according to the results.