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S. Delignat et al.
and localization and depending on the stage of the immune response, B cells play different roles as (i) antigen- presenting cells either in the initiation phase of an immune response (i.e., MZ B cells involved in FVIII capture and trafficking in the spleen),11 or in recall responses (higher number of FVIII-specific memory B cells with high-affini- ty FVIII-specific BCR), and (ii) precursors of antibody- secreting plasma cells (that produce FVIII inhibitors) local- ized in the spleen or in the bone marrow. Therefore, B cells represent an ideal therapeutic target either to prevent the initiation of anti-FVIII immune responses or to eradi- cate ongoing responses in inhibitor-positive patients. Incidentally, the only strategies known to eradicate FVIII inhibitors in patients’ target B cells. ITI was shown to favor the development of anti-idiotypic antibodies that neutralize FVIII inhibitors3,4 and, at least in mice, eliminate FVIII-specific memory B cells.5 Likewise, the therapeutic anti-CD20 antibody rituximab specifically targets CD20- expressing naïve and memory B cells, thus preventing the replenishment of the pool of plasma cells upon re-chal- lenge with therapeutic FVIII. Both strategies fail to target long-lived plasma cells that reside in niches in the bone marrow and lack expression of CD20 and of the BCR.28 Attempts to target plasma cells in FVIII-deficient mice using either inhibitors of the proteasome29 or cocktails of immunosuppressive agents30 have met with limited suc- cess or lack of antigen specificity. Here we investigated a new approach to prevent the activation of antigen-specific B cells by inhibiting BTK, a kinase involved in upstream BCR signaling. Inhibition of BTK targets only those B cells that are activated at the time of administration of thera- peutic FVIII, thus providing some degree of specificity for FVIII.
We first validated that, upon BCR triggering, PF- 06250112 blocks BCR signaling in vitro and in vivo by meas- uring the induction of CD86 expression, a co-stimulation molecule involved in anti-FVIII immune responses.31 PF- 06250112 inhibited BCR signaling in vitro with an IC50 of about 1 nM, a value close to that reported previously.18 PF- 06250112 used at 15 mg/kg also inhibited the induction of CD86 in vivo following stimulation of splenic B cells with an anti-IgD serum. A marginal effect of PF-06250112 was observed on B cells stimulated by CD40 ligation, a BCR- independent pathway. This moderate alteration of CD40 signaling had also been reported in the case of PCI-32765, another BTK inhibitor; a possible interference of BTK inhibitors on CD40-induced NF-κB-signaling was evoked.32
As previously reported in an experimental model of sys- temic lupus erythematosus18 or using immunization of mice with the T-dependent model antigen SRBC,19 the reg- ular per os administration of PF-06250112 to FVIII-deficient mice led to a statistically significant decrease in the per- centages of MZ and follicular B cells. In contrast to the lat- ter reports however, there was no change in percentages of splenic germinal center B cells, possibly owing to the use of a different antigen or of a different disease model. In our hands, treatment of FVIII-deficient mice with PF- 06250112 failed to prevent the onset of a primary anti- FVIII immune response. Exogenously administered FVIII was previously shown to closely associate with MZ
11,33,34
macrophages and MZ B cells. The depletion of MZ
macrophages or MZ B cells was independently shown to delay the onset of an anti-FVIII immune response.11,33 However, it is not clear whether reduced immune
responses to FVIII following depletion of MZ macrophages or B cells result from the direct involvement of these cells in FVIII internalization and presentation to T cells, or trafficking to the germinal center, or from a mere disruption of the splenic architecture.35 The lack of effect of chronic BTK inhibition on the development of primary anti-FVIII humoral responses, despite an alteration of peripheral B-cell populations, including MZ B cells, sug- gests that MZ macrophages are the principal antigen-pre- senting cells at play in the onset of primary anti-FVIII immune responses, at least in mice. PF-06250112 feeding was also associated with a modest decrease in percentages of CD4+ T cells. PF-06250112 has been shown to be 10,000-fold more specific for BTK than for interleukin-2- inducible T-cell kinase (ITK).18 In addition, there is no modulation of splenic T-cell populations in BTK-deficient mice.14 Hence, the decrease in the percentage of CD4+ T cells probably does not reflect a direct effect of PF- 06250112 on T cells in FVIII-sensitized mice, but rather a modulation of T-cell homeostasis consequent to a decrease in splenic follicular and MZ B cells. This illus- trates a possible moderate off-target effect of PF-06250112 in naïve mice. Signaling through CD40 and CD40L is essential for the onset of primary immune responses, including in the case of therapeutic FVIII.36,37 The lack of effect of PF-06250112 on the primary anti-FVIII immune response presumably reflects the lack of effect of BTK inhibition on CD40 signaling.
The inhibition of BTK using PF-06250112 was able to
Figure 5. PF-06250112 inhibits the ex vivo differentiation of FVIII-specific memory B cells into antibody-secreting plasma cells. Splenocytes from FVIII- sensitized FVIII-deficient mice having developed a humoral response to FVIII were isolated 14 days after the last injection of FVIII. Pooled splenocytes were cultured for 6 days in the presence of 1 mg/mL FVIII. Splenocytes were incubat- ed with vehicle or PF-06250112 2 h before adding FVIII. At day 6, newly differ- entiated anti-FVIII antibody-secreting plasma cells (ASCs) were detected by an enzyme-linked immunospot assay. The graph depicts means±SEM.
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haematologica | 2019; 104(5)