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M. Mahevas et al.
AB
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Figure 1. Serum level of total γ-globulins and immunoglobulin isotypes (IgG, IgA, IgM) during the study of rituximab and belimumab combined. Serum level of total γ-globulins (A) and IgG (B), IgM (C), and IgA (D) were assessed by nephelometry at week 12 (W12), W24, W36, W52. Dotted line represents normal threshold for each isotype. *P<0.05, **P<0.01, ***P<0.001; ns: not significant.
an of seven serotypes (range, 4-8). Overall, at W52, 11 (73%) patients had protective titers for at least 11 serotypes. All patients had received vaccination with tetanus and measles at different times before enrollment. Fourteen patients (93%) had no significant change in anti- tetanus antibody titers between W0 and W52. There were also no significant changes in anti-measles antibody titers, which remained at protective level >200 UA/m (protective >16.5 UA/m) for all patients at W52.
Antiplatelet antibody testing
Direct MAIPA was performed at inclusion in all but one patient and was positive in ten (71%) patients, including nine with glycoprotein IIb/IIIa (GpIIb/IIIa) specificity, and one with GpIb/IX specificity (Online Supplementary Table S4). Among these patients, seven (70%) achieved response and had negative MAIPA results at W52, two achieved response (one CR and one R) and still had anti- GpIIb/IIIa antibodies at W52, and one did not respond but had negative MAIPA results at W52.
Immunological analysis
In order to precisely assess the impact of blocking BAFF concomitantly with B-cell depletion on B- and T-cell sub- sets, we analyzed in parallel a prospective cohort of 12 ITP patients who received RTX without belimumab as a stan- dard of care (Online Supplementary Table S5). As previously reported, we observed a significant increase in BAFF serum levels in patients receiving RTX alone at W12, W24, W36 and W52 (all P<0.001) after RTX, as compared to baseline. In the RITUX-PLUS study, belimumab treatment effectively reduced BAFF levels at W12 as compared to
baseline (1,210±248 vs. 90±38 pg/mL, P<0.0001). BAFF levels started to return to baseline at W24 (730±294 pg/mL, P<0.01), then strongly increased at W36 (2,199±1,498 pg/mL, P<0.0001) and reached a plateau at W52 (2937±1561 pg/mL P<0.0001) (Figure 3A). BAFF lev- els at W36 and W52 did not significantly differ between patients receiving belimumab + RTX in the study and con- trol patients receiving RTX alone.
In order to evaluate the effect of the RTX and belimum- ab combination on B-cell depletion and re-appearance, we analyzed circulating CD19+ B cells in both cohorts at base- line, W4, W12, W24, W36, and W52 (Figure 3B). All patients showed complete depletion of circulating CD19+ B cells at W4 and W12 (Figure 3C).
Reappearance of B cells in the peripheral blood varied among patients. We observed no significant delay in B-cell reconstitution in patients receiving belimumab plus RTX versus controls receiving RTX alone, despite a slight differ- ence at W36. Transitional B cells (CD19+IgD+ CD24+CD38+CD10+), which are precursors of naïve B cells in peripheral blood,14,15 emerged early during B-cell reconstitution (Figure 3D to E). Indeed, two of 15 patients receiving belimumab showed transitional B cells in the peripheral blood at W24 as compared with five of 12 patients receiving RTX alone. All but one patient in both groups showed naïve and transitional B cells at W52. The absolute number of CD19+ cells remained significantly decreased at W52 in both groups as compared to baseline, and B-cell depletion mainly affected memory B cells and IgD+CD27+ B cells (Figure 3F and G).
As previously reported, the number of circulating CD27highCD38high cells (mainly plasmablasts expressing
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