B lymphocyte-typing for prediction of clinical response to rituximab

This work was funded by an unrestricted grant from Roche (Vienna, Austria). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Fifty-two patients undergoing de novo treatment with rituximab for active RA were included in the national 'B Cell surveillance' registry. The participating clinical rheumatologists from local and remote hospitals judged the need for the routine administration of rituximab. Informed consent was obtained from all patients before entering the study, in accordance with the protocol approved by the local ethics committee of the Medical University of Graz. All patients received two 1,000 mg infusions of rituximab preceded by the administration of 100 mg of prednisolone [15]. The characteristics of all patients are shown in Table 1. Disease activity score using 28 joint counts (DAS28) using the erythrocyte sedimentation rate was determined before and 2 and 24 weeks after rituximab application in order to determine the European League Against Rheumatism (EULAR) response. Peripheral blood samples from 17 healthy donors (15 females and two males; mean age of 64 years) were used to determine the normal range for the different B-cell subsets.

Peripheral blood samples were drawn before and 15 days after the first rituximab infusion. Peripheral mononuclear cells were prepared as described [4] and stained with the following antibodies: fluorescein isothiocyanate-labeled IgD, phycoerythrin (PE)-conjugated CD24, allophycocyanin (APC)-conjugated CD27, PE-Cy7-labeled CD38, APC-H7-conjugated CD45, horizon Blue-labeled CD19, pyridine-chlorophyll-protein (PerCP)-conjugated CD3 and CD14, and PE-labeled CD80 and CD95 (all mAbs were obtained from BD Biosciences, Schwechat, Austria). Five hundred thousand CD45⁺ cells were acquired and analyzed by using a seven-channel flow cytometry (BD Canto II cytometer, Software FACSDiva; BD Biosciences). According to their surface staining in the CD27/IgD blot, B cells were classified as naïve (CD19⁺, IgD⁺, and CD27^-), pre-switch memory (CD19⁺, IgD⁺, and CD27⁺), post-switch memory (CD19⁺, IgD^-, and CD27⁺), and double-negative (CD19⁺, IgD^-, and CD27^-) cells. Plasmablasts were classified as CD19⁺, IgD^-, and CD27⁺⁺ [12, 16]. In addition, the intensity of the CD38 expression was determined in the post-switch memory and plasmablast populations since this molecule was recently shown not to be constantly expressed within the populations [16, 17]. The distribution of co-stimulatory molecules and the FAS receptor on the different B-cell subsets was determined by replacing CD24 with CD80 or CD95. Complete depletion of B cells was defined as CD19 values of below 0.0001 × 10⁹ cells per liter. The gating strategy and representative dot blots can be found in Figure S1 of Additional file 1.

Since 41% of the blood samples coming from peripheral hospitals had to be transported for more than 12 hours, we compared 15 samples of patients with RA in a pair-wise fashion (2 and 24 hours after blood letting) and found no significant change in absolute counts of B-lymphocyte subsets after this time.

Table 1 summarizes the baseline demographics and clinical characteristics of the enrolled patients (n = 52), and Figure 1 shows the DAS28 values at baseline and 24 weeks after rituximab application. At baseline, EULAR responders and non-responders were significantly different with regard to the DAS28 only. Interestingly, this difference was due to elevated DAS28 values in RA patients with moderate EULAR response (6.2 ± 0.2; P ≤ 0.0004), whereas patients with good response were not statistically different from EULAR non-responders (5.7 ± 0.3 versus 4.9 ± 0.3, respectively). In our small cohort, the autoantibody status had no influence on the effect of the treatment with rituximab. The few patients with double-seronegative RA were all in the responder group.

Two weeks after the first rituximab infusion, there was a dramatic reduction in the number of B cells in all patients with RA. When high-sensitivity flow cytometry was used, the difference in the number of B cells 15 days after rituximab did not reach statistical significance between responders and non-responders. The median numbers (interquartile ranges) of B cells per 10⁹/L were 0.022 (0.008 to 0.043) in RA patients with good EULAR response, 0.022 (0.011 to 0.059) in patients with a moderate response, and 0.008 (0.003 to 0.035) in patients with no response (Figure 2).

At baseline, the differences in the frequency of naïve, double-negative, pre-switch memory, and post-switch memory B cells between EULAR responders and non-responders did not reach significance. In addition, the expressions of CD80 or CD95 on the B-cell subsets were similar between RA patients and healthy controls. Only a few naïve B cells in patients with RA expressed the co-stimulation and activation markers (1.8% (1.1% to 3.5%) and 4.9% (3.0% to 8.5%), respectively), whereas the highest frequency was found in post-switch memory B cells (58.0% (42.0% to 94.1%) and 65.8% (55.1% to 79.0%), respectively). In all populations analyzed, there was a significant correlation between the frequency of CD95⁺ and CD80⁺ B-cell subsets. In responders, this correlation was highest in the double-negative subset (R² = 0.71; P ≤ 0.0001) but was lowest in non-responders and controls (R² = 0.520; P ≤ 0.0437 and R² = 0.384; P ≤ 0.0080, respectively).

When the frequencies of B cells in responders and non-responders at baseline were compared with those in healthy controls, significant dissimilarities were seen (Figure 3). Thus, responders had significantly more double-negative B cells (Figure 3a) and fewer plasmablasts (Figure 3b) in comparison with controls: 6.6% (4.1% to 11.1%) versus 4.4% (2.8% to 6.9%), P ≤ 0.02 and 0.6% (0.3% to 1.3%) versus 1.3% (0.5% to 2.8%), P ≤ 0.03, respectively. The frequencies of these B-cell subsets in non-responders were 6.2% (2.1% to 10.7%) and 0.9% (0.3% to 2.8%), respectively.

The total cohort of patients with RA had a significantly lower frequency of CD38^hi B cells within the post-switch memory subset (P ≤ 0.0001). Thus, the median (interquartile range) frequencies of this B-cell subset were 0.9% (0.3% to 2.1%) in responders, 1.3% (0.3% to 3.9%) in non-responders, and 18.2% (14.7% to 34.5%) in controls (Figure 3c). Furthermore, the frequency of CD38^hi B cells within plasmablasts was significantly diminished in EULAR responders compared with controls: 28.1% (9.6% to 54.3%) versus 61.1% (44.1% to 66.0%), P ≤ 0.001 (Figure 3d). In contrast, only non-responders had a significantly higher frequency of CD95⁺ pre-switch memory B cells than controls: 52.6% (37.9% to 62.6%) versus 37.9% (26.5% to 49.3%), P ≤ 0.023 (Figure 3e). The frequency of CD95⁺ cells in the other B-cell subsets is depicted in Figure S2 of Additional file 2. As indicated in Figure 3, no significant differences were found between EULAR responders and non-responders.

When univariate logistic regression analysis was used (Table 2), only a low frequency of plasmablasts was a valid predictor for EULAR responsiveness (odds ratio of 2.22; P ≤ 0.04). In our small cohort, complete depletion on day 15 let us calculate an odds ratio of 2.33 for the lack of a therapeutic response to rituximab (P = 0.516).