Expansion of Activated Peripheral Blood Memory B Cells in Rheumatoid Arthritis, Impact of B Cell Depletion Therapy, and Biomarkers of Response

Abstract

Although B cell depletion therapy (BCDT) is effective in a subset of rheumatoid arthritis (RA) patients, both mechanisms and biomarkers of response are poorly defined. Here we characterized abnormalities in B cell populations in RA and the impact of BCDT in order to elucidate B cell roles in the disease and response biomarkers. In active RA patients both CD27+IgD- switched memory (SM) and CD27-IgD- double negative memory (DN) peripheral blood B cells contained significantly higher fractions of CD95+ and CD21- activated cells compared to healthy controls. After BCD the predominant B cell populations were memory, and residual memory B cells displayed a high fraction of CD21- and CD95+ compared to pre-depletion indicating some resistance of these activated populations to anti-CD20. The residual memory populations also expressed more Ki-67 compared to pre-treatment, suggesting homeostatic proliferation in the B cell depleted state. Biomarkers of clinical response included lower CD95+ activated memory B cells at depletion time points and a higher ratio of transitional B cells to memory at reconstitution. B cell function in terms of cytokine secretion was dependent on B cell subset and changed with BCD. Thus, SM B cells produced pro-inflammatory (TNF) over regulatory (IL10) cytokines as compared to naïve/transitional. Notably, B cell TNF production decreased after BCDT and reconstitution compared to untreated RA. Our results support the hypothesis that the clinical and immunological outcome of BCDT depends on the relative balance of protective and pathogenic B cell subsets established after B cell depletion and repopulation.

Fig 1. Expansion of activated memory B cells in active RA patients.

(A) Multicolor flow cytometry of PBMCs from healthy controls (n = 24) and untreated RA (n = 16) reveals a similar distribution of core total naïve (IgD+CD27-: includes transitional B cells), unswitched memory (USM) (IgD+CD27+), switched memory (SM) (IgD-CD27+: includes plasmablasts) and double negative (DN) memory (IgD-CD27-) B cells. CD19+ B cells gated B cells are shown. (B) The expression of CD95 (up-regulated during activation) and CD21 (down-regulated during activation) on each of the SM and DN memory B cells is defined. The CD95+ B cells from both the SM and DN are significantly higher in active RA before rituximab. There is also a significantly higher representation of CD21 negative B cells in the SM and DN subpopulations in baseline RA (Mann-Whitney) (*p<0.05, **p<0.005, ***p<0.0005). Data is expressed as the median +/- interquartile range.

Fig 2. Memory B cell populations become dominant following rituximab treatment.

Dot plots show a comparison of B cell subsets in RA at baseline and 1-month post-BCDT for the canonical B cell subsets (A) and (B) the expansion of CD95+ and CD21- B cells in the SM and DN memory B cell populations 1 month after BCDT. (C) The figures depict the change in SM and DN memory over time after BCDT for all subjects. The percentage of both SM and DN B cell populations increase in the earlier months after BCDT as compared to both healthy control and baseline. Percentage of SM B cells subsequently decreases beginning at 12 month. The percentage of DN began to decrease at 8 months. (D) Kinetics of change in the cohort as a whole for CD95 and CD21 expression. *significantly different from HC; # significantly different from baseline p values ranging from <0.05 to <0.0005 (Mann-Whitney). Data is expressed as the median +/- interquartile range.

Fig 3. Residual memory B cells in RA following BCDT upregulate Ki67.

(A) Dot plots show the expression of intracellular Ki67 (a proliferation antigen) in the gated SM and DN memory B cells. Dramatic up-regulation is seen in the RA patient after BCD compared to a healthy control. (B) Ki67 expression is significantly different in the DN and SM for RA post-BCD (n = 8) compared to untreated RA (n = 6) and healthy controls (n = 4) (*p<0.05, **p<0.005, ***p<0.0005) (Mann-Whitney) (3 group comparison DN p = 0.05, SM p = 0.0017) (Kruskal-Wallis). (C) CD95- B cells have very low expression of Ki67 in HC and untreated RA. Ki67 expression is higher in CD95+ B cells in the healthy controls in all 4 subsets (data not shown for Naïve and USM) (paired t-test). An increase in Ki67 expression is seen post-BCD in both the CD95+ and CD95- populations (*p<0.05, ***p<0.0005) (Mann-Whitney) (DN: 3 group comparison Kruskall-Wallis p = 0.0255 for CD95+ and p = 0.0420 for CD95-; SM: p = 0.0018 for CD95+ and p = 0.0284 for CD95-). The RA subjects post-BCD (n = 8) are B cell depleted (data not shown) and ranged from 3 to 6 months post-treatment. Data is expressed as the median +/- interquartile range. (D) Increase in Ki67 expression after BCD in matched samples (paired t test: *p<0.05, **p<0.005, ***p<0.0005). Post samples are 5 months after rituximab treatment.

Fig 4. Sequential repopulation of B cells in RA patients after B cell depletion.

(A) Naïve/transitional B cell subsets are gated as described as IgD+CD27- (expressed as a % of the total B cell compartment). These B cells are rapidly depleted and remained significantly lower than baseline (***p<0.0001 compared to baseline) until 8 months when reconstitution begins (**reconstitution higher than baseline, p = 0.0137 for 16 month, 0.0062 for 20 month, 0.0037 for 24 month). (B) T1/T2, T3, and mature naïve B cells are gated within the IgD+CD27- population based on MTG, CD38, and CD24 expression and show sequential repopulation. The transitional B cells are rapidly depleted by 1 month and remained significantly lower than baseline (***p<0.0001 compared to baseline) until 8 months when reconstitution begins. The transitional B cells became significantly higher than baseline with reconstitution (*p value: T1/T2 .047916 month 16; T3: 0.0062, 0.0161, 0.0029 at 16, 20, 24 months). The mature naïve significantly decrease at months 1 through 12 (*p ranging from <0.0001 to 0.0426) but re-equilibrate to baseline at months 16 through 24. P values are calculated by Mann-Whitney. Data is expressed as the median +/- interquartile range.

Fig 5. Biomarkers of clinical response.

The RA patients are separated into groups based on DAS response at 4 months. (A) The DAS28 is depicted over time in the 3 different clinical response groups. (B) The depletion in total absolute B cells over time is depicted in the 3 different clinical response groups, where globally a similar depth of depletion and kinetics of reconstitution is observed. In the middle graph absolute numbers of switched memory B cells remain low at 8 month post-depletion in all 3 response groups. In the right graph the ratio of absolute numbers of transitional B cells to memory B cells is significant higher in the good responders compared to RA baseline, moderate, and non-responders. (C) CD95+ and CD21- subsets from DN and SM B cells are elevated in non- and moderate responders at 4 months as compared to healthy controls. Notably, the CD95+ from the SM and DN subsets are significantly higher in the moderate and non-responders than the good responders. (Mann-Whitney) (*p<0.05, **p<0.005, ***p<0.0005). Data is expressed as the mean +/- SEM.

Fig 6. B cell cytokine expression.

(A) B cell subsets are sort purified from healthy control peripheral blood and then stimulated for 4 days with 2.5 μg/ml CPG 2006, 2.5 μg/ml anti-CD40, and 50 U/ml IL-2 followed by PMA/ionomycin for 4.5 hours. Intracellular expression of TNF versus IL10 in each subset is quantitated. TNF expression is significantly higher in the USM and SM than the transitional B cells. IL-10 is significantly lower in the SM than in naïve, transitional, and USM B cells. SM B cells have a significantly higher ratio of TNF+ to IL10+ B cells than naïve and transitional (*p<0.05, **p<0.005) (Paired t-test). N = 6 healthy controls. (B) Total PBMCs are stimulated for 4 hours with PMA/ionomycin to compare B cell cytokine expression in untreated RA (n = 6) versus RA after BCD and reconstitution (n = 6) ranging from 15 to 32 months after rituximab (mean = 22 months) versus HC (n = 8). The intracellular expression of TNF, IL10, and IL6 is examined in CD19 gated B cells. B cell expression of TNF is significantly reduced in RA after reconstitution (**p<0.005) (Mann-Whitney). There are no significant differences in IL10 and IL6 producing B cells. The ratio of TNF to IL10 producing B cells is significantly lower in RA after BCD/reconstitution (*p<0.05). Data is expressed as the mean +/- SEM.