B cells from rheumatoid arthritis patients show important alterations in the expression of CD86 and FcgammaRIIb, which are modulated by anti-tumor necrosis factor therapy

Abstract

Introduction: Several molecules help preserve peripheral B cell tolerance, but when altered, they may predispose to autoimmunity. This work studied the expression of the costimulatory molecule CD86 and the inhibitory receptor for IgG immune complexes FcgammaRIIb (CD32b), on B cells from rheumatoid arthritis (RA) patients, and the influence of anti-tumor necrosis factor (TNF) therapy.

Methods: Peripheral B cells from 18 RA patients and 13 healthy donors were characterized using flow cytometry. Eleven patients who underwent a six-month adalimumab therapy were further assessed for phenotypic changes on their B cells.

Results: RA patients exhibited a high percentage of naïve and memory B cells expressing CD86. In contrast, expression of FcgammaRIIb was significantly reduced on RA memory B cells and plasmablasts as compared to healthy donors, probably due to downregulation of this receptor when differentiating from naïve to memory cells. These alterations on FcgammaRIIb were associated with high levels of anti-citrullinated vimentin autoantibodies. In addition, treatment with adalimumab normalized the expression of CD86 on memory B cells and reduced the expression of FcgammaRIIb, mainly on naïve B cells.

Conclusions: Our findings show that peripheral B cells from RA patients have an altered expression of key molecules, such as CD86 and FcgammaRIIb. Because this latter receptor is required for feedback inhibition, a deficient expression might contribute to humoral autoimmune responses. Furthermore, these molecules are likely to be influenced by inflammatory factors, since they were modulated by TNF inhibition.

Figure 1

Characterization of B-cell subpopulations from rheumatoid arthritis (RA) patients and healthy controls (HCs). (a) Dot plot representing the distribution of naïve B cells (R3), memory B cells (R4), and plasmablasts (R5) which was used on further analyses. No differences in the percentages of naïve B cells (b), memory B cells (c), and plasmablasts (d) between 18 RA patients and 13 HCs were detected. P > 0.05, two-tailed unpaired Student t test. Horizontal lines represent mean values.

Figure 2

Increased expression of CD86 on naïve and memory B cells from rheumatoid arthritis (RA) patients. (a) Dot plots representative of the expression of CD86 on B cells from an RA patient (left) and a healthy control (HC) (right). The number in the quadrant represents the percentage of CD19⁺ CD86⁺ cells. Graphics summarizing the percentages of CD86⁺ cells among naïve B cells (b), memory B cells (c), and plasmablasts (d) from 18 RA patients and 9 HCs. *P < 0.05, two-tailed Mann-Whitney U test. Horizontal lines represent mean values.

Figure 3

Decreased expression of FcγRIIb on memory B cells and plasmablasts from rheumatoid arthritis (RA) patients. Graphics summarize the expression of FcγRIIb on naïve B cells (a), memory B cells (b), and plasmablasts (c) from 18 RA patients and 13 healthy controls (HCs). Expression was quantified as mean fluorescence intensity (MFI). **P < 0.01, ***P < 0.001, two-tailed unpaired Student t test. Horizontal lines represent mean values.

Figure 4

Altered regulation of FcγRIIb on B cells from rheumatoid arthritis (RA) patients. (a) Representative histograms of FcγRIIb (CD32b) expression on naïve B cells (gray line), memory B cells (black line), and plasmablasts (dotted line) from an RA patient (left) and a healthy control (right). The shaded curve represents the isotype control. Graphics show a comparison of FcγRIIb expression between naïve B cells, memory B cells, and plasmablasts from 18 RA patients (b) and 13 healthy controls (c). Expression was quantified as mean fluorescence intensity (MFI). The differences in the FcγRIIb expression levels between B-cell subpopulations were analyzed with the two-tailed paired Student t test; ***P < 0.001. Horizontal lines represent mean values. PE, phycoerythrin.

Figure 5

FcγRIIb expression levels on rheumatoid arthritis (RA) patients' memory B cells are inversely associated with anti-modified and citrullinated vimentin (anti-MCV) titers. (a) FcγRIIb expression on memory B cells from RA patients with high titers (at least 50 U/mL) and from those with no or low titers (less than 50 U/mL) of serum anti-MCV antibodies. Expression was quantified as mean fluorescence intensity (MFI). *P < 0.05, two-tailed Mann-Whitney U test. (b) Anti-MCV antibody titers in patients who downregulated the expression of FcγRIIb (the difference between MFI of naïve B cells and MFI of memory B cells was greater than 10 for downregulators) and in those who upregulated or maintained it almost invariable (the difference between MFI of naïve B cells and MFI of memory B cells was not greater than 10 for non-downregulators). *P < 0.05, two-tailed Mann-Whitney U test. (c) FcγRIIb expression on memory B cells from RA patients with normal levels (less than 1,350 mg/dL) and high levels (at least 1,350 mg/dL) of total serum IgG. Expression was quantified as MFI. P > 0.05, two-tailed Mann-Whitney U test. Horizontal lines represent mean values.

Figure 6

B-cell phenotype and anti-modified and citrullinated vimentin (anti-MCV) titers on rheumatoid arthritis (RA) patients after 6 months of adalimumab therapy. After 6 months of anti-tumor necrosis factor (anti-TNF) therapy, B-cell phenotype and serum anti-MCV antibodies from 11 RA patients, who exhibited at least a moderate response to the treatment, were reassessed. (a) Graphics summarizing the percentages of CD86⁺ cells among naïve B cells, memory B cells, and plasmablasts from RA patients before and after 6 months of anti-TNF therapy. *P < 0.05, two-tailed Wilcoxon signed-rank test. (b) Graphics summarizing FcγRIIb expression on naïve B cells, memory B cells, and plasmablasts from RA patients before and after 6 months of anti-TNF therapy. Expression was quantified as mean fluorescence intensity (MFI). **P < 0.01, two-tailed paired Student t test. (c) Comparison of FcγRIIb expression between naïve B cells, memory B cells, and plasmablasts from RA patients after anti-TNF therapy. The differences in FcγRIIb expression levels between B-cell subpopulations were analyzed with the two-tailed paired Student t test; *P < 0.05, **P < 0.01, ***P < 0.001. (d) Comparison of serum anti-MCV antibody levels before and after 6 months of anti-TNF therapy. P > 0.05, two-tailed paired Student t test. Horizontal lines represent mean values.