Potential of B-cell-targeting therapy in overcoming multidrug resistance and tissue invasiveness associated with P-glycoprotein expressing-B cell compartments

Figures & data

Table 1. Relation of P-glycoprotein with disease modifying antirheumatic drugs and immunosuppressants.

Drug	Pharmacological substrates of P-glycoprotein	Competitive inhibitor of P-glycoprotein
Corticosteroids	YES	NO
Immunosuppressants
Cyclophosphamide	NO	NO
Mycophenolate mofetil	NO	NO
Azathioprine	NO	NO
tsDMARDs
Tofacitinib	YES	NO
Baricitinib	YES	NO
Peficitinib	YES	unknown
csDMARDs
Methotrexate	NO	NO
Cyclosporine	YES	YES
Tacrolimus	YES	YES
Leflunomide	NO	NO
Hydroxycloroquine	YES	YES
Sulfasalazine	YES	unknown
D-penicillamine	YES	unknown
Colchicine	YES	NO
bDMARDs	NO	NO

tsDMARDs: Targeted synthetic disease-modifying antirheumatic drugs; csDMARDs: conventional synthetic DMARDs; bDMARDs: biological DMARDs.

Figure 1. Schematic diagram of induction of P-gp expression on B cells and P-gp⁺ B-cell targeting therapies in RA. (A) YB-1, which is located in the cytoplasm of resting B cells, is translocated by various signal transductions associated with activation of B cells, which results in overexpression of P-gp. (B) P-gp-related treatment resistance in RA can be probably overcome with P-gp⁺ B-cell targeting therapies as follows; inhibitors of B cell activation and differentiation (JAK inhibitors, TNF antagonists, IL-6 blockade, Anti-CD20 Monoclonal Antibodies), P-gp induction inhibitors (Methotrexate, TNF antagonists, IL-6 blockade, CXCR4 Antagonist), P-gp function inhibitors (P-glycoprotein Competitive Inhibitors) and P-gp⁺ B-cells depletors (Anti-CD20 Monoclonal Antibodies).

Figure 2. Relations among P-gp⁺CXCR4⁺ B cells, P-gp⁺ CD27^-IgD⁺B cells and RA organ involvement. (A) Correlation between the proportions of P-gp⁺CXCR4⁺ B cells and P-gp⁺ CD27^-IgD⁺B cells in RA patients. Statistical analysis was performed by Person’s correlation analysis. (B) Flow cytometric analysis identified P-gp⁺ CD27^-IgD⁺B cells in the representative RA patient with rheumatoid vasculitis. Values at the top of each section are percentages of CD19⁺B cell subpopulations based on CD27/IgD classification. Flow cytometric analysis showed P-gp expression on each B cell subpopulation (blue lines). Data represent the percentages of P-gp-positively stained B cell subpopulations. Red: isotype-control FITC-conjugated anti-mouse IgG Ab. The number of CD27⁺IgD⁺CD19⁺ B cells was lower than that was available for the histogram exhibition and P-gp expression analysis. (C) Flow cytometry for P-gp⁺ CD27^-IgD⁺B cells in 17 RA patients, including 10 with (closed bar) and 7 without (hatched bar) organ involvement. Values are mean ± SD of independent experiments. *p < .05, by non-paired t-test. RA disease activity, as estimated by the SDAI score, was not significantly different between the two groups (with: 26.5% ± 8.3, without: 30.9% ± 12.9; p = .41). Organ involvement included interstitial pneumonia (n = 2), interstitial pneumonia with rheumatoid vasculitis (n = 3), Felty syndrome (n = 1), amyloidosis (n = 1) and lymphadenopathy (n = 1). (A–C) Specific antibodies used for staining and flow cytometric analysis, including MRK16 for P-gp (a specific mAb against P-gp; Kyowa Medex, Tokyo) with FITC-conjugated goat anti-mouse IgG mAb, cy-chrome-conjugated CD19 mAb, APC-conjugated CD27 mAb and PE-conjugated IgD and CXCR4 mAb (BD Biosciences Pharmingen).

Figure 3. Tacrolimus inhibits excretion of intracellular dexamethasone through P-glycoprotein. 1 × 10⁶ of PBMCs were pre-incubated with (solid circles) or without (open circle) 10 ng/mL of IL-2 for 4 h. Then, 20-min after the addition of [6,7-³H(N)]-dexamethasone and [¹⁴C] n-butanol, the cell to medium ratio (C/M ratio; an index of intracellular and extracellular dexamethasone concentration ratio) was evaluated in the presence of the indicated concentrations of tacroliums. The C/M ratio was computed using the following formula: C/M ratio = [(³H in cell fraction/¹⁴C in cell fraction)/(³H in medium fraction/¹⁴C in medium fraction)]. Data are mean ± SD of five independent experiments. *p < .05, **p < .01, by one-way ANOVA.

Figure 4. Tocilizumab downregulates P-gp⁺CXCR4⁺B cells and P-gp⁺CD27^-IgD⁺B cells in RA with microscopic polyangiitis. Flow cytometric analysis identified P-gp⁺CXCR4⁺B cells and P-gp⁺CD27^-IgD⁺B cells in the RA patient. Values above the chart are the proportions of P-gp⁺CXCR4⁺ B cells just at start (A) and 4 weeks after (B) of tocilizumab therapy. Values at the top of each section are percentages of CD19⁺B cell subpopulations based on CD27/IgD classification. Flow cytometric analysis showed P-gp expression on each B cell subpopulation (blue lines). Data represent the percentages of P-gp-positively stained B cell subpopulations. Red: isotype-control FITC-conjugated anti-mouse IgG Ab. The number of CD27⁺IgD⁺CD19⁺ B cells was lower than that was available for the histogram exhibition and P-gp expression analysis. Specific antibodies used for staining and flow cytometric analysis, including MRK16 for P-gp (a specific mAb against P-gp; Kyowa Medex, Tokyo) with FITC-conjugated goat anti-mouse IgG mAb, cy-chrome-conjugated CD19 mAb, APC-conjugated CD27 mAb and PE-conjugated IgD and CXCR4 mAb (BD Biosciences Pharmingen).