Background: The role of B cells in the pathogenesis of rheumatoid arthritis (RA) is acknowledged and they represent a well defined therapeutic target. ZAP-70 has been demonstrated to be involved in B cells' activation and survival in CLL. Its expression in B cells of RA synovial fluid suggest a gain of survival for these cells. (1) Objectives: To assess repopulation of B cells and to evaluate ZAP-70 protein expression in B cells at baseline and after treatment with RTX in a cohort of RA patients during a 24 months follow-up. To evaluate these biomarkers for the possible correlations with clinical response. Methods: 15 RA patients received treatment with RTX (1 g two weeks apart). Immunofluorescence labeling for multicolor flow cytometric analysis was performed by incubating PBMCs with anti-human monoclonal antibodies. The staining was performed using surface markers (CD19, IgD, CD38, CD27, CD5, CD23) and intracellular ZAP-70. For each patient clinical and demographic data were collected at baseline and every three months. DAS score was used to evaluate the clinical activity. 15 healthy controls were enrolled in the study. To date 14 patients reached 6 months follow-up, 13 and 12 reached 12 and 24 months, respectively. Results: The assessment of B cells subpopulation according to both IgD-CD38 and IgD-CD27 staining did not reveal significant differences in B cells distribution in patients at baseline and controls. We observed a significant decrease of CD19+% at 6 (3.9±7.8% vs 10.2±3.4% at baseline, p<0.001), 12 (2.5±2.9%, p<0.001) and 24 months follow-up (2.0±2.4%, p<0.001) as well as an increase of the ratio Bm2+Bm2'/eBm5+Bm5 (baseline 2.6±2.7 vs 10.8±11.0 at 6 months FU, p=0.003 and 5.6±5.0 at 12 months, p=0.06). These findings were confirmed by the IgD-CD27 staining that revealed a significant decrease of IgD-CD27+% at 6 months FU (19.4±15.3 at baseline vs 7.7±6.7%,p=0.01), IgD-CD27- (12.0±5.6 at baseline vs 7.3±5.3 at 6 months, p=0.05 and 8.0±6.7 at 12 months, p=0.04). Moreover, there was a parallel decrease of CD27+CD38+% during FU (9.1±6.1 at baseline vs 2.9±2.1% at 6 months, p=0.01; 5.7±6.7% at 12 months, p=0.06; 4.6±4.8% at 24 months, p=0.03). The percentage of CD19+/ZAP-70+ cells increased significantly at 6 months (1,7±1,9% at baseline vs 7,6±11,4%, p= 0,002), 12 months (12,6±20,2%, p=0,001) and 24 months (18,5±20,3%, p=0,003) after RTX treatment. Moreover, the percentage of CD19+/ZAP-70+ tended to be lower in good responders (6.0±2.1%) than in poor responders at 6 months FU (9.2±17.9%, p=0.06) even if the difference was not confirmed at 12 months analysis. Poor responders showed higher baseline values of CD19+CD27+% (36.7±17.6%), CD19+IgD+CD27+% (10.9±9.8%), CD19+CD27+CD38+ (11.8±6.1%) compared to poor responders (CD19+CD27+%:13.8±8.3%, p=0.02; CD19+IgD+CD27+%: 2.9±2.3%, p=0.04; CD19+CD27+CD38+: 5.3±4.7% p=0.04). Conclusion: The kinetic of B cell repopulation in RA patients after B cell depletion seems to be characterized by a significant reduction of memory B cells coupled with the increase of a subset of B cells expressing ZAP-70, mainly in those patients with a poor response to therapy. References: • Tolusso B, et al. Clin Immunol (2009): 131:98-108.
ZAP-70 expression in B-cells during repopulation of peripheral blood following B-cell depletion in rheumatoid arthritis
Gremese E;
2010-01-01
Abstract
Background: The role of B cells in the pathogenesis of rheumatoid arthritis (RA) is acknowledged and they represent a well defined therapeutic target. ZAP-70 has been demonstrated to be involved in B cells' activation and survival in CLL. Its expression in B cells of RA synovial fluid suggest a gain of survival for these cells. (1) Objectives: To assess repopulation of B cells and to evaluate ZAP-70 protein expression in B cells at baseline and after treatment with RTX in a cohort of RA patients during a 24 months follow-up. To evaluate these biomarkers for the possible correlations with clinical response. Methods: 15 RA patients received treatment with RTX (1 g two weeks apart). Immunofluorescence labeling for multicolor flow cytometric analysis was performed by incubating PBMCs with anti-human monoclonal antibodies. The staining was performed using surface markers (CD19, IgD, CD38, CD27, CD5, CD23) and intracellular ZAP-70. For each patient clinical and demographic data were collected at baseline and every three months. DAS score was used to evaluate the clinical activity. 15 healthy controls were enrolled in the study. To date 14 patients reached 6 months follow-up, 13 and 12 reached 12 and 24 months, respectively. Results: The assessment of B cells subpopulation according to both IgD-CD38 and IgD-CD27 staining did not reveal significant differences in B cells distribution in patients at baseline and controls. We observed a significant decrease of CD19+% at 6 (3.9±7.8% vs 10.2±3.4% at baseline, p<0.001), 12 (2.5±2.9%, p<0.001) and 24 months follow-up (2.0±2.4%, p<0.001) as well as an increase of the ratio Bm2+Bm2'/eBm5+Bm5 (baseline 2.6±2.7 vs 10.8±11.0 at 6 months FU, p=0.003 and 5.6±5.0 at 12 months, p=0.06). These findings were confirmed by the IgD-CD27 staining that revealed a significant decrease of IgD-CD27+% at 6 months FU (19.4±15.3 at baseline vs 7.7±6.7%,p=0.01), IgD-CD27- (12.0±5.6 at baseline vs 7.3±5.3 at 6 months, p=0.05 and 8.0±6.7 at 12 months, p=0.04). Moreover, there was a parallel decrease of CD27+CD38+% during FU (9.1±6.1 at baseline vs 2.9±2.1% at 6 months, p=0.01; 5.7±6.7% at 12 months, p=0.06; 4.6±4.8% at 24 months, p=0.03). The percentage of CD19+/ZAP-70+ cells increased significantly at 6 months (1,7±1,9% at baseline vs 7,6±11,4%, p= 0,002), 12 months (12,6±20,2%, p=0,001) and 24 months (18,5±20,3%, p=0,003) after RTX treatment. Moreover, the percentage of CD19+/ZAP-70+ tended to be lower in good responders (6.0±2.1%) than in poor responders at 6 months FU (9.2±17.9%, p=0.06) even if the difference was not confirmed at 12 months analysis. Poor responders showed higher baseline values of CD19+CD27+% (36.7±17.6%), CD19+IgD+CD27+% (10.9±9.8%), CD19+CD27+CD38+ (11.8±6.1%) compared to poor responders (CD19+CD27+%:13.8±8.3%, p=0.02; CD19+IgD+CD27+%: 2.9±2.3%, p=0.04; CD19+CD27+CD38+: 5.3±4.7% p=0.04). Conclusion: The kinetic of B cell repopulation in RA patients after B cell depletion seems to be characterized by a significant reduction of memory B cells coupled with the increase of a subset of B cells expressing ZAP-70, mainly in those patients with a poor response to therapy. References: • Tolusso B, et al. Clin Immunol (2009): 131:98-108.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
