sj-doc-1-tab-10.1177_1759720X221124028 – Supplemental material for Predictive model to identify multiple failure to biological therapy in patients with rheumatoid arthritis

Supplemental material, sj-doc-1-tab-10.1177_1759720X221124028 for Predictive model to identify multiple failure to biological therapy in patients with rheumatoid arthritis by Marta Novella-Navarro, Diego Benavent, Virginia Ruiz-Esquide, Carolina Tornero, Mariana Díaz-Almirón, Chafik Alejandro Chacur, Diana Peiteado, Alejandro Villalba, Raimon Sanmartí, Chamaida Plasencia-Rodríguez and Alejandro Balsa in Therapeutic Advances in Musculoskeletal Disease</p

sj-docx-2-tab-10.1177_1759720X221124028 – Supplemental material for Predictive model to identify multiple failure to biological therapy in patients with rheumatoid arthritis

Supplemental material, sj-docx-2-tab-10.1177_1759720X221124028 for Predictive model to identify multiple failure to biological therapy in patients with rheumatoid arthritis by Marta Novella-Navarro, Diego Benavent, Virginia Ruiz-Esquide, Carolina Tornero, Mariana Díaz-Almirón, Chafik Alejandro Chacur, Diana Peiteado, Alejandro Villalba, Raimon Sanmartí, Chamaida Plasencia-Rodríguez and Alejandro Balsa in Therapeutic Advances in Musculoskeletal Disease</p

Numbers of circulating CD19+CD24^hiCD38^hi B cells in AS patients naïve for TNF blockers (AS/nb).

<p>PBMCs isolated from the peripheral blood of AS/nb patients (n = 42) and from age and gender-matched healthy controls (HC) (n = 42) were stained with fluorochrome-labeled antibodies against cell surface markers and examined by flow cytometry. AS/nb patients showed an increased proportion and absolute numbers of circulating CD19+CD24^hiCD38^hi B cells. A. Representative dot plots demonstrate CD24 and CD38 expression in cells gated for CD19. B, C. Frequency (B) and absolute numbers (C) of circulating CD19+CD24^hiCD38^hi B cells in HC and AS/nb patients. Bars represent the median and interquartile range. *p<0.01.</p

Clinical characteristics of AS/nb patients.

<p>Clinical characteristics of AS/nb patients.</p

Suppressive capacity of CD19+CD24^hiCD38^hi B cells in healthy subjects.

<p>Total CD19+ or B cells depleted of CD19+CD24^hiCD38^hi cells (Breg-depleted CD19+ B cells), isolated from the peripheral blood of three healthy controls (HC), were established in culture, and stimulated with anti-IgM+IgG. Secretion of IFNγ was determined in culture supernatants by sandwich ELISA at 2, 5 or 7 days. Bar graphs represent the mean and SEM for IFNγ concentrations of 3 independent experiments.</p

Treatment with anti-TNF agents is associated with a significant reduction of circulating CD19+CD24^hiCD38^hi B cell numbers in AS patients.

<p>A. Frequency of CD19+CD24^hiCD38^hi B cells in six AS patients before and six months after initiating treatment with anti-TNFα agents, and in their 6 age and gender-matched healthy controls at the basal and 6 month study. (*p<0.05) B. CRP, calprotectin and ASDAS-CRP values in these 6 AS/nb patients before and six months after initiating treatment with anti-TNFα. (*p<0.05)</p

Frequency of circulating Tfh subsets in patients with AS.

<p>A. AS/nb, but not AS/b patients, demonstrate an increased frequency of circulating CD4+CXCR5+CXCR3+CCR6- (Tfh-Th1) cells and a decreased frequency of CD4+CXCR5+CXCR3-CCR6+ (Tfh-Th17) cells as compared with HC, whereas the frequency of CD4+CXCR5+CXCR3-CCR6- (Tfh-Th2) cells is not different among the three groups. Representative dot plots demonstrate CXCR3 and CCR6 expression in cells gated for CD3, CD4 and CXCR5. B. Underrepresentation of cTfh subsets with a B cell helper phenotype (Tfh-Th17 plus Tfh-Th2 cells) in AS/nb but not in AS/b patients. Box and whiskers plots represent the median, interquartile range, maximum and minimum values calculated from 25 AS/nb patients, 25 AS/b patients and 50 HC. *p<0.05 vs HC, ^†p<0.005 vs AS/nb.</p

Functional capacity of circulating CD4+CXCR5+ T cells.

<p>CD4+CXCR5+ T cells isolated from peripheral blood of HC are more efficient than cells from AS/nb patients at inducing maturation of cocultured autologous naïve B cells. A. CD19+CD20-CD38^high plasmablasts in 9-day cocultures of naïve B cells from AS/nb patients or HC with autologous CXCR5+ or CXCR5- CD4+ T cells. B, C. Concentrations of IgG, A and M at different time points (B) or at 13 days (C) in cocultures of naïve B cells from AS/nb patients or HC with autologous CXCR5+ or CXCR5- CD4+ T cells. D. Recovered viable B and T cells in 9-day cocultures of naïve B cells from AS/nb patients or HC with autologous CXCR5+ or CXCR5- CD4+ T cells. Line and bar graphs represent the mean and SD of 3 independent experiments.</p

Serum levels of IgG, IgA and IgM in patients with AS.

<p>A. IgG, IgA and IgM were determined by nephelometry in the serum of AS/nb and AS/b patients. For AS/b patients, determinations were not only done in sera collected on the day when phenotypical studies were done, but also on sera that had been taken just before initiation of treatment with TNF blockers. B. Relation of serum Ig concentrations with cTfh, circulating plasmablasts and disease activity. Shown are cTfh proportions, cTfh subset ratio and circulating plasmablast proportions together with serum IgG, IgA and IgM concentrations in HC, AS patients with inactive disease or moderate disease activity (ASDAS-CRP < 2.1) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107086#pone.0107086-Machado1" target="_blank">[38]</a> and AS patients with high or very high disease activity (ASDAS-CRP > 2.1) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107086#pone.0107086-Machado1" target="_blank">[38]</a>. Normal or elevated serum Ig levels are observed despite the presence of decreased or normal cTfh and plasmablasts, respectively. Note that serum Ig concentrations vary with disease activity whereas cTfh and plasmablast numbers do not. Box and whiskers plots represent the median, interquartile range, maximum and minimum values. *p<0.05 vs HC, ^†p<0.05 vs AS/nb.</p

Numbers of circulating Tfh counterparts (cTfh) in patients with AS.

<p>AS/nb but not AS/b patients demonstrate decreased frequencies and absolute numbers of cTfh. A, B. Frequency of cTfh in HC, AS/nb and AS/b patients. Representative dot plots demonstrate ICOS and CXCR5 expression (A) or ICOS and PD-1 expression (B) in cells gated for CD3, CD4 and CXCR5. C. Absolute numbers of circulating Tfh counterparts (cTfh) in HC, AS/nb and AS/b patients. Box and whiskers plots represent the median, interquartile range, maximum and minimum values calculated from 25 AS/nb patients, 25 AS/b patients and 50 HC. *p<0.0001 vs HC; † p<0.01 vs AS/nb patients.</p