Activated T-Follicular Helper 2 Cells Are Associated With Disease Activity in IgG4-Related Sclerosing Cholangitis and Pancreatitis

Abstract

Objectives: Immunoglobulin G4-related sclerosing cholangitis (IgG4-SC) and autoimmune pancreatitis (AIP) are characterized by an abundance of circulating and tissue IgG4-positive plasma cells. T-follicular helper (Tfh) cells are necessary for B-cell differentiation into plasma cells. We aimed at elucidating the presence and phenotype of Tfh cells and their relationship with disease activity in IgG4-SC/AIP.

Methods: Circulating Tfh-cell subsets were characterized by multiparametric flow cytometry in IgG4-SC/AIP (n = 18), disease controls with primary sclerosing cholangitis (n = 8), and healthy controls (HCs, n = 9). Tissue Tfh cells were characterized in IgG4-SC/AIP (n = 12) and disease control (n = 10) specimens. Activated PD1+ Tfh cells were cocultured with CD27+ memory B cells to assess their capacity to support B-cell differentiation. Disease activity was assessed using the IgG4-responder index and clinical parameters.

Results: Activated circulating PD-1+CXCR5+ Tfh cells were expanded in active vs inactive IgG4-SC/AIP, primary sclerosing cholangitis, and HC (P < 0.01), with enhanced PD-1 expression on all Tfh-cell subsets (Tfh1, P = 0.003; Tfh2, P = 0.0006; Th17, P = 0.003). Expansion of CD27+CD38+CD19lo plasmablasts in active disease vs HC (P = 0.01) correlated with the PD-1+ Tfh2 subset (r = 0.69, P = 0.03). Increased IL-4 and IL-21 cytokine production from stimulated cells of IgG4-SC/AIP, important in IgG4 class switch and proliferation, correlated with PD-1+ Tfh2 (r = 0.89, P = 0.02) and PD-1+ Tfh17 (r = 0.83, P = 0.03) subsets. Coculture of PD1+ Tfh with CD27+ B cells induced higher IgG4 expression than with PD1- Tfh (P = 0.008). PD-1+ Tfh2 cells were strongly associated with clinical markers of disease activity: sIgG4 (r = 0.70, P = 0.002), sIgE (r = 0.66, P = 0.006), and IgG4-responder index (r = 0.60, P = 0.006). Activated CXCR5+ Tfh cells homed to lymphoid follicles in IgG4-SC/AIP tissues.

Conclusions: Circulating and tissue-activated Tfh cells are expanded in IgG4-SC/AIP, correlate with disease activity, and can drive class switch and proliferation of IgG4-committed B cells. PD1+ Tfh2 cells may be a biomarker of active disease and a potential target for immunotherapy.

Figure 1.

Circulating Tfh cells have an activated phenotype in IgG4-SC/AIP. Circulating Tfh-cell phenotype was analyzed by flow cytometry. (a) Contour plot showing CXCR3 and CCR6 expression on CD45RA-CD4+CXCR5+ Tfh cells from a representative donor. Three populations within the Tfh cells were identified: CXCR3+CCR6− Tfh1 cells, CXCR3−CCR6− Tfh2 cells, and CXCR3−CCR6+ Tfh17 cells. Gating for PD1 high cells shown. (b) Dot plot showing the percentage of CD4+CXCR5+ cells in active IgG4-SC/AIP, inactive IgG4-SC/AIP, disease controls with PSC, and healthy donors (HCs). (c) Dot plot showing the percentage of Tfh1, Tfh2, and Tfh17 subsets of blood Tfh cells in the 4 groups. (d) Dot plot showing the percentage of Tfh1, Tfh2, and Tfh17 cells expressing PD-1 in the 4 groups. (e) Dot plot showing absolute numbers of Tfh1, Tfh2, and Tfh17 cells expressing PD-1 in active and inactive IgG4-SC/AIP and HC groups. Median shown for all plots, Mann-Whitney and ANOVA with multiple comparisons, P values: NS P ≥ 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. AIP, autoimmune pancreatitis; HC, healthy control; IgG4-SC, IgG4-related sclerosing cholangitis; PD-1, programmed cell death protein 1; PSC, primary sclerosing cholangitis; Tfh, T-follicular helper.

Figure 2.

Circulating PD-1+ Tfh2 cells correlate with plasmablasts in active IgG4-SC/AIP. Circulating plasmablasts and Tfh cells were analyzed by flow cytometry. (a) Contour plot showing CD38+ CD20− on CD27+ CD19+(low) cells identifying a plasmablast population from a representative donor. (b) Dot plot showing the percentage of CD27++CD38++CD20−CD19low plasmablasts in active IgG4-SC/AIP and HC. Median values and Mann-Whitney P values as in Figure 1. Correlation plots showing (c) PD-1+ Tfh cells and (d) PD-1+ Tfh2 cells plotted against the percentage of plasmablasts. Spearman rank correlation (r) and P values are expressed as NS ≥ 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001. AIP, autoimmune pancreatitis; HC, healthy control; IgG4-SC, IgG4-related sclerosing cholangitis; PD-1, programmed cell death protein 1; Tfh, T-follicular helper.

Figure 3.

Circulating PD1+ Tfh cells preferentially help B cells to produce IgG4. Circulating CD27+ memory B cells and PD-1+ or PD-1− Tfh cells from healthy donors were isolated and cocultured in the presence of staphylococcal enterotoxin B (SEB). (a) IgG4 concentration (log¹⁰ ng/mL) detected in cell culture supernatant after day 7 of coculture by IgG4 ELISA (3 values are not shown on the logarithmic scale). Data from 9 donors. (b) CD27+ CD38hi antibody–secreting cells as a log¹⁰ percentage of CD19lo cells after days 6 and 7 of the coculture. (c) IL-10, IL-21, IL-17A, and IL-4 cytokine concentration in pg/mL detected in cell culture supernatant after days 6 and 7 of the coculture. **Median values and Mann Whitney P values < 0.01. Data from 10 donors. PD-1, programmed cell death protein 1; Tfh, T-follicular helper.

Figure 4.

Circulating PD-1+ Tfh1 and Tfh2 subsets correlate with serum IgG4 and IgE levels in IgG4-SC/AIP. Serum IgG4 and IgE levels were measured by nephelometry and automated ImmunoCAP, respectively. Correlation plots showing serum IgG4 (g/L) plotted against (a) PD-1+ Tfh1 cells, (b) PD1+ Tfh2 cells, and (c) PD1+ Tfh17 cells, and serum IgE (kIU/L) plotted against (d) PD-1+ Tfh1 cells, (e) PD1+ Tfh2 cells, and (f) PD1+ Tfh17 cells. Spearman rank correlation and *P < 0.05, **P < 0.01. AIP, autoimmune pancreatitis; IgG4-SC, IgG4-related sclerosing cholangitis; PD-1, programmed cell death protein 1; Tfh, T-follicular helper.

Figure 5.

Circulating PD-1+ Tfh2 and Tfh17 subsets correlate with disease activity in IgG4-SC/AIP. Disease activity was measured using the IgG4-RI. Correlation plots showing disease activity (DA) (log 10) plotted against the percentage of (a) CXCR5+ Tfh cells, (b) Tfh2 cells, (c) PD-1+ Tfh cells, (d) PD1+ Tfh1 cells, (e) PD1+ Tfh2 cells, and (f) PD1+ Tfh17 cells. Spearman rank correlation and P values as per Figure 2. AIP, autoimmune pancreatitis; IgG4-RI, IgG4-responder index; IgG4-SC, IgG4-related sclerosing cholangitis; PD-1, programmed cell death protein 1; Tfh, T-follicular helper.

Figure 6.

CD4+ T cells and Tfh cells are present in IgG4-RD tissue specimens and home to lymphoid follicles. Paraffin-embedded tissue specimens were stained for Tfh-cell markers. (a) Representative panels from patients with IgG4-SC/AIP stained for CD4 (liver, top left panel), CXCR5 (pancreas, top right panel), ICOS (pancreas, bottom left panel), and CXCL13 (pancreas, bottom right panel). Positively staining cells stain DAP-positive (brown). (b) Histogram shows the percentage of positively staining specimens for CD4+, CXCR5+, CXCR5+ICOS+, and CXCL13+ cells. Data from 12 patients with IgG4-SC/AIP (red bars) and 10 disease controls (gray bars). **P values as in Figure 2. AIP, autoimmune pancreatitis; DC, disease control; IgG4-RD, IgG4-related disease; IgG4-RI, IgG4–responder index; IgG4-SC, IgG4-related sclerosing cholangitis.