Unique expansion of IL-21+ Tfh and Tph cells under control of ICOS identifies Sjögren's syndrome with ectopic germinal centres and MALT lymphoma

Abstract

Objectives: To explore the relevance of T-follicular-helper (Tfh) and pathogenic peripheral-helper T-cells (Tph) in promoting ectopic lymphoid structures (ELS) and B-cell mucosa-associated lymphoid tissue (MALT) lymphomas (MALT-L) in Sjögren's syndrome (SS) patients.

Methods: Salivary gland (SG) biopsies with matched peripheral blood were collected from four centres across the European Union. Transcriptomic (microarray and quantitative PCR) analysis, FACS T-cell immunophenotyping with intracellular cytokine detection, multicolor immune-fluorescence microscopy and in situ hybridisation were performed to characterise lesional and circulating Tfh and Tph-cells. SG-organ cultures were used to investigate functionally the blockade of T-cell costimulatory pathways on key proinflammatory cytokine production.

Results: Transcriptomic analysis in SG identified Tfh-signature, interleukin-21 (IL-21) and the inducible T-cell co-stimulator (ICOS) costimulatory pathway as the most upregulated genes in ELS+SS patients, with parotid MALT-L displaying a 400-folds increase in IL-21 mRNA. Peripheral CD4⁺CXC-motif chemokine receptor 5 (CXCR5)⁺programmed cell death protein 1 (PD1)⁺ICOS⁺ Tfh-like cells were significantly expanded in ELS+SS patients, were the main producers of IL-21, and closely correlated with circulating IgG and reduced complement C4. In the SG, lesional CD4⁺CD45RO⁺ICOS⁺PD1⁺ cells selectively infiltrated ELS+ tissues and were aberrantly expanded in parotid MALT-L. In ELS+SG and MALT-L parotids, conventional CXCR5⁺CD4⁺PD1⁺ICOS⁺Foxp3^- Tfh-cells and a uniquely expanded population of CXCR5^-CD4⁺PD1^hiICOS⁺Foxp3^- Tph-cells displayed frequent IL-21/interferon-γ double-production but poor IL-17 expression. Finally, ICOS blockade in ex vivo SG-organ cultures significantly reduced the production of IL-21 and inflammatory cytokines IL-6, IL-8 and tumour necrosis factor-α (TNF-α).

Conclusions: Overall, these findings highlight Tfh and Tph-cells, IL-21 and the ICOS costimulatory pathway as key pathogenic players in SS immunopathology and exploitable therapeutic targets in SS.

Keywords: autoimmune diseases; cytokines; sjogren's syndrome; t-lymphocyte subsets.

Figure 1

IL-21 and T follicular helper (Tfh) cells in peripheral blood of SS and NSCS patients. (A) ELISA quantification of IL-21 serum level (pg/mL) from HD (n=12), NSCS (n=37) and SS (n=37). (B) Correlations of IL-21 serum level (pg/mL) with serum level of IgG, IgM, IgA, C3, C4 and ESSDAI in NSCS (n=37) and SS (n=37) patients. Spearman and p value are shown for the significant correlation (IgG, in red). (C) IL-21 serum level (pg/mL) in SS (n=31) according to ESSDAI domains. Box and whiskers plot show median and 5–95 percentile. Statistical analysis by Kruskal-Wallis-test with Dunn’s post-test correction for multiple comparison (A). (D) Average frequencies of Tfh-cell subsets, identified on the basis of ICOS and PD-1 expression and their frequencies distribution (E) in NSCS (white dots, n=10) and SS (dark grey dots, n=42). (F) Frequency of IFN-γ⁺, IL-21⁺ and IFN-γ⁺-IL-21⁺ double producing cells as percentage of PD-1⁺ICOS⁺, gated on CXCR5⁺CD4⁺ cells, detected by flow-cytometry on PBMC stimulation with PMA and ionomycin. Frequency of IL-21⁺ cells segregating the SS cohort for Ro (G) (Ro- (n=17) and Ro+ (n=20)) and La presence (H) (La- (n=27) and La+ (n=10)). Spearman correlation of IL-21⁺ cell (I, J, O), IFN-γ⁺ (K, L, P) or IL-21⁺-IFN-γ⁺ cell frequency (M, N, Q) with IgG (g/L) and C4 (g/L) serum levels and SG focus score in NSCS (n=10) and SS (n=42) patients. (R) Frequency of Tfh-cells subsets identified on the basis of ICOS and PD-1 expression in SS cohort segregated for ELS presence (ELS-, light grey dots (n=17), ELS+, dark grey dots (n=25)). (S) Frequency of CXCR5^-PD-1^hi cells, as percentage of CD4⁺ cells, in NSCS (n=10) and SS (n=42), and segregating the SS cohort for the presence of ELS. Statistical analysis by Mann-Whitney U t-test in (C), (E), (F), (G), (H), (R), (S). All graphs represent mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ELS, ectopic lymphoid structure; HD, healthy donor; ICOS, inducible T-cell costimulator; IFN-γ, interferon-γ; IL-21, interleukin-21; NSCS, non-specific chronic sialoadenitis; PD-1, programmed cell death protein 1; PNS, peripheral nervous system; SS, Sjogren’s syndrome.

Figure 2

Tfh-cell signature and IL-21 expression correlate with lymphocytic infiltration, ELS formation and GC B-cell genes in SS SGs. (A) Unsupervised whole-genome microarray analysis from SG RNA of NSCS (n=15) and SS (n=30), segregated for the absence (ELS-, n=15) and the presence of ELS (ELS+, n=15). (B) Supervised whole-genome microarray analysis of cohort described in (A), with the signature pathways rank-ordered for expression intensity. (C–E) GSVA for indicated signatures and (F–I) gene expression intensity comparison of Tfh-cell signature pathway genes (encoding respectively for CXCR5, ICOS, SAP and PD-1) in NSCS (n=15), ELS- (n=15) and ELS+ (n=15) SG. Linear regression model statistics. All graphs represent mean±SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Real-time PCR expression for IL-21 (J) and IL-21 receptor (IL21R) (K) on total RNA from SG biopsies from NSCS (n=37) and SS (n=29) patients, segregated on the basis of absence (ELS-, n=7) and presence of ELS (ELS+, n=22). Quantification of IL-21 mRNA (L–O) and IL-21R expression (P–S) in SG tissue biopsies, according to histological semi-quantitative score (0 to 3) of B (CD20), T (CD3), plasma cells (CD138) and macrophages (CD68). Statistical analysis by Kruskal-Wallis-test with Dunn’s post-test correction for multiple comparison. (T–Y) Spearman correlation analysis between IL-21 mRNA in SG and indicated lymphoid and GC B-cell genes. All graphs represent mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. CXCR5, CXC-motif chemokine receptor 5; ELS, ectopic lymphoid structure; GC, germinal centres; GSVA, Gene Set Variation Analysis; ICOS, inducible T-cell co-stimulator; IL-21, interleukin-21; NSCS, non-specific chronic sialoadenitis; PD-1, programmed cell death protein 1; SAP, SLAM-associated protein; SG, Salivary gland; SS, Sjogren’s syndrome; Tfh, T-follicular-helper.

Figure 3

PD-1⁺ICOS⁺CD45RO⁺CD4⁺ cells are increased within SG with ELS in SS and produce IL-21. (A) Representative immunofluorescence detection of CD4⁺CD45RO⁺ (top row), PD1⁺CD45RO⁺ (middle row) and ICOS⁺PD1⁺ cells (bottom row) in SG biopsy tissues with ELS. (B–D) Quantification (mean counts per field, a minimum of 5 random fields) of the double positive cells for each double immunofluorescence combination in SG biopsy tissues from NSCS (n=8) and SS (n=12) patients. Images displayed at x20 magnification. (E–H) ICOS⁺PD1⁺ cell count was segregated according to histological semi-quantitative score (0 to 3) of B (CD20), T (CD3) plasma cells (CD138) and macrophages (CD68). Statistical analysis by Kruskal-Wallis-test with Dunn’s post-test correction for multiple comparison (B–H). (I) Representative fluorescent in situ hybridisation (FISH) detection of IL-21 RNA, costained with CD4 and ICOS in SG biopsy tissues with ELS. (J) Spearman correlation analysis between SG real-time PCR IL-21 mRNA expression with ICOS⁺PD1⁺ cell count. All graphs represent mean±SEM. *p<0.05, **p<0.01, ***p<0.001. ELS, ectopic lymphoid structure; ICOS, inducible T-cell co-stimulator; NSCS, non-specific chronic sialoadenitis; PD-1, programmed cell death protein 1; SG, salivary gland; SS, Sjogren’s syndrome.

Figure 4

Development of parotid malt lymphomas is associated with elevated SG IL-21 and IL-21R expression. real-time PCR expression for IL-21 (A) and IL21R (B) on RNA extracted from SS labial SG biopsies with ELS (ELS+, n=22), SS parotid SG MALT-lymphoma (n=15) and parotid adenocarcinoma (n=10). Quantification (mean counts per field) of (C) CD4⁺CD45RO⁺, (D) PD1⁺CD45RO⁺, (E) PD1⁺ICOS⁺ cells in SS labial SG biopsies with ELS (ELS+, n=10) and SS parotid SG MALT-lymphoma (n=7). Mann-Whitney U t-test statistics. (F) Mean counts per field of ICOS⁺BCL6⁺ cells between SS parotid SG MALT-lymphoma (n=23), SS labial SG biopsies with ELS (ELS+, n=21) and tonsils (n=3). Statistical analysis by Kruskal-Wallis-test with Dunn’s post-test correction for multiple comparisons (A, B, F). (G) Representative immunofluorescence detection of ICOS⁺BCL6⁺ cells relative to B (CD20+) cell aggregates in SG biopsy tissues with ELS, parotid SG MALT-lymphoma and tonsil. All graphs represent mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ELS, ectopic lymphoid structures; ICOS, inducible T-cell costimulator; IL-21, interleukin-21; MALT, mucosa-associated lymphoid tissue; SG, Salivary gland; SS, Sjögren’s syndrome.

Figure 5

SG inflammation and development of parotid MALT lymphomas is associated with increased Tfh-cell numbers with increased production of IL-21 and IFN-γ. (A) Pie chart showing cytokines production by T helper (CD4⁺) cells isolated from parotid SG MALT-lymphoma (n=1), on stimulation with PMA and ionomycin and flow-cytometry analysis. (B) Representative flow-cytometry dot plots for cytokines production by T helper (CD4⁺) cells and (C) tSNE plots for indicated markers in T helper (CD4⁺) cells from parotid SG MALT-lymphoma. Flow-cytometry gating strategy for the identification of (D) IL-21 and IFN-γ producing Tfh-cells (identified as CD4⁺CD25^-Foxp3^- CXCR5⁺ICOS⁺PD-1⁺) and (E) pathogenic T peripheral helper cells (identified as CD4⁺CD25^-Foxp3^-CXCR5^-ICOS⁺PD-1⁺) in parotid SG MALT-lymphoma (n=1). ELS, ectopic lymphoid structure; ICOS, inducible T-cell costimulator; IFN-γ, interferon-γ; IL-21, interleukin-21; MALT, mucosa-associated lymphoid tissue; NSCS, non-specific chronic sialoadenitis; PD-1, programmed cell death protein 1; SG, Salivary gland; SS, Sjogren’s syndrome; Tfh, T-follicular-helper.

Figure 6

The blockade of ICOS-ICOS ligand signalling pathway reduces pro-inflammatory cytokines level in SG with ELS and MALT-lymphoma organ culture in SS. (A) Ingenuity Pathway Analysis (IPA) of microarray data obtained from the analysis of RNA extracted from ELS- and ELS+ SG. The orange-coloured bars (ICOS-ICOSL, CD28, CD40) show predicted pathway activation (with positive z-score), while the white bar (OX40 signalling pathway) indicates a z-score at or very close to 0 and the grey bar (CTLA4) pathways where no prediction can be made. (B) Schematic representation of a labial minor SG lobule cut longitudinally in half for the organ culture experiment. (C) representative histological images (H&E and IHC for CD20) of inflammatory infiltration in a parotid SG with B-cell non-Hodgkin MALT-lymphoma from a patient with SS. (D) Multiplex antibody array for cytokine analysis in the supernatant of a parotid MALT-lymphoma (n=1) organ culture, treated with an anti-ICOS blockade or its isotype control. The array template (white panel on the left) shows the coordinate reference of analytes with IL-6 spots highlighted (black circle). Black panels show the arrays incubated with organ culture supernatants treated with isotype control or anti-ICOS blockade with white circles around IL-6 spots. ELISA quantification of IL-8 and IL-6 in the supernatants of MALT-lymphoma organ culture, treated with an anti-ICOS blockade (grey dots) or its isotype control (white dots). Each dot represents a technical replicate. (E) Levels of TNF-α, IL-21, IL-8 and IL-6 detected in the supernatant of minor SG lobule organ culture, treated with an anti-ICOS blockade or its isotype control. The same colour dots represent minor SG lobules (from 2 to 8 lobules) from the same patient with SS (patients with SS, n=3). The lines link the two halves of the same lobules treated with the anti-ICOS blockade or its isotype control. Limit of detection (LD) for each cytokine is highlighted with a dashed line. ELISA quantification of IL-8 and IL-6 in the supernatants of minor SG lobules organ culture, treated with an anti-ICOS blockade or its isotype control, for cytokine levels that reach the Legendplex’ upper detection limit. Statistical analysis by Wilcoxon t-test. *p<0.05, **p<0.01. (F) All graphs represent mean±SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. ELS, ectopic lymphoid structure; ICOS, inducible T-cell costimulator; IFN-γ, interferon-γ; IHC, immunohistochemistry; IL-21, interleukin-21; MALT, mucosa-associated lymphoid tissue; NSCS, non-specific chronic sialoadenitis; PD-1, programmed cell death protein 1; SG, Salivary gland; SS, Sjogren’s syndrome; Tfh, T-follicular-helper.