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. 2020 Sep 22;79(12):1588–1599. doi: 10.1136/annrheumdis-2020-217646

Figure 6.

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.