Fig 2: 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.

Fig 3: ICOS expression pattern in tumor sites and peripheral blood. (A) CRC surgical tumor tissues were enzymatically digested and stained with fluorochrome labeled Abs, followed by flow cytometry analysis. (i) Representative figures of ICOS expression on CD4+ and CD8+ T cells, analyzed by flow cytometry, in tumor-infiltrated leukocytes. (ii) Quantitation of the percentage of ICOS+CD4+ or ICOS+CD8+ T cells among all of the CD4+ or CD8+ T cells gotten from tumor-infiltrated leukocytes, respectively (n = 26). (B) The similar protocol is as (A), except for the specimens from pericarcinous tissues (i) or distal normal tissues (ii). (C) The similar protocol is as (A), except for the specimens from peripheral blood. (D) CRC samples were stained with ICOS antibody by immunohistochemistry. At the same time, the samples were enzymatically digested to get the TILs and ICOS expression on CD4+ T cells were analyzed by flow cytometry. (E) Correlation between percentage of ICOS+ among CD4+ T cells and ICOS staining scores in the CRC tumor tissue (n = 13). Data are presented as averages ± SEM, statistical difference was evaluated by Student's t test. **p < 0.01; ***p < 0.001.

Fig 4: The percentage of ICOS+ T cells in PBMC is a good marker for prognosis of CRC. (A) Correlation between percentage of ICOS+ T cells in peripheral blood and that in primary tumor tissues. (B) Quantity of ICOS+ T cells in peripheral blood of luminal breast cancer patients by flow cytometry. Data are presented as averages ± SEM, statistical difference was evaluated by Student's t test. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 5: 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-1hi 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.