Fig 1: 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-a, 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-?, interferon-?; 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 2: Characterization of acute HIV-1 infection in ‘BLT' humanized mice.(a) Generation and infection of BLT humanized mice schema. (b) Proportion of human CD4 T cells in peripheral blood, spleen and colon lamina propria (LP). (c) Flow cytometry analysis of intracellular IL-17A and IFN? protein expression in CD3+CD4+ T cells from the indicated tissues from uninfected (top) or HIV-1-infected (bottom) humanized mice after 6 weeks. Single cell suspensions from the indicated tissues were stimulated with PMA and ionomycin for 5 h. Numbers represent proportion of cells within each gate. (d) Quantification of IL21, IL17A and IFNG mRNA (normalized to GAPDH) in splenic CD4 T cells from BLT mice 6 weeks post HIV-1 infection (four mice per group). *P<0.05; ***P<0.0001; unpaired Students t-test.
Fig 3: IL-21 suppresses initial HIV-1 infection in lymphoid CD4 T cells.(a) GFP versus CD4 expression on gated CD3+ cells in untreated (medium) or IL-21-treated HLACs 72 h post infection with GFP-tagged HIV-1NL4-3. (b) X4-HIV-1 infection (GFP+ cells gated as in a) in HLAC across multiple donors after 72 h. Infection in IL-21 treated cultures is expressed relative to untreated (medium, 100%). Each data point represents infection in HLAC prepared from individual donors (n=12) and compared by a paired Wilcoxon signed-rank test (P<0.0005). (c) HIV-1 p24 in HLAC supernatant at 3 and 5 days post infection. (d) HIV-1 p24 protein in HLAC supernatants infected with primary HIV-1 clades 72 h post infection. (e) Frequency of GFP+ cells in HLACs infected for 12 h with HIV-1 and washed extensively to remove viral supernatants before IL-21 treatment. Each data point represents a single donor. (f) Proportion of X4-HIV–GFP+ T cells in NK, NKT and/or CD8 T-cell-depleted HLAC determined as in b. Specific cell populations were depleted using commercially available anti-CD8 or anti-CD56 depleting microbeads as described in Methods. (g,h) Representative CD4 versus CD8 flow cytometry plot of CD3+ human splenocytes (g) and CD4/CD8 T-cell ratio (h) in uninfected (‘Uninf.') or HIV-1-infected HLACs in the presence or absence (Medium, Med) of IL-21 6 days after HIV-1 inoculation. Each data point represents one donor. Data in c–e and g are means (± s.e.m.) of triplicate wells and are representative of three donors. *P<0.05; **P<0.005, ***P<0.0005; unpaired Students t-test.
Fig 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.
Fig 5: 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.
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