CD28 co-stimulation inhibits <i>in vitro</i> generation of IL-17 producing CD4⁺ T cells.

<p>Purified CD4⁺ T (1×10⁶) cells from spleen (A–C) and LNs (A) were stimulated with plate-bound anti-CD3 with or without anti-CD28 under pro-Th17 conditions for 4–5 days and restimulated for 6 h with PMA/Ionomycin in the presence of brefeldin A. (A) CD4⁺ T cells were stained intracellularly for IL-17 and IFN-γ. Numbers represent the percentage of events in each quadrant. (B) Cumulative data of the percentage of CD4⁺IL-17 and IFN-γ positive T cells after restimulation as assessed by intracytoplasmic staining. Data represent the mean±SEM of at least ten independent experiments. (C) The ratio of the percentage of IL-17⁺ to IFN-γ⁺ cells among CD4⁺ T cells. (D) qRT-PCR analysis of IL-17 and IL-22 mRNA expression in purified CD4⁺ T cells cultured as described in A. Data are expressed as mean±SEM of four experiments. P values were calculated using the two-tailed, paired Student's <i>t</i> test, *, P<0.05; ***, P<0.001.</p

CD28 co-stimulation did not modulate memory Th17 cells.

<p>(A) FACS sorted naïve CD4⁺ (CD4⁺CD62L^highCD44^low), CFSE-labeled naïve CD4⁺ CD25⁻ (CD4⁺CD62L^highCD44^lowCD25⁻) and memory CD4⁺ (CD4⁺CD62L^lowCD44^high) T cells were cultured with anti-CD3 in the presence or absence of anti-CD28 mAb under pro-Th17 conditions for 4–5 days. Shown is one representative experiment out of at least three. (B) CD4⁺ T cells were stimulated with anti-CD3 under Th17 polarizing conditions for 4–5 days and restimulated with plate-bound anti-CD3 in the presence or absence of IL-23, and with or without anti-CD28 mAb for 2 days. Data represent the mean±SD of five to seven independent experiments. (C) Purified CD4⁺ T cells were cultured with anti-CD3 in the presence or absence of anti-CD28 mAb without adding Th17 differentiation cytokines for 4–5 days. Data are the mean±SD of 2 independent experiments. (A–C) Cells were restimulated for 6 h with PMA/Ionomycin in the presence of brefeldin A. Naïve and memory CD4⁺ T cells were stained intracellularly for IL-17 and IFN-γ and CFSE-labeled naïve CD4⁺CD25⁻ for Il-17. Numbers represent the percentage of positive cells in each gate. P value were calculated using the two-tailed, paired Student's <i>t</i> test, **, P<0.01; ***, P<0.001.</p

Inhibition of Th17 differentiation by anti-CD28 mAb is not correlated with a expansion of Foxp3⁺ T cells.

<p>Purified CD4⁺ T cells were stimulated with plate-bound anti-CD3 in the presence or the absence of soluble anti-CD28 mAb under Th17 polarizing conditions. (A) The proportion of Foxp3⁺CD4⁺ T cells. Data from 8 independent experiments are shown. (B and C) CD4⁺, CD4⁺CD25⁻ T cells and CD4⁺CD25⁻ T plus CD4⁺CD25⁺ T cells (at 1/1, 4/1 CD25⁻/CD25⁺ T cell ratios) were stimulated as described. Numbers indicate the percentage of IL-17 and IFN-γ-expressing CD4⁺ T cells (B) and IL-17 and Foxp3-expressing cells (C). One of 2 representative experiments is shown. P values were calculated using the two-tailed, paired Student's <i>t</i> test, **, P<0.01.</p

TCR avidity and CD28 co-stimulation signals regulate Th17 development.

<p>(A) Purified CD4⁺ T were stimulated with plate-bound anti-CD3 (5 µg/ml) and titrated doses of soluble anti-CD28 mAb under Th17 polarizing conditions. Cells were stained intracellularly for IL-17 and IFN-γ expression. The data shown is one representative experiment out of two. (B) CD4⁺ T cells were stimulated with titrated doses of plate-bound anti-CD3 in the presence or absence of soluble anti-CD28 mAb (2 µg/ml). Data represent the mean±SD of four independent experiments. (C) Cumulative data of viable CD4⁺ T cell recovery after stimulation with anti-CD3 (5 µg/ml) the presence or absence of anti-CD28 mAb (2 µg/ml). Data are the mean±SD of 3 (LNs) to 20 (spleen) independent experiments. (D) CD4⁺ T cells were stimulated with coated anti-CD3 (10 µg/ml) in the presence or absence of soluble anti-CD28 under Th17 conditions and analyzed at different time points for the percentage of apoptotic cells (Annexin V binding positivity). Mean values±SD obtained from at least three separate experiments are presented. P values were calculated using the two-tailed, paired Student's <i>t</i> test, *, P<0.05; **, P<0.01.</p

CTLA4-Ig favours Th17 development.

<p>(A) CD4⁺ T cells were cultured with mature BMDC at three different CD4⁺/mBMDC ratios (2/1; 1/1; 1/2) in the presence of anti-CD3 under Th17 conditions. CTLA4-Ig (20 µg/ml) was added to cultures with a 1/1 CD4⁺/mBMDC cell ratio. Cells were stained intracellularly for IL-17 and IFN-γ. Shown is one representative experiment out of 4. (B) CD4⁺ T cells were stimulated as in A. Data represent the mean±SD of 4 experiments. (C) Human naïve CD4⁺ T cells were co-cultured with activated monocyte-derived DC in the presence or absence of CTLA4-Ig (5 µg/ml), expanded in IL-2 and restimulated. IL-17, IL-22 and IFN-γ secretion was assessed in the culture supernatants and cells were stained for intracellular IL-17. Shown are 3 independent experiments (ELISA, left panel) and 1 representative out of 3 experiments (intracytoplasmic staining, right panel). P values were calculated using the two-tailed, paired Student's <i>t</i> test, *, P<0.05; **, P<0.01.</p

The CD28-driven inhibition of Th17 differentiation is mediated by IL-2 and IFN-γ production.

<p>Purified CD4⁺ T cells were stimulated with plate-bound anti-CD3 in the presence or the absence of soluble anti-CD28 mAb under Th17 polarizing conditions. (A) The percentages of IL-2⁺CD4⁺ T cells from 7 independent experiments (mean±SEM). (B) CD4⁺ T cells were cultured in the presence or absence of anti-mouse IL-4, IFN-γ or IL-2 alone or in combination. The data shown are representative of 1 experiment out of 3 (dot plots, left panel). The cumulative data from 2–6 independent experiments are shown in the right panel. P values were calculated using the two-tailed, paired Student's <i>t</i> test, *, P<0.05; **, P<0.01; ***, P<0.001.</p

Mature BMDC inefficiently support Th17 differentiation.

<p>CD4⁺ T cells were cultured with immature or mature BMDC at a 2/1 CD4⁺/BMDC ratio in the presence of anti-CD3 under Th17 conditions. (A) BMDC were stimulated with LPS (1 µg/ml) and stained for CD40, CD86, or MHCII (IA/IE) expression. The mean fluorescence intensity are shown. Data are representative of one experiment out of three. (B) Cells were stained intracellularly for IL-17 and IFN-γ. Shown is one representative experiment out of 6. (C) Cumulative data of the percentage of CD4⁺IL-17 and IFN-γ positive T cells after restimulation as assessed by intracytoplasmic staining. Data represent the mean±SEM of 6 independent experiments. (D) The ratio of the percentage of IL-17⁺ to IFN-γ⁺ cells among CD4⁺ T cells. (E) CD4⁺ T cells (2.5×10⁵ cells) were cultured with immature or mature BMDC at three different CD4⁺/BMDC ratios (2/1; 1/1; 1/2) as described in A. Data represent the mean±SD of 4 independent experiments. P values were calculated using the two-tailed, paired Student's <i>t</i> test, *, P<0.05; **, P<0.01; ***, P<0.001.</p

Topoisomerase I peptide-loaded dendritic cells induce autoantibody response as well as skin and lung fibrosis

<p>DNA Topoisomerase I (TopoI) is a candidate autoantigen for diffuse cutaneous systemic sclerosis (dcSSc) associated with fatal lung disease. Dendritic cells (DCs) contribute to bleomycin-induced lung fibrosis. However, the possibility that TopoI-loaded DCs are involved in the initiation and/or perpetuation of dcSSc has not been explored. Here, we show that immunization with TopoI peptide-loaded DCs induces anti-TopoI autoantibody response and long-term fibrosis. Mice were repeatedly immunized with unpulsed DCs or DCs loaded with either TOPOIA or TOPOIB peptides, selected from different regions of TopoI. At week 12 after initial DC immunization, TOPOIA DCs but not TOPOIB DCs immunization induced mixed inflammation and fibrosis in lungs and skin. At a late time point (week 18), both TOPOIA DCs and TOPOIB DCs groups displayed increased alpha-smooth muscle actin expression in lungs and dermis along with skin fibrosis distal from the site of injection when compared with unpulsed DCs. Both TopoI peptide-DC-immunized groups developed IgG2a anti-TopoI autoantibody response. At week 10, signs of perivascular, peribronchial, and parenchymal pulmonary inflammation were already observed in the TOPOIA DCs group, together with transient elevation in bronchoalveolar lavage cell counts, IL-17A expression, and CXCL4 production, a biomarker of early human dcSSc. Collectively, TopoI peptide DCs induce progressive autoantibody response as well as development of protracted skin and lung dcSSc-like disease. Pronounced lung inflammation, transient IL-17A, and CXCL4 expression precede fibrosis development. Our immunization strategy, that uses self immune system and autoantigen, will help to further investigate the pathogenesis of this complex autoimmune disorder with unmet medical needs.</p

CD47 on CD4 T cells regulates the contraction of the immune response <i>in vivo</i>.

<p>One day after adoptive transfer of CD47^+/+ or CD47^−/− Tg T cells isolated from DO11.10 mice into CD47^−/− BALB/c hosts, mice were immunized s.c. with CFA-OVA or DEC205-OVA. (<b>A and C</b>) Kinetic of the recovery of viable Tg T cells is shown. (<b>B</b>) CD47 status (SIRP-α-Fc protein) gated on CD4 KJ126⁺(Tg) T cells post immunization. Data are representative of 4 to 6 independent experiments, student <i>t</i> test was performed on 8 to 12 mice. *p<0.05, ***p<0.001.</p

CD4 effectors display a CD47^high status in lymph nodes and lamina propria of patients with Crohn’s disease.

<p>(<b>A</b>) CD4 T cell subsets were examined in PBMC, mLNs and LPMC of patients with Crohn’s disease. (<b>B</b>) TSP-1 concentration in human colon biopsies. (<b>C</b>) CD47 expression (SIRP-α-Fc protein) after gating on memory (CCR7⁺) and effector (CCR7⁻) CD45RA⁻CD4⁺ T cells in PBMC, mLNs and LPMC. Data are representative of 4 to 6 independent experiments. The CD47 Mean Fluorescence Intensity (MFI) CCR7⁻T/CCR7⁺ T cell ratio was calculated for patients with CD and unrelated IBD patients. (<b>A and C</b>) The mean ± standard deviation (SD) for 5 to 6 independent experiments. Student <i>t</i> test was performed: *p<0.05.</p