Fig 2: CD155-CD96 interaction promotes differentiation of IL-9-producing effector T cellsMonocyte-derived Mφs were generated from patients with GCA or healthy controls and used as antigen-presenting cells (APCs) for CD4+ T cells. Induction of T cell effector cytokines was assessed by RT-PCR or flow cytometry.(A) CD4+ T cells from the Mφ-T cell co-cultures were isolated after 72 h. Transcripts for 15 pro-inflammatory effector cytokines were quantified by RT-PCR. Each column represents Mφs from one patient or one control.(B–E) Frequencies of IL-9-producing (B and C), IFN-γ-producing (D), or TNF-α-producing (E) CD4+ T cells induced by healthy, GCA, or GPA (C) Mφs. Effector cytokines were measured by flow cytometry. Frequencies of cytokine-producing CD4+ T cells from 6 to 12 independent experiments are presented.(F) CD4+ T cells were stimulated with healthy Mφs in the absence and presence of anti-CD155 antibody. Intracellular IL-9 protein expression in T cells was analyzed by flow cytometry. Frequencies of IL-9-producing CD4+ T cells from 8 experiments.(G) CD4+ T cells were stimulated with healthy Mφs in the absence and presence of anti-CD96 antibody. Induction of IL-9-producing T cells was determined by flow cytometry. Frequencies of IL-9-producing CD4+ T cells from 6 experiments.Mean ± SEM with individual values shown. (A and C–G) Two-tailed paired t test. (B) Wilcoxon matched-pairs signed rank test. ∗∗p < 0.01, ∗∗∗p < 0.001, n.s., not significant.

Fig 3: CD155 is trapped in the ER of GCA MφsMφs were induced from peripheral blood precursor cells as in Figure 3.(A) Dual-color immunofluorescence imaging of Mφs stained for CD155 and cellular organelles. Markers represent the following organelles: ER, calnexin; ERGIC, P58; Golgi body, GM130; early endosome, EE1; late endosome, CD63; lysosome, LAMP2a; autophagosome, LC3; mitochondria, HSP60. Cellular stains were analyzed by imaging software to reveal co-localization as white dots (bottom panel).(B) Co-localization coefficient between CD155 and individual cell organelles in GCA Mφs. At least 25 cells were quantified from each independent experiment.(C) The ER fraction was isolated from healthy and GCA Mφs and CD155 protein expression analyzed by immunoblotting. Calnexin served as control. Western blot and quantitative data are shown.(D) ER stress signatures compared in control and patient-derived Mφs. Transcript levels of ER stress markers were quantified by RT-PCR. z scores are shown (n = 12).(E) Quantification of the ER stress-related protein BiP in control and GCA Mφs. Representative immunoblot and results from 3 independent experiments.(F) Dual-color immunofluorescence staining of tissue sections for the Mφ marker CD163 (red) and the ER stress protein BiP (green). Nuclei marked with DAPI (blue). Temporal arteries were collected from patients with GCA. Sinonasal biopsies from patients with GPA served as disease control.(G) Mφs were treated with the ER stress-inducer tunicamycin or DMSO. ER fractions were isolated, and CD155 was quantified by immunoblotting. Calnexin served as a control. Representative immunoblot and data from 4 independent experiments.Mean ± SEM with individual data shown. Scale bars: 5 μm (A) and 20 μm (F). (B–E) Two-tailed unpaired t test. (G) Two-tailed paired t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.