Extended Data Fig. 2: Anti-PILRα monoclonal antibody C21 specifically targets the stalk region of PILRα.
From: PILRα on tumor cells interacts with the T cell surface protein CD99 to suppress antitumor immunity
a,j, CD25+ quantification in Fig. 3d (a) and 3n (j) (n = 5 biologically independent experiments). b, Expression kinetics of CD99, PD-1, and TIM-3 in T cells with or without αCD3-stimulation for 48 h (n = 3 biologically independent experiments). c, Representative flow cytometry of CD99-Fc (10 μg ml−1) binding to WT or PILRAOE-MDA-MB-231 cells using Dylight 650-conjugated anti-human IgG Fc antibody (n = 3 biologically independent experiments). d, ELISA of hybridoma supernatants binding to PILRα-coated plates (OD450/OD630) (n = 3 biologically independent experiments). e,f, PILRα-expressing U87 cells incubated with hybridoma supernatants were stained with Alexa Fluor 488-conjugated anti-mouse IgG (H + L) antibody for high-content imaging (e), and immunofluorescence (IF) quantification (f) (n = 3 biologically independent experiments). g, Primary T cells were co-cultured with aAPC-293T cells expressing PILRα, with or without hybridoma supernatants for 72 h, followed by ELISA for IFNγ (n = 3 biologically independent experiments). Red clones were further purified. h,i, αCD3-stimulated (1 μg ml−1) NFAT-Lucia reporter Jurkat T cells were treated with or without PILRα (ECD)-hIg (20 μg ml−1) and anti-PILRα antibodies (50 μg ml−1), for luciferase activity (h) (n = 6 biologically independent experiments) and IFNγ detection (i) (n = 3 biologically independent experiments). k, PILRα (ECD)-hIg (2 μg ml−1) was pre-incubated with His-CD8-coated plates, followed by blocking ELISA using isotype, C21, or C126 (n = 3 biologically independent experiments). l, ELISA of PILRα (IgV)-hIg binding to C126-coated plate (n = 3 biologically independent experiments). m,n, αCD3-stimuated (1 μg ml−1) T cells were treated with or without PILRα (ECD)-hIg (20 μg ml−1) and isotype, C21, or C126 (50 μg ml−1) for 72 h, followed by flow cytometry for CD25 expression (m), and ELISA for IFNγ, granzyme B, and IL-2 (n) (n = 5 biologically independent experiments). Data are presented as mean ± s.e.m. a, P values were calculated using a two-way ANOVA with Tukey’s multiple comparisons test. j,n, P values were calculated using a one-way ANOVA with Tukey’s multiple comparisons test.
