a, hTIM-3 does not co-immunoprecipitate (co-IP) with ITGA5 despite interactions with hCEACAM1. HEK293T cells transfected with Flag–ITGA5 and HA–TIM-3 (ITGA5Tw) or Flag–CEACAM1 and HA–TIM-3 (CwTw). Immunoprecipitation with anti-HA antibody and immunoblotted (IB) with anti-Flag antibody are shown. Input represents anti-Flag immunoblot of lysates. b, Co-immunoprecipitation of human TIM-3 and CEACAM1 from activated primary human T cells after N-glycanase treatment of lystates followed by immunoprecipitation with anti-human TIM-3 antibodies (2E2, 2E12 or 3F9) or IgG as control and immunoblotted with anti-human CEACAM1 antibody (5F4). Protein lystates from HeLa-CEACAM1 transfectants treated with N-glycanase followed by immunoprecipitation with 5F4 and the immune complex used as positive control (pos). c, mTIM-3 interacts with mCEACAM1 in mouse T cells. Splenocytes from Ceacam1-4STg Ceacam1−/− and Ceacam1-4LTg Ceacam1−/− mice cultured with anti-CD3 (1 μg ml−1) or anti-CD3 (1 μg ml−1) and anti-CD28 (1 μg ml−1) or medium for 96 h. Cell lysates immunoprecipitated with anti-mCEACAM1 antibody (cc1) or with mIgG and IB with 5D12 (anti-mTIM-3 antibody) are shown. Locations of mTIM-3 protein variants are indicated. CHO, carbohydrate. d, Immunoprecipitation and immunoblot as in a with tunicamycin treated, wild-type HA–hTIM-3 and Flag–hCEACAM1 co-transfected HEK293T cells. Arrowhead denotes core CEACAM1 protein. e, Potential hCEACAM1-interacting residues on hTIM-3 highlighted in blue. f, HEK293 T cells transiently co-transfected with Flag–hCEACAM1 and HA–hTIM-3 mutants. Immunoblotting of anti-HA were used to analyse hTIM-3 expression in HEK293T transfectants. Except for Pro50Ala mutation displaying enhanced overall protein expression, all other mutations in the IgV domain of hTIM-3 are equally detected by anti-HA antibody. g, Quantification of association of hTIM-3 mutants associated with wild-type hCEACAM1 shown in Fig. 2c summing all experiments performed. Association between wild-type hCEACAM1 and hTIM-3 core protein are depicted as reference (set as 1, n = 3, mean ± s.e.m. shown, unpaired Student’s t-test). h, Immunoprecipitation with anti-Flag (hCEACAM1) and immunoblot with anti-HA (hTIM-3) or anti-Flag of wild-type hCEACAM1 and mutant hTIM-3 proteins are shown. i, Quantification of h as performed in g. j, HEK293T cells co-transfected with Flag–hCEACAM1 wild-type and HA–hTIM-3 mutants and immunoprecipitation/immunblot as in h revealing no effects of Cys52Ala or Cys63Ala mutations in hTIM-3 in affecting association with hCEACAM1 in contrast to Cys109Ala mutation of hTIM-3 that disrupts interactions with hCEACAM1. k, Potential hTIM-3-interacting-residues around the FG–CC′ cleft of hCEACAM1 highlighted in red. l, HEK293T cells transiently co-transfected with Flag–hCEACAM1 mutants and wild-type HA–hTIM-3. Immunoblot with anti-Flag antibody was used to analyse hCEACAM1 expression in HEK293T co-transfectants. All hCEACAM1 mutations in IgV domain equally detected. m, Densitometric quantification of IgV domain hCEACAM1 mutations associating with wild-type HA–hTIM-3 described in Fig. 2d. n–p, Analysis of Gly47Ala mutation of hCEACAM1 in hTIM-3 co-transfected HEK293T cells by immunoprecipitation with anti-HA (hTIM-3) and immunoblot with anti-Flag (hCEACAM1) to detect association (n), IB with anti-Flag to confirm similarity of hCEACAM1 transfection (o) and quantification of associated hCEACAM1 of n as shown in m. q–s, Analysis of hCEACAM1 mutants Asn42Ala and Arg43Ala association with hTIM-3 (q), similarity of transfections (r) and quantification of q as in n–p. Representative of four (d, h), three (f, g, i, l–s), two (a–c) and one (j) independent experiments. *P < 0.05; **P < 0.01; ***P < .001.