a, Schematic diagram of OVA antigen-specific tolerance induction model. b, Schematic diagram of OVA immunization. c, Tracking in vivo antigen-specific T-cell responses of CFSE-labelled OT-II transgenic Rag2−/− T cells in total lymphocyte gate of mesenteric lymph nodes, peripheral lymph node or spleen of wild-type or Ceacam1−/− recipients after gating on CFSE-positive cells and staining for CEACAM1 in PBS and OVA323–339 immunized mice. Hyper-responsiveness of OT-II transgenic Rag2−/− T cells in Ceacam1−/− mice was not due to decreased regulatory T-cell induction (data not shown) or increased initial parking on the basis of cell numbers shown. d, TIM-3 expression on CEACAM1-positive and -negative CFSE+ cells as in c. e, Schematic diagram of SEB-induced T-cell tolerance model. f, mCEACAM1 and mTIM-3 expression on CD4+ Vβ8+ T cells after SEB tolerance induction. g, hCEACAM1 and hTIM-3 expression on activated primary human T cells defined by staining with indicated antibodies. h, CEACAM1 expression on TIM-3-silenced primary human T cells after re-activation by flow cytometry. Relative TIM-3, CEACAM1 or CD4 expression on T cells expressing control shRNA (lacZ control, red) or three independent shRNAs directed at TIM3 (overlay, blue). shRNA target sequences shown. i–l, CEACAM1 and TIM-3 expression and functional consequences on T cells in HIV infection. CD4+ IFN-γ+ T cells are decreased among CEACAM1+ TIM-3+ CD4+ T cells in HIV infection in response to Gag peptides (i). Although proportions of CEACAM1+ TIM-3+ CD8+ T cells are similar in HIV-infected and -uninfected subjects (j), CEACAM1+ TIM-3+ CD8+ T cells express little IFN-γ after stimulation with HIV Gag peptides or SEB relative to TIM-3+ CEACAM1− CD8+ T cells (k, l). C, hCEACAM1; T, hTIM-3 (n = 4 per group, mean ± s.e.m.). m–o, In situ proximity ligation analysis (PLA) of CEACAM1 and TIM-3. m, HEK293T cells transiently co-transfected with Flag–hCEACAM1 or HA–hTIM-3. Cells stained with DAPI (left), anti-tubulin (middle), anti-HA (rabbit) and anti-Flag (mouse) (middle right) or merged (right). Several examples of a positive PLA signal (middle right and right panels: red fluorescent dots) indicative of a maximum distance of 30–40 nm between hCEACAM1 and hTIM-3. n, Negative control, co-expression of Flag–PLK1 (protein kinase I) and HA–TIM-3 failed to generate fluorescent dots (that is, PLA negative). Cells stained with DAPI, anti-tubulin, anti-HA/anti-Flag or merged as in m. o, Negative control, co-expression of HA–ADAP (adhesion and degranulation promoting adaptor protein) failed to show a signal (that is, PLA negative) with staining as in m. p, q, CEACAM1 and TIM-3 colocalization at immunological synapse of primary human CD4 and CD8 T cells. Confocal microscopy of hTIM-3+ hCEACAM1+ primary CD4+ and CD8+ T cells forming conjugates with SEB-loaded B cells. DIC, differential interference contrast. Blue denotes B cell; red denotes CD3; purple denotes CEACAM1; green denotes TIM-3. White indicates colocalization between CEACAM1 and TIM-3 (p). Average Pearson correlation coefficients for CD4+ and CD8+ T cells were 0.543 and 0.566, respectively, representing strong co-localization (q). Data are mean ± s.e.m. and representative of five (f, g), four (p, q), three (c, d, m–o) and two (h) independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.