Extended Data Figure 5 : CEACAM1 determines TIM-3 expression and function.

From: CEACAM1 regulates TIM-3-mediated tolerance and exhaustion

Extended Data Figure 5

a, HEK293T cells transiently co-transfected with Flag–hCEACAM1 and wild-type or mutants of HA–hTIM-3. Flow cytometry detecting HA–hTIM-3 (detected with anti-HA) and Flag–hCEACAM1 (detected with 5F4) proteins at cell surface (top), Golgi apparatus (middle) or endoplasmic reticulum (bottom) using monensin and brefeldin A, respectively. b, Cellular distribution of wild-type or mutant hTIM-3 when co-expressed with wild-type hCEACAM1. Total counts of hTIM-3 at surface, Golgi apparatus and endoplasmic reticulum summed up to 100%. Depicted as percentage of hTIM-3. c, HEK293T cells transiently co-transfected with wild-type HA–hTIM-3 (detected with 2E2) and wild-type or mutant Flag–hCEACAM1 (detected with anti-Flag). Flow cytometry analyses as in a. d, Cellular distribution of c, as in b. Depicted as percentage of hTIM-3. e, Immunoblot for wild-type or Thr101Ile variant of hTIM-3 showing maturation status in presence of wild-type or mutated (Gln44Leu) hCEACAM1. f, Normal association of Thr101Ile variant of hTIM-3 with hCEACAM1. g, Analysis of CD4+ Vβ8+ T cells after SEB tolerance induction from experimental mice of indicated genotypes. h, Galectin-9 induction of apoptosis. Annexin V+ propidium iodide staining of TH1 cells polarized from Tim3Tg or Tim3Tg Ceacam1−/− mice after treatment with galectin-9 (2 μg ml−1) for 8 h. Note decreased apoptosis in Tim3Tg Ceacam1−/− T cells. i, Schematic diagram of protocol used for protein pull-down using in-column IgV domain of GST–hTIM-3 incubated with hCEACAM1 protein derived from transfected HEK293T cells as in Fig. 2m. j, GST or GST–hTIM-3 staining of hCEACAM1-4L-transfected Jurkat T cells. k, Wild-type CD4+ T cells stimulated with anti-CD3 and/or anti-CD28 in the presence or absence of mCEACAM1 NFc, or IgG1-Fc as control, and cells analysed for secretion of IFN-γ and IL-2. l, m, Characterization of tolerance in SEB model. Tim3Tg (l) and Tim3Tg Ceacam1−/− (m) mice treated with SEB with schedule described in Extended Data Fig. 1e. Lymph node cells collected after SEB treatment and re-stimulated with soluble anti-CD3 at indicated doses and IL-2 measured by ELISA after 72 h. Note tolerance in Tim3Tg but not Tim3Tg Ceacam1−/− mice. n = 3 per group. n, Anti-mTIM-3 blockade with 2C12 antibody of mCEACAM1 NFc or control IgG-Fc staining of CD4+ T cells from indicated genotypes expressed as levels relative to Ceacam1/ mice. o, p, Analysis of mTIM-3 cytoplasmic tail function in transmitting mCEACAM1-induced signals. Activated mouse CD4+ T cells from wild-type (o) or Ceacam1−/− (p) mice were retrovirally transduced, sorted and stimulated with anti-CD3 with either human IgG-Fc (IgG, control) or mCEACAM1 N-terminal domain as NFc and TNF-α secretion assessed by ELISA after 72 h. Note ability of CEACAM1 N-terminal domain to transduce a signal associated with inhibition of TNF-α secretion in wild-type but not Ceacam1−/− T cells. n = 3 per group. Data are mean ± s.e.m. and represent three (f, g, kp) and two (ae, h, j) independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.