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. 2014 Apr 17;40(4):542-53.
doi: 10.1016/j.immuni.2014.03.004. Epub 2014 Apr 3.

TIM-1 glycoprotein binds the adhesion receptor P-selectin and mediates T cell trafficking during inflammation and autoimmunity

Stefano Angiari  1 , Tiziano Donnarumma  1 , Barbara Rossi  1 , Silvia Dusi  1 , Enrica Pietronigro  1 , Elena Zenaro  1 , Vittorina Della Bianca  1 , Lara Toffali  2 , Gennj Piacentino  1 , Simona Budui  1 , Paul Rennert  3 , Sheng Xiao  4 , Carlo Laudanna  2 , Jose M Casasnovas  5 , Vijay K Kuchroo  4 , Gabriela Constantin  6
Affiliations

TIM-1 glycoprotein binds the adhesion receptor P-selectin and mediates T cell trafficking during inflammation and autoimmunity

Stefano Angiari et al. Immunity. .

Abstract

Selectins play a central role in leukocyte trafficking by mediating tethering and rolling on vascular surfaces. Here we have reported that T cell immunoglobulin and mucin domain 1 (TIM-1) is a P-selectin ligand. We have shown that human and murine TIM-1 binds to P-selectin, and that TIM-1 mediates tethering and rolling of T helper 1 (Th1) and Th17, but not Th2 and regulatory T cells on P-selectin. Th1 and Th17 cells lacking the TIM-1 mucin domain showed reduced rolling in thrombin-activated mesenteric venules and inflamed brain microcirculation. Inhibition of TIM-1 had no effect on naive T cell homing, but it reduced T cell recruitment in a skin hypersensitivity model and blocked experimental autoimmune encephalomyelitis. Uniquely, the TIM-1 immunoglobulin variable domain was also required for P-selectin binding. Our data demonstrate that TIM-1 is a major P-selectin ligand with a specialized role in T cell trafficking during inflammatory responses and the induction of autoimmune disease.

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Figures

Figure 1
Figure 1. TIM-1 interacts with selectins in vitro requiring a specific glycosylation profile and mediates tethering and rolling on endothelial selectins under flow conditions
(A and B) Microtiter plates were coated with 5 µg/ml murine or human P-selectin, E-selectin or L-selectin, TIM-4 (positive control) (Meyers et al., 2005) or ICAM-1 (negative control) (Santiago et al., 2007B), and tested for their ability to bind recombinant mouse or human TIM-1 respectively. In some experiments, 10 mM EDTA was added to chelate divalent cations. Both murine (A) and human (B) TIM-1 bound to all three selectins, and binding was dependent on the presence of divalent cations (*P < 0.0001 compared to ICAM-1 binding). (C and D) Microtiter plate assays show the binding of recombinant mouse TIM-1 protein from CHO (shown in C) and 293T cells (shown in D) to P- and E-selectin after treatment with α1,(3,4)-fucosidase, tyrosine sulfatase, PNGase, OSGE and neuraminidase treatment (*P < 0.001). (E) Protein A-covered microspheres were coated with a murine TIM-1 Fc-chimera (Sizing et al., 2007) or a control mouse IgG Fc-chimera (control beads) and were infused into glass capillary tubes pre-coated with P-selectin, E-selectin or L-selectin, under physiological shear stress conditions (2 dyne/cm2) (*P < 0.002, **P < 0.0001). See also Movies S1 and S2. (F) The rolling velocity of TIM-1-covered beads declines as the concentration of P-selectin and E-selectin increases under physiological flow conditions. Data represent the means ± standard error of the mean (SEM) of three independent experiments. In (A), (B), (C) and (D), data represent means ± SEM of at least three independent experiments performed in triplicate for each condition.
Figure 2
Figure 2. The TIM-1 mucin domain is required for the capture and rolling of Th1 and Th17 cells on P-selectin under physiological flow conditions in vitro
(A–E) Treg cells or CD4+ T cells stimulated with concanavalin A (ConA) or polarized toward Th1, Th17 or Th2 phenotypes were infused into capillary tubes pre-coated with 5 µg/ml P-selectin or E-selectin under shear stress conditions (2 dyne/cm2) (*P < 0.002; **P < 0.0003; ***P < 0.0001). Bone marrow-derived PMNs, which do not express TIM-1, were used as negative controls. (B) Representative images of wild-type and Havcr1Δmucin Th1 cells rolling on P-selectin, showing a consistently lower number of rolling Havcr1Δmucin Th1 cells. See also Movies S3 and S4. (C and E) Rolling velocities of wild-type and Havcr1Δmucin ConA blasts, Th1, Th17, Th2 and Treg cells on P- and E-selectin. Data represent means ± SEM of four independent experiments. For rolling velocities, data represent means ± SEM of at least 100 cells per condition.
Figure 3
Figure 3. TIM-1 mediates tethering and rolling of Th1 and Th17 cells in vivo
Th1 and Th17 cells were generated from murine wild-type and Havcr1Δmucin CD4+ cells. Bone marrow-derived PMNs were used as negative controls. Cells were injected intravenously into the exposed mesenteric vessels of mice pre-treated with bovine thrombin to upregulate P-selectin on the vascular endothelium. (A) Havcr1Δmucin T cells have a significantly reduced ability to roll on P-selectin in vivo (*P < 0.002 compared to wild-type cells). See also Movie S5. (B) Representative images of Th1 cells rolling in mesenteric vessels. Cells are the white spots inside the venules (arrow tips). Scale bar: 100 µm. (C) The number of tethers (each new interaction with the vessel wall) are shown. (*P < 0.03 and **P < 0.004 compared to the corresponding wild-type cells). Data in (A) and (C) represent means ± SEM of 13-15 independent experiments for a total of 15-20 total venules/condition. (D and E) Havcr1Δmucin Th1 (D) and Th17 (E) cells display no defects in their rolling velocity in mesenteric venules, compared to wild-type cells. Rolling velocities represent the mean ± SEM of at least 100 cells per condition (left panel). The distribution of leukocyte rolling velocities is also shown (right panel).
Figure 4
Figure 4. The TIM-1 IgV domain is required for P-selectin-dependent rolling in vivo
(A–D) Wild-type Th1 cells were treated with control rat IgG or the blocking anti-TIM-1 antibodies RMT1-10 or 4A2.2, which recognize epitopes in the TIM-1 IgV domain (Xiao et al., 2007; Sizing et al., 2007). (A) Cells treated with RMT1-10 and 4A2.2 showed a strongly reduced ability to roll on P-selectin (*P < 0.005 and **P < 0.04 compared to cells treated with rat IgG). (B) RMT1-10 and 4A2.2 treatments also reduced the number of total tethers (*P < 0.008 and **P < 0.03 compared to control cells). Data in (A) and (B) represent means ± SEM of 3-4 independent experiments for a total of 6-8 total venules per condition. (C and D) Cells treated with both RMT1-10 (C) and 4A2.2 (D) showed no defects in their rolling velocity in mesenteric venules. Rolling velocities represent the mean ± SEM of at least 50 cells per condition. The distribution of leukocyte rolling velocities is also shown (right panels). (E) Microtiter plate assays showing the binding of full-length versus truncated TIM-1 proteins to P-selectin (*P < 0.001 compared to entire TIM-1 protein). ICAM-1 was used a negative control for TIM-1 binding. (F) Effect of RMT1-10 antibody on Havcr1Δmucin Th1 cell tethering and rolling in thrombin-treated mesenteric venules. Data represent means ± SEM of two experiments for a total of 5–6 venules per condition.
Figure 5
Figure 5. TIM-1 mediates T cell recruitment in the inflamed skin
Mice were sensitized with 1-fluoro-2,4-dinitrobenzene (DNFB) to induce cutaneous hypersensitivity (CHS) and were challenged again 5 days later on each side of the right ear pinnae with DNFB. The ear thickness was measured 0, 24 and 48 h after challenge. (A) 10 × 106 CFSE-labeled Th1 cells from wild-type and Havcr1Δmucin mice were transferred to CHS mice 24 h after the ear pinnae were painted, and Th1 accumulation was evaluated 24 h later by flow cytometry (*P < 0.02). (B) The injection of wild-type but not Havcr1Δmucin Th1 cells increased the inflammation response in the challenged ear (*P<0.01). In both A and B, results represent the mean ± SEM of 9-11 mice per condition.
Figure 6
Figure 6. TIM-1 controls T cell trafficking in the inflamed CNS and the induction of EAE
(A) Havcr1Δmucin Th1 and Th17 cells ability to roll and adhere firmly in LPS-inflamed brain pial vessels, in intravital microscopy experiments (**P < 0.008; *P < 0.01; ***P < 0.007). Data are mean ± SEM of three experiments for a total of 8-9 venules per condition. (B) Representative images of brain venules showing adhered Th1 cells as white spots inside the vessels (arrow tips). Scale bar = 100 µm. (C) 5 × 106 MOG35-55-specific Th1 cells were labeled with CFSE and injected into C57BL/6J wild-type mice treated 5 h previously with 20 ng pertussis toxin (PTX). The accumulation of CSFE+ cells in the CNS was evaluated 60 h after cell transfer. Data represent means ± SEM of 12 mice per condition from two experiments (*P < 0.006). (D) 5 × 106 MOG35-55-specific Th1 cells were transferred in C57BL/6J wild-type mice. Data represent the mean ± SEM of 10 mice per condition from two experiments (*P < 0.05). (E) EAE was actively induced by immunization with the MOG35-55 peptide. Data represent the mean ± SEM of 10 mice per condition from two independent experiments with similar results (*P < 0.05). (F) Hematoxylin & eosin and Spielmeyer stainings are shown at disease peak. (G) CD4+ T cells were isolated from the draining lymph nodes 7 days post-immunization with the MOG35-55 peptide and the proliferative response was determined. Data are shown as counts per minute (CPM) of [3H]-thymidine radioactivity and represent the mean ± SD of five mice per condition.
Figure 7
Figure 7. TIM-1 and PSGL-1 concur to control activated T cell rolling on P-selectin and T cell trafficking in inflamed tissues
(A) Th1 cells were infused into capillary tubes pre-coated with P-selectin or E-selectin (*P < 0.00001; **P<0.0003) Data represent the mean ± SEM of four experiments. (B) Rolling velocities in capillary tubes coated with P-selectin or E-selectin are shown (*P < 0.001 and **P < 0.008 compared to WT cells). Rolling velocities represent the mean ± SEM of at least 50 cells per condition. (C) Shown is the ability to tether and roll in thrombin-treated mesenteric venules. Data obtained by treating Havcr1Δmucin cells with antibody 4RA10 are also shown (*P < 0.001 and **P < 0.02 compared to WT cells). Data represent the mean ± SEM of 10 independent experiments for a total of 16-17 total venules per condition. (D) Rolling velocities are shown in mesenteric venules. Rolling velocities represent the mean ± SEM of at least 50 cells per condition. The distribution of leukocyte rolling velocities is also shown in vivo (right panel). (E) Selplg−/− Th1 cells treated with RMT1-10 showed a reduced ability to tether ad roll on P-selectin in vivo, compared to cells treated with the control rat IgG (*P < 0.05, left panel; *P < 0.002, right panel). Data represent the mean ± SEM of three experiments for a total of seven venules per condition. (F) Rolling and arrest interactions are shown in intravital microscopy experiments in LPS-treated cerebral vessels (*P < 0.001; **P < 0.04). (G and H) Accumulation of wild-type, Havcr1ΔmucinSelplg−/− and Selplg−/− Havcr1Δmucin Th1 cells in the inflamed ear pinnae of CHS mice (G) and in the CNS of pertussis toxin-treated mice (H) (*P < 0.01, **P < 0.001 and ***P < 0.03). Data represent the means ± SEM of five mice per condition from one representative experiment from a series of two with similar results. (I) Rolling and arrest of naïve CD4+ T cells in Peyer’s patches HEVs (*P < 0.03 compared to WT cell arrest). Data represent the mean ± SEM of four mice per condition for a total of nine venules per condition. (J) Accumulation of exogenous naïve CD4+ T cells in lymphoid organs of wild-type resting mice at 2h after transfer (*P < 0.003 and **P < 0.04 compared to WT cells). Data represent the mean ± SEM of six mice per condition from one representative experiment from a series of three with similar results.
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