TIM genes: a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity

Abstract

The TIM (T cell/transmembrane, immunoglobulin, and mucin) gene family plays a critical role in regulating immune responses, including allergy, asthma, transplant tolerance, autoimmunity, and the response to viral infections. The unique structure of TIM immunoglobulin variable region domains allows highly specific recognition of phosphatidylserine (PtdSer), exposed on the surface of apoptotic cells. TIM-1, TIM-3, and TIM-4 all recognize PtdSer but differ in expression, suggesting that they have distinct functions in regulating immune responses. TIM-1, an important susceptibility gene for asthma and allergy, is preferentially expressed on T-helper 2 (Th2) cells and functions as a potent costimulatory molecule for T-cell activation. TIM-3 is preferentially expressed on Th1 and Tc1 cells, and generates an inhibitory signal resulting in apoptosis of Th1 and Tc1 cells. TIM-3 is also expressed on some dendritic cells and can mediate phagocytosis of apoptotic cells and cross-presentation of antigen. In contrast, TIM-4 is exclusively expressed on antigen-presenting cells, where it mediates phagocytosis of apoptotic cells and plays an important role in maintaining tolerance. TIM molecules thus provide a functional repertoire for recognition of apoptotic cells, which determines whether apoptotic cell recognition leads to immune activation or tolerance, depending on the TIM molecule engaged and the cell type on which it is expressed.

Fig. 1. Schematic representation of TIM protein structures

Positions of the glycosylation sites in IgV domains are approximately positioned according to the crystal structures. Glycosylation sites in the mucin domain were predicted with NetOglyc and NetNglyc and are positioned approximately.

Fig. 2. Reciprocal regulation of Th1 and Th2 activity by TIM-1 and TIM-3: a Yin-Yang model

Fig. 3. Structures of the TIM Ig variable (IgV) domains and the unique conformation of the CC’ loop in each

(A) Ribbon diagram of the mTIM-1 IgV domain (PDB 2OR8). β-strands of the GFC β-sheet are red and those in the BED face are pink, coil is orange, and helices are in light-blue. The tip of the unique loop between β-strands C and C’ (CC’ loop) is colored yellow. Cys residues and disulphide bonds are shown as green cylinders. Strands are labeled with uppercase letters and terminal ends (n and c) are in lowercase. (B-E) Detailed view of the CC’ loop in the structures of the four murine TIM IgV domains (PDB 2OR8, 2OR7, 3KAA, and 3BI9). The tip of the loop, comprising residues between the two disulfide bonds bridging the loop to the GFC β-sheet, and residues at the β-sheet interacting with the loop are shown with a ball and stick drawing and with carbons in yellow. Oxygen and nitrogen atoms are in red and blue, respectively. Hydrogen bonds between the conserved Arg and Lys residues and the tip of the loop are shown as pink dashed cylinders. (F) Surface representation of the mTIM-4 IgV domain structure determined in the absence of ligand (PDB 3BI9). Residues at the tip of the CC’ and FG loops building a narrow cavity are colored in yellow and orange, respectively.

Fig. 4. Binding of PtdSer to the MILIBS in the TIM IgV domain

Structures of mTIM-3 (PDB 3KAA) and mTIM-4 (PDB 3BIB) IgV domains in complex with PtdSer are shown in panels (A) and (B), respectively. Stick drawing of the MILIBS pocket with the bound PtdSer molecule and the metal ion (green sphere). The amino acid residues at the CC’ and FG loops building the MILIBS are shown with carbons in gray, whereas the PtdSer is shown with carbons in yellow and phosphate in orange. Oxygens are red and nitrogens blue. The fatty acid (fAc), glycerol (Gl), phosphate (Ph), and serine (S) moieties of PtdSer are labeled. Amino acid residues contacting the hydrophobic moiety of PtdSer and the conserved Asn and Asp coordinating the metal ion are labeled. Coordinations are shown as dashed red lines, whereas hydrogen bonds between the PtdSer molecule and the protein are orange. (C) Model for TIM protein binding to PtdSer in a cell membrane. Surface representation of the mTIM-4 IgV domain bound to PtdSer is shown with a phospholipid bilayer membrane. Side chains at the tip of the CC’ (Asn-Ser) and FG loops (Trp-Phe) are shown. The PtdSer and phospholipids are shown in stick representation as described above. The hydrophilic head moiety of PtdSer penetrates into the MILIBS where the phosphate coordinates with the metal ion (green sphere), whereas hydrophobic residues of the TIM-4 CC’ and FG loops penetrate the lipid bilayer. The tip of the BC loop, shown in blue, comes close to the charged head of adjacent phospholipids.

Fig. 5. A model of TIM-1 on a T cell interacting with PtdSer

IM-1 can interact directly with PtdSer on an apoptotic cell or alternatively, TIM-1 and TIM-4 molecules can interact with PtdSer on an exosome, forming a bridge. An exosome might bridge any two TIM proteins except TIM-2.

Fig. 6. PtdSer and galectin-9 binding regions in the mTIM-3 IgV domain

Ribbon diagram of the mTIM-3 IgV domain, with β-strands of the GFC β-sheet labeled. The opposite sides of the IgV domain engaged in PtdSer or galectin-9 binding are indicated. The residues at the FG and CC’ loops interacting with PtdSer and coordinating the metal ion (green sphere) are shown with stick drawing and with the same color coding as in Fig. 4A. The side chains of two Asn residues which can be N-glycosylated are shown as spheres and with carbons in orange, nitrogen in blue, and oxygen in red. The complex carbohydrate modifications would extend outwards from these residues. Galectin-9 can bind to some specific motif of these N-linked carbohydrates. Residues polymorphic between BALB/c and HBA in the β-strand A and the BC loop have been colored pink.

Fig. 7. Model of phagocytosis of apoptotic cells by TIM-3 or TIM-4⁺ APCs and antigen cross-presentation

APCs phagocytose apoptotic cells via TIM-3 or TIM-4 recognition of PtdSer. The apoptotic cells are transported to an acidic phagolysosome, and antigen is processed and presented to T cells. We believe the outcome of phagocytosis may be different for TIM-3 or TIM-4 cells, as TIM-3 has a tyrosine kinase signaling motif in the cytoplasmic domain but TIM-4 does not. Engagement of TIM-3 on APCs, particularly in conjunction with TLR stimulation, may enhance inflammatory responses by inducing inflammatory cytokine production (70).

Fig. 8. Model of TIM-3 on a T cell interacting with PtdSer and/or Galectin-9

TIM-3 can interact directly with PtdSer on an apoptotic cell, or two TIM-3 molecules can be cross-linked by Galectin-9. TIM-3 may also interact with PtdSer on an exosome. Galectin-9 may also link TIM-3 to an N-linked glycan on another protein.

Fig. 9. The dimeric conformation of mTIM-2

Representation of a dimeric mTIM-2 on the cell surface. The dimeric mTIM-2 IgV domain structure (PDB 2OR7) is shown with surface representation. One domain is in red and the other in yellow, with the location of the N-linked glycans marked in dark green. The glycosylated mucin domain that follows the IgV domain is shown in green.