gene family was identified using a congenic mouse model in which polymorphisms in both TIM-1 and TIM-3 were associated with Th1/Th2 differentiation and AHR between BALB/c and congenic HBA mice. The HBA alleles of TIM-1 and TIM-3 originated from a DBA/2 ancestor (28
) and are identical to C57BL/6 alleles. Here, we show that the BALB/c allele of TIM-3 bound PtdSer and mediated the recognition and phagocytosis of apoptotic cells more efficiently than the HBA allele of TIM-3. Polymorphisms in the BC loop of the IgV domain were responsible for these differences. This is the first report of a functional difference between the BALB/c and HBA alleles of TIM-3 or TIM-1 and suggests that functional differences in the TIM-3 alleles may contribute to the Th1/Th2 phenotypic differences between the strains. Further support for a role for TIM-3 in these differences is provided by the observation that blockade of TIM-3 with mAb in BALB/c modulates Th1/Th2 responses and AHR (29
). We also showed that cells expressing hTIM-3 bound and phagocytosed apoptotic cells. Similarly in humans, one study has linked asthma to both TIM-1 and TIM-3 polymorphisms (30
), suggesting that TIM-1 and TIM-3 may contribute together to Th1 and Th2 differentiation. However, precise mechanisms whereby recognition of apoptotic cells by TIM-3 variants mediates differences in immune responsiveness require further investigation.
In this study, we determined the structure of the mTIM-3/PtdSer complex, which showed that except for mTIM-2, the TIM proteins have a MILIBS motif designed for recognition of PtdSer, thus demonstrating a conserved binding mode in TIM family members. In the two TIM/PtdSer structures solved to date, the hydrophobic region of PtdSer interacts with hydrophobic residues on the tips of loops building the MILIBS, whereas the hydrophilic moiety of PtdSer penetrates into the binding pocket. The metal ion coordination must provide the specificity for phospholipid recognition. The ligand binding cleft on the GFC β-sheet defined in the ligand-free mTIM-3 structure overlaps with the PtdSer binding pocket and is distinct from the galectin-9 binding site on an N-linked glycan on the opposite side of the IgV domain from the MILIBS (10
). Regions near the MILIBS, such as the BC loop, also contribute to the binding of TIM proteins to PtdSer in a membrane. According to our model, this additional interaction site is likely to interact with the membrane surface and not directly with PtdSer, such as proposed for the binding of protein kinase C to PtdSer (31
). Differences in the cavity size and the membrane-interacting residues, at both the tips of the CC′ and FG loops and the regions surrounding the MILIBS, could modulate the TIM binding affinity for PtdSer and account for the differences observed among TIM proteins. The highly conserved TIM-PtdSer interactions observed in the TIM/PtdSer complex structures prove that the TIM proteins are pattern recognition receptors specialized for recognition of the PtdSer death signal.
Identification of TIM-3 as an additional receptor for PtdSer adds to the versatility of the host in recognition of apoptotic cells. PtdSer is normally localized to the inner leaflet of the plasma membrane, but it is redistributed or exposed to the outer leaflet when the cell undergoes apoptosis, injury, or cell activation. Recognition of PtdSer on the surfaces of apoptotic cells provides a key signal to the phagocyte that triggers engulfment of apoptotic cells, which can result in potent anti-inflammatory effects (32
) and protection from autoimmune disease (34
). However, phagocytosis of apoptotic cells in association with danger signals can induce immune responses and inflammation (35
DCs, macrophages, and human monocytes (36
), including microglial cells in the CNS, all express TIM-3. These APCs have been suggested to regulate immunity by phagocytosing apoptotic cells and cross-presenting Ags in tolerogenic pathways (37
), which may be mediated in part by TIM-3 (23
). Consistent with this idea, mice deficient in TIM-3 were resistant to development of transplant tolerance (38
). In experimental autoimmune encephalomyelitis and multiple sclerosis, there is an expanded number of TIM-3+
microglial cells, which may function to down modulate disease, because treatment with a blocking TIM-3 mAb-enhanced demyelination and the severity of experimental autoimmune encephalomyelitis (15
) and induced TNF-α production by microglial cells (36
). In some systems, engagement of TIM-3 on APCs may enhance inflammation by increasing inflammatory cytokine production; however, this may be due to coactivation with TLR agonists (36
). Considering that myelin is a membrane with normal compartmentalization of PtdSer (40
), future studies on multiple sclerosis will need to clarify the role of TIM-3+
microglial cells in the phagocytosis of apoptotic myelin and cross-presentation of myelin Ags to T cells.
In contrast to APCs, we showed that T cells that express TIM-3 form conjugates with but do not phagocytose apoptotic cells. The failure of T cells to engulf apoptotic cells was probably not due to the small size of the T cells, because small eryptotic RBCs or fragments of U937 cells were not phagocytosed and large T cell hybridoma cells or pre-B cells transfected with TIM-3 were still unable to phagocytose apoptotic cells. It is possible that lymphocytes lack some cellular machinery required for engulfment or alternatively have an active “do not eat” receptor system (41
). Cross-linking of TIM-3 on T cells by apoptotic cells instead may provide a proapoptotic signal to the T cell, as is induced by binding of TIM-3 on Th1 or Th17 cells by galectin-9, another ligand of TIM-3 (7
). TIM-3 has an intracellular tyrosine phosphorylation motif, and cross-linking TIM-3 with mAb induces tyrosine phosphorylation. The induction of a proapoptotic signal by TIM-3 in T cells would also be consistent with a negative regulatory role for TIM-3 in immunity (23
). This effect may depend on the affinity of the interaction, which we have shown is different for BALB/c versus HBA alleles.
In contrast, TIM-1 is expressed on Th2 cells, invariant NK T cells, and mast cells (43
) and costimulates T cell activation and cytokine production (6
). TIM-1–expressing T cells avidly form conjugates with apoptotic cells, suggesting that this interaction provides a signal to the T cell that could result in T cell expansion, cytokine production, or both.
The finding that TIM gene family members TIM-1, TIM-3, and TIM-4, which have distinct patterns of expression on distinct cell types or on cells at specific stages of activation or differentiation, are a family of pattern recognition receptors for PtdSer suggests that the TIM proteins provide a functional repertoire for recognition of apoptotic cells. For example, TIM-1 may bind PtdSer on apoptotic cells and mediate T cell activation, whereas TIM-3 on T cells may mediate T cell elimination, and TIM-4 on APCs may mediate apoptotic cell clearance resulting in tolerance. Previous work that identified several receptors mediating apoptotic cell recognition and clearance by phagocytes (45
) led to the speculation that the repertoire might provide specificity in the phagocyte response. However, PtdSer receptors such as milk fat globule–EGF-factor 8 and growth arrest-specific gene 6 (46
) are widely expressed in somatic cells and do not appear to specify phagocyte behavior following phagocytosis of apoptotic cells (21
). We suggest that the TIM molecules evolved as a family of pattern recognition receptors for PtdSer that determine 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.
In summary, we have shown using structural and functional approaches that TIM-3 is a receptor for PtdSer and that polymorphic variants of TIM-3 differ functionally in their recognition of PtdSer and clearance of apoptotic cells. These findings establish a new paradigm for TIM proteins as PtdSer receptors and unify the function of the TIM gene family, which has been associated with asthma and autoimmunity and shown to modulate peripheral tolerance. Demonstration of functional differences in TIM-3 alleles has important implications for understanding of autoimmune disease mechanisms and development of therapeutic approaches.