In these studies, we demonstrated that TIM-4, a cell surface PtdSer receptor expressed on APC and macrophage populations, but not T cells, regulates T cell responses through, to our knowledge, a novel mechanism involving the clearance of PtdSer-expressing, apoptotic-predisposed T cells. Using both protein Ag and infection models, we found that treatment with a TIM-4 mAb that blocked TIM-4 binding to PtdSer greatly increased secondary T cell responses as measured by proliferation and cytokine production, as well as the number of Ag-specific CD4 and CD8 T cells. Conversely, overexpression of TIM-4 on APCs in a Tg mouse model resulted in decreased secondary T cell responses and decreased Ag-specific T cell numbers. We also showed that TIM-4–expressing phagocytic cells rapidly engulfed apoptotic T cells, a process that could be specifically blocked by mAb against TIM-4. These results strongly suggest that TIM-4 regulates immunity through a pathway in which TIM-4–expressing APCs control the clearance of Ag-specific T cells, thereby regulating the size of the Ag-specific memory T cell population.
Engulfment of apoptotic cells has been studied previously in the context of self-tolerance, in which the prevention of autoimmunity requires engulfment of autoantigen-expressing somatic cells by DCs, which then induce T cell tolerance to self Ags (32
). Removal of cells undergoing programmed cell death has also been studied in the context of normal physiological processes in which dying cells are silently removed without inflammation. In contrast, little is known of the molecular mechanisms by which apoptotic T cells are cleared during an Ag-specific immune response (34
). It is well known that after immunization, Ag-reactive T cells expand exponentially, but that >90% of the T cells die by apoptosis during the contraction phase, mediated by expression of Bim and Fas (36
), leaving a population of memory T cells, generally ~10% of the peak number, but still >100-fold above the initial frequency (39
). The number of remaining Ag-reactive memory T cells depends on the extent of the expansion and contraction phases of the immune response, and determines the rapidity and strength of subsequent memory responses. We suggest that TIM-4 and PtdSer control immune responses by regulating the clearance of Ag-specific T cells during the late expansion and early contraction phases of immune responses.
During the course of the immune response, expression of PtdSer by T cells occurs at two stages, as follows: shortly after cell activation (42
), when it is exposed and reinternalized, and during apoptosis at levels that remain high as a consequence of signaling through receptors of apoptosis, such as FAS and TRAIL, or due to insufficient survival signals via common γ-chain cytokine receptors. The expression of PtdSer on the external surface of the plasma membrane is a key signal for recognition of cells by phagocytes, and T cells expressing PtdSer over a threshold level are rapidly removed by phagocytic cells (3
). T cells expressing levels of PtdSer below this threshold would normally continue to survive and progress into the memory T cell pool, but in TIM-4 Tg mice, even T cells expressing low levels of PtdSer are likely to be removed by phagocytes overexpressing TIM-4. We believe that TIM-4–mediated engulfment of PtdSer-expressing cells is an important mechanism that controls the size of the Ag-specific memory T cell pool, by regulating the fraction of Ag-specific cells that are selected for removal.
The marginal zone of the spleen is a major site of clearance of PtdSer-expressing cells (30
), in which TIM-4–expressing CD169+
metallophilic macrophages reside (14
). These macrophages play a critical role in regulating immune responses because the failure of these macrophages to remove apoptotic cells impairs tolerance (30
). In our studies, we demonstrated that transferred apoptotic cells localized to the splenic marginal zone, which also stained with TIM-4 and CD169 mAbs, and treatment with anti–TIM-4 mAb in vivo delayed clearance of apoptotic cells in this site. In contrast, a recent study found that in vitro uptake of apoptotic cells by isolated splenic macrophages or DCs was not affected by TIM-4 deficiency, although engulfment of apoptotic cells by isolated peritoneal cells from TIM-4–deficient mice was greatly reduced compared with WT (16
). However, in these studies, isolated splenic cells are not likely to replicate the complex events that occur in the tissue microenvironment of the splenic marginal zone, where a specific population of TIM-4+
marginal metallophilic macrophages engulfs apoptotic cells.
The restricted expression of TIM-4 may explain the role of TIM-4 on regulating the number of Ag-specific T cells. TIM-4 is expressed by CD8+
DCs, as well as by macrophages (12
) in the marginal zone of the spleen (14
), and in lymph nodes (data not shown), but not in resting or differentiated T cells (14
) (Supplemental Fig. 2
). In contrast, other PtdSer receptors, such as MFG-E8 and GAS6, are widely expressed by somatic cells (32
). This suggests that TIM-4 differs from MFG-E8 and GAS6 by specifically regulating immune function by targeting T cells passing through the lymphoid tissues, where TIM-4 is preferentially expressed.
We showed that secondary T cell responses in TIM-4 Tg mice were diminished compared with that in WT mice and remained low following multiple Ag boosts. Moreover, we demonstrated that the reduced secondary T cell responses in TIM-4 Tg mice were not due to the development of increased numbers of either Ag-specific adaptive or natural Treg cells. Because secondary responses were decreased in TIM-4 Tg mice, but enhanced with a blocking TIM-4 mAb (), we suggest that TIM-4–expressing macrophages inhibit immune responses by removing Ag-specific apoptotic T cells during the contraction phase of the immune response.
We and others demonstrated that both human and mouse TIM-4 bind PtdSer (12
), a finding confirmed by the cocrystallization of TIM-4 with PtdSer (29
). However, TIM-4 was previously reported to be a ligand for TIM-1 (11
). Both TIM-1 and TIM-4 may have several different ligands depending on conditions (45
), and the physiological ligands and interactions of TIM-1 and of TIM-4 under different conditions are still being determined. Miyanishi et al. (13
) have shown that TIM-1 and TIM-4 can bind to separate sites on the surface of an exosome, and this bridge can give the appearance of an interaction. Exosomes are nanovesicles secreted by many cell types and expose PtdSer on their outer leaflet and are derived from intracellular structures of the endosomal pathway called multivesicular bodies (46
). Thus, TIM-4 binds PtdSer, but also may bind to TIM-1, although the TIM-1–TIM-4 binding may be of lower affinity (47
) and/or mediated by bridging PtdSer on exosomes or apoptotic bodies (13
Because both TIM-1 and TIM-4 bind PtdSer (12
), the possibility exists that TIM-1 might also be involved in engulfment of apoptotic cells in vivo. Only APCs, such as peritoneal macrophages and DCs, express high levels of TIM-4, whereas activated T cells and differentiated Th2 cells express TIM-1 and do not express TIM-4 (6
). Whereas expression of TIM-1 by kidney epithelial cells facilitates phagocytosis (12
), we found that expression of TIM-1 by T cells allows T cells to bind apoptotic cells, but does not enable T cells to engulf apoptotic cells (24
), suggesting that the engulfment machinery that is required for phagocytosis is not present in T cells. The tyrosine-signaling motifs present in the cytoplasmic domain of TIM-1 might allow the T cell to respond to the health of the APCs or surrounding tissue by signaling in response to the level of PtdSer on interacting cells. These observations suggest that TIM-4, but not TIM-1, mediates the clearance of apoptotic T cells and regulates the size of the T cell memory population.
We and others recently showed that TIM-3 is also a receptor for PtdSer (24
), which binds PtdSer with a lower affinity than TIM-1 and TIM-4 (24
), and that TIM-3–expressing cells could bind and/or engulf apoptotic cells (24
). TIM-3, which also binds galectin-9 (50
), is expressed on differentiated Th1 cells (7
), and engagement of TIM-3 by galectin-9 has been shown to lead to Th1 cell death and decreased IFN-γ production (50
). TIM-3 is also expressed by mast cells (51
) and by subpopulations of DCs and macrophages (52
). TIM-3–expressing DCs were shown to phagocytose apoptotic cells and cross-present apoptotic cell-associated Ag to CD8+
T cells (49
). Outcome of phagocytosis mediated by TIM-3– or TIM-4–expressing APCs may differ, as TIM-3 has a tyrosine kinase-signaling motif in the cytoplasmic domain, but TIM-4 does not (5
). Further studies are needed to elucidate the role of TIM-3–mediated recognition of apoptotic cells in regulation of immune responses.
TIM-4 lacks the intracellular tyrosine phosphorylation motifs present in mouse TIM-1, TIM-2, and TIM-3 that have been shown to participate in T cell signaling (53
). The role of TIM-4, which regulates the extent of secondary T cell responses, is quite distinct from that of TIM-2 and TIM-3, which provide down-modulatory signals that directly attenuate Th2- and Th1-type responses, respectively (7
), and is distinct from that of TIM-1, which costimulates and enhances T cell proliferation and cytokine production. Nevertheless, each of the TIM
family members regulates some aspect of T cell survival, suggesting that the TIM
gene family evolved to regulate immune responses by determining the fate of T cells.