Differential engagement of Tim-1 during activation can positively or negatively costimulate T cell expansion and effector function (1)
The Tim family consists of eight genes in mice (Tim-1–8) and three genes in humans (TIM-1, -3, and -4). Tim family members are cell surface glycoproteins that share a common motif, including an IgV domain, a mucin-like domain, a transmembrane domain, and an intracellular tail. Tim family members are differentially expressed on Th1 cells, Th2 cells, or DCs, and they are implicated in the regulation of asthma and autoimmunity. Tim-4, which is expressed on APCs, is a natural ligand for Tim-1. Tim-4 binding to Tim-1 has different effects on T cell proliferation. A higher dose of Tim-4-Ig consistently led to an increase in T cell proliferation upon TCR ligation, whereas a lower concentration of Tim-4-Ig inhibited T cell proliferation. A higher dose of Tim-4-Ig consistently led to an increase in T cell proliferation upon TCR ligation, whereas a lower concentration of Tim-4-Ig inhibited T cell proliferation. All known costimulatory molecules have been categorized as positive costimulators (e.g., CD28 and ICOS) or negative costimulatory molecules (e.g., CTLA-4 and PD-1), although some reports suggested that CTLA4, which is an inhibitory molecule, can up-regulate LFA-1.
Proliferation assays
Female SJL mice were immunized with PLP139-151 (HSLGKWLGHPDKF) /CFA and treated with anti–Tim-1 or control antibodies. Mice were killed at the time of disease onset (on day 10 for 3B3 treatment and on day 14 for RMT1-10 treatment) and spleens were removed. Spleen cells were isolated and plated in round-bottomed 96-well plates in culture medium with various concentrations of PLP139-151. After 48 h, Plates were pulsed for 16 h with 1 μCi [3H]thymidine per well. Proliferation was measured as counts per minute by using a Wallac Liquid Scintillation Counter.
When spleen cells isolated from SJL mice immunized with the encephalitogenic peptide proteolipid protein (PLP)139-151 in CFA were treated with antigen in vitro, addition of an agonistic anti–Tim-1 antibody (clone 3B3) to the cultures significantly increased T cell proliferation at all doses of antigens. Interestingly, in contrast to 3B3, addition of another anti–Tim-1 antibody (RMT1-10) reduced T cell proliferation in the cultures. The addition of 3B3 increased the production of both IFN-γ and IL-17 with no detectable production of IL-4 or -10. However, the addition of RMT1-10 to the cultures resulted in inhibition of IFN-γ and IL-17, but induced the production of the Th2 cytokines IL-4 and -10.
Both 3B3, which was generated by immunizing rats with Tim-1 IgV-only fusion protein, and RMT1-10 bound to both full-length and mucinless forms of Tim-1, suggesting that both the antibodies are specific for the IgV domain of Tim-1. The binding of RMT1-10 to EL-4–Tim-1 cells was also strongly blocked by 3B3 anti–Tim-1 antibody but not by anti–Tim-3 antibody. When we used 3B3 to stain the EL-4–Tim-1 cells, addition of unlabeled 3B3 strongly inhibited the labeled 3B3 binding to the Tim-1 transfectants, whereas addition of RMT1-10 in a 1:1 ratio weakly blocked the 3B3 binding.
EAE is mediated by myelin- reactive CD4+ T cells, and many studies suggest that both Th1 and Th17 cells are crucial for its development. whereas Th2 cells that produce IL-4 and -10 have been shown to inhibit and reverse EAE. SJL mice were immunized with the encephalitogenic PLP139-151 peptide, and treated with 3B3 or control reagents. Whereas the control groups (rIgG and PBS) showed a typical EAE course, treatment with 3B3 dramatically altered the course of EAE. 80% of 3B3-treated mice died 3–4 d after the onset of disease.
In contrast to treatment with 3B3, administration of RMT1-10 inhibited the development of EAE. Only 30% of RMT1-10–treated mice developed EAE, whereas all of the rIgG-treated mice developed severe EAE. Furthermore, RMT1-10 treatment not only dramatically decreased the severity of EAE but also delayed the disease onset.
Higher-avidity antibodies like 3B3 could enhance T cell activation by forming a stable Tim-1 complex and bringing Tim-1 into the TCR–CD3 complex, and they also help form large supramolecular activation clusters for full T cell activation. On the other hand, low-avidity antibodies like RMT1-10 have a significantly faster off-rate and may not support the formation of stable Tim-1–TCR–CD3 complexes. Monovalent Fab′ fragments of 3B3, like bivalent 3B3, enhanced Th1 and Th17 responses, whereas Fab′ fragments of RMT1-10, such as bivalent RMT1-10, decreased Th1 and Th17 responses and increased Th2 responses. However, the Fab′ fragments of both anti–Tim-1 antibodies did not change Th cell proliferation significantly upon antigen restimulation.