Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity (1)
PD-1 expression is found on tumor-infiltrating CD8+ T cells in multiple solid tumors and on antigen-specific CD8+ T cells in hosts with nonsolid tumors. Second, these PD-1+ T cells are dysfunctional. Third, PD-L1 is expressed at high levels in several different cancers, and high expression of PD-L1 on tumors is strongly associated with poor prognosis. Fourth, interference with PD-1–PD-L signaling, either through antibody blockade or PD-1 deficiency, has been shown to improve clinical outcome and restore functional T cell responses in several cancers. Tim-3 is a molecule originally identified as being selectively expressed on IFN-γ–secreting Th1 and Tc1 cells. Interaction of Tim-3 with its ligand, galectin-9, triggers cell death in Tim-3+ T cells. Thus, both Tim-3 and PD-1 can function as negative regulators of T cell responses.
In mice bearing B16F10 melanoma, all three populations of CD8+ tumor-infiltrating lymphocytes (TILs) (Tim-3−PD-1−, Tim-3−PD-1+, and Tim-3+PD-1+) are present at roughly equal frequency. Interestingly, in all three of the tumor models examined we did not observe any Tim-3+PD-1− TILs. the abundance of Tim-3+PD-1+ and Tim-3−PD-1+ cells in G0, G1, and S-M phases of cell cycle. We found that Tim-3+PD-1+ cells are the most abundant population that is stuck in G0, outnumbering Tim-3−PD-1+ cells by 5 to 1.
The expression of the PD-1 and Tim-3 ligands (PD-L1 and galectin-9, respectively) on CT26 tumor was confirmed. 5 × 10^5 CT26 were implanted into the right flank of wild-type BALB/c mice. Mice were treated with 100 µg of anti–Tim-3, i.p. on days 0, 2, and 4, 200 µg of anti–PD-L1 on days 0, 3, 6, 9, and 12, or isotype control immunoglobulins. Tumor surface was measured in two dimensions using a caliper. CT26 tumor-bearing mice were treated with an anti–Tim-3 antibody, which was previously described to have blocking function in vivo, anti-PD-L1 antibody, anti–Tim-3 plus anti–PD-L1 antibodies, or control immunoglobulins. The treatment with anti–Tim-3 alone had little or no effect and treatment with anti–PD-L1 alone showed a trend toward delayed tumor growth, but this varied between experiments and did not reach statistical significance. combined treatment with anti–Tim-3 and anti–PD-L1 resulted in a dramatic reduction in tumor growth, with 50% of the mice exhibiting complete tumor regression. Indeed, the mice from the combined anti–Tim-3 plus anti–PD-L1 group that exhibited complete regression remained tumor free even after rechallenge.
TILs were isolated by dissociating tumor tissue in the presence of 2.5 mg/ml collagenase D for 20 min before centrifugation on a discontinuous Percoll gradient. TILs were harvested as described and cultured (1–3 × 10^5/well) in the presence of 5 µg/ml of soluble anti-CD3 and 10 µg/ml of anti–Tim-3, anti–PD-L1, anti–Tim-3 plus anti–PD-L1, or control immunoglobulins. After 96 h, culture supernatant was collected and IFN-γ measured by cytometric bead array. TILs from mice bearing CT26 tumor were isolated and cultured in the presence of anti–Tim-3, anti–PD-L1, anti–Tim-3 plus anti–PD-L1 antibodies, or control immunoglobulins. Although both anti–Tim-3 and anti–PD-L1 alone were able to augment IFN-γ production from TILs, this effect was variable and often weaker when compared with the increase in IFN-γ production observed in TILs treated with both anti–Tim-3 and anti–PD-L1 antibodies.
The Tim-3–Tim-3L pathway and PD-1–PD-L pathways, two pathways which likely evolved to limit tissue pathology after infection, have been co-opted in chronic disease states to promote a state of functional impairment in T cells.