The researchers analyzed proteins and protein fragments in patients' serum by mass spectrometry in collaboration with the laboratory of Dr. Samir Hanash (Public Health Sciences Division). Several proteins were detected at high levels only in patients who later developed severe GVHD compared to serum of patients without GVHD. A soluble fragment of the protein T cell Ig and mucin domain 3 (Tim-3) was one of the proteins identified. Importantly, increased levels of Tim-3 were detected before clinical onset of severe mid-gut GVHD compared to upper-gut GVHD. A microbead assay for detecting Tim-3 in individual serum samples was developed by the FHCRC Cytokine Laboratory Shared Resource, led by Rick Lawler. Using a larger 185-patient cohort, Hansen et al. detected significantly higher levels of soluble Tim-3 in the plasma of patients with the more severe mid-gut GVHD compared to those with upper-gut GVHD (P=0.005), those without GVHD (P=0.002) and normal controls (P<0.0001, see figure). In a 64-patient cohort with serum samples three weeks after tranplant, Tim-3 was the strongest univariate risk factor examined to predict mid-gut GVHD development. The sample size was too small to perform multivariate analysis with other GVHD risk factors.
The Tim-3 receptor regulates the immune response by suppressing the activation of T lymphocytes. Tim-3 is expressed on the cell surface of activated effector T cells and mediates its suppressive function by binding the ligand galectin-9. Tim-3 is also expressed on activated B cells, dendritic cells, and natural killer (NK) cells. The researchers detected a soluble form of Tim-3 in circulation that contained the extracellular domain of the receptor, but lacked the transmembrane and intracellular portions of the protein. In addition, cell-surface expression of Tim-3 was significantly increased in CD8+ T lymphocytes from patients with more severe GVHD (P=0.01) as determined by flow cytometry.
While the mechanism for generating soluble Tim-3 is unknown, previous studies have shown that defective Tim-3 signaling exacerbates autoimmune reactivity. In a mouse model of GVHD, blocking the interaction of Tim-3 with its ligand galectin-9 resulted in a more severe GVHD reaction (Oikawa et al., 2006). Results from a different mouse model using soluble a Tim-3 fusion protein further suggest that soluble Tim-3 can inhibit the negative regulatory activity of Tim-3 on immune cells and inflammation, resulting in more severe GVHD (Veenstra et al., 2012). Conversely, reducing soluble Tim-3 levels in plasma could serve as a treatment to reduce severity of GVHD reactions by allowing normal function of Tim-3 in regulating the immune response.
"One key concept here is that measuring Tim-3 before a patient becomes sick with GVHD is a little like having a crystal ball -- to predict the future before it happens," according to Dr. McDonald. The goal is to develop a panel of biomarkers to treat patients for GVHD even before the disease appears. Current and future studies will examine how well serum levels of Tim-3 perform as a predictor of the onset of more severe GVHD, in comparison or in combination with twenty-two other putative biomarkers of GVHD previously reported in the literature. Dr. McDonald surmises that "because Tim-3 plays a role in immune regulation, the correlation of blood Tim-3 levels with more severe GVHD might lead to deeper insight into the cellular mechanisms of GVHD and possibly to novel therapies."
Hansen JA, Hanash SM, Tabellini L, Baik C, Lawler RL, Grogan BM, Storer BE, Chin A, Johnson M, Wong CH, Zhang Q, Martin PJ, McDonald GB. 2013. A Novel Soluble Form of Tim-3 Associated with Severe Graft-versus-Host Disease. Biology of Blood and Marrow Transplantation. Epub ahead of print, doi: 10.1016/j.bbmt.2013.06.011.
Also see: Oikawa T, Kamimura Y, Akiba H, Yagita H, Okumura K, Takahashi H, Zeniya M, Tajiri H, Azuma M. 2006. Preferential involvement of Tim-3 in the regulation of hepatic CD8+ T cells in murine acute graft-versus-host disease. Journal of Immunology 177, 4281-4387.
Veenstra RG, Taylor PA, Zhou Q, Panoskaltsis-Mortari A, Hirashima M, Flynn R, Liu D, Anderson AC, Strom TB, Kuchroo VK, Blazar BR. 2012. Contrasting acute graft-versus-host disease effects of Tim-3/galectin-9 pathway blockade dependent upon the presence of donor regulatory T cells. Blood 120:682-690.