The Tim-3-galectin-9 Secretory Pathway is Involved in the Immune Escape of Human Acute Myeloid Leukemia Cells (1)
Galectin-9 is a β-galactoside-binding lectin, which has a tandem structure and contains two carbohydrate recognition domains (CRDs) fused together by a peptide. Galectin-9 has a specific receptor on acute myeloid leukemia (AML) cells known as Tim-3 which also could act as its possible trafficker (galectin-9 as all other galectins lacks a signal sequence required for transport into the endoplasmic reticulum (ER) and thus requires a trafficking protein for its secretion.
THP-1 human myeloid leukemia monocytes were cultured with or without 16 h exposure to 100 nM phorbol 12-myristate 13-acetate (PMA) known to activate proteolytic shedding of Tim-3.
Specific galectin-9 bands appeared at around 32 kDa (molecular weight of galectin-9) as well as 52 kDa. Interestingly, the 52 kDa band was also detectable by anti-Tim-3 antibody. This band corresponds to the unbroken Tim-3-galectin-9 complex. Furthermore, specific Tim-3 bands appeared at around 33 kDa (molecular weight of soluble Tim-3 – sTim-3) and around 20 kDa. This 20 kDa band is likely to be a fragment of Tim-3 shed together with galectin-9 being released from the complex during the Western blot procedure. PMA treatment significantly upregulated sTim-3 release as well as the release of galectin-9. GI254023X (ADAM 10 and 17 inhibitor) or BB-94, a matrix metalloproteinase inhibitor, decreased PMA-induced sTim-3 release but did not affect the release of either galectin-9 or the Tim-3-galectin-9 complex.
Despite the levels of released sTim-3, galectin-9 and Tim-3-galectin-9 complex were increased in PMA-treated cells, the levels of respective cell-associated proteins decreased. A specific band in the range of 70 kDa detectable by both anti-Tim-3 and anti-galectin-9 antibodies was present in all the assays. Given that PMA, a specific PKC activator, significantly increases Tim-3 and galectin-9 secretion, it is likely that PKC is involved in the Tim-3 and galectin-9 co-secretion process.
Fibronectin leucine rich transmembrane protein 3 (FLRT3), which is one of physiological ligands of LPHN1 induced significant upregulation of galectin-9 and sTim-3 release. The mBM extracts significantly upregulated galectin-9 and sTim-3 secretion in THP-1 cells. FLRT3 neutralizing mouse antibody reduced the effects of mBM extracts but did not block them. With co-cultured THP-1 cells with RCC-FG1 renal carcinoma cells (which are highly adherent) in the ratio 1 THP-1:2 RCC-FG1, RCC-FG1 cells express high levels of FLRT3 and release almost undetectable amounts of galectin-9.
LAD2 human mast cell sarcoma cells express both Tim-3 and galectin-9 with both proteins located mostly on the cell surface. Resting LAD2 cells do not release detectable amounts of galectin-9 and sensitization with IgE (which was used in order to label the cells for visualization) does not augment galectin-9 secretion considerably.
sTim-3 is capable of binding a target protein (or a group of target proteins) and reducing IL-2 production thus preventing induction of NK cell and T lymphocyte anti-cancer activities. Human AML cells possess a secretory pathway which leads to the production and release of sTim-3 and galectin-9. Both proteins prevent the activation of NK cells and impair their AML cell-killing activity. This pathway, which involves the LPHN1-dependent activation of Tim-3 and galectin-9 production is summarized. The described pathway presents both biomarkers for AML diagnostics and potential targets (both sTim-3 and galectin-9) for AML immune therapy and thus can be considered as a fundamental discovery.
