FasL: a double-edged sword for tumor formation Differential effects of FasL on tumor formation Cell-cell contact suppresses Fas-mediated apoptosis Unconventional Fas signal for gene expression FasL reverse signaling 楊倍昌 國立成功大學醫學院 微生物及免疫學研究所 BC Yang

Fight cancer, But, sometimes we lose BC Yang

The great escape: Resistance to killing Defective Fas pathway Resistance to Granzyme Fas-L and Neutrophil Cytotoxic T cells Innate immunity BC Yang

Immune privilege or inflammation? Joe O'Connell et al. Nature Medicine 7, (2001) BC Yang

Caspase 3 Apaf-1 Caspase 9 Death substrates Apoptosis Nature 407, 789 – 795(2000) Nagata, S. (Fas); Krammer, P. (Apo1) CD95/CD95-L Transducing death signal Involving in cytotoxicity, immune homeostasis, tumor formation, etc. BC Yang

In vivo consequences of FasL expression by tumors (using over-expression system) Enhanced rejection Delayed rejection Renca, MH134, L5178Y, B16/BL6, CT26* Note: *:syngenic, nude, SCID # : allogenic, lpr Ref: Lejeune FJ et al., 1998, Curr. Op. Immunol. CT26 # B16-F10 BC Yang

FasL ribozyme /EGFP plasmid Hammerhead ribozyme. 54-mers. Inserted into the pEGFP-N1. Driven by CMV promoter. Chio CC, Wang YS, Chen YL, Lin SJ and Yang BC (2001) Down-regulation of Fas-L in glioma cells by ribozyme reduces cell apoptosis, tumor infiltrating cells, and liver damage but accelerates tumor formation in nude mice. Br J Cancer 85: BC Yang

U-373MG cells transfected with FasL ribozyme /EGFP plasmid Light microscopy Fluorescent microscopy Immunohistochemistry (tumor cells in tissue)  -tubulin Fas-ligand U373(v) U373(R) U118(v) U118(R) BC Yang

Tumor formation in nude mice FasL high FasL low Br J Cancer (2001) 85, 1185 Tumor cells T, B, Macrophages, monocytes, granulocytes, etc…. Stroma cells Blood vessels BC Yang

Q: How about the action of tumor FasL in immune intact mice? A: It is organ dependent! BC Yang

Animal model C57B/6 Melanoma cell line B6F10 Gene knockdown by FasL ribozyme Subcutis/lung BC Yang

Effect of FasL on growth and death of B16F10 cells Apoptosis in 3-day culture Cell growth rate BC Yang

Granulocytes mediates the Fas-L-associated apoptosis during lung metastasis of melanoma that determines the metastatic behavior (B16F10 in C57BL/6) Br J Cancer, 87: 359 (2002) BC Yang

Depletion of CD4, CD8 T cells and PMNs affected lung metastasis Vector controlsFas-L ribozyme What happened here? Br J Cancer, 87: 359 (2002) BC Yang

Control Fas-L ribozyme In subcutaneous environment: FasL on tumor cells mediates inactivation of neutrophils 5x10 5 cells subcutaneous injection FasL on tumor cells mediates inactivation of neutrophils. J. Immunol. (2003) 171: BC Yang

Vector controlsFas-L-ribozyme Depletion of CD4, CD8 T cells and PMNs affected subcutaneous tumor formation BC Yang

Tumor FasL high impairs neutrophil activation: gelatinase B, ROS, CD-11b, and FasL (In coculture for 24 h). Cell death Gelatinase B ROS J. Immunol. (2003) 171: BC Yang

Puzzles on tissue specificity (to be answered) Factors responsible for tissue specificity Function of immune cells in different tissues BC Yang

No death occurred in Jurkat cells during coculture with gliomas PI-staining AB CDE A: Jurkat cells alone B: CH-11 (1ng/ml) C: with U-118MG(V) D: with U118MG (R) E: ZB4 (100ng/ml)/CH-11 免疫細胞沒那麼容易死亡 : BC Yang

Coculture with tumor cells inhibits the Fas- mediated apoptosis. Can ECM play a role in this protection? BC Yang

Death pathway inhibited DISC formation reduced

U 118(V/R) Jurkat cells BC Yang Cell-cell contact is required and integrins involved.

Coculture with glioma cells/conditioned media did not reduced the surface Fas protein. BC Yang

Intracellular signaling induced by cell-cell contact ： PI3K/AKT is important for suppression of Fas-mediated apoptosis in T cells

BC Yang Other anti-apoptosis genes

J Immunol. 2007, in revision BC Yang Phosphatidylinositol 3-Kinase/Akt activation by integrin-tumor matrix interaction suppresses Fas-mediated apoptosis in T cells

Fas/FasL interaction may create an immunosuppresive environment Induction of IL-10 in T cell lines upon contact with human glioma cells, that is mediated by Fas signaling through a PKA-independent pathway. J Immunol (2003) 171: BC Yang

Jurkat Molt IL-10  -actin Glioma-associated IL-10 induction in T cell lines BC Yang 1. control, Jurkat alone 2. Jurkat + U118MG 3. Jurkat + U373MG

U373(V/R) or U118(V/R) Jurkat cells IL-10  -actin Direct cell-to-cell contact is required 1. Jurkat alone 2. Jurkat separate chamber 3. Jurkat in upper chamber /coculture in low chamber 4. Jurkat in coculture U118(V) BC Yang

Fas/Fas-L signal-associated IL-10 induction Jurkat U-373MG U-118MG - V R V R IL-10  -actin Molt-4 U-373MG U-118MG - V R V R IL-10  -actin BC Yang

Fas FasL T cells Tumor Tissue environment ? Cytokines ? IL-10, IL-6, IL-8 Neutrophils To help or not to help ? To be solved BC Yang

FasL reverse signaling Suzuki I. and Fink PJ.. Maximal proliferation of cytotoxic T lymphocytes requires reverse signaling through Fas ligand. J. Exp. Med., 187:123, Boursalian TE, Fink PJ, Mutation in Fas Ligand Impairs Maturation of Thymocytes Bearing Moderate Affinity T Cell Receptors, J. Exp. Med., 198, 349, 2003 Sun M, Ames KT, Suzuki I,. Fink PJ, The Cytoplasmic Domain of Fas Ligand Costimulates TCR Signals, J.Immunol., 177, 1481, 2006 BC Yang

3T3-N13T3-FasL 3T3- △ 33 3T3-Fas 3T3- △ 70 FasL on lung metastases Manuscript in preparation; 2007 BC Yang