1 Shaping immune responses by extracellular matrix. Bei-Chang Yang, Ph.D. ( 楊倍昌 ) Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University (NCKU), Tainan, Taiwan. Research center for Society, Technology and Medicine (STM center); NCKU Tainan, Taiwan.

2 NPC (CD3 stain) Why do T cells infiltrate and accumulate here? H/E

3 Tumor structure and extracellular matrix as a possible barrier for therapeutic approaches using immune cells or adenoviruses in colorectal cancer. Peter J.K. Kuppen et al, Histochem Cell Biol (2001) 115:67–72 Immune cells were abundantly present in tumors from colorectal origin. These cells were, however, not found in direct contact with tumor cells, but mainly in the stromal part of the tumor. Brest cancer (invasive) Not only nasopharyngeal carcinoma

4 Questions: Why are T cells trapped in peripheral area of tumor?  Hwang JY, et al. (2010) J Immunol. 185: Are those infiltrating immune cells alive?  Su CC, et al (2007) J Immunol. 179:  Hor WS, et al. (2003) J Leukocyte Biol. 73:  Lin YP, et al. (2010) Mol Immunol. 47: 林裕萍，黃俊淵，蘇重禎

5 Why are T cells trapped in peripheral area of tumor mass? Tenascin-C of glioma. Are those T cells alive? Two type of ECMs mediate the reduced apoptosis! Stepwise hijacking of infiltrating T cells by tumor: Paralyzing → Keeping alive → modifying (IL-10) Take home message:

6 NPC (CD3 stain) Why do T cells infiltrate and accumulate here? Nature Med. 1999, 5:874 Traditional view: cell-cell interaction

7 Leukocytes migration through endothelia cells

8 Molecules involve in cell-cell interaction

9 Conformational change of integrin (LFA-1) from bent to extended triggers an activation signal to the I domain which binds ICAM-1. Note: copy right belongs to

10 When released into the cell membrane, newly synthesized integrin dimers are speculated to be found in the same "bent" conformation (low affinity).  (This bent form prevents them from interacting with their ligands, although bent forms can predominate in high-resolution EM structures of integrin bound to an ECM ligands). By activation, integrin dimers must be 'unbent' in order to prime them and allow their binding to the ECM (high affinity).  (In cells, the priming is accomplished by a protein named Talin, which binds to the β tail of the integrin dimer and changes its conformation. Talin proteins are able to dimerize and thus to intervene in the clustering of integrin dimers which leads to the formation of a focal adhesion. ) Integrin activation:

11 Consist of different combinations of fibrous collagen proteins, hyaluronic acid, fibronectin, etc. Bind to cell-surface receptor: integrin, laminin receptors, etc. Are required for cell proliferation, differentiation, morphogenesis, etc. Tumor extracellular matrix may play a role.

12 Transmigration of T cells across the tumor monolayers. Extracellular matrix recaptures the effect of tumor monolayers. It is integrin-dependant.

13 Extracellular matrix recaptures the effect of tumor monolayers. It is integrin-dependant. ECM was extracted by 0.5% Triton X-100.

14 Jurkat cells spread poorly on glioma ECM. (F-actin accumulated at front of lamellipodia)

15 The transcripts of collagen IV (COL IV), fibronectin (FN), laminin-g1 (LAM) and tenascin-C (TN-C) are detected by RT-PCR. Glioma cells express high amount of tenascin-C. Immunohistochemical stain for tenascin-C on a glioma tumor sample.

16 Tenascin-C can interact with perlecan and fibronectin, and cell surface receptors including integrins  2  1,  v  3 and  9  1, and annexin I. Nature Reviews Cancer 5, (2005)

17 Tenascin-C gene of glioma cells is knocked down by shRNA method. Tenascin-C-low tranfectant cells allow better transmigration of Jurkat cells.

18 A better spreading of Jurkat cells on monolayer and ECM of U-118MG(TNCshRNA) cells. Conclusion: Tenascin-C inhibits the polarization and transmigration of T cells, that consequently prevents a direct contact of tumor and T cells.

19 Tumor contact activates ERK activity in Jurkat cells, which is required for the transmigration of Jurkat cells.

20 Hwang JY, et al. (2010) Extracellular matrix of glioblastoma inhibits polarization and transmigration of T cells: a role of tenascin-C in immune suppression. J Immunol. 185:

21 Are the tumor infiltrating T cells alive ? Lin YP, et al. (2010) Mol Immunol. 47: Su CC, et al (2007) J Immunol. 179: Hor WS, et al. (2003) J Leukocyte Biol. 73:

22 No death occurs in Jurkat cells during coculture with gliomas. 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 PI-staining

23 Coculture with tumor cells inhibits the Fas-mediated apoptosis.

24 Caspase 3 Apaf-1 Caspase 9 Death substrates Apoptosis Apoptosis program is inhibited.

25 Direct cell contact is required.

26 Integrin signal is required Coculture with U118

27 How Fas signal-mediated death is suppressed in T cells?

28 Fas-mediated death is suppressed by inhibitor of PI3K but not by inhibitors of MAPK, NF  B, PKA, HSP70.

29 Su CC, et al Phosphatidylinositol 3-Kinase/Akt activation by integrin-tumor matrix interaction suppresses Fas-mediated apoptosis in T cells. J Immunol 179:

30 Clinical feature: stiffness joint swelling pain Incidence: years female/male ratio 3/1 Mechanism: TNF-  MMP (synovial fibroblast) Reduced apoptosis Nature review 2, , 2003 Extracellular matrix-related inflammation ( Rheumatoid arthritis )

31 Enhanced p38 phosphorylation in rheumatoid arthritis synovial T cells.

32 Aberrant activation of integrin by soluble collagen induced the p38 phosphorylation. G429N: autoclustering mutant V737N: autoactivation mutant

33 SB: inhibitor of p38 MAPK Z-VAD: pan-caspase inhibitor PD: inhibitor of ERK1/2 SN-50 : inhibitor of NF-kB Inhibition of p38 MAPK activity sensitized T cells to Fas-induced apoptosis.

34 Resistance to Fas-mediated apoptosis.

35 Lin YP, et al. (2010) Soluble collagen induced the phosphorylation of p38 MAPK in Jurkat T cells and conferred those cells resistance to Fas-mediated apoptosis. Mol Immunol. 47:

36 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 Yang BC, et al. (2003) Stimulation of IL-10 expression in T cell lines upon contact with human glioma cells, that is mediated by Fas signaling through a PKA-independent pathway. J. Immunol. 171:

37 Conclusion: Stepwise hijacking of infiltrating T cells by tumor: Paralyzing → Keeping alive → modifying (IL-10)