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Shaping T cell functions by extracellular matrix.

Bei-Chang Yang, Ph.D. (楊倍昌 )

Director, Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University (NCKU), Tainan, Taiwan.

Director, Department of Parasitology, College of Medicine, NCKU, Tainan, Taiwan.

Director, Research center for Society, Technology and Medicine (STM center); NCKU Tainan, Taiwan.

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

Control

Fas-Lribozyme

In subcutaneous environment: FasL on tumor cells mediates inactivation of neutrophils

5x105cells subcutaneous injection

FasL on tumor cells mediates inactivation of neutrophils. J. Immunol. (2003) 171:1183-1192

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NPC Done by 張逸如 (1998)

Hematoxylin/eosin staining CD3+ cells

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)

This is not a single case

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Questions:

Why are T cells trapped in peripheral area of tumor? Tenascin C ? Lin YP, et al. (2009) Mol Immunol. In press.

Are those infiltrating T cells alive ? (PI3K) Su CC, et al (2007) J Immunol. 179:4589-4597. Hor WS, et al. (2003) J Leukocyte Biol. 73: 363-368.

Do they affect tumor growth or metastasis? (depletion study) Chen YL, et al (2002) Br J Cancer 87:359-365. Chen YL, et al. (2003) J Immunol. 171:1183-1192.

Can they modulate consequent local immune reaction? IL-10 & TGF-; Yang BC, et al. (2003) J Immunol. 171:3947-3954.

etc.

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A: Tumor extracellular matrix may play a role.

collagen proteins, hyaluronic acid, fibronectin,tenascin-Cetc…..

Q: Why do immune cells appear only in peripheral area of tumor nest?

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Transmigration of T cells through the tumor monolayers.

Extracellular matrix recaptures the effect of tumor monolayers.

It is integrin-dependant.

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Jurkat cells spread poorly on glioma ECM.

(F-actin accumulated at front of lamellipodia)

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The transcripts of collagen IV (COL IV), fibronectin (FN), laminin-g1 (LAM) and tenascin-C (TN-C) were detected by RT-PCR.

Glioma cells express high amount of tenascin-C.

Immunohistochemical stain for tenascin-C on a glioma tumor sample.

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Tenascin-C can interact with perlecan and fibronectin, and cell surface receptors including integrins 21, v3 and 91, and annexin I.

Nature Reviews Cancer 5, 436-446 (2005)

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Tenascin-C gene of glioma cells is knocked down by shRNA method.

Tenascin-C-low tranfectant cells allow better transmigration of Jurkat cells.

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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.

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Tenascin-C inhibits the polarization and transmigration of T cells, that consequently prevents a direct contact of glioma tumor and T cells. J Immunol (2010, in revision)

There are two ways to keep T cells alive.

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Caspase 3 Apaf-1Caspase 9Death substrates

Apoptosis

Nature 407, 789 5(2000)

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No death occurs in Jurkat cells during coculture with gliomas.

A B

C D E

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

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Coculture with tumor cells inhibits the Fas-mediated apoptosis.

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Caspase 3 Apaf-1Caspase 9

Death substrates Apoptosis

Apoptosis program is inhibited.

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Direct cell contact is required.

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

25 M LY294002 for 1 h

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Fas-mediated death is suppressed by inhibitor of PI3K but not by inhibitors of MAPK, NFB, PKA, HSP70.

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Su CC, et al. 2007 Phosphatidylinositol 3-Kinase/Akt activation by integrin-tumor matrix interaction suppresses Fas-mediated apoptosis in T cells. J Immunol 179:4589-4597.

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Clinical feature:

• stiffness

• joint swelling

• pain

Incidence:

• 25-65 years

• female/male ratio 3/1

Mechanism:

• TNF-• MMP (synovial fibroblast)

• Reduced apoptosis

Nature review 2, 473-86, 2003

Extracellular matrix-related inflammation (Rheumatoid arthritis)

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Lin YP, et al. (2009) Soluble collagen induced the phosphorylation of p38 MAPK in Jurkat T cells and conferred those cells resistance to Fas-mediated apoptosis. Mol Immunol. 46:3328-3335.

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Aberrant activation of integrin by soluble collagen induced the p38 phosphorylation.

G429N: autoclustering mutantV737N: autoactivation mutant

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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.

Jurkat Molt-4 1 2 3 1 2 3 IL-10

-actin

Glioma-associated IL-10 induction in T cell lines

BC Yang

Yang BC, Hor WS, Lin HK, Hwang JY, Lin YP, Liu MY, and Wang YJ (2003) Mediation of enhanced transcription of the IL-10 gene in T cells, upon contact with human glioma cells, by Fas signaling through a protein kinase A-independent pathway. J Immunol. 171:3947-3954.

U373(V/R) or U118(V/R)

Jurkat cells 1 2 3 4

IL-10

-actin

Direct cell-to-cell contact is required

1. Jurkat alone2. Jurkat separate chamber3. Jurkat in upper chamber /coculture in low chamber4. Jurkat in coculture

U118(V)

BC Yang