# Presentation: 727006

## Actions of Interferon- on the Immune System

**Slide courtesy of Dr. Veronica Hall**

**Notes:**
IFNg is a pleuotropic cytokine produced by Natural Killer cells which is part of the innate immune response and by T-cells which is part of adaptive immune response. The IFNg receptor is expressed on many cell types not just immune cell which when IFNg bind intitates a signaling cascade 

## IFN- Signaling

**IFN-******** Signaling**

**SH2**

**Stimulation**

**Inhibition**

**      ****GAS****    **

**PIAS1**

**Box**

**Proline****rich box**

**      ****GAS****    **

**      ****GAS****    **

**CBP/p300    **

## Inducers/Co-Inducers of IFN-

**Inducers/Co-Inducers of IFN-******

**Interleukin 1********            **
**Interleukin 2**
**Interleukin 4                **
**Interleukin 6               **
**Interleukin 7               **
**Interleukin 10              **
**Interleukin 12**
**Interleukin 15**
**Interleukin 18**
**Interleukin 21**
**Interleukin 23**
**Interleukin 27**
**Interferons ********/******

**TNF******
**Cross-linking Cell Surface           Receptors **
**LY49 Activating Receptors**
**NKp46**
**CD3/CD28**
**CD16 **
**CD45**
**CD69 **
**CD161**
******1 integrin**

## IFN- Expression-6hr Stimulation

**0**

**10000**

**20000**

**30000**

**40000**

**50000**

**60000**

**70000**

**80000**

**90000**

**Media**

**IL2**

**IL12**

**IL18**

**IL2+IL12**

**IL2+IL18**

**IL12+IL18**

**IL2+IL12+IL18**

**P/I**

**NK92 cells**

**IFN-******** Expression-6hr Stimulation**

**PMA**

**+**

**+**

**+**

**+**

**+**

**+**

**+**

**+**

**-**

**-**

**-**

**-**

**-**

**-**

**-**

**-**

## Commentary:Yin and yang interplay of IFN- in inflammation and autoimmune diseaseJingwu Zhang 

*J. Clin. Invest.* 117:871-873 (2007)

## The paradoxical roles of IFN- in inflammation and autoimmune disease 

-  Zhang J.

*J. Clin. Invest.* **117**:871-873 (2007)

**The paradoxical roles of IFN-******** in inflammation and autoimmune disease**

## J. Exp. Med., Volume 188, Number 9 
November 2, 1998 1603-1610
Immune Suppression by Recombinant Interleukin (rIL)-12 Involves Interferon Induction of Nitric Oxide Synthase 2 (iNOS) Activity: Inhibitors of NO Generation Reveal the Extent of rIL-12 Vaccine Adjuvant Effect 
Holly Kurzawa Koblish,* Christopher A. Hunter,§ Maria Wysocka,  Giorgio Trinchieri, William M.F. Lee   

 “These results support the view that suppression of T cell responses is due to NO produced by macrophages responding to the high levels of IFN- induced by rmIL-12. When a NOS inhibitor was given with rmIL-12 during vaccination of A/J mice with irradiated SCK tumor cells, immunosuppression was averted and the extent of rmIL-12's ability to enhance induction of protective antitumor immunity was revealed.” 

- November 2, 1998 1603-1610
**Immune Suppression by Recombinant Interleukin (rIL)-12 Involves Interferon Induction of Nitric Oxide Synthase 2 (iNOS) Activity: Inhibitors of NO Generation Reveal the Extent of rIL-12 Vaccine Adjuvant Effect **
**Holly Kurzawa Koblish,* Christopher A. Hunter,§ Maria Wysocka,  Giorgio Trinchieri, William M.F. Lee   **
-  “These results support the view that suppression of T cell responses is due to NO produced by macrophages responding to the high levels of IFN- induced by rmIL-12. When a NOS inhibitor was given with rmIL-12 during vaccination of A/J mice with irradiated SCK tumor cells, immunosuppression was averted and the extent of rmIL-12's ability to enhance induction of protective antitumor immunity was revealed.”

## Science 7 May 1999:       Vol. 284. no. 5416, pp. 951 - 955Requirement for Type 2 NO Synthase for IL-12 Signaling in Innate Immunity 

***Science*** 7 May 1999:       Vol. 284. no. 5416, pp. 951 - 955_**Requirement for Type 2 NO Synthase for IL-12 Signaling in Innate Immunity**__** **_
**Andreas Diefenbach, Heike Schindler, Martin Röllinghoff, Wayne M. Yokoyama, Christian Bogdan****   **
- In the absence of NOS2 activity, IL-12 .....did not stimulate NK cells for IFN- release, and failed to activate Tyk2 kinase and to tyrosine phosphorylate Stat4 in NK cells. Thus, NOS2-derived NO is a prerequisite for cytokine signaling and function in innate immunity.

**Biochemical and Biophysical Research Communications** Volume 355, Issue 4, 20 April 2007, Pages 956-962
_**Interferon-γ and NF-κB mediate nitric oxide**_
_**production by mesenchymal stromal cells**__ _
**Oh, K. Ozaki**_[ ](http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WBK-4N3PY1K-4&_user=10843&_coverDate=04%2F20%2F2007&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000000150&_version=1&_urlVersion=0&_userid=10843&md5=db59cce30762cb2caa39e8704a9591f4#cor1)_**, K. Sato, A. Meguro, R. Tatara, K. Hatanaka, **
**T. Nagai, K. Muroi and K. Ozawa**_**[ ](http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WBK-4N3PY1K-4&_user=10843&_coverDate=04%2F20%2F2007&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000000150&_version=1&_urlVersion=0&_userid=10843&md5=db59cce30762cb2caa39e8704a9591f4#cor1)**_

**Inhibition of NO by L-NMMA restores T cell proliferation**
**and IFN-******** production**

## IFN- and Cancer

## IFN- Receptor -/- Mice are more Susceptible to Chemical Carcinogenesis

**Demonstration of an interferon ******** -dependent tumor surveillance system in immunocompetent mice**
- Kaplan DH et al., *PNAS*  95:7556, 1998

**MCA dose**

## The immunobiology of cancer immunosurveillance and immunoediting 

**The immunobiology of cancer immunosurveillance and immunoediting**
- Dunn GP, Old LJ, Schreiber RD.  *Immunity*. 21:137-48,2004

## IFN-: Good ?

**Interferon-gamma induces **_**regression**_** of epithelial cell carcinoma: critical roles of IRF-1 and ICSBP transcription factors.**Egwuagu CE et al.** ***Oncogene*** **25:3670, 2006.**   **
_**Regression**_** of AK7 malignant mesothelioma established in immunocompetent mice following intratumoral gene transfer of interferon gamma.**Cordier Kellerman L et al. *Cancer Gene Ther*. 10:481, 2003.
**Production of interferon-gamma by tumor-sensitized T cells is essential for interleukin-12-induced complete tumor **_**eradication**_**.**Lee NC et al. *Surgery*. 132:365, 2002.

## Effect of IL-12 on B16 Lung Metastasis

_**Effect of IL-12 on B16 Lung Metastasis**_

**Control cDNA**

**IL-12 cDNA**

**Data generated by Dr. Cecilia Rodriquez-Galan**

## IFN-: or Bad?

**Inhibition of tumor growth and **_**enhancement**_** **_**of metastasis**_** after transfection of the γ-interferon gene.****   **
- 	Lollini P-L et al. *Int. J. Cancer* 55:320, 1993.
**Sustained low-level expression of interferon-gamma **_**promotes tumor development**_**: potential insights in tumor prevention and tumor immunotherapy.**He Y-F et al. *Cancer Immunol Immunother*. 54:891, 2005.

## Host and Tumor Cell Responses to IFN-

** ****Recruitment and activation**
**   ****of effector cells**
** ****Anti-angiogenic effects**
**Apoptosis**

**Host**

**Tumor**

** ****Recruitment of regulatory**
**   ****cells**
** ****Enhanced growth **
**   ****environment**
** ****Matrix remodeling**

**Advantage:**

**Host and Tumor Cell Responses to IFN-******

**Tumor**

**Host**

**Advantage:**

**Slide courtesy of Drs. Robert Wiltrout and Dr. Veronica Hall**

## Orthotopic mouse RCC Model (Intra-renal)

**Orthotopic mouse RCC Model (Intra-renal)**

_**2-3 weeks**_

**Monitor for progression of primary vs metastases**

**Assess immune responses**

**Various treatment approaches**

**Tumor**

_**11 days**_

**Slide courtesy of Drs. Robert Wiltrout and Dr. Veronica Hall**

**Notes:**
Our lab uses an orthotopic renal cell carcinoma model. This model mimics more closely to a clinical model. Tumor is inserted under the renal cap and allowed to grow, the tumor is resected away which similar to clinic with the primary tumor being able to be removed surgically, and we treat metastasis with various immuno-therapies

## Cytokine Therapy Elicits Anti-Tumor Responses

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**Control**

**IL-12/IL-2**

**Control**

**IL-18/IL-2**

**Days Post  Implant**

**Slide courtesy of Drs. Robert Wiltrout and Dr. Veronica Hall**

## Immunotherapy Fails in IFN- KO mice

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**Control**

**IL-12/IL-2**

**Days Post  Implant**

**Control**

**IL-18/IL-2**

**WT**

**IFN-******** Knockout**

**% Survival**

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**Slide courtesy of Drs. Robert Wiltrout and Dr. Veronica Hall**

## IFN- As A Sole Therapeutic Doesn’t Work!

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**Days Post Implant**

**% Survival**

**IFN-******** As A Sole Therapeutic Doesn’t Work!**

**Slide courtesy of Drs. Robert Wiltrout and Dr. Veronica Hall**

## IFN- Inhibits Primary Tumor Progression

**IFN-******** Inhibits **_**Primary**_** Tumor Progression**

**0**

**20**

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**60**

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- tumor area (mm2)

- IFNR -/-

**Untreated**

**10000 U**

**50000 U**

- Wildtype

**Slide courtesy of Drs. Robert Wiltrout and Dr. Veronica Hall**

## *

**The IFN-******** Paradox: **
**IFN-******** Inhibits **_**Primary**_** Tumor Progression**

- Wildtype

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- tumor area (mm2)

- IFN-R -/-

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**Untreated**

**10000 U**

**50000 U**

- # lung mets

**Dose of IFN-******** (2x/week)**

**Untreated**

**10000 U**

**50000 U**

**Slide courtesy of Drs. Robert Wiltrout and Dr. Veronica Hall**

## Implications

- IFN- is required for host anti-tumor response
- Goal: Enhance these effects

**Slide courtesy of Drs. Robert Wiltrout and Dr. Veronica Hall**

## Implications

- IFN- may enhance/mediate tumor metastasis?
- Goal: Separate tumor effect from host effect

- IFN- is required for host anti-tumor response
- Goal: Enhance these effects

**Slide courtesy of Drs. Robert Wiltrout and Dr. Veronica Hall**

## IFN- cooperates with other signals to influence tumor gene expression

**J Biol Chem**. 2007 Apr 16
- Ziesche E, Bachmann M, Kleinert H, Pfeilschifter J, Muhl H.
**The IL-22/STAT3 pathway **
**potentiates expression of iNOS **
**in human carcinoma cells**** **

## Development of a new 

**mouse model **
**of dysregulated **
**IFN-******** expression**

**Dr. Debbie Hodge**
**Staff Scientist**

**With the critical assistance of Dr. Cyril Berthet and Dr. Lino Tessarollo**

## IFN- mRNA stability

**LY-49 Activating receptor crosslinking, PKC activators**

**IFN-******** mRNA stability**

**IFN-******** mRNA transport**

## Structure of Pre-IFN- mRNA

- (AAAAA)n

- m7Gppp

- E1

- E2

- E3

- E4

- (AAAAA)n

- E4

- AUUUAn5AUUUAn5AUUUA

- AUUUA

- AUUUA

- 3’ UTR

- Structure of Pre-IFN- mRNA

## Evolutionary Comparison of the IFN- 3’ Untranslated Region

**Evolutionary Comparison of the IFN-******** 3’ Untranslated Region**

## Slide 29

## KO Mouse - 3’ conserved nucleotide sequence

**KPN 1**

**KPN 1**

**1           2   3                 4**

**Neo**

**KPN 1**

**KPN 1**

**1           2   3                 4**

**KPN 1**

**7.7 Kb**

**5.75Kb**

**5.7 Kb**

- 7.7 Kb
- 5.7 Kb

**Parental**

**Recombined locus**
**       ****Cre-deleted **
**    ****recombined locus**

**Progeny w/o Cre**
**deletion**

**Progeny w/ Cre**
**deletion**

**5’ probe**

**KO Mouse - 3’ conserved nucleotide sequence**

**CRE Deletion**

**KPN 1**

- Collaboration with Cyril Berthet, NCI

## Serum IFN- Expression 

**Serum IFN-******** Expression**

**18 hours**

**ELISA performed by Mike Sanford**

## WT

- CD4

- CD8

**Thymic CD4+ and CD8+ Populations are Increased in KO mice**

**WT**

**KO**

**Data courtesy of Dr. Veronique Pascal**

## Liver CD4 SP & CD8 SP are Increased in KO mice

**Liver CD4 SP & CD8 SP are Increased in KO mice**

**Data courtesy of Jeff Subleski **

## Tregs from KO mice are more Suppressive

**Tregs from KO mice are more Suppressive**

**Data courtesy of Dr. Xin Chen**

## KO CD4+ Cells Exhibit Reduced Proliferation 

**KO CD4+ Cells Exhibit Reduced Proliferation **
**                     ****Due to NO production?**

**Data courtesy of Dr. Xin Chen**

## Animal Pathology Report

- Phenotyping of IFN- knockout mice
- Born 07/14/2006
- Died 03/01/2007
_**Cause Of Death**_**: **Immune dysfunction
_**Femur, Marrow**_
- Hypoplasia, granulocytic moderate
_**Heart**_
- Inflammation, chronic severe multifocal
- Degeneration, myocyte moderate multifocal
- Thrombus, atrium severe diffuse
_**Lymph Node**_
- Hyperplasia, atypical, lymphoid severe
- Arteritis moderate focal
_**Spleen**_
- Arteritis moderate multifocal
- Depletion, lymphoid moderate
- Extramedullary hematopoiesis, decreased mild
- Necrosis severe focal
- Plasmacytosis moderate multifocal
- Syncytia mild multifocal
_**Liver**_
- Lymphocytic infiltrate moderate multifocal
- Atrophy, hepatocellular, centrilobular moderate multifocal
- Hyperplasia, bile ductule minimal multifocal
- Hypertrophy, hepatocellular moderate multifocal
- Necrosis, hepatocellular severe multifocal
- Pigmented macrophages mild multifocal

## Conclusions

- IFN- is critical and essential for immune modulated tumor regression
- IFN- may promote tumor escape from immune attack through selection of non-immunogenic tumor variants
- IFN- may enhance tumor growth, possibly through suppression of the immune response by induction of NO production, induction of IDO or enhanced Treg development

## Collaborators

- Dr. Xin Chen – Tregs
- Dr. Dennis Klinman – Ab response
- Dr. Veronique Pascal – NK cells, Thymic populations
- Dr. Remy Bosselut – Thymic Development
- Jeff Subleski/Dr. Bob Wiltrout – NKT cells, tumor models
- Dr. David Wink – NO expression and physiology
- Many others planned
**            ****Contact: younghow@mail.nih.gov**