Actions of Interferong on the Immune System

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Actions of Interferon-g on the Immune System
Slide courtesy of Dr. Veronica Hall
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IFN-g Signaling
Inhibition
SH2
Prolinerich box
Stimulation
Box
PIAS1
CBP/p300
GAS
GAS
GAS
3
Inducers/Co-Inducers of IFN-g

• Interleukin 1b
• Interleukin 2
• Interleukin 4
• Interleukin 6
• Interleukin 7
• Interleukin 10
• Interleukin 12
• Interleukin 15
• Interleukin 18
• Interleukin 21
• Interleukin 23
• Interleukin 27
• Interferons a/b

• TNFa
• Cross-linking Cell Surface Receptors
• LY49 Activating Receptors
• NKp46
• CD3/CD28
• CD16
• CD45
• CD69
• CD161
• b1 integrin

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IFN-? Expression-6hr Stimulation
90000
NK92 cells
80000
70000
60000
50000
40000
30000
20000
10000
0
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-
-
-
-
-
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PMA
P/I
IL2
IL12
IL18
Media
IL2IL12
IL2IL18
IL12IL18
IL2IL12IL18
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CommentaryYin and yang interplay of IFN-g in
inflammation and autoimmune diseaseJingwu Zhang
J. Clin. Invest. 117871-873 (2007)
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The paradoxical roles of IFN-g in inflammation
and autoimmune disease
Zhang J.
J. Clin. Invest. 117871-873 (2007)
7

• 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-g 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.

8
Science 7 May 1999 Vol. 284. no. 5416,
pp. 951 - 955Requirement 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-g 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-? and NF-?B
mediate nitric oxide production by mesenchymal
stromal cells Oh, K. Ozaki , K. Sato, A. Meguro,
R. Tatara, K. Hatanaka, T. Nagai, K. Muroi and
K. Ozawa
Inhibition of NO by L-NMMA restores T cell
proliferation and IFN-g production
9
IFN-g and Cancer
10
IFN-g Receptor -/- Mice are more Susceptible to
Chemical Carcinogenesis
MCA dose
Demonstration of an interferon g -dependent tumor
surveillance system in immunocompetent
mice Kaplan DH et al., PNAS 957556, 1998
11
The immunobiology of cancer immunosurveillance
and immunoediting Dunn GP, Old LJ, Schreiber RD.
Immunity. 21137-48,2004
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IFN-g Good ?

• Interferon-gamma induces regression of epithelial
  cell carcinoma critical roles of IRF-1 and ICSBP
  transcription factors.Egwuagu CE et al. Oncogene
  253670, 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. 10481, 2003.
• Production of interferon-gamma by
  tumor-sensitized T cells is essential for
  interleukin-12-induced complete tumor
  eradication.Lee NC et al. Surgery. 132365,
  2002.

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Effect of IL-12 on B16 Lung Metastasis
Control cDNA
IL-12 cDNA
Data generated by Dr. Cecilia Rodriquez-Galan
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IFN-g or Bad?

• Inhibition of tumor growth and enhancement of
  metastasis after transfection of the ?-interferon
  gene.
• Lollini P-L et al. Int. J. Cancer 55320, 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. 54891, 2005.

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Host and Tumor Cell Responses to IFN-g
Advantage
Advantage
Host
Tumor
Tumor
Host

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

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

Slide courtesy of Drs. Robert Wiltrout and Dr.
Veronica Hall
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Orthotopic mouse RCC Model (Intra-renal)
Monitor for progression of primary vs metastases
Various treatment approaches
Tumor
11 days
2-3 weeks
Assess immune responses
Slide courtesy of Drs. Robert Wiltrout and Dr.
Veronica Hall
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Cytokine Therapy Elicits Anti-Tumor Responses
WT
100
80
60
Control
40
IL-12/IL-2
20
0
Survival
0
10
20
30
40
50
60
70
80
100
80
60
Control
40
IL-18/IL-2
20
0
0
10
20
30
40
50
60
70
80
Slide courtesy of Drs. Robert Wiltrout and Dr.
Veronica Hall
Days Post Implant
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Immunotherapy Fails in IFN-g KO mice
WT
IFN-? Knockout
100
100
80
80
60
60
Control
40
IL-12/IL-2
40
20
20
0
0
Survival
0
10
20
30
40
50
60
70
80
0
10
20
30
40
50
60
70
80
100
100
80
80
60
60
Control
40
40
IL-18/IL-2
20
20
0
0
0
10
20
30
40
50
60
70
80
80
0
10
20
30
40
50
60
70
Days Post Implant
Slide courtesy of Drs. Robert Wiltrout and Dr.
Veronica Hall
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IFN-g As A Sole Therapeutic Doesnt Work!
100
80
60
Survival
40
20
0
0
10
20
30
40
50
60
Days Post Implant
Slide courtesy of Drs. Robert Wiltrout and Dr.
Veronica Hall
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The IFN-g Paradox IFN-g Inhibits Primary Tumor
Progression
Wildtype
80
IFNgR -/-
60
tumor area (mm2)
40
20
0
Untreated
10000 U
50000 U
Slide courtesy of Drs. Robert Wiltrout and Dr.
Veronica Hall
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The IFN-g Paradox IFN-g Inhibits Primary Tumor
Progression
Wildtype
80
IFN-gR -/-
60
tumor area (mm2)
40
20
0
Untreated
10000 U
50000 U
But can also increase the number of lung
metastases

120
80
lung mets

40
0
Untreated
10000 U
50000 U
Dose of IFN-g (2x/week)
Slide courtesy of Drs. Robert Wiltrout and Dr.
Veronica Hall
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Implications

• IFN-g is required for host anti-tumor response
• Goal Enhance these effects

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

• IFN-g is required for host anti-tumor response
• Goal Enhance these effects

• IFN-g may enhance/mediate tumor metastasis?
• Goal Separate tumor effect from host effect

Slide courtesy of Drs. Robert Wiltrout and Dr.
Veronica Hall
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IFN-g 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

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Development of a new mouse model of
dysregulated IFN-g expression
Dr. Debbie Hodge Staff Scientist
With the critical assistance of Dr. Cyril Berthet
and Dr. Lino Tessarollo
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LY-49 Activating receptor crosslinking, PKC
activators
IFN-? mRNA transport
IFN-? mRNA stability
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Structure of Pre-IFN-? mRNA
E4
E1
E2
E3
m7Gppp
(AAAAA)n
3 UTR
E4
(AAAAA)n
AUUUAn5AUUUAn5AUUUA
AUUUA
AUUUA
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Evolutionary Comparison of the IFN-g 3
Untranslated Region
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(No Transcript)
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KO Mouse - 3 conserved nucleotide sequence
KPN 1
KPN 1
1 2 3 4
KPN 1
Neo
Recombined locus Cre-deleted
recombined locus
7.7 Kb
5.75Kb
KPN 1
KPN 1
1 2 3 4
KPN 1
5.7 Kb
Progeny w/o Cre deletion
Progeny w/ Cre deletion
Parental
7.7 Kb 5.7 Kb
CRE Deletion
5 probe
Collaboration with Cyril Berthet, NCI
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Serum IFN-? Expression
18 hours
ELISA performed by Mike Sanford
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Thymic CD4 and CD8 Populations are Increased in
KO mice
WT
KO
CD4
CD8
Data courtesy of Dr. Veronique Pascal
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Liver CD4 SP CD8 SP are Increased in KO mice
Data courtesy of Jeff Subleski
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Tregs from KO mice are more Suppressive
Data courtesy of Dr. Xin Chen
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KO CD4 Cells Exhibit Reduced Proliferation
Due to NO production?
Data courtesy of Dr. Xin Chen
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Animal Pathology Report Phenotyping of IFN-g
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 Lymphocy
tic 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
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Conclusions

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

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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_at_mail.nih.gov