The InterferonSTAT1 axis

1
The Interferon/STAT1 axis in T Cell
Development
2
Interferon Signaling The Jak/STAT Pathway
3
How it started.

• Type I Interferons Keep Activated T Cells Alive
  (Marrack et al., 1999)
• IL-6 Promotes Survival of Resting T Cells and
  Induces Tyrosine Phosphorylation of STAT1 in
  Resting, but not Activated T Cells (Teague et
  al., 2000)
• BUT
• STAT1-Deficient Lymphocytes Show Reduced
  Apoptosis in Vitro (Lee et al., 2000)
• ? Role of STAT1 in T Cell Survival ?

4
Effects of IFNb on Resting and Activated STAT1-/-
CD4 T Cells
Anti-CD3
Anti-CD3IFNb
5
Consequences of STAT1-Deficiency?
6
Consequences of STAT1-Deficiency?
Approach

• Cross STAT1-/- Mice to TCR-Transgenic Animals
• Choose Transgenic TCR that Provides Autoimmune
  Disease Model
• Multiple Sclerosis Demyelinating T
  cell-mediated Autoimmune Disease

7
Experimental Autoimmune Encephalomyelitis (EAE)

• Murine model for Multiple Sclerosis (MS)
• Transgenic I-Au restricted TCR against Myelin
  Basic Proteins (MBP) (Baron et al., 1993)
• Requires H2u/u background to develop EAE after
  immunization with MBP (H2b/u or H2b/b mice are
  resistant)

8
STAT1- Deficiency is Associated with Impaired
Treg Cell Function
J. Exp. Med., Vol 199, Number 1, January 5, 2004
2534
STAT1-/- TCRMBP H2b/u
9
Thymic T cell Development
Annual Review of Immunology Vol. 21 139-176
(2003)
10
The H-Y-Specific Transgenic TCRHY

• The minor histocompatibility (H) male-specific
  (Y) antigen (H-Y) is expressed solely in male
  tissues (derived from a protein encoded by
  the Smcy gene located on the Y chromosome)
• The MHC class I H-2Db-restricted transgenic TCRHY
  specifically recognizes an antigen-derived
  peptide (KCSRNRQYL) presented on H-2Db molecules.

CD8
CD4
H von Boehmer and P Kisielow, 1990Science vol
248 1370
11
Thymic T Cell Populations in HYSTAT1-/- Mice
?
2.65
8.42
HYSTAT1/
14.8
74.1
CD8
24.0
45.1
HYSTAT1-/-
23.8
7.12
Gated on HY
CD4
12
Thymic T Cell Populations in HYMale Mice
HY WT
HY STAT1-/-
24.0
45.1
8.42
2.65
7.12
23.8
14.8
74.1
CD8
HY IFN?R-/-
HY IFN??R-/-
34.3
44.3
4.11
1.44
6.66
14.8
8.96
85.5
Gated on HY
CD4
13
Thymic T Cell Populations in HYFemale Mice
HY WT
HY STAT1-/-
31.4
50.5
12.5
80.3
2.14
16
4.02
3.1
CD8
HY IFN?R-/-
HY IFN??R-/-
32.8
52.8
27.4
62.9
3.07
11.3
3.39
6.24
Gated on HY
CD4
14
Peripheral T Cell Populations in HYMale Mice
HY WT
HY STAT1-/-
CD8
CD4
15
Summary of Thymic T Cell Selection in HYMice
16
Thymic T Cell Apoptosis in HY- STAT1-/- Mice
17
BM Transplants (male) Recipient 129WT
Donor male
HY-WT
HY-STAT1-/-
Inject 1x107 bone marrow cells
Inject 1x107 bone marrow cells
1,000 rad
Recipient male
129WT
129WT
4 weeks
4 weeks
Reconstituted thymus includes
Thymocytes HY-STAT1-/- DCs STAT1-/- TECs STAT1
Thymocytes HY-STAT1 DCs STAT1 TECs STAT1
18
BM Transplants (male) Recipient STAT1-/-
Donor male
HY-WT
HY-STAT1-/-
Inject 1x107 bone marrow cells
Inject 1x107 bone marrow cells
1,000 rad
Recipient male
STAT1-/-
STAT1-/-
4 weeks
4 weeks
Reconstituted thymus includes
Thymocytes HY-STAT1-/- DCs STAT1-/- TECs STAT1-
/-
Thymocytes HY-STAT1 DCs STAT1 TECs STAT1-/-
19
BM Transplants (male) Summary
20
MHC Class I Expression on Thymic Cell Populations
21
BM Chimeras (male recipients) Overview
Donor female
129WT
Inject mixed bone marrow cells
Inject mixed bone marrow cells
5x106 cells
5x106 cells
5x106 cells
5x106 cells
1,000 rad
1,000 rad
Recipient male
4 weeks
4 weeks
Reconstituted thymus includes
Thymocytes STAT1 / HY-STAT1-/- DCs STAT1 /
HY-STAT1-/- TECs STAT1
Thymocytes HY-STAT1-/- DCs STAT1 /
HY-STAT1-/- TECs STAT1
22
BM Chimeras (male recipients) Summary
23
Reduced Bim Expression in STAT1-/- T cells
24
BM Chimeras (male recipients) Summary
25
Altered Thymic Architecture in STAT1-/- Cells
Keratin 5
EpCAM
HE
WT
STAT1-/-
26
Reduced AIRE Expression in STAT1-/- mTECs
35
WT
13
STAT1
AIRE
EpCAM
27
Constitutive Thymic IFNb Production
Lienenklaus et al, J Immunol, 2009, 183, 3229
28
Conclusions

• IFNa/b act as Potent Mitogens and Anti-apoptotic
  Factors on STAT1-Deficient T cells
• STAT1-/- Mice Display a Significant Impairment in
  the Number and Function of CD4CD25 Regulatory T
  Cells
• STAT1- Deficient Mice Show Significant Defects in
  Positive and Negative Selection of T Cells,
  whereas in IFNAR1-/- Mice Only Negative Selection
  is Impaired
• IFN/STAT1 Appear to Influence Thymic Selection
  via Thymocyte - mTEC - Thymocyte communication

29
THANKS...
David Lab
Robert Rickert
Hiroshi Moro Dennis Otero Irene Pedersen Takeshi
Nakatsue Hilary Sandig Ville Veckman Manqing
Li Kirsten Vroom Elaine Kao
Steve Hedrick Craig Walsh Ananda Goldrath
30
(No Transcript)
31
Thymic T Cell Populations in HYFemale Mice
HY WT
HY STAT1-/-
31.4
50.5
12.5
80.3
2.14
16
4.02
3.1
CD8
HY IFN?R-/-
HY IFN??R-/-
32.8
52.8
27.4
62.9
3.07
11.3
3.39
6.24
Gated on HY
CD4
32
BM Transplants (female) Summary
33
BM Chimeras (female recipients) Summary
A.
B.
C.
D.
34
Maturation of mTECs
TNF family members expressed by
previously selected Thymocytes influence mTEC
Development.
Interferons are known to modulate expression of
CD40 and other TNF family members.
What is the role of STAT1 in thymic architecture?
Immunity, Volume 29, Issue 3, pg386-388
35
RANK IFN Crosstalk ?
RANKL induces Osteoclast maturation.
Interferon Beta is expressed following c-Fos
dependent RankL signaling.
Interferon signaling inhibits c-Fos and
Osteoclast maturation as a Negative feedback
mechanism.
Nature 416, 686-687
36
Negative Selection in OT I / Rip-OVA Chimeras