Enhancing anti-tumor immunity to MHC class I-deficient tumors [Elektronische Ressource] : role of regulatory T cells and type I IFN / presented by Ioanna Evdokia Galani

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Enhancing anti-tumor immunity to MHC class I-deficient tumors: role of regulatory T cells and type I IFN Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University Of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Ioanna Evdokia Galani born in Acharnes, Greece Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University Of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Ioanna Evdokia Galani born in Acharnes, Greece Oral examination: 20.06.2008 Enhancing anti-tumor immunity to MHC class I-deficient tumors: role of regulatory T cells and type I IFN 1. Referee: Prof. Dr. G. Hämmerling 2. Referee: PD Dr. A. Cerwenka The work described in this thesis was performed from September 2004 to April 2008 at the German Cancer Research Center, Germany, in the laboratory of PD Dr. Adelheid Cerwenka. Parts of this thesis have been published in: Galani I.E., M. Wendel, C. Schellack, E. Suri-Payer and A. Cerwenka. Treg suppress leukocyte accumulation and macrophage activation in lymphoma. Manuscript in preparation. Nausch N., I.E. Galani and A. Cerwenka. Myeloid-derived ‚suppressor’ cells express RAE-1 and activate NK cells.

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Enhancing anti-tumor immunity to MHC class I-deficient
tumors: role of regulatory T cells and type I IFN





Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University Of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences










presented by
Ioanna Evdokia Galani
born in Acharnes, Greece

Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University Of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences















presented by
Ioanna Evdokia Galani
born in Acharnes, Greece


Oral examination: 20.06.2008



Enhancing anti-tumor immunity to MHC class I-deficient
tumors: role of regulatory T cells and type I IFN





















1. Referee: Prof. Dr. G. Hämmerling
2. Referee: PD Dr. A. Cerwenka


The work described in this thesis was performed from September 2004 to April 2008 at the
German Cancer Research Center, Germany, in the laboratory of PD Dr. Adelheid Cerwenka.


Parts of this thesis have been published in:

Galani I.E., M. Wendel, C. Schellack, E. Suri-Payer and A. Cerwenka. Treg suppress leukocyte
accumulation and macrophage activation in lymphoma. Manuscript in preparation.

Nausch N., I.E. Galani and A. Cerwenka. Myeloid-derived ‚suppressor’ cells express RAE-1 and
activate NK cells. Manuscript in revision.

Wendel M., I.E. Galani, E. Suri-Payer and A. Cerwenka. NK cell accumulation in tumors is
dependent on IFN- γ and CXCR3 ligands. Manuscript submitted.

Kiessling M., C.D. Klemke, M. Kami ński, I.E. Galani, P.H. Krammer and K. Gülow. Survival of
primary T cell lymphomas is dependent on regulation of Ferritin Heavy Chain by constitutively
activated NF- κB. Manuscript submitted.


Conference presentations:

Galani I.E., M. Wendel, C. Schellack, E. Suri-Payer and A. Cerwenka. IFN- γ dependent rejection
thof the NK cell dependent RMA-S tumor in the absence of regulatory T cells. 16 European
Congress of Immunology, September 2006, Paris, France.

Galani I.E., M. Wendel, C. Schellack, E. Suri-Payer and A. Cerwenka. Regulatory T cells
suppress IFN- γ dependent rejection of the NK cell sensitive RMA-S tumor. Natural Killer Cell
Symposium 2006 Heidelberg, December 2006, Heidelberg, Germany.
Galani I.E., M. Wendel, C. Schellack, E. Suri-Payer and A. Cerwenka. Rejection of the NK cell
sensitive tumor RMA-S via IFN- γ in the absence of regulatory T cells. Keystone Symposia
meeting ‘Mechanisms Linking Inflammation and Cancer’, February 2007, Santa Fe, New
Mexico, USA.

Galani I.E., M. Wendel, C. Schellack, E. Suri-Payer and A. Cerwenka. Treg suppress leukocyte
thaccumulation and macrophage activation in RMA-S lymphoma. 10 Meeting of the Society for
Natural Immunity, April 2007, Cambridge, UK.

Galani I.E., M. Wendel, C. Schellack, E. Suri-Payer and A. Cerwenka. Regulatory T cells
th suppress IFN- γ dependent leukocyte accumulation and macrophage activation in lymphoma. 37
Annual Meeting of the German Society for Immunology (DGfI), September 2007, Heidelberg,
Germany.


















CONTENTS

1 ZUSAMMENFASSUNG .......................................................................................... 1
2 SUMMARY................................................................................................................ 3
3 INTRODUCTION..................................................................................................... 5
3.1 THE IMMUNE SYSTEM....................................................................................................5
3.1.1 The immune response to tumors ............................................................................5
3.1.2 The innate immune system.....................................................................................7
3.1.2.1 Natural Killer cells...........................................................................................7
3.1.2.1.1 NK cells and tumor......................................................................................11
3.1.2.1.2 NK cells as regulatory cells.........................................................................13
3.1.2.2 Macrophages..................................................................................................14
3.1.2.2.1 Tumor associated macrophages...................................................................15
3.1.2.2.2 Myeloid-Derived Suppressor Cells.............................................................16
3.1.3 The adaptive immune system...............................................................................17
3.1.3.1 Mechanisms of T cell tolerance.....................................................................18
3.1.3.2 Regulatory T cells..........................................................................................20
3.1.3.2.1 Phenotypic characterization of Treg............................................................20
3.1.3.2.2 Treg and autoimmunity .........................................22
3.1.3.2.3 Treg and tumor immunity.........................................................22
3.1.3.2.4 Treg and innate immunity.........................23
3.1.3.2.5 Manipulation of the suppressive function of Treg.......................................24
3.2 TYPE I IFN.....................................................................................................................25
3.2.1 Regulatory effects of type I IFN...........................................................................26
3.2.2 Type I IFN and tumor...........................................................................................27
4 AIM OF THE STUDY.....................................................................................................29
5 MATERIAL AND METHODS .....................................................................................30
5.1 MATERIALS..................................................................................................................30
5.1.1 Laboratory equipment..........................................................................................30 5.1.2 Cell culture products ............................................................................................31
5.1.3 Cell culture media................................................................................................32
5.1.4 Solutions...............................................................................................................33
5.1.5 Chemicals.............................................................................................................34
5.1.6 Antibodies............................................................................................................35
5.1.6.1 Antibodies for FACS analysis .......................................................................35
5.1.6.2 Antibodies for in vitro activation...................................................................35
5.1.6.3 In vivo administered antibodies .....................................................................35
5.1.7 Cell lines...............................................................................................................38
5.1.8 Magnetic Cell Sorting (MACS) beads and columns ............................................38
5.1.9 Kits.......................................................................................................................39
5.2 MICE .............................................................................................................................40
5.3 METHODS .....................................................................................................................40
5.3.1 Cell culture methods.............................................................................................40
5.3.1.1 Determination of cell number........................................................................40
5.3.1.2 Freezing and thawing of cells ........................................................................40
5.3.1.3 Splitting of adherent cells ..............................................................................41
5.3.1.4 Splitting of suspension cells ..........................................................................41
5.3.2 Mouse tumor model .............................................................................................41
5.3.2.1 Isolation of organs and preparation of single cell suspensions......................42
5.3.2.1.1 Blood...........................................................................................................42
5.3.2.1.2 Spleen and LN .............................................................................................42
5.3.2.1.3 Tumor infiltrating leukocytes.....................................................................42
5.3.2.1.4 Bone marrow (BM) cells.............................................................................42
5.3.2.2 In vivo depletion and neutralization experiments ..........................................43
5.3.2.3 Bone marrow transplantation.........................................................................43
5.3.2.4 Memory experiments.....................................................................................44
5.3.2.4.1 In vivo memory experiments .......................................................................44
5.3.2.4.2 Adoptive transfer memory experiments ......................................................44
5.3.3 Antibody purification...........................................................................................44
5.3.3.1 Purification of the anti-CD25 mAb ...............................................................44
5.3.3.2 Purification of the anti-IFN- β mAb ...............................................................45
5.3.4 Fluorescence-activated cell sorting (FACS).........................................................45
5.3.4.1 FACS staining and analysis ...........................................................................45
5.3.4.2 FACS sorting.................................................................................................46 5.3.5 MACS sorting ......................................................................................................46
5.3.6 Determination of cytokines ..................................................................................46
5.3.6.1 Intracellular FACS staining ...........................................................................46
5.3.6.2 Enzyme-Linked ImmunoSorbent Assay (ELISA).........................................46
5.3.6.3 In vivo IFN- γ capture assay.........................................................47
5.3.6.4 Bio-plex protein array assay for cytokine measurement................................47
5.3.7 Proliferation and suppression assays....................................................................47
5.3.7.1 Treg suppression assay..................................................................................47
5.3.7.2 Tumor cell growth inhibition assay ...............................................................48
5.3.7.3 Documentation of proliferation by CFSE dilution.........................................49
5.3.8 Killing assays .......................................................................................................49
515.3.8.1 Cr release cytotoxicity assay .......................................................................49
5.3.8.2 In vitro induction of apoptosis by death ligands............................................49
5.3.9 Visualization of angiogenesis by confocal microscopy .......................................50
5.3.10 Statistical analysis ................................................................................................50
6 RESULTS ...........................................................................................................................51
6.1 REGULATORY T CELLS SUPPRESS ANTI-TUMOR IMMUNITY AGAINST A MHC
CLASS I DEFICIENT LYMPHOMA ..................................................................................51
6.1.1 Regulatory T cells suppress IFN- γ dependent leukocyte accumulation in
lymphoma ............................................................................................................51
6.1.1.1 Accumulation of Treg in RMA-S tumor-bearing mice..................................51
6.1.1.2 Depletion of Treg abrogates RMA-S tumor growth ......................................52
+ + -6.1.1.3 NK1.1 cells and CD4 CD25 T cells mediate the rejection of RMA-S
tumors in the absence of Treg........................................................................53
6.1.1.4 Depletion of Treg leads to the generation of anti-tumor memory responses
against RMA-S tumor cells............................................................................55
6.1.1.5 Neutralization of IFN- γ abrogates RMA-S tumor rejection mediated by Treg
depletion in vivo.............................................................................................58
6.1.1.6 Perforin, FasL and inhibition of angiogenesis are not involved in the rejection
of RMA-S tumor in the absence of Treg .......................................................60
6.1.1.7 In the absence of Treg, increased numbers of leukocytes accumulate in the
RMA-S tumor tissue in an IFN- γ dependent manner ....................................62 +6.1.1.8 Increased numbers of NK cells, conventional CD4 T cells and macrophages
are detected in the RMA-S tumors in the absence of Treg ............................63
6.1.1.9 Treg suppress RMA-S anti-tumor immunity independently of TGF- β or IL-10
.......................................................................................................................65
6.1.2 Regulatory T cells suppress macrophage activation in lymphoma ......................66
6.1.2.1 Increased amounts of MHC class II on macrophages in the absence of Treg
.......................................................................................................................66
6.1.2.2 Enhanced macrophage activity in the absence of Treg..................................68
6.1.2.3 Macrophages kill RMA-S cells independently of the enzymatic activity of
iNOS, PGE2 and IDO....................................................................................70
+6.1.3 Regulatory T cells suppress CD8 T cell recognition of TAP-deficient tumors ..74
+6.1.3.1 CD8 T cells contribute to RMA-S tumor rejection in the absence of Treg by
direct recognition of RMA-S cells.................................................................74
6.1.4 Immune intervention in established RMA-S tumors............................................78
6.1.4.1 Depletion of Treg in established RMA-S tumors .......................78
6.1.4.2 Depletion of NK cells in established RMA-S tumors....................................78
6.2 CONTROL OF ANTI-TUMOR IMMUNITY BY TYPE I IFN...............................................81
6.2.1 Enhanced RMA-S tumor growth in the absence of type I IFN signaling ..81
6.2.2 Requirement of hematopoietic and non-hematopoietic compartment for
responsiveness to type I IFN............................................................................82
6.2.3 Contribution of the hematopoietic compartment for responsiveness to type
I IFN ....................................................................................................................83
-/-6.2.3.1 NK cell cytotoxicity is impaired in IFNAR1 mice......................................83
-/-6.2.3.2 Macrophage tumoristatic activity is not impaired in IFNAR1 mice ...........83
-/-6.2.3.3 Intratumoral leukocyte accumulation is not impaired in IFNAR1 mice .....84
Contribution of the non-hematopoietic compartment for responsiveness 6.2.4
to type I IFN .......................................................................................................85
-/-6.2.4.1 Angiogenesis is impaired in IFNAR1 mice.................................................85
7 DISCUSSION.....................................................................................................................87
7.1 SUPPRESSION OF INNATE IMMUNE CELLS BY TREG ..................................................87
7.2 THE ROLE OF ENDOGENOUS TYPE I IFN IN THE ANTI-TUMOR RESPONSE.................92
8 REFERENCES..................................................................................................................96