Effects of intracellular and extracellular heat shock proteins on anti-tumor immune responses [Elektronische Ressource] : implications for the treatment of solid tumors using hyperthermia and HSP-based vaccines / vorgelegt von Valeria Milani

Milani Giuliano, Valeria

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2005
• Nombre de lectures: 23
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Aus der Medizinischen Klinik und Poliklinik III-Großhadern
der Ludwig-Maximilians-Universität München
Vorstand: Prof. Dr. med. W. Hiddemann






Effects of intracellular and extracellular heat shock proteins
on anti-tumor immune responses

Implications for the treatment of solid tumors using hyperthermia and
HSP-based vaccines




Dissertation
zum Erwerb des Doktorgrades der Medizin
an der Medizinischen Fakultät der
der Ludwig-Maximilians-Universität zu München





vorgelegt von

Valeria Milani

aus
Rom


2005







Mit Genehmigung der Medizinischen Fakultät
der Universität München












1. Berichterstatter: Prof. Dr. med. dipl. biochem. Rolf Dieter Issels

2. Berichterstatter: Prof. Dr. A. Wagner
Prof. Dr. W. Machleidt
Priv. Doz. Dr. G. Hartmann

Mitbetreuung durch den
promovierten Mitarbeiter: PD Dr. rer. nat. Elfriede Nössner


Dekan: Prof. Dr. med. Dr. h. c. K. Peter


Tag der mündlichen Prüfung: 13.01.2005
2








Dedicated to
Titti
and
Maria-Giulia


















3
INDEX

ABBREVIATIONS................................................................................................................................................................. 7
1 INTRODUCTION........................ 9
1.1 THE CELLULAR ANTITUMOR IMMUNE RESPONSE: EFFECTOR CELLS AND
MECHANISMS..................................................................................................................................................................10
1.1.1 Dendritic cells: the link between the innate and adaptive immune response....10
1.1.2 Maturation of dendritic cells......................................................................................................................10
1.1.3 DC/T cell interaction...................................12
1.1.4 Antigen presentation....................................................................................................12
1.1.5 T cell mediated anti-tumor immune response..........................15
1.1.6 Failure of tumor control by the immune system: tumor immune escape mechanisms .....................16
1.2 EFFECTS OF HYPERTHERMIA ON TUMOR ANTIGENICITY AND SUSCEPTIBILITY TO
IMMUNE EFFECTOR MECHANISMS.......................................................................................................................18
1.3 HEAT SHOCK PROTEINS AND THEIR ROLE IN INNATE AND ADAPTIVE IMMUNE
RESPONSE.........................................................................................................................................................................19
1.3.1 Biochemistry of the HSP70 family.............21
1.3.2 Receptors and signal mediators for heat shock proteins.......21
1.3.3 Heat shock protein-peptide complexes (HSP-PC): a tool for immunization.....................................23
2 GOAL OF THE STUDY ..........................................................................................................24
2.1 THE MODEL SYSTEM....................................25
2.1.1 The human melanoma model......................................................................................................................25
2.1.2 Melanogenesis and antigenic profile associated with progression of malignant melanoma..........26
2.2 DESIGN OF THE STUDY................................................................................................................................28
3 MATERIALS ..............................................................30
3.1 CHEMICALS......................................................................................................................31
3.2 KITS......................................................................................................................................................................32
3.3 LABORATORY EQUIPMENT, CONSUMABLES....................32
3.4 CELLS..33
3.4.1 Tumor cell lines ............................................................................................................................................33
3.4.2 Cytotoxic lymphocyte clones......................34
3.4.3 Primary cells (PBMC).................................................................................................................................34
3.5 MATERIALS USED FOR CELL CULTURE...............................34
3.5.1 Cell culture media........................................................................................................................................34
3.5.2 Culture vessels and plastic ware................35
3.5.3 Solutions and antibodies for flow cytometry............................................................................................36
3.5.4 Fluorescence labeling kits..........................................................37
3.5.5 Buffer and antibodies for ELISA................................................................................37
4
3.6 MATERIALS USED FOR BIOCHEMISTRY..............................................................................................38
3.6.1 Buffers and antibodies for Western Blot and Silver Stain.....................................38
3.6.2 Buffers for cell lysis and Western Blot......................................................................38
3.6.3 Buffers and column materials for purification of HSP70-PC...............................41
3.6.4 Plastic ware...................................................................................................................................................42
3.7 SOFTWARE........................42
4 METHODS...................................................................................................................................................................43
4.1 CELL CULTURE METHODS.........................44
4.1.1 Cell culture techniques................................................................................................................................44
4.1.2 Heat shock treatment...46
4.1.3 Clonogenic assay..........................................................................................................................................46
4.1.4 Thermal isoeffect doses and cell viability47
4.1.5 Flow cytometry analysis..............................................................................................................................47
4.1.6 T cell stimulation assay...............................48
4.1.7 Cell mediated cytolysis (CML)...................................................................................................................49
4.1.8 Cross-presentation assay............................49
4.1.9 ELISA..............................................................................................................................................................50
4.1.10 Confocal microscopy51
4.2 BIOCHEMICAL METHODS..........................52
4.2.1 Fluorescence labeling of proteins (Cy5) ..................................................................................................52
4.2.2 Biochemical analysis of protein expression.............................52
4.2.3 Purification of HSP70-PC ..........................................................................................................................53
4.3 STATISTICAL ANALYSIS.............................61
5 RESULTS .....................................................................................................................................................................62
5.1 EFFECTS OF HYPERTHERMIA TREATMENT ON TUMOR ANTIGENICITY AND
SUSCEPTIBILITY TO IMMUNE EFFECTOR MECHANISMS.............................................................................63
5.1.1 Selection of two thermal isoeffect doses in 624.38-MEL cells..............................63
5.1.2 Thermal dose-related differential kinetics of HSP70 protein expression...........65
5.1.3 Thermal-dose related changes in expression levels of immunologically relevant proteins: MHC
class I, HLA-A2, tyrosinase and Melan-A/MART-1 antigens during the heat shock response.......................67
5.1.4 Thermal dose-related endogenous HLA-A2-restricted tyrosinase and Melan-A/MART-1 peptide
presentation...................................................................................................................................................................71
5.1.5 Thermal dose-related susceptibility of heat treated melanoma cells to cytotoxic effector
mechanisms....74
5.2 TUMOR-DERIVED HSP70-PEPTIDE COMPLEXES MEDIATE CROSS-PRESENTATION IN
HUMAN DENDRITIC CELLS ........................................................................................................................................77
5.2.1 Purification and characterization of HSP70 from melanoma cell lines.............77
5.2.2 DCs bind and uptake tumor