Recognition of renal cell carcinoma by CD8_1hn+ and CD4_1hn+ TCR-engineered T lymphocytes [Elektronische Ressource] / vorgelegt von Adriana Turqueti Neves

Turqueti Neves, Emiliozzi Adriana

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 20
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Recognition of renal cell carcinoma by
+ +CD8 and CD4 TCR-engineered T lymphocytes



Dissertation der Fakultät für Biologie der
Ludwig-Maximilians-Universität München





vorgelegt von


Adriana Turqueti Neves
aus Porto Alegre, Brasilien



München, 26. August 2010





















Erstgutachterin: Prof. Dr. Elisabeth Weiß
Zweitgutachter: PD. Dr. rer. nat. Daniel Krappmann
Drittgutachter: PD. Dr. rer. nat. Josef Mautner
Viertgutachter: Prof. Dr. Angelika Böttger
Betreuerin der Arbeit: PD. Dr. rer. nat. Elfriede Nößner
(Sondergutachterin)

Eingereicht am: 26.08.2010
Tag der mündlichen Prüfung: 21.02.2011






Diese Dissertation wurde angefertigt am:

Institut für Molekulare Immunologie
Helmholtz Zentrum München
unter der Leitung von Prof. Dr. Dolores Schendel
und der Betreuung von PD Dr. Elfriede Nößner


















Die in dieser Arbeit vorgestellten Ergebnisse flossen in folgende Publikationen ein:

Leisegang M.*, Turqueti-Neves A.*, Engels B., Blankenstein T., Schendel D.J.,
Uckert W. and Noessner E. T-cell receptor gene-modified T cells with shared renal
cell carcinoma specificity for adoptive T-cell therapy. Clin Cancer Res 16:2333-43,
2010.

*Contributed equally to this work

















To my parents
_____________________________________________________Table of Contents
Table of Contents

Abbreviations .......................................................................................................... VI
Abstract .................. VIII
Zusammenfassung ... X
1 Introduction ..................................................................................................... 1
1.1 The T lymphocytes ........................ 1
1.1.1 The TCR complex ......................................................................................... 1
1.1.2 Effector functions of T lymphocytes .............................. 3
1.1.3 Secretion of cytokines ................... 3
1.1.3.1 The interferon family .................................................................................. 4
1.1.3.2 TNF ........................................... 4
1.1.3.3 IL-2 and IL-15 ............................ 5
1.1.4 Cytotoxicity .................................................................... 6
1.1.5 Lytic granule: perforin and granzymes .......................... 7
1.2 T lymphocytes in cancer therapy... 8
1.2.1 TCR optimization strategies ........................................................................ 11
+1.2.2 The importance of CD4 in the tumor immunotherapy 12
1.3 Renal cell carcinoma ................... 13
1.3.1 Tumor-associated antigens and antigen specific T cells for the immune
therapy of RCC ........................................................................................... 14
2 Rationale of the PhD project ........................................ 16
3 Results ........................................................................... 18
3.1 The B3Z-TCR53m indicator cell line for the analysis of the TCR53-pMHC
ligand prevalence among tumors and non-malignant cell lines ....................... 18
3.1.1 High incidence of TCR53-pMHC ligand in RCC cells and in tumor cells of
other histologies .......................................................................................... 19
3.2 Expression of TCR53 in PBLs and functional analysis ................................ 25
3.2.1 Cross-pairing of TCR53mc with endogenous TCR of PBLs. ....................... 26
3.2.2 Transduction with pMP71-TCR53mc endows PBL with HLA-A2 restricted
specific tumor recognition ........................................................................... 28
3.2.3 PBL-TCR53mc can kill RCC targets ........................................................... 28
3.2.4 The antigen specificity of PBL-TCR53mc is HLA-A2 restricted ................... 29
3.2.5 PBL-TCR53mc cells are cytotoxic toward tumors of other histology but not
normal kidney .............................................................................................. 31
+ +3.3 TCR53mc is expressed on both CD4 and CD8 T cells but only functional in
+CD8 T cells. ................................... 32
3.3.1 PBL-TCR53mc cells are polyfunctional upon target recognition ................. 37
I
Table of Contents_______________________________________________________
3.3.2 TCR53mc-mediated killing of RCC-26 cells in a spheroid model mimicking
the tumor environment ................................................................................ 43
3.3.3 TCR53mc expression and functional performance after retroviral
transduction of PBLs of RCC patients ........................ 46
3.3.3.1 Expression of TCR53mc in PBLs of RCC patients .................................. 47
3.3.3.2 Expansion capacity of PBLs of RCC patients 9 days after stimulation and
supplementation with IL-2 ....................................... 48
3.3.3.3 Expansion capacity of PBLs of RCC patients 20 days after stimulation in
medium-containing IL-2 or IL-15 ............................. 49
3.3.3.4 CD28 expression on PBLs cultured in medium supplemented with IL-2 or
IL-15 ........................................................................................................ 50
3.3.3.5 Cytotoxic capacity of PBLs of RCC patients transduced with pMP71-
TCR53mc 51
3.3.3.6 PBLs of RCC patients transduced with pMP71-TCR53mc are
polyfunctional .......................................................................................... 53
3.4 Maintenance of functionality of PBLs transduced with pMP71-TCR53mc .. 55
3.4.1 Cytotoxic response of PBLs of RCC patients and a healthy donor
expressing TCR53mc at day 22 after stimulation. ...................................... 56
3.4.2 Polyfunctional response of PBL-TCR53mc of RCC patients and a healthy
donor at day 20 after stimulation ................................ 57
3.4.3 Comparison of the functional capacity of PBL-TCR53mc of RCC patients
and a healthy donor at day 9 and 20 after stimulation ................................ 58
3.4.4 IFN-  treatment of target cells enhances TCR53-associated recognition . 60
3.4.5 B3Z-TCR53m cells can be used to detect TCR53-pMHC ligand expression
on fresh tissue ............................................................................................ 62
3.5 T cells develop deficits when exposed to spheroids ................................... 63
3.5.1 T cell survival after 4 h and 24 h in spheroids............. 63
3.5.2 Cytotoxic proteins in T cells exposed to spheroids ..... 66
+ +3.5.3 CD28 expression on CD4 and CD8 T cells exposed to spheroids ........... 67
3.5.4 Functional performance of PBL-TCR53mc cells in 3-D tumor cell spheroids .
................................................................................................................... 68
3.6 Perforin deficits are seen in CD8 T cells in tumor tissues. .......................... 69
+ +3.7 The role of CD4 T cells in supporting CD8 CTLs ..................................... 73
+ +3.7.1 TCR26 is expressed on CD8 and CD4 T cells and is functional in both .. 73
+3.7.2 CD4 T cells expressing TCR26 are HLA-A2 restricted .............................. 77
+3.7.3 CD4 T cells exp TCR26 are lytic against RCC-26 ......................... 78
+ +3.7.4 CD4 T cells facilitate CD8 T cell recruitment into spheroids .................... 79
+ +3.7.5 CD4 T cells support the functional response of CD8 T cells 81
II _____________________________________________________Table of Contents
4 Discussion .................................................................................................... 84
5 Material .......... 94
5.1 Equipment ... 94
5.2 Consumable material .................................................................................. 94
5.3 Reagents ..................................... 95
5.4 Cell culture basis-medium and supplements ............... 96
5.5 Cytokines and growth factors ...................................................................... 96
5.6 Commercial kits........................................................... 97
5.7 Human cell lines .......................................................... 97
5.7.1 RCC cell lines ............................. 97
5.7.2 Tumor cell lines ........................................................... 97
5.7.3 Normal kidney cell lines .............................................. 99
5.7.4 Other normal cell lines ................ 99
5.8 RCC patient samples ................................ 100
5.9 Blood samples .......................................................... 100
5.10 Bacteria strain ........................... 100
5.11 Murine cells .....