Peptide Dose, MHC Affinity, and Target Self-Antigen Expression are Critical for Effective ...

85 Pages·2001·4.3 MB·English

Peptide Dose, MHC Affinity, and Target Self-Antigen
Expression are Critical for Effective Immunotherapy of
Nonobese Diabetic Mouse Prediabetes
Shawn Winer
A thesis submitted in confomity with the requirernents
for the degree of Master of Science
Graduate Department of Imrnunology
University of Toronto
O Copyright by Shawn Winer 200 1
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Peptide Dose, MHC Affinity, and Target Self-Antigen Expression are
Critical for Effective Immunotherapy of Nonobese Diabetie Mouse Prediabetes
Shawn W iner
Master of Science
Department of Immunology
University of Toronto
Abstract
Cross-reactive T cells which recognize both. Tep69- and ABBOS-epitopes in
the islet autoantigen. ICA69 and bovine serum albumin (BSA). are routinely generated
during human and nonobese diabetic (NOD) mouse prediabetes. Here we analyzed how
systemic administration of these peptides affects progressive autoimmunity in
adoptively transferred and cyclophosphamide-accelerated NOD mouse diabetrs.
Unexpectedly. high dose i.v. ABBOS prevented. while Tep69 exacerbated. disease in
both models. Peptide effects required cognate recognition of the endogenous self-
antigen. The affinity of ABBOS for NOD I-A~'w as significantly higher than that of
Tep69. This explained 1) the expansion of the ABBOS and Tep69 T ce11 pools
following i.v. Tep69. 2) long term unresponsiveness of these cells after i.v. ABBOS.
and 3) precipitation of disease after low dose i.v. ABBOS. ABBOS or ABBOS-
analogs with even higher MHC-affinity may be candidates for experimental
intervention strategies in human prediabetes. but the dose translation from NOD mice
to humans requires caution.
Contributors
The data presentrd in the thesis have been published in the Journal of Immunology:
Winer. S.. Gunaratnam, L.. Astsatourov, 1.. Cheung, R.K.. Kubiak. V.. Karges. W..
Hammond-McKibben. D.. Gaedigk. R.. Graziano, D.. Trucco. M.. Becker, D.J.. and Dosch,
H.-M. 2000. Peptide Dose. MHC Affinity. and Target Self-Antigen Expression Are Critical
For the Effective Immunotherapy of Nonobese Diabetic Mouse Prediabetes. J Immunol.
16 54086, Copyrighr 2000. The .-Imerican rlssocinrion of Immunologisrs (.LU).
Each author made the following contribution to the paper:
Shmvn Winer: 1 was in overall cornrnand of the project and performed and analyzed the bulk
of the data. including Figures 1-5. 7-9. The data described in Figure 1 was gathered during
my founh year undergraduate research project, when 1 worked with a former MSc. student of
the Immunology depanment, Lakshman Gunaratnam.
Lakshman Gzinarornam did the initial adoptive transfer experiments. which 1 reproduced and
expanded. As a summer student he contributed to data in Figures 1.2A-B. M.7 :
Igor Astsatoirror did al1 of the MHC binding experiments (Figure 6):
Roger Gaedigk. Violerta Kubiak, lvolfram Karges. Roy K. Chetrng, and Denise Hammond-
ICA^^"'"
McKibben developed the 129-line mice and generated the NOD congenic ICA69
knockout;
Daniel Graziano. Massirno Trucco: taught us MHC binding assays and provided reagents:
Dorothy J. Becker: intellectuai input.
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Acknowledgements
I would like to thank my supervisor Dr. H.-Michael Dosch for his motivation,
contagious optimism. advice. and support throughout the project.
1 thank the members of my thesis cornmittee. Dr. Tania Watts and Dr. Jayne Danska
for their patience. guidance and inteliectual discussions of this work.
I thank al1 CO-authors for their valuable contributions. 1 would also like to
acknowledge al1 of the members of the Dosch lab for their insight at lab meetings and for
creating a friendly atmosphere in the lab. 1 also recognize and thank Juan Carlos Ortiz for
help in some in vivo experiments. Finally. 1 would like to thank my family for their support
throughout the years of rny study.
Table of Contents
..
Abstract ................................................................................................................ II
...
Contributors ..............................................................................................................
iii
Copyright Statement. The Journal of Immunology ................................................. iv
Acknowledgements .................................................................................................... v
Table of Contents ...................................................................................................... vi
...
List of Figures ......................................................................................................... viit
Abbreviations ............................................................................................................ ix
.
I introduction .......................................................................................................... 1
1.1. Autoimmune Diabetes: A Clinical Perspective ................................... 1
1.11. Epidemiology .......................................................................................... -7
1.111. GeneticsofHumanTlDM ..............................................
I N. The NOD Mouse: A Brief Introduction ...........................
I.V. Pathogenesis ...................................................................
[.VI. Cow Milk and Diabetes: Does Bovine Serum Aibumin P ay a Role? ...... 11
I.VI 1. Antigen-Based Immunotherapy .............................................................. 15
.
11 Sudy Objectives .................................................................................................... 16
III . Materials and Methods ....................................................................................... 17
Mice ....................................................................................................... 17
Peptides .................................................................................................. 17
E 12.3 T ce11 Hybridoma ......................................................................... -18
Adoptive Trans fer .................................................................................. -19
C y clophosphamide-Accelerated Diabetes ............................................... 19
Histology ................................................................................................ 19
Tolerance Induction and Proliferation Recall Assay ................................'O
LA'' Purification and Affini ty Studies ................................................... 20
1II.IX. Statistics ................................................................................................ .7--3
.
IV Results .............................................................. 33
...................................................
1V.I. BSA- and ICA69-S~ecificR eactivity in Cloned T ce11 Hvbridomas ........ 22
IV.11. Irnmunotherapy with Tep69 and ABBOS has Opposite Outcornes ..........2 5
IV.111. Peptide Effects Require Expression of Cognate Self-Antigen .................. j0
IV.IV. ABBOS and Tep69 Differ in MHC Affinity ........................................... -32
1V.V. Tolerance Induction Following i.v. Peptide Treatment ............................ 34
.
V Discussion ............................................................................................................ -39
.
VI Future StudieslSynopsis ..................................................................................... 44
.
VI1 References .......................................................................................................... 46
vii
List of Figures/Tables
Table 1. Linear Sequence Homology Behveen ABBOS and Tep69 Peptides ............. 13
Figure 1. Cross-reactivity between Tep69 and ABBOS in the E 12.3 hybridoma and NOD
splenocytes .. . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . , . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .X
Figure 2. Tep69 and ABBOS peptides have opposing effects on adoptively transferred IDD
.... .. .... .. . .. ... .. . . . . . . .. . ..... .. . .. . . . . ... .. . .... .. .... .. . .. .. ....... .... .. ........ ...... ....... .. ......... .. ... .. .. .. . .. .. . .- 26
Figure 3.1. v. Tep69 exacerbates and i.v. ABBOS protects from cyclophosphamide-induced
IDD ............................................................................................................................ 28
Figure 4. High dose i.v. ABBOS reduces insulitis ......... ...... ..... ....... .. ......... .. .............. 29
Figure 5. Peptide-mediated modification of adoptively transferred IDD requires expression
of the cognate self-antigen .. ....... ... ... .. ... .. .. ....... .. . .. .. . .......... . . . . .. . .....3 1
Figure 6. Tep69 and ABBOS have different affnity for MHC ............. .... ....... ......... ... 3 3
xi
Figure 7. Short and long terrn unresponsiveness following i.v. peptide treatments ......3 5
Figure 8. High dose i.v. Tep69 expands ABBOS and Tep69 cross-reactive T cells in
adoptive transfer recipients .. ...... ... .. .. ......... .... ... .... ... . .. .. .. . . ..-....... *.... . ..... .. . ....... ... ........ 3 7
Figure 9. Low dose i.v. ABBOS exacerbates prediabetes. ....... .................. .. ............... .3 8
Figure 10. Proposed role of mimicry peptides in diabetes development ...................... 43
viii
Abbreviations
aa: Amino acid
ABBOS: An irnmunodominant epitope in BSA
ALA: Alanine
APC: htigen Presenting Ceil
BB: BioBreeding
BCG: Bacillus Calmette-Guerin
BS.4: Bovine Serum Albumin
CFA: Complete Freund's Adjuvant
CY: Cyclophosphamide
DPT-1:D iabetes Prevention Trial 1
EBV: Epstein-Barr Virus
FDR: First Degree Relative
FITC: Fluoroscein Isothiocyanate
GAD65i67: Glutamic Acid Decarboqlase (65 or 67 kD isoform)
HPLC: Hi& Performance Liquid Chromatography
HLA: Human Leukocye Antigen
HSP6O: Heat Shock Protein (60 kD)
142: A protein tyrosine-phosphatase
I-A~~M:H C class II molecule in NOD mice
ICA: lslet Ce11 Antibodies
ICA69: Islet Cell Antigen (69 kD)
IDD(iM): Insulin Dependent Diabetes (Mellitus)