New protocol for the generation of insulin-specific T …...New protocol for the generation of...
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New protocol for the generation of insulin-specific T regulatory cells Ivana Stojanović, PhD Diabetology group leader Institute for Biological Research “Sinisa Stankovic”, University of Belgrade 8th European Immunology Conference , Madrid, 29.6.2017.
Transcript of New protocol for the generation of insulin-specific T …...New protocol for the generation of...
New protocol for the generation of insulin-specific T regulatory cells
Ivana Stojanović, PhD Diabetology group leader
Institute for Biological Research “Sinisa Stankovic”, University of Belgrade
8th European Immunology Conference , Madrid, 29.6.2017.
Mills KHG, Nature Reviews Immunology 2004
Vignali et al., How regulatory T cells work? 2008 Nature Reviews Immunology 8, 523-532
Stability of FoxP3 expression
Notley & Ehrenstein, Nature Reviews Rheumatology 6, 572-577
Treg application in therapy
• 702 clinical studies: - Type 1 diabetes - Multiple sclerosis - Rheumatoid arthritis - Lupus - GVHD - Asthma - Kidney transplantation
• Type 1 diabetes trials: - Gdansk University (Poland) - University of California, San Francisco (USA)
Dilema – non-specific or antigen-specific action?
Treg Teff
Teff
DĆ
Functionality of Tregs
• Autoimmunity might arise as a consequence of Treg defects
Cellular & Molecular Immunology (2012) 9, 455–463
Clin Transl Immunology. 2016 Jan; 5(1): e56.
Premisses
• Autoantigen-specific Treg are more efficient than polyclonal
• Individuals with autoimmunity might have a “defect” in Treg function
• Autoantigen-specific Treg are difficult to isolate due to their low numbers
• Autoantigen-specific T effectors are more abundant
• Due to cell plasticity, it is possible to convert effector into regulatory cells
Li Q, Nature Communication 2014
Alchemy of CD4+ cells
Premisses
• Autoantigen-specific Treg are more efficient than polyclonal
• Individuals with autoimmunity might have a “defect” in Treg function
• Autoantigen-specific Treg are difficult to isolate due to their low numbers
• Autoantigen-specific T effectors are more abundant
• Due to cell plasticity, it is possible to convert effector into regulatory cells
CD4+ T cells Dendritic cells
48 h
Isolation of insulin-specific CD4+ T lymphocytes
Cultivation with anti-CD3, anti-CD28, TGF-beta, IL-2
Regulatory T lymphocytes
0,1% insulin-specific CD4+ T lymphocytes
3%-8% insulin-specific CD4+ T lymphocytes
Insulin-specific CD4+ cells are effector cells
3.1
82
0
10
20
30
40
50
60
70
80
90
prekultivacije sa
DĆ
nakonkultivacije sa
DĆ
CD
69
(%
)
Treg
0
500
1000
1500
2000
2500
IL-17 IFN-gamma
Cyt
oki
ne
se
cret
ion
(p
g/m
l)
Before cultivation with DC
After cultivation with DC
CD4+ T cells Dendritic cells
48 h
Isolation of insulin-specific CD4+ T lymphocytes
Cultivation with anti-CD3, anti-CD28, TGF-beta, IL-2
Regulatory T lymphocytes
0,1% insulin-specific CD4+ T lymphocytes
3%-8% insulin-specific CD4+ T lymphocytes
Conversion of insulin-specific CD4+ into insulin-specific regulatory T cells
0
20
40
60
80
100
120
prekonverzije
2 3 5 7 9
Tre
g (%
)
Time (days)
CD4+CD25highFoxP3+
Treg
Before conversion
From inflammatory to anti-inflammatory phenotype
0
500
1000
1500
2000
2500
pre konverzije nakonkonverzije
IFN
-gam
a (p
g/m
l)
Treg
0
50
100
150
200
250
300
350
400
prekonverzije
3 5 7
IL-1
0 (
pg/
ml)
Time (days)
Before conversion
After conversion
Before conversion
*
* *
*
% s
up
ress
ion
of
Teff
pro
life
rati
on
0
5
10
15
20
25
30
35
40Insulin-specific Treg
Polyclonal Treg
1:32 1:16 1:8 1:4 1:2 1:1
Treg:Teff ratio
Treg supressive function
Treg supressive function
*
* *
*
Shortcomings...
• During the conversion lot of cells dye
0
50000
100000
150000
200000
250000
0 2 5
Nu
mb
er
of
insu
lin-s
pec
ific
T c
ells
Days after sorting
Shortcomings...
• % of Tregs decrease significantly over time
Tre
g (%
)
Iacocca Family Foundation project (2016-2018)
Department of Immunology, IBISS, Serbia
Can tolerogenic DCs direct naive CD4+ cells into Tregs in vitro?
%
Insulin-specific CD4+ 9±0,4
Treg 2,6±0,4
Treg
