Download Investigating Human T cells Using Multicolor Flow Cytometry

Investigating Human T cells
Using Multicolor Flow Cytometry
Insoo Kang
Section of Rheumatology
Aging and IL-7-mediated CD8+ T cell
survival
• IL-7, largely produced from thymus, is critical
for the development and survival (homeostasis)
of CD8+ T cells.
• Decreased naïve CD8+ T cells and impaired
memory CD8+ T cell responses occur with
aging.
• Decreased plasma levels of IL-7 have been
observed with aging.
• IL-7 has been used to rejuvenate T cell
immunity in aged mice and non-human
primates, without significant success.
• It is critical to determine whether aging also
affects other steps involved in IL-7-mediated
CD8+ T cell survival pathway.
• Objective:
Investigating IL-7 receptor expression on
CD8+ T cell subsets and their signaling and
survival responses to IL-7 in healthy young (≤
40 years) and elderly (≥ 65 years) people.
Exclusion criteria: Individuals who were
taking immunosuppressive drugs or who had a
disease potentially affecting the immune
system including infection, cancer, asthma,
autoimmunity and diabetes were excluded
from the study
IL-7 receptor (R) signal transduction pathways
IL-7
gc
a
P
Jak1
+ STAT5
X
?
P
?
Jak3
Box1 region
@Y449, STAT5 docking sites
PI3 kinase P
PTEN
BCL-2
Retention
of Bax
Akt
Survival (major)
P
P
BAD inactivation
FOXO inactivation
GSK inhibition
Survival
Proliferation
Glucose use
MCB 2004, 24(14):6501-6513
J Exp Med. 2004,200(5):659-69
Immunol Rev. 2003,192:7-20
Flow cytometry is the key step for experiments
Measure IL-7Rα and γC expression by
different CD8+ T cell subsets as well as their
cellular characteristics using
flow cytometry (FACSCalibur® and LSRII®).
Peripheral blood
mononuclear cells
(PBMCs)
from human subjects
Stain cells with
Abs
Stimulate
cells with IL-7 or
PBS.
Using FACSAria®
Sort cells into
different cell
subsets
Measure cell
signaling using flow
cytometry
(FACSCalibur®)
Conduct functional
studies
IL-7Ra expression by subsets of CD8+ T cells
PBMCs
600
400
58
200
Elderly
400
101
100
0
200
400
600
800
FSC-H: FSC-Height
CM
1000
100
EM
284
101
102
103
FL4-H: APC CD8
104
EMCD45RA+
286
Naive
102
24.5
0
252*
600
47.1
CM
103
EM
200
0
Naive
5.23
CD8+ T cells
CCR7
800
104
FL2-H: PE CCR7
800
FSC-H: FSC-Height
1000
SSC-H: SSC-Height
Stain with Abs
to CD8, CD45RA,
CCR7 and IL-7Rα,
γC or isotype
1000
EM
CD45RA+
8.99
38.6
100
101
102
103
FL3-H: Cyc CD45RA
104
CD45RA
55
Isotype
anti-IL-7Ra Ab
281
Young
271
306
low
267
high
*median
fluorescen
t intensity
IL-7Ra
More than 4 markers can be simultaneously measured
using LSRII®
Young
0
Late
0.212
0 10
3
4
103
102
0
4.722
10
10
10
<FITC-A>: CD28
0 10
2.61
5
105
47.7
104
103
105
102
102
0
0
73
0 10
105
39.7
104
103
103
105
102
102
0
0
0 10
103
10
104
<FITC-A>: CD28
44.72
0 10
0.27
5
103
104
10
<FITC-A>: CD28
CD28
5.49
103
104
105
<FITC-A>: CD28
IL-7Ralow
105
3.44
1.47
103
102
0
28
3.69
5
94.22
103
104
10
<FITC-A>: CD28
9.93
0 10
105
12.3
0.86
103
104
105
<FITC-A>: CD28
1.16
104
103
0
1.4
5
30.3
104
102
5.622
0 10
104
0 102
15.2
104
12
54
103
104
10
<FITC-A>: CD28
<APC-A>: CD27
19.7
<APC-A>: CD27
105
103
104
10
<FITC-A>: CD28
0.81
5
<APC-A>: CD27
0 10
22.52
10
10
10
<FITC-A>: CD28
103
0
32.62
0.4
5
4
104
102
5.03
5
0
3
IL-7Rahigh
104
7.712
102
0 10
18.8
64.6
7.58
103
2.612
10
10
10
<FITC-A>: CD28
<APC-A>: CD27
67.7
103
EMCD45RA
4
IL-7Ralow
<APC-A>: CD27
EM
19.8
3
105
104
103
0
IL-7Rahigh
105
104
102
0.043
5
89.2
7.8
<APC-A>: CD27
Early
105
<APC-A>: CD27
104
103
102
80.9
11.6
CM
<APC-A>: CD27
Int
105
<APC-A>: CD27
104
96.6
3.18
<APC-A>: CD27
105
<APC-A>: CD27
Markers used
CD8, CD45RA,
CCR7, IL-7Ra
CD27 and CD28
Elderly
Naive
CM
<APC-A>: CD27
Naive
103
102
0
61
0 102
1.09
5
103
104
10
<FITC-A>: CD28
85.92
0 10
0.6
103
104
105
<FITC-A>: CD28
P-STAT5 in subsets of CD8+ T cells in response to IL-7*
can be measured using flow cytometry
Stain cells with
CD8,CD45RA
and CCR7
Stimulate
cells with IL7 or PBS.
Naive
CM
% of Max
60
40
20
0
80
80
60
60
60
40
10 1
10 2
FL1-H: FITC
10 3
10 4
10 1
% of Max
60
40
20
10 2
FL1-H: FITC
10 3
10 4
10 1
10 2
FL1-H: FITC
10 3
10 4
10 1
10 2
FL1-H: FITC
10 3
10 4
80
60
60
60
40
20
10 1
10 2
FL1-H: FITC
10 3
10 4
10 1
10 2
FL1-H: FITC
10 3
10 4
Phospho-STAT5
40
*10 min at 10 ng/ml
20
0
10 0
IL-7Ra expression
0
10 0
100
80
0
10 0
EMCD45RA+
40
80
20
0
10 0
100
40
Measure expression of
P-STAT5 by CD8+ T
cell subsets using flow
cytometry
(FACSCalibur®)
20
0
10 0
100
IL-7
PBS
40
20
0
10 0
% of Max
80
20
100
80
100
% of Max
a-IL-7Ra
IgG
100
% of Max
Elderly
% of Max
80
EM
100
% of Max
100
Permeabilize and
stain cells with
Abs to P-STAT5
or isotype.
% of Max
PBMCs
0
10 0
10 1
10 2
FL1-H: FITC
10 3
10 4
10 0
10 1
10 2
FL1-H: FITC
10 3
10 4
Advantage of flow cytometry: Cell signaling can be measured in a small number
of cells (<100,000) and quantitative analysis is possible.
IL-7Ralow cells have decreased survival in
response to IL-7
Sorted into subsets
Using FACSAria®
Naive
EM
104
0.05
19.4
26.6
16.6
101
102
103
FL1-H: FITC annexin
104
104
63.8
9.54
101
102
103
FL1-H: FITC annexin
104
104
0.14
4.48
3
0.01
FL3-H: 7 AAD
89.8
100
100
5.59
101
102
103
FL1-H: FITC annexin
104
0.32
10.6
77.2
12.6
101
102
103
FL1-H: FITC annexin
104
104
0.51
55.5
19.5
24.5
10
102
101
100
100
17.6
101
102
103
FL1-H: FITC annexin
3
102
101
13.7
100
100
104
10
102
101
104
FL3-H: 7 AAD
10
102
13.5
101
102
103
FL1-H: FITC annexin
3
FL3-H: 7 AAD
10
58.4
100
100
10.1
68.4
101
104
3
0.29
102
101
100
100
EMCD45RA+
IL-7Ralow
103
102
101
63.9
104
28
FL3-H: 7 AAD
102
100
100
0.11
103
FL3-H: 7 AAD
102
101
IL-7
104
0.06
103
FL3-H: 7 AAD
103
EMCD45RA+
IL-7Rahigh
Measure live cells
using flow cytometry
FL3-H: 7 AAD
104
PBS
Incubate
cells with IL-7
FL3-H: 7 AAD
PBMCs
101
79.8
100
100
9.37
101
102
103
FL1-H: FITC annexin
104
100
100
101
102
103
FL1-H: FITC annexin
104
FACS sorting can be used to measure gene
expression in a small number (100,000) of
specific cell subsets
P < 0.05
50
40
30
P > 0.05
20
Naive
EMCD45RA+ IL-7Rahigh
10
EMCD45RA+ IL-7Ralow
0
IL-7Ra
GABPa
GFI-1
Elderly (n=5)
Conclusions
• Our findings suggest that aging affects
IL-7Ra expression by CD8+ T cells
leading to impaired signaling and survival
responses to IL-7.
• Flow cytometry is valuable in investing
the phenotypes and functions of human
immune cells.
Kim et al, Blood 06
Acknowledgements
• Hang-Rae Kim
• Myung Sun Hong
• Kyung-A Hwang
• Ping Zhu
• Chris Bailey
• Barbara Foster
• Lynne Iannone
• Yale Flow Cytometry
Facility
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Hartford Foundation
Arthritis Foundation
NIH/NIAMS
Lupus Foundation
AFAR
Yale Pepper Center