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
600
400
10
2
10
1
0
200
400
600
800
FSC-H: FSC-Height
CM
1000
10
10
0
EM
284
10
1
2
10
10
FL4-H: APC CD8
3
10
4
EMCD45RA+
286
47.1
CM
3
Naive
EM
24.5
0
252*
10
200
0
Naive
4
5.23
CD8+ T cells
CCR7
800
10
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
0
10
0
10
1
2
10
10
FL3-H: Cyc CD45RA
3
10
CD45RA
55
Isotype
anti-IL-7Ra Ab
281
Young
271
306
low
267
high
*median
fluorescen
t intensity
IL-7Ra
4
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
40
20
100
80
80
80
60
60
60
40
20
0
10
1
10
3
10
4
0
10
1
% of Max
60
40
20
2
10
FL1-H: FITC
10
3
10
4
0
10
1
2
10
FL1-H: FITC
10
3
10
4
0
10
1
2
10
FL1-H: FITC
10
3
10
4
80
60
60
60
40
20
0
10
1
2
10
FL1-H: FITC
10
3
10
4
10
1
2
10
FL1-H: FITC
10
3
10
4
Phospho-STAT5
40
*10 min at 10 ng/ml
20
0
10
IL-7Ra expression
0
0
10
100
80
0
10
EMCD45RA+
40
80
20
0
10
100
40
Measure expression of
P-STAT5 by CD8+ T
cell subsets using flow
cytometry
(FACSCalibur®)
20
0
10
100
IL-7
PBS
80
2
10
FL1-H: FITC
% of Max
0
40
20
0
10
100
% of Max
100
% of Max
60
% of Max
% of Max
Elderly
a-IL-7Ra
IgG
EM
100
% of Max
100
80
Permeabilize and
stain cells with
Abs to P-STAT5
or isotype.
% of Max
PBMCs
0
10
0
10
1
2
10
FL1-H: FITC
10
3
10
4
10
0
10
1
2
10
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