Investigating Human T cells Using Multicolor Flow Cytometry Insoo Kang Section of Rheumatology.
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Investigating Human T cells Using Multicolor Flow Cytometry Insoo Kang Section of Rheumatology
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Transcript of Investigating Human T cells Using Multicolor Flow Cytometry Insoo Kang Section of Rheumatology.
Investigating Human T cells Using Multicolor Flow
CytometryInsoo 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
STAT5X
c
Jak1Jak3
PI3 kinase P
PTEN
Akt P BAD inactivation
FOXO inactivation
GSK inhibition
SurvivalProliferationGlucose use
P
+
Survival (major)
Retention of Bax
Box1 region
P
BCL-2
??
IL-7
@Y449, STAT5 docking sites
P
MCB 2004, 24(14):6501-6513J Exp Med. 2004,200(5):659-69Immunol Rev. 2003,192:7-20
Peripheral blood mononuclear cells
(PBMCs)from human
subjects
Measure IL-7Rα and γC expression bydifferent CD8+ T cell subsets as well as
their cellular characteristics using flow cytometry (FACSCalibur® and LSRII®).
Measure cell signaling using flow
cytometry (FACSCalibur®)
Stimulate cells with IL-7 or
PBS.
Stain cells with Abs
Using FACSAria®Sort cells into different cell
subsets
Flow cytometry is the key step for experiments
Conduct functional studies
0 200 400 600 800 1000FSC-H: FSC-Height
0
200
400
600
800
1000
SS
C-H
: SS
C-H
eig
ht
58
100 101 102 103 104
FL4-H: APC CD8
0
200
400
600
800
1000
FS
C-H
: FS
C-H
eig
ht
24.5
100 101 102 103 104
FL3-H: Cyc CD45RA
100
101
102
103
104
FL
2-H
: PE
CC
R7
5.23 47.1
8.9938.6
CD8+ T cells
CD45RA
CC
R7
CM Naive
EMCD45RA+
EM
Naive CM EM EMCD45RA+
IL-7R
Eld
erl
yYou
ng
PBMCs
IL-7R expression by subsets of CD8+ T cells
Isotype
anti-IL-7R Ab
271 281 306 267
252* 284 286 55
*median fluorescent intensity
highlow
Stain with Abs to CD8, CD45RA,
CCR7 and IL-7Rα, γC or
isotype
0 102 103 104 105<FITC-A>: CD28
0
102
103
104
105
<A
PC
-A>
: C
D2
7
39.7 15.2
0.2744.70 102 103 104 105
<FITC-A>: CD28
0
102
103
104
105
<A
PC
-A>
: C
D2
7
19.7 73
1.45.62
0 102 103 104 105<FITC-A>: CD28
0
102
103
104
105
<A
PC
-A>
: C
D2
7
47.7 18.8
0.8132.60 102 103 104 105
<FITC-A>: CD28
0
102
103
104
105
<A
PC
-A>
: C
D2
7
19.8 67.7
5.037.71
0 102 103 104 105<FITC-A>: CD28
0
102
103
104
105
<A
PC
-A>
: C
D2
7
11.6 80.9
2.614.720 102 103 104 105
<FITC-A>: CD28
0
102
103
104
105
<A
PC
-A>
: C
D2
7
3.18 96.6
0.0430.21
EM
CD
45
RA
EM
0 102 103 104 105<FITC-A>: CD28
0
102
103
104
105
<A
PC
-A>
: C
D2
7
12.3 1.16
0.685.90 102 103 104 105
<FITC-A>: CD28
0
102
103
104
105<
AP
C-A
>:
CD
27
28 9.93
1.0961
0 102 103 104 105<FITC-A>: CD28
0
102
103
104
105
<A
PC
-A>
: C
D2
7
3.44 1.47
0.8694.20 102 103 104 105
<FITC-A>: CD28
0
102
103
104
105
<A
PC
-A>
: C
D2
7
12 30.3
3.6954
0 102 103 104 105<FITC-A>: CD28
0
102
103
104
105
<A
PC
-A>
: C
D2
7
7.58 64.6
5.4922.50 102 103 104 105
<FITC-A>: CD28
0
102
103
104
105
<A
PC
-A>
: C
D2
7
7.8 89.2
0.42.61
Naive CM
IL-7Rhigh IL-7Rlow
Young Naive CM
IL-7Rhigh IL-7Rlow
Elderly
CD28
CD
27
Late
Early
Int
More than 4 markers can be simultaneously measured using LSRII®
Markers usedCD8, CD45RA, CCR7, IL-7RCD27 and CD28
P-STAT5 in subsets of CD8+ T cells in response to IL-7* can be measured using flow cytometry
IL-7R expression
Phospho-STAT5
100 101 102 103 104
FL1-H: FITC
0
20
40
60
80
100
% o
f M
ax
100 101 102 103 104
FL1-H: FITC
0
20
40
60
80
100
% o
f M
ax
100 101 102 103 104
FL1-H: FITC
0
20
40
60
80
100
% o
f M
ax
100 101 102 103 104
FL1-H: FITC
0
20
40
60
80
100
% o
f M
ax
IgG -IL-7R
PBS IL-7
100 101 102 103 104
FL1-H: FITC
0
20
40
60
80
100
% o
f M
ax
100 101 102 103 104
FL1-H: FITC
0
20
40
60
80
100
% o
f M
ax
100 101 102 103 104
FL1-H: FITC
0
20
40
60
80
100
% o
f M
ax
100 101 102 103 104
FL1-H: FITC
0
20
40
60
80
100
% o
f M
ax
*10 min at 10 ng/ml
Eld
erl
y
PBMCs Measure expression of P-STAT5 by CD8+ T cell subsets using
flow cytometry (FACSCalibur®)
Stimulate cells with IL-
7 or PBS.
Permeabilize and stain cells with
Abs to P-STAT5 or isotype.
Stain cells with CD8,CD45RA
and CCR7
Advantage of flow cytometry: Cell signaling can be measured in a small number of cells (<100,000) and quantitative analysis is possible.
Naive CM EM EMCD45RA+
100 101 102 103 104
FL1-H: FITC annexin
100
101
102
103
104
FL
3-H
: 7 A
AD
0.51 55.5
24.519.5
100 101 102 103 104
FL1-H: FITC annexin
100
101
102
103
104
FL
3-H
: 7 A
AD
0.29 68.4
17.613.7
100 101 102 103 104
FL1-H: FITC annexin
100
101
102
103
104
FL
3-H
: 7 A
AD
0.32 10.6
9.3779.8100 101 102 103 104
FL1-H: FITC annexin
100
101
102
103
104
FL
3-H
: 7 A
AD
0.01 10.1
12.677.2100 101 102 103 104
FL1-H: FITC annexin
100
101
102
103
104
FL
3-H
: 7 A
AD
0.14 4.48
5.5989.8
100 101 102 103 104
FL1-H: FITC annexin
100
101
102
103
104
FL
3-H
: 7 A
AD
0.11 28
13.558.4100 101 102 103 104
FL1-H: FITC annexin
100
101
102
103
104F
L3-
H: 7
AA
D
0.06 26.6
9.5463.8100 101 102 103 104
FL1-H: FITC annexin
100
101
102
103
104
FL
3-H
: 7 A
AD
0.05 19.4
16.663.9
Naive EM
PBS
IL-7
EMCD45RA+
IL-7Rhigh
EMCD45RA+ IL-7Rlow
IL-7Rlow cells have decreased survival in response to IL-7
PBMCs Sorted into subsetsUsing FACSAria®
Incubate cells with IL-7
Measure live cellsusing flow cytometry
Elderly (n=5)
IL-7R GABP GFI-10
10
20
30
40
50
Naive
EMCD45RA+ IL-7Rhigh
EMCD45RA+ IL-7Rlow
FACS sorting can be used to measure gene expression in a small number (100,000) of
specific cell subsets
P > 0.05
P < 0.05
Conclusions• Our findings suggest that aging affects
IL-7R 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
• Yale Flow Cytometry Facility
• Hartford Foundation• Arthritis Foundation• NIH/NIAMS• Lupus Foundation• AFAR• Yale Pepper Center
• Hang-Rae Kim• Myung Sun Hong• Kyung-A Hwang
• Ping Zhu
• Chris Bailey • Barbara Foster• Lynne Iannone
