Effect of Strain, Sex and
Age on Immunophenotyping
Parameters in the Rat.
Authors: B. Finney*, K. Belsham and N. Downes. * Corresponding author: [email protected]
Sequani Ltd, Ledbury, UK
Printed: 22/02/2016 16:06:13
Assay: T-Cell Subsets UD
Sample ID: 38T
Cytometer settings and performance was maintained by use of
CS&T Beads (Cat#650621, BD Biosciences, Oxford, UK).
Fig.2
Immunophenotyping of lymphocyte subsets can be a useful
Examples of the gating strategy and hierarchy for each assay
Tube_001
- Lymphocytes
addition to toxicological studies where there is the potential for
are shown
in Fig.1 and Fig.2. The T-, B- and NK-cell analysis
x1000
Show Statistical Gates/Populations
250
effects on the immune system. At Sequani we are using two
was set to analyse up to 30000 events in the lymphocyte gate
Gate
Hierarchy
commercially available staining cocktails for rat lymphocytes
and data was accepted as long as at least 5000 cells were
200
All
Events
with a FACSVerse flow cytometer and FACSuite software. The
analysed in this gate. The T-lymphocyte subset analysis was
Rat T-, B- and NK-cell cocktail uses CD3, CD45RA and CD161a
Lymphocytes
set to analyse up to 5000 events in the T-cell gate and results
150
for the specific labelling of each cell type, while the
T-Cells (CD3+)
are considered
acceptable as long as at least 2500 cells were
T-Cells (CD3+)
T-lymphocyte cocktail uses CD3, CD4 and CD8 to label T-cells,
analysed in this gate. Data is reported as the % of each cell type
T-Helper (CD4+)
100
T-helper cells and T-cytotoxic cells respectively. Blood samples
from its parent gate, i.e. % B-cells/Lymphocyte or %
T-Cytotoxic (CD8+)
from 2 month old males and females from both the Han Wistar
T-helper/T-cells. Results were plotted as individual data points
50
and Sprague Dawley strains were analysed.
Samples
from older 16:06:13
for each group with the mean±SD shown as black bars.
Population
View
Printed:
22/02/2016
Assay: T-Cell
Subsets
UD
Sample ID: 38T
Sprague Dawley post-parturition females were also analysed.
Statistical
analysis was
conducted
A.
Printed: 22/02/2016 16:06:13
Assay: T-Cell Subsets
UD
Sample
ID: 38T between the groups using a
0
Data were calculated as the cell percentage within the parent
non-parametric,
two-tailed
t-test with Mann-Whitney.
0
102
103
104
105
population, i.e. Lymphocytes or T-cells depending on the assay.
Statistical
significance was reached when p≤0.05 and is
CD3 APC-A
Tube_001
Lymphocytes
In general, there was a low amount of variation
theGates/Populations
x1000
indicated on graphs by asterisks.
Showwithin
Statistical
Tube_001 - T-Cells (CD3+)
Tube_001 - T-Cells (CD3+)
250
results for each group. Data for Sprague Dawley samples were
Tube_001 - Lymphocytes
x1000
Show
Statistical
Gates/Populations
5
Gate Hierarchy
5
10
250
similar regardless of sex (although this data set was small)
10
T-Helper (CD4+)
T-Cytotoxic (CD8+)
Gate Hierarchy
200
Events
except in %NK-cells. Han Wistar animals showedAll
some
Abbreviations used in the figures: HW-Han Wistar,
4
4
Lymphocytes
200
All
statistically significant differences between the sexes
in the
10Events
10
SD-SpragueDawley,
M-male, F-female.
150
T-Cells
(CD3+)
%B-cells, %NK-cells and % of T-cytotoxic cells. Both sexes
LymphocytesT-Cells (CD3+)
Statistically significant differences were seen in the % of
3
150
showed highly significant strain differences at 2 months T-Helper
of age (CD4+)
3
T-Cells (CD3+)
10
T-Cells
(CD3+)
10
B-cells
(Fig.3
A.) between strains with HW males having
100
in the percentage of either T-helper or T-cytotoxic cells T-Cytotoxic (CD8+)
T-Helper (CD4+)
25.74±5.18% which was lower than SD males with
100
counted although the percentage of total T-cells counted in
2
T-Cytotoxic
(CD8+)
2
10
30.41±4.51%
(p=0.0382) and HW females having
10
50
the Lymphocyte population was similar. WhilePopulation
this database
View is
21.39±6.42% which was lower than SD females with
0
0
50
Population View
28.87±4.88% (p=0.016). These differences were not
0
B. 2 UDT-Cell
Assay:16:06:13
Rat Lymphs No CD45
5
Printed: 22/02/2016
Assay:
Subsets
UD 102103
38T
Sample ID: 35L
3
103 Sample
104 ID:
0
102
104
105
0
102 0
104
105 10
significant
within10each strain
between
the sexes. There was
CD4
PE-A
0
CD8 FITC-A
CD3 APC-A
2
3
4
5
0
10
10 a statistically
10
10
also
significant
decrease in the % of B-cells in
Fig.1
CD3 APC-A
Tube_001 - T-Cells (CD3+)
Tube_001 - T-Cells (CD3+)
post-parturition SD females with 15.19±5.39% in comparison
Tube_001 - All Events
x1000
5
5
toTube_001
the
younger
SD females
- T-Cells (CD3+)
Tube_001Tube_001
-10Lymphocytes
Statistics
10
- T-Cells
(CD3+) (p=0.0087).
x1000
Show Statistical
Gates/Populations
250
Show Statistical Gates/Populations
Fig.4
FSC-A
Abstract and Introduction:
A.
T-Helper (CD4+)
CD3 APC-A
150
Lymphs
0
Assay: Rat Lymphs No CD45
2 UD
0
50
T-Cells (CD3+)
T-Helper (CD4+)
2
50
Sample
ID: 35L
Lymphs
10
10
100
T-Cytotoxic (CD8+)
x1000
102
103
PE-A
Tube_001 - AllCD4
Events
104
x1000
200
All Events
200
Lymphs
150
100
FSC-A
NK
T cells
2 0 102
250
-10
103
104
B. CD3 APC-A
SSC-A
150
Tube_001 - Lymphs
100
50
0
100
All Events
50
00
50
B cells100
150
x1000
Tube_001 - Lymphs
250
200
150
200
Acquisition Date (Tube_001):
22-Feb-2016
B cells
Tube ID (Tube_001): M35L
C.
0
102
103
T cells
50
150 100
200
250
Acquired
(Tube_001):
119000
x1000
104
105
Tube_001:All
103
ShowCD4
Statistical Gates/Populations
104 PE-A 105
CD3 APC-A
Gate Hierarchy
Statistics
0
103
9,975
63.00 B cells
63.00
***
Name
Tube_001:T-Cells (CD3+) 4,996
31.55 NK 50.09
31.55
Signature: Katie Belsham
Tube_001:Lymphocytes
Tube_001:T-Helper
(CD4+) 3,807
24.04
76.20
38.17
100
3
10Comments:
Tube_001:T-Cytotoxic
(CD8+) 1,201
7.58
24.04 Tube_001:T-Cells
12.04 (CD3+)
Tube_001:T-Helper (CD4+)
NK
250
Population ViewTube_001:T-Cytotoxic (CD8+)
50
x1000
10
150
Tube_001:Lymphocytes
150
100
T cells
having 31.88±3.67% (p=0.056) and HW females having
Page 1 of 1 which was lower than SD females having
23.93±3.22%
33.0±2.16% (p<0.0001). The differences were also statistically
significant between the sexes for HW animals (p=0.078).
NK
100
expression scatter plot with T-cytotoxic cell gate (red).
Events % Total %50Parent % Grandparent
NK
22/02/2016 16:06:12
0
2
3
0 10
EventsGate
34,634
291.04 10
Hierarchy
4
10
***
Tube_001 CD45RA
- Lymphs
- Lymphs
FITC-A
CD161a
PE-A
Tube_001:Lymphsx1000
30,000Tube_001
252.10
86.62
105
22/02/2016 16:06:12
0
Mean
Mean
Page
1 of 1
when
looking atMean
results from
Immunophenotyping
analysis or
*** 100.00
713
2,780
2,379
planning
studies where
may be an immunological effect.
Tube_001
- Lymphs
***x1000 86.62
619
3,037 there2,605
Events 98.13
Tube_001:T cells250All
11,678
38.93
33.72250 33.72
187
Tube_001:B cells
8,438
70.91
28.13
24.36
24.36
1,586
Statistics
Lymphs
Tube_001:NK200 1,914
16.08
6.38
5.53200 5.53
309
TName
cellsof Experiment or Worklist (Tube_001): Lymph control data 5
266
83
40,995
Page 1 of 1
6,480
151
136
Staining Method:
A.
Blood samples were collected from animals (HanWistar or
SpragueDawley Males and Females of approximately 2 months
be_001): Lymph control data 5
Sample ID (Tube_001): 35L
NK
of age or SpragueDawley post-parturition Dams greater than 3
100
100
-2016
Tube ID (Tube_001): M35L
months ofNK
age) killed by Schedule 1 methods into K2EDTA
T cells
Sample
B Volume
cells Acquired (Tube_001): 119000
50
50
150
200
250
Population
View
tubes and placed on a roller at room temperature. Whole blood
Operator (Tube_001): Katie Belsham
x1000
D.
counts
using
an 5Advia 120 (Siemens UK) were used to
0
0
:-A
119000
CD45RA FITC-A CD161a
PE-A CD3 APC-A
-102 0 102
103
104
105
0
102
103
104
105
0 102
103
104
10
Name
Events Events/uL % Parent normalise
% Grandparent
% Total
lymphocytes
per mL
to 1 x 106 Mean
Mean
Mean
CD3 APC-A
CD45RA FITC-A
CD161a PE-Aan aliquot of blood
Signature:
Katie
Belsham
Phosphate Buffered
(PBS). Staining
34,634
291.04
*** with addition
*** of
100.00
713 Saline2,780
2,379 22/02/2016 16:06:12
CD45RA FITC-A Tube_001:All
CD161a PE-AEvents
CD3 APC-A
s/uL % Parent % Grandparent % Total
Tube_001:Lymphs
30,000
252.10
86.62 cocktails used
*** were
86.62the T, B, NK619
3,037
2,605 and
Cell cocktail
(Cat#558495)
MeanComments:
Mean
Tube_001
- Lymphs
Tube_001
- Lymphs
Signature:
Katie Belsham Mean
x1000
x1000
Tube_001:T
cells 11,678
98.13
38.93
33.72
33.7222/02/2016 16:02:47
187
266
6,480
1.04
***
*** 100.00
2,780 cells 2,379
cocktail1,586
(Cat#558493)83both purchased
250
250 713
Tube_001:B
8,438
70.91
28.13 the Rat T-Lymphocyte
24.36
24.36
151
Comments:
2.10
86.62
***
86.62
619
3,037
2,605
Statistics
Tube_001:NK
1,914
16.08
6.38 from BD Biosciences
5.53
5.53 (Oxford, UK).
30910 μL of
40,995
136
the
T-,
B-,
and
8.13
38.93
33.72
33.72
266
6,480
Page 1 of 1
200
200 187
of Experiment
or Worklist
Lymph control data
NK-Cell cocktail was used to stain 100 μL of normalised blood,
0.91 Name
28.13
24.36
24.36(Tube_001): 1,586
835
151
6.08
6.38
5.53
5.53
309
40,995
136
Page 1 of 1
Acquisition Date (Tube_001): 22-Feb-2016150
150
while 10 μL of the Rat T-Lymphocyte cocktail was used to stain
Sample ID (Tube_001): 35L
200 μL of normalised blood. Both were incubated for 15
100
100
Tube ID (Tube_001): M35L
minutes in the dark at RT before lysing (FACSLyse
NK
Sample
Volume
Acquired
(Tube_001):
119000
B cells
50
50
Cat#349202, BD Biosciences, Oxford, UK) and washing
Operator (Tube_001): Katie Belsham
according to instructions for the FACSLyse solution. Samples
E. 5
0
0
2
3
4
2
3
4
5
CD45RA
FITC-A
CD161a
PE-A then
CD3 APC-A
were
resuspended in 300 μL PBS prior to analysis.
0
10 Name
10
10 Events
10
0 10% Grandparent
10
10 % Total
10
B.
Events/uL % Parent
150
Acquisition
Date (Tube_001): 22-Feb-2016
B
cells
SSC-A
150
SSC-A
SSC-A
Statistics
CD45RA FITC-A
CD161a PE-A
Mean
Tube_001:All Events 34,634
291.04
***
*** 100.00
713
Tube_001:Lymphs 30,000
252.10Signature:
86.62 Katie Belsham***
86.62
619
Tube_001:T cells 11,678
98.13
38.93
33.72
33.72
187
Comments:
Tube_001:B cells
8,438
70.91
28.13
24.36
24.36
1,586
22/02/2016
16:02:47
strategy
and Hierarchy
T-, B- and NK-cells.
Tube_001:NK
1,914
16.08
6.38 for Lymphocytes
5.53 and
5.53
309
Statistics Gating
A. Initial data collection with Lymphocyte gate (orange). B. Gate hierarchy. C. CD3
01): Lymph control data 5 expression scatter plot with T-cell gate (purple). D. CD45RA expression scatter plot
with B-cell gate (blue) E. CD161a expression scatter plot with NK- cell gate (green).
6
Mean
Mean
2,780
2,379
3,037
2,605
22/02/2016 16:02:47
266
6,480analysed using a FACSVerse flow cytometer with
Samples
were
83
151
associated FACSuite
software (BD Biosciences, Oxford, UK). An
40,995
136
Page 1 of 1
Analysis Method:
assay was developed for each staining cocktail to allow for
Page
1 of 1 of samples to minimise differences between
routine
analysis
operators and staining runs.
Graphical data for % B- and NK-cells. A. B-cells as % of Lymphocytes, * =
p≤0.016, **=p≤0.0087 B. NK-cells as % of Lymphocytes * = p≤0.026, **=p≤0.0051
9000
L % Parent % Grandparent % Total
***
***
nature:
Katie Belsham ***
86.62
38.93
33.72
mments:
28.13
24.36
6.38
5.53
100.00
86.62
33.72
24.36
5.53
CD45RA FITC-A CD161a PE-A CD3 APC-A
Mean
Mean
Mean
713
2,780
2,379
619
3,037
2,605
187
266
6,480
1,586
83
151
309
40,995
136
22/02/2016 16:02:47
Sequani Limited
Page 1 of 1
Bromyard Road, Ledbury, HR8 1LH, United Kingdom
website: www.sequani.com
22/02/2016 16:02:47
Page 1 of 1
Graphical data for % T-, T-helper and T-cytotoxic cells. A. T-cells as % of
Lymphocytes B. T-cytotoxic cells as % of T-cells, **** = p<0.0001, **=p≤0.0078.
C. T-helper cells as % of T-cells, ** = p≤0.0053, ***=p≤0.0008.
This difference was not seen between the sexes in SD animals
or in different age ranges for SD animals as post-parturition SD
females had 36.2±5.62% T-cytotoxic cells.
Fig.3
200
(purple).
C. CD4 Tube_001
expression scatter plot with T-helper cell gate (green). D. CD8
Tube Name
(Tube_001):
B cells
Population View Name
NK
(Fig.4 B.), statistically
C.
22/02/2016 16:06:12
9,975
63.00
63.00
***
significant
differences
were
seen
between
strains with HW
4,996
31.55
50.09
31.55
3,807
24.04
38.17which was lower than SD males
males
having76.20
27.76±3.36%
1,201
7.58
24.04
12.04
150 control data 5
Name
of
Experiment
or Worklist
(Tube_001):for
Lymph
B cells
Gating
strategy
and Hierarchy
T-cells,
T-helper and T-cytotoxic cells.
Gate(Tube_001):
hierarchy for
data collection. B. CD3 expression scatter plot with T-cell gate
FCS FileA.Name
4c0d188a-a16f-492a-b11a-01162eaf1d19.fcs
Signature: Katie Belsham
50
Comments:
Statistics
Events
% Total
% Parent %cell
Grandparent
In the
T-cytotoxic
population
Tube_001:Lymphocytes
9,975 Katie
63.00
63.00
***
Signature:
Belsham
0results
still
being
compiled,
these
initial
show
that
there
are5
Tube_001:T-Cells
(CD3+) 4,996
31.55
50.09
31.55
50 2 0 102
4
5
3
4
-10
103
104
105
0 Tube_001:T-Helper
102
103
10Comments:
10
0 102
10
10
(CD4+)
3,807
24.04
76.20
38.17 10
CD45RA
FITC-A
CD161a
PE-A
CD3
APC-A
strain
andFITC-A
sex
differences
that should
into
Show Events
Statistical
Gates/Populations
CD3 APC-A
CD45RA
CD161a
PE-Aaccount
Tube_001:T-Cytotoxic
(CD8+)
1,201
7.58
24.04 be taken
12.04
Events/uL
% Parent % Grandparent % Total
Operator (Tube_001):
FSC-A 0Katie Belsham
Name
102
0
102
Lymphs
Statistics
Lymphs
Name
of Experiment or Worklist (Tube_001): Lymph control data 5
0Sample
50 Volume
100
200
102
0
D.
(Tube_001):
35L
Assay: RatSample
LymphsIDNo
CD45 100
2 UD
150
150
0
0
0
10
0
0
SSC-A
SSC-A
200
0
50
C.
0
4
10
2
FSC-A
10
0
Population
View
105
0
102
103 Show
104 Statistical
105
0 102
103
104
105
Gates/Populations
0
CD45RA FITC-A
CD161a PE-A
5
0
102
103 Gate Hierarchy
104
105
102
103
104
Tube_001 - Lymphs
Tube_001 - Lymphs 0
Tube_001
-10Lymphs
x1000
x1000
CD4 PE-Ax1000
CD8
FITC-A
All
250
250Events
250
200
SSC-A
250
Lymphs
2
Tube_001 - All Events
x1000
200
x1000
100
T-Cells (CD3+)
SSC-A
250 0
x1000
T-Helper (CD4+)
SSC-A
200
4
150
50
T cells
Assay: Rat Lymphs No CD45 2 UD
3
B cells 100
10
50
150
Tube_001 -- Lymphs
T-Cells (CD3+)
Tube_001
10
150
3
10
105
Population
View
SSC-A
2505
10
SSC-A
Gate Hierarchy
SSC-A
CD3 APC-A
SSC-A
mple ID: 35L
250
50
significant differences were seen between the sexes. HW
Events % Total % Parent % Grandparent
males had 6.84±2.86% NK-cells as opposed to females with
Tube_001:Lymphocytes 9,975
63.00
63.00
***
9.72±1.72%
In SD
males there were 8.00±2.02%
Tube_001:T-Cells (CD3+)
4,996
31.55 (p=0.0051).
50.09
31.55
3
10
Tube_001:T-Helper (CD4+)
3,807
24.04
76.20
38.17
in comparison
to24.04
females which,
similar to the HW strain had a
Tube_001:T-Cytotoxic (CD8+) 1,201
7.58
12.04
higher percentage of NK cells at 11.4±2.53% (p=0.035). The
2
10
post-parturition SD females showed a statistically significant
0
increase over the younger SD females with 17.87±5.17%
103
104
105
CD8
FITC-A105
(p=0.026).
104
0
102
103
104
105
Name
CD8
There were
noFITC-A
statistically significant differences in the %
T-cells (Fig.4 A.) between sexes or strains with a mean %
range of 39.02±4.31 to 46.78±9.18%. However, when
Tube_001
- T-Cells
Tube_001
- Lymphs
Name
of Experiment
or Worklist
(Tube_001):
Lymph (CD3+)
control data 5
x1000
All
Events
Statistics
250
investigated further, statistically significant differences were
5
FCS File Name (Tube_001):
4c0d188a-a16f-492a-b11a-01162eaf1d19.fcs
10
Lymphs Name of Experiment or Worklist (Tube_001): Lymph control data 5
Tube Name (Tube_001): Tube_001
seen in T-cytotoxic (Fig.4 B.) and T-helper (Fig.4 C.) cell
T-CytotoxicFCS
(CD8+)
File Name (Tube_001): 4c0d188a-a16f-492a-b11a-01162eaf1d19.fcs
200
T cells
Tube%Name
(Tube_001): Tube_001 populations which make up the total T-cell population.
Name 4
Events % Total % Parent
Grandparent
200
Show Statistical Gates/Populations
x1000
Tube Name (Tube_001): Tube_001
B cells
2
Assay: Rat Lymphs No10CD45 2 UD
NK 2
0
250
1 - All Events
x1000
3
T cells
10
10
10
View0
150Population
200
250
FSC-A
Tube_001 - Lymphs
150
0
100
CD3 APC-A
Lymphocytes
3
100
A.
All Events104
200
FSC-A
SSC-A
All Events
4
CD3 APC-A
10
CD3 APC-A
200
B.
T-Cytotoxic
Name of(CD8+)
Experiment or Worklist
Lymph control data 5
5
In (Tube_001):
the NK-cell
population (Fig.3 B.) there were no significant
10
T-Helper (CD4+)
FCS File Name (Tube_001): 4c0d188a-a16f-492a-b11a-01162eaf1d19.fcs
T-Cytotoxic
differences between the(CD8+)
strains, rather the statistically
5
10
Gate Hierarchy
CD3 APC-A
250
Gate Hierarchy
4
CD3 APC-A
FSC-A
FSC-A
CD3 APC-A
Results:
phone: +44 (0)1531 634121
For T-helper cells, statistically significant differences were seen
between strains with HW males having a higher % of T-helper
cells, 72.12±3.12%, than SD males with 68.31±2.85%
(p=0.0053) and HW females following the same trend with
74.20±4.99% compared to SD females at 67.35±2.22%. These
differences were not significant within each strain between the
sexes. There was no apparent effect of age as SD females had
63.55±5.69% T-helper cells, which was not significantly
different to the younger SD females.
Discussion and Conclusion:
Although these data sets are relatively small, there are some
interesting patterns emerging which indicate that strain, sex
and age differences are relative to cell type. Generally where
there were strain differences the sexes showed the same
trends with HW animals having lower B-cell and T-cytotoxic cell
percentages and higher T-helper cell percentages than SD
animals of the same age.
Interestingly, there are no differences seen in T-cell
percentages, however when the constituents of this population
are investigated, the relative percentages of T-helper and
T-cytotoxic cells can vary by sex or strain. Although the actual
percentage changes appear small, they are highly significant
with very little overlap between the different populations.
This data shows that there is potential for standard total
lymphocyte counts to be overly simplistic where there is
potential for effects on the immune system. Discrimination of
the relative percentages of each sub-population could indicate
an aspect of toxicity or potential mechanisms of action not
highlighted by the total lymphocyte count, which may not
change. Also, the use of both sexes in investigations which
may have effects on lymphocytes is an important part of study
design dependent upon the strain chosen.