4
CD6: New low/negative surface marker forFigure
human
FOXP3+ naturally-occurring regulatory T-cells
Lymphocytes
Carlos A. Garcia Santana M.D., Ph.D. , James W. Tung Ph.D.2, and Sergei Gulnik Ph.D.1
1
1
FIG. 4
Figure
7A
B
CD4+ cells
Life Science Research; 2Life Science Development, Beckman Coulter, Inc., Miami, FL., USA
CD4+CD25hi cells
CD6-nTreg CD127-nTreg
CD4+CD25hi
CD4+CD25hi
subsets
CD4 CD25
1. CD6
lo/-CD127lo/+
CD6lo/-CD127lo/-
CD6+CD127+
94.9%
FIG. 4
B D
A
CD4+ cells
C
CD4 CD25
FIG. 4
FIG. 4
CD6-nTreg CD127-nTreg
CD25
Figure
7A
Figure
7 CD4
+
B
B
A
CD4
cells
CD4 cells
+
Treg transcript
BioLegend
18
0
(Positive
Control)
A
18
nTreg: T-responder ratio
0
CD127-nTreg
1:1
CD4+ cells
CD4+ cells
C
1:1
C
1:2
FULL STAIN
CD25
FSC-W
FSC-W
SSC-A
SSC-A
SSC-W
SSC-W
Figure 1. Fluorescense minus one for FOXP3 and CD6 staining
CD25
SSC-A
CD6-nTreg
CD127-nTreg
CD4+ cells
CD39
CCR4
B
CTLA-4
CTLA-4
CD127-nTreg
CD39
CCR4
CCR4
CD4+CD25hi
% positive cells
% positive cells
% positive cells
% positive cells
CD127
CD127-nTreg
CD6
CD6-nTreg
1:4
% FOXP3+
% FOXP3+
cells
cells
+
% FOXP3
% FOXP3++
% FOXP3
cells cells
cells
%FOXP3
FOXP3++
%
cells
cells
% FOXP3+ cells
% FOXP3+ cells
HLA-DR
HLA-DR
HLA-DR
HLA-DR
HLA-DR
HLA-DR
HLA-DR
HLA-DR
HLA-DR
HLA-DR
HLA-DR
HLA-DR
HLA-DR
Note: arrows show
B marker
N/R
CD392.42.44.84.8 expression pathway
N/R 86.1
Note:
6.76.7 arrows show marker
I/Me
2.4 4.8 86.1
CD39
31.8%
expression pathway
20.8%
86.1 6.7
could
Dexpression
TD pathway 2.4
CD39
4.8
CD45RA
Pop2
I/Me
3D:
19.1%
CCR4
N/R
I/Me
N/R I/Me
HLA-DR
HLA-DR
Pop3
3D: 16.8%
3D: 16.8%
3D: 16.8%
GARP
Pop1
3D: 16.8%
B
Pop2
GARP
Ma/E
Pop3
Pop2
GARP
-CCR4hiHLA-DR+
lo
lo
hi
lo
Ma/E
lo
hi
hi
Pop2
lo
lo
GARP
CCR4
Differentiation?
CD39+
memory
effectors
CCR4
Terminal
Naïve/
Immature/
Mature/ Regulation blocking
Treg
3D:
19.1%
3D: 16.8%
hi ++ hi
Regulation
Treg
FOXP3
++ CCR4
FOXP3
FOXP3 CD39
FOXP3
Differentiation?
R
R
Terminal
suppression
?
-D
hi
resting
memory
effectors
suppression
?
-D HLA
CCR4
Naïve/
Mature/
CCR4
LA
Figure 10Immature/
CCR4
lo
lo
lo
H
CD25 hi Differentiation?
CD25
blocking
+ CD39+
CD25
resting
memory
effectors
+
CD39
CCR4
FOXP3hi
+
FOXP3
CD45RA
FOXP3
FOXP3 Terminal
CD45RA
CD6Naïve/
CD127
CD6 CD127
Immature/
Mature/
blocking
FOXP3
FOXP3
FOXP3
FOXP3
CD39
hi
CD39
hi
Differentiation?
CCR4
Regulation
Treg
+
CCR4
+effectors
CCR4
resting
memory
hiCCR4 hi
R
CCR4
CCR4
suppression ?
-D
lo
CD25++
lo/-
lo
CCR4lo
CD25++
lo/-lo/+ lo/CD6
CD127
CD6lo/-CD127
CD45RA
lo
FOXP3hi
CD25++
FOXP3
CD25++
CD6lo/-CD127lo/-
CD45RA+
CD4+CD25hi
CD6-Treg
-Treg
CD127
+
hi
CD45RA
CD6lo/-CD127lo/-
Explains
anergy in nTreg
cells
CD4 CD25
CD6-Treg
CD6lo/-CD127lo/CD25++
hi
CD25++
CD6 Treg
CD127-Treg
lo
CD25++
+CD39loCCR4lo
CD25++
CCR4 hi
Figure
10
CD39
CD25++
CCR4lo
lo
CD25++
CD6lo/-CD127lo/-
CD4+CD25hi
CD127-Treg
CD6-Treg
CD127-Treg
CD39lo
CCR4
lo
CD39
CD25++
lo
blocking
FOXP3lo
CD25 + CD25 +
CCR4
CD25 hi
CCR4
CD25 hi
Naïve/
resting
CCR4lo
CD39+
hi
CD25CCR4
hi
CD25++
CD6lo/-CD127lo/-
lo
hiH-L
CCR4
HLA
CCR4lo
CD45RA+
+
CD39+CD6+CD127
CD6+ +CD127+
blocking
Regulation
FOXP3+hi+ ++
Regulation
TregTreg
FOXP3lo
R
suppression ?
DAR-D
suppression
?
hi
CCR4
Regulation
Treg
+
++
CD39
suppression ?
+
-DR
hi
CD25 +
HLA CD25CD39
Regulation Treg
++
CCR4Rhi
hi
?
-D CD6+CD127+ CCR4suppression
CD39+
HLAhi
FOXP3lo
CD25++ +
CD45RA
LA
H
hi
CD25 hiCD25
FOXP3lo
++
CD25++ CD25
+
hi
lo
lo
lo
FOXP3lo
+
++
lo
lo/-
lo
CD4+CD25hi
CD6-Treg
CD127-Treg
FOXP3lo
CD6lo/-
CD25
hi +
CCR4
CD6+CD127+
hi
CD25 +
CCR4
CD25 +
hi
CCR4
+
+
CD6 CD127
Immature/
CD25
memory
CD6 CD127
+
hi
CD6+CD127
CD25
+
+
SSC-A
CD6
CD45RA
lo
hi
T
Diffe
FOXP3lo
++
-DR
HLA
CD39+
CCR4 hi
lo
+
CD6
CD6
Sorted Populations in
Sorted Populations in
supplemented
media
supplemented
media
Mature/
effectors
FOXP3hi
FOXP3lo
Mechanism
to induce
tolerance
Mechanism
to induce
tolerance
nTreg
FS INT
FSC-W
CD25
CD25
FS INT
CD25
FSC-W
CD25
SSC-A
Treg cells
CD4
CD4
CD25
SSC-A
CD4+
+
Explains anergy in nTreg cells
untouched
nTreg without
cells without
loosing
untouched
nTreg cells
loosing
lo/lymphoid
progenitors
and could
deplet
+
progenitors
and
could
deplet
In vitro suppression assay. Sorting regulatory T-suppressor
cells.
After PBMC staining,lymphoid
CD4+CD25-CD6
Depletion
of
bone
marrow
with
anti-CD6
Depletion
of bone
marrow
with anti-CD6
inflammatory/
cytotoxic
T-cells
PBMC
mL cellcells
culture media. Stained cellsinflammatory/
cells were
pelleted and resuspended at 10 × 106 perNon-Treg
cytotoxic
T-cells
PBMC
cells c
Terminal
CCR4 CCR4
Immature/
Mature/
blocking
Regulation
Treg
++
blocking
FOXP3
FOXP3
FOXP3 FOXP3
R
Differentiation?
Pop3
suppression
?
-DFOXP3
Terminal
memory
effectors
HLA
Immature/
Mature/
FOXP3lo
CD6lo/-: New insights into nTreg Explains
cell biology anergy
lo/-lo/-in nTreg cells
-CD6+cells
CD4+CD25
+CD6
Non-Treg
Figure 2. Gating strategy for 10-color
panel
analysis.
CD4+CD25
PBMC nTreg
Non-Treg cells
Note: ar
express
CD39
GARP
CCR4
CD39lo
CD127-nTreg
HLA-Dr
Thymus
Treg
selection
Thymus
Treg
selection
2.4 4.8
86.1 6.7
16.8%
3D: 16.8% 3D:Pop3
lo
+
Pop2
Pop1
Pop3
lo
FOXP3CD4 CD25
Pop3
GARP
GARP
Pop1B
TD
60.0% could
cells
3D: 16.8%
Pop2Pop3
Pop3
GARP
CCR4
CCR4
Naïve/
FOXP3
resting
Naïve/
Figure
10
CD4+CD25hi
CD6-Treg
CD127-Treg
CD6-nTreg
CD25
Lymph
Lymph
FSC-W
SSC-A
SSC-A
SSC-W
SSC-W
SSC-A
SSC-W
FS TOF
SSC-A
SS TOF
Lymph
CD25
FSC-W
SSC-A
SS TOF
SSC-A
SSC-W
FS INT
SSC-A
SS INT
FS INT
SS INT
Lymph
FS TOF
SSC-A
FSC-W
SSC-W
SSC-A
+
66.8 31.3
HLA-DR
CD45RA
CD45RA
Ma/E
hiPop3
Pop2
CD6
CD6 nTreg
nTreg
Depletion of bone marrow with anti-CD6
Identification and isolation of high
CD39
CD4 population was analyzed (Figure 2)
CTLA-4
+
Treg selection
will enrichThymus
the cell preparation
with
CD45RA
pure
FOXP3+ population
Mechanism
to induce
tolerance
Figure 3. Treg isolation by MoFlo XDP high speed cell sorting
Thymus
Treg
selection
untouched
nTreg
cells
without
loosing
CD39
CD4 PC7
Explains
anergy inIdentification
nTregMechanism
cells and isolation
Figure 3. Treg isolation by MoFlo XDP high speed cell sorting
to induce
tolerance
Depletion
of of
bone
marrow
with
anti-CD6
Staining
of high
FSC-A
Depletion
bone
marrow
with
anti-CD6
CD6
SSC-H
FSC-H
speed
cellstrategy
sorting
Figure
3. Treg isolation
by APC
MoFlo XDP high
Explains
in
nTreg cells
CD25 PE
Sorting
Gate
Identification
and isolation
of highCCR4
lymphoid
progenitors
and
could
deplet anergy
Staining
+CD25+CD6CD4
+
will
enrich
the
cell
preparation
with
Explains
anergy
in
nTreg
cells
Sorting
Gate
strategy
Accurate
evaluation
of nTreg
FSC-A
CD6 APC
SSC-H
FSC-H
population
pure
FOXP3
CD4 CD25
+ population
willinflammatory/
enrich the cell
preparation
with
Treg cells
Staining
CD25++
CD25++
pure
FOXP3
lo/- nTreg
cytotoxic
T-cells
+CD6CD25
Sorting Gate strategy CD4+CD4+
CD6-Treg
SSC-Gate
FSC-Gate
CD6
untouched
nTreg
cells
without
loosing
subset
Treg cells
CD6lo/-CD127lo/-Treg
untouched
nTreg
cells
without
loosing
lo/SSC-Gate
FSC-Gate CD4+CD25-CD6+
CD127
Thymus
Treg
selection
CCR4
CD4+
CD4
PC7
CD6
nTreg
Mechanism
to induce
tolerance
Non-Treg cells
Thymus
Treg
selection
lymphoid
progenitors
and
could
deplet
CD4 PC7 PBMC
CD4+CD25-CD6+
Mechanism
induce
tolerance
lymphoid
progenitors
and
could
deplet
Accurate
evaluation
of to
nTreg
CD25
PE
SSC-Gate
FSC-Gate Non-Treg
cells
CD4
CD6
Accurate
evaluation
of nTreg
PBMC
CD25 PE
inflammatory/
cytotoxic
T-cells
Sorted Populations in
Thymus
Treg
selection
FSC-A
CD6
APC
SSC-H
FSC-H
CD4
CD6
CD4 PC7
inflammatory/ cytotoxic T-cells
subset
Mechanism
to
induce
tolerance
media
FSC-ASortedSSC-H
Populations
in supplemented
CD6 APC
FSC-H
subset
+CD25+CD6CD4CD4-KrO
Depletion of bone marrow with anti-CD6
SS INT supplemented
FS INT
7AAD
media
+CD6
Identification
and isolation of high
CD25 PE FS INT
CD4+CD25
Treg
cells
Depletion of bone marrow with anti-CD6CD6lo/- lo/Treg cells
Identification
and isolation of high
SS INT
FS INT CD4+
7AAD
CD4-KrO
FS INT
nTreg
CD4+
will
enrich
the
cell
preparation
with
FSC-A
CD6 APC
SSC-H
FSC-H
Figure
2. Gating
strategy
for 10-color
nTreg
panel analysis.
CD6
nTreg
+
+
pure
FOXP3+ population
CD4 CD25 CD6 will enrich the cell preparation with
+
selected as 7AAD negative
cells, side and forward scatter doublets were excluded, and
Staining
Sorting Gate strategy
90.5 7.9
Pop2
+
Pop1
Pop2
+CD127
+
GARP
CD6Pop1
-B
hiHLA-DR
+
CCR4
Pop1
Thymus Treg
selection
Explains
anergy
in nTreg
cells
Explains
anergy
in nTreg
cells
Mechanism
to induce tolerance
DISCUSSION & CONCLUSION
0.5 1.4
B
CCR4
HLA-DR cells could
CD4 CD25
3D: 19.1%
Ma/E
resting
Figure
10 CD45RA+
CD127
CD6
I/Me
1.1 0.4
HLA-DR
HLA-DR
CD45RA
CD45RA
CD45RA
N/R I/Me Ma/E
FOXP3lo
1:8
C
35.8
Terminal
3D:Immature/
19.1%
3D: 16.8%
Naïve/
Mature/
Differentiation?
resting
memory
effectors
Figure
10
Figure 10
FigureA9maturation pathway
Postulated
nTreg functional
modelCCR4
B
Terminal
Pop1 blocking
Terminal
Naïve/
Immature/
Mature/
N/R
FOXP3
FOXP3 Immature/
FOXP3
FOXP3
Naïve/
Mature/
I/Me
Differentiation?
Differentiation?
resting
memory
effectors
Figure 10 memory
resting
effectors
CD4hi+CD25hi
CD4+CD25
CD6-Treg
CD6-Treg
CD127-CD127
Treg -Treg
CTLA-4
B
lo
HLA-Dr
CTLA-4
HLA-Dr
CTLA-4
Ma/E
Ma/E
CCR4
Figure 10
CTLA-4
CTLA-4
HLA-Dr
1.1
1.114.0
35.8 47.1
14.0 47.1
66.8 31.3
HLA-DR cells could
60.0%
N/R I/Me
Figure 10
CTLA-4
% positive cells
CD45RA
CD45RA
CD45RA
CD127
CD127-nTreg
90.5 7.9
86.1 6.7
CCR4 HLA-DR
cells
could
CD39
expression pathway
3D: 19.1%
3D: 19.1%
nTreg: T-responder ratio
FOXP3
HLA-Dr
FOXP3
HLA-Dr
CD127-nTreg
CD6
CCR4
CCR4CCR4
CD4+CD25hi
FOXP3
CD127-nTreg
% positive cells
% positive cells
GARP
CCR4
SSC-H
FSC-HCD4-KrO
FS INT
SS INTFS INT
7AAD
SSC-Gate
SSC-Gate FSC-Gate
FSC-Gate
CD4+CD25+CD6SS for
INT 10-color FS
INT
7AAD
CD4-KrO
FS INT Figure
Treg cells
CD4
PC7
Gating
strategy
nTreg
panel analysis.
CD42.
PC7
CD4+
CD25
PE
Lymph
Figure 2. Gating
CD6 Lymph nTreg panel analysis. CD4+CD25-CD6+
CD25strategy
PECD4 for 10-color
CD4
Non-Treg cells
CD6
SSC-H
FSC-H
PBMC FSC-A
FSC-A
CD6APC
APC
SSC-H
FSC-H
+CD25+CD6CD4
CD4
CD6
+Sorted
+CD6CD4
CD25
in
Treg
cellsPopulations
Treg cells
CD4+
supplemented
media
CD4+
-CD6+
CD4+CD25
-CD6+
CD4+CD25
SSC-Gate
FSC-Gate
Non-Treg
cells
PBMC
Non-Treg cells
PBMC
CD4 PC7
CD4
CD6
SSC-Gate
FSC-Gate
CD4
CD6 Sorted Populations in
Sorted Populations
CD25 PE
Lymph
supplemented
media in
supplemented media
Figure 2. Gating strategy for 10-color nTreg panel analysis.
Figure 3. Treg isolation by MoFlo XDP high speed cell sorting
Lymphocytes
were
gatedStaining
based
on forward
and
side light
scatter,
viable
cells were
XDP
high
speed
cell
sorting
Figure
3. Treg
isolation
byminus
MoFlo
Figure
1. Fluorescense
one
for
FOXP3
and
CD6
staining
Sorting
Gate
strategy
CD127
CD127-nTreg
GARP
FSC-A
SSCD4-KrO
INT
CD6-nTreg
CD6-nTreg
FS INT
FS INT
FSC-W
FS TOF
SSC-W
SSC-A
CD6 APC
7AAD
FS INT
FS TOF
SS TOF
SS
FSTOF
INT
FS INT
FS TOF
FS INT
FS INT
FOXP3
FS TOF
FOXP3
SS TOF
SS INT
SS INT
SS TOF
FS INT
FS INT
FOXP3
FS INT
CD4
Lymph
CD4+CD25hi
CD39
GARP
CD45RA
GARP
CD45RA
CD45RA
FS INT
FS TOF
FS INT
SS INT
Figure 6
CD39
CD39
A
FMOXDP high speed cell sorting
Figure 3. Treg isolation by MoFlo
FMO
SS INT
INT
7AAD
Staining
CD4-KrO
FS INT
SortingFSGate
strategy
CCR4
A. FOXP3 FMOFigure 2. Gating
B. CD6
FMO
strategy for
nTreg panel analysis.
+ cells
CD410-color
+ cells
CD4
SS
INT
FS
INT
7AAD
CD4-KrO
SSC-Gate
FSC-Gate
FS INT
XDP
high
speed
cell
sorting
Figure
bybyMoFlo
XDP
high
speed
cell
sorting
Figure3.3.Treg
Tregisolation
isolation
MoFlo
FULL STAIN
CD4
PC7panel
FMO
Figure 2. Gating
for
nTreg
analysis.
FULLstrategy
STAIN
Staining
FMO10-color
Staining
Sorting
Gate
CD6
Sorting
Gatestrategy
strategy
CD25 PE
CD4
FULL STAIN
FOXP3
SS INT SS INT
SS TOF
B.Gating
CD6
FMOfor10-color
Figure
strategy
nTreg
panel
analysis.
Figure2.2.Gating
strategyfor
10-color
nTreg
panel
analysis.
CD4+ cells
FULL STAIN
B
CD4-KrO
CD4 2. Gating strategy for 10-color nTreg panel analysis.
Figure
CD6FOXP3 and CD6 staining
Figure
1. Fluorescense minus
CD4 one for
CD4
CD4+ cells
CD6-nTreg
B
+
CD127
CD39CD39
CD6-nTreg
CD4+CD25hi
INTINT
INT
7AAD
SS
FS INT staining
7AAD
CD4-KrO
FSFS
INT
CD4-KrO
INT
Figure 1. Fluorescense
minus
one for SS
FOXP3
andFSCD6
A. FOXP3 FMO
CD4+CD25hi
CD25
FS INT
FS INT
FS TOF
FS TOF
SS TOF
SS TOF
FS INT
FS INT
FS INT
CD4+CD25hi
GARP
GARP
FMO
SS INT
7AADstaining
Figure1.
1.Fluorescense
Fluorescenseminus
minusone
onefor
forFOXP3
FOXP3
andCD6
CD6
staining
FS INT and
Figure
CD6
CD4
SS INT
SS INT
FS INT
FS TOF
SS TOF
CD6
FULL STAIN
CD6 CD39
CD127
Ma/E
Ma/E
1.4
expression pathway
Pop1
CCR4
HLA-DR
cellsCD39could
hi
+
N/R I/Me
1:8
hi
CD6-nTreg
CD6
CD4+ cells
FOXP3
FS INT
FOXP3
SS INT
FOXP3
FOXP3
CD6
CD6
FMO
B
B. CD6 FMO
FULL STAIN
A. FOXP3 FMO
B. CD6 FMO
B
FMO
FMO
+ cells
CD4+ cells
Figure 1. Fluorescense
minus
one for CD4
FOXP3
and CD6 stainingB
FULL
STAIN
CD4
CD4
CD4 flow cytometry panels CD4
Table 2. Multicolor
CD6
CD45RA
FOXP3
FOXP3
FOXP3
FOXP3
FOXP3
FOXP3
A. FOXP3 FMO
1:4
FOXP3
hi
hi
TD
TD
3D: 19.1%
FigureA9Ma/E
Ma/E
(n=5)
+
GARP
A
CD25
FMO
CD6
CD6
CD4
CD4
Table
Multicolor
flow cytometry
A. 2.
FOXP3
FMO
B.panels
CD6
FMO
Figure
Fluorescenseminus
minus
one
FOXP3
and
CD6
staining
Figure
1.1.Fluorescense
one
forfor
FOXP3
and
CD6
staining
+
+
CD45RA
FULL STAIN
CD25
+
D
TD
TD
0.5
0.5
66.81.431.3
86.1 6.7
60.0%
-CCR4hiHLA-DR
+ cells could
2.4 4.8
hi TD + cells
+ Note:
arrows show
86.1 marker
6.7
CCR4hiHLA-DR
could
FigureA9 CCR4
B
FigureA9
3D: 19.1%
19.1%
N/R
3D:
+
GARP
CD25
FOXP3
FOXP3
B. CD6 FMO
+
TD
-CCR4
N/R I/Me
A
FOXP3
+
CD25
GARP
FOXP3
FOXP3
FOXP3
FOXP3
A. FOXP3 FMO
+
FULL STAIN
FULL STAIN
Figure 9
FigureA9 A
Figure 9
1:8
70
nTreg:
T-responder ratio
FOXP3
35
the percentage of cells above
CD6-nTreg
CD127-nTreg
CD6-nTreg
CD6
FMO
CD127-Treg
Figure 6
CD127-nTreg
B.B.
CD6
FMO
CD6-nTreg
CD127-nTreg
HLA-Dr
CD4 cells for each
C
the
negative
Figure 6 B CD4 CD25
CD4region
cells
18
(Positive
CD4 cells
A
FOXP3
C
CD6
CD127
FMO
CD6
CD127
CD6
FMO
CD4
CD4 cells Control)
mAb; alternatively,
results
were
A
FOXP30
-nTreg
CD6
6
CD127 nTreg
A. FOXP3 FMO 1. Fluorescense
B. CD6 FMO
minus one
for FOXP3 and CD6 staining Figure B
C
-nTregCD6-nTreg
CD25
CD6-nTregCD127-nTreg
CD127-nTreg
HLA-Dr
expressed as the mean of fluorescenceFigure
intensity (MFI) of discrete
CD6
CD4 populations.
cells
1:1
1:2
CD4 CD4
CD25
HLA-Dr
B
CD127-nTreg
CD4 cells
FOXP3FMO
FMO
A.A.FOXP3
DD
I/Me
N/R
1.1 0.4
1.1 0.4
90.5
7.9
2.4 4.8
CD6nTreg
hi
CD39
Note: arrows
show
marker
HLA-DRB+ cells could represent
a mature
effector
CD45RA
FigureCCR4
A9
CD39
expression pathway
Pop1
N/R I/Me
Figure
C
B
Note: arrows D
show marker
stageA9of the nTreg
population
TD
hi
+
(n=9)
Figure
6
CD6
No differences
in-nTreg
the expression
markers in
CD127-nTreg of nTreg-associated
R² = 0.9927
CD6-nTreg
C
CD127-nTregC
53
CD4+ cells
(n=9)
A
CD4+ cells and CD127-Treg
CD6-Treg
populations
CD6
CD127
+
CD4+CD25+
lo/CD127
1.1
35.847.1
14.0
Ma/E
31.3
14.0 47.1
1.1 35.8
Note: arrows
show marker
Ma/E
hi
+ CD39
show marker
D 86.1
TDNote: arrows
TD
pathway
CD6lo/-CD127lo/- CD39
6.7expression
arrows
show 33.8%
marker
CD39Note:
expression
pathway
60.0%
C
(n=5)
1:8
60.0%
60.0%
60.0%
CCR4 CCR4
1:4
CD39
CD39
lo/-
CD127
Ma/E
CD6
1.1lo/-35.8
CD39
-CCR4
hiHLA-DR
+
hi
2.4 4.8 cells
CD4+CD25
1:8
1:2
nTreg
14.0 47.1
nTreg
0.5
1.431.3
Ma/E
66.8
I/Me
66.8
TD
B
TD
D
33.8%
CD39
TD
TD
CD4+CD25hi CD6+CD127+
(n=9)
30.0000
N/R
90.5 7.9
0.4 D
nTreg
66.8 31.3
I/Me
CD1270.5 1.4
nTreg
1.1 0.4
7.9 I/Me
N/R 90.5
Figure
33.8%
A 8
D Ma/E
TD
lo/-CD127lo/CD6
+CD25
+CD25
hihi
CD4
CD4
CD6 CD127
CD4+CD25hi
CD6+CD127+
R² = 0.9914
35
CD6-Treg
0
CD127-Treg
15.0000
1:4 C
1:2 1:1
1:41:2
1:8
18
+ cells
(Positive
CD41:1
0
Control)
nTreg: T-responder
ratio
nTreg: T-responder
ratio
CD6-nTreg
35.8
14.0 47.1
lo/-
CD4+CD25+ CD6CD127CD6lo/lo/1.1 35.8
nTreg
lo/Ma/E nTreg CD127
N/R
90.5 7.9
+CD25hi
B1.1
CD39
Ma/E
60.0%
CD6+CD127+
1:4
1:8
nTreg: T-responder ratio
60.0000
B
33.8%
31.8% 20.8%
TD
+
C +CD25hi
CD4
0 0
% =Suppression
1:4 1:1
1:8
R²
0.9927
1:2
1:4
1:8
1:1
1:2
1:4
1:8
R² 1:1
= 0.9927
1:2
1:4
1:8(n=9)
53
hi
Ma/E
CD6lo/-
66.8 - 31.3
CD127
CD6lo/-
+
0.5+CD25
CD4
I/Me1.4
1.1 0.4
lo/-
N/RCD61.1
0.4
CD39
I/Me
lo/CD6lo/-CD127
CD4+CD25hi
C
(n=9)
(n=9)
1:8(n=9)
1:4
1:4
1:2
CD4
Ma/E
CD45RA
CD127-Treg
% FOXP3+ cells
% FOXP3+ cells
% FOXP3+ cells
1:8
I/Me
B
60.0%
N/R Ma/E
I/Me
hi
CC
CD4+CD6
CD25
CD127
C
1:4(n=5) 1:8
1:4
lo/-
CD45RA
53
0
CD6-
*
CD127-
lo/nTreg - CD127
CD127
0.5
1.4 CD6 1.1
nTreg CD127lo/-
CD127
nTreg
- CD6lo/- nTreg
CD127
CD4+CD25+ CD6
CD6lo/lo/CD4+CD25+ CD6nTregCD127
CD127
nTregCD127
lo/nTreg
nTreg
33.8%
31.8%
N/R
20.8%
CD6+CD127+
(n=9)
nTreg: T-responder
ratio
70
nTreg: T-responder
ratio
1:1
1:2
1:4
1:8
35
nTreg: T-responder ratio
35 CD127-Treg
45.0000 R² = 0.9927
(Positive
Control)
Figure
6
Figure 6
FOXP3 FMO
B. CD6 FMO
BDA.
PharMingen
CD4 cells
CD4 cellsThe results were expressed
BD PharMingen
as
A
R&D Systems
FMO
FOXP3-Pacific
Blue signal
206D
Inhibitory
0
Control)
1:2
1:1
+
D
I/Me
CD6-
CD4+CD25+
CD4 CD25
lo/-
20.8%
I/Me
+CD127
+hi
++
CD6
CD4+CD25
R² = 0.9914
R² = 0.9927
1:2
8
+
(n=5)
R²
R² ==0.9927
0.9927
1:1
18
(Positive
70
Control)
18
(Positive
Control)
70
1:1
1:2
53
Figure 6
Figure 6
A A
0
53
CD127
CD4+CD25hi
CD25
+CD127
+
CD6CD4
2.
CD6+CD127+
FOXP3
CD45RA
BNI3
53
45.0000
Control)
53
0
15.0000
22.6%
nTreg
CD6
+
CD4+CD25
1.1
0.4 p<0.009
nTreg
lo/-
31.8% 20.8%
CD4+CD25hi
(n=5)
1:2
1:4
1:81:8
1:1
1:2
1:4
R² 1:1
= 0.9914
(n=9)
(n=5)
35
CD6-Treg 70
R²R²
==
0.9927
70
0.9914
35 30.0000
(n=5)
15.0000
18
(Positive
0
CD25
CTLA-4-PCy5 (CD152)
CD6
CD127
+CD25
CD4Figure
CAhi
CD6lo/-CD127lo/-
CD127-Treg
60.0000
35
30.0000
(Positive 18
0 CD127-Treg
35
CD6-Treg
CD127-Treg
35 15.0000
18
1:1
1:2
Control)
FigureCD127-Treg
5 (Positive
BCI
1:1
2.
22.6%
0.5 1.4
I/Me
Ma/E
31.8%
+CD25lo/hi
lo/-CD127
CD4
7.9 0.4
CD6
N/R 20.8%
66.8 0.5
31.31.4
14.0 1.1
47.1 35.8
Ma/E
33.8% 90.5 1.1
I/Me
CD6lo/CD127lo/-31.8%
2.4 4.8
+CD25
hi
+CD25
hi
20.8%Ma/E 33.8%
CD4
90.5 7.9
66.8 31.3
14.0 47.1
CD4
CD39
Figure
8
lo/TD
CD6lo/ACD127
B
N/R
+
+
33.8%
86.1 6.7I/Me
CD45RA
CD127-Treg
CD6-Treg
53
0
hi
Figure
CA 8 + hi N/R
CD4 CD25
= 0.9914
1:2
hi
+
Figure
A 8 +
N/R
CD4 CD25hi
Figure
N/R
A 8 CD6lo/-CD127lo/- 31.8%
30.0000
70
45.0000
CD6+CD127+
FOXP3
90.5+ 7.9+
CD127CD6
CD4 CD25
N/RCD6-I/MenTreg
CD127
Figure
A 8 CD4 CD25nTreg
B
CD127
nTreg
nTreg
31.8%
20.8%
+
0
R²
= 0.9927
45.0000
% Suppression
53 60.0000
CD45-PE A1
J.33
Pan Leukocyte
BCI
CD39-APC
Ecto-nucleotidase
BioLegend
CD45-PE
J.33
Pan Leukocyte
BCI
R
BCI
R34.34
IL-7J.33
Pan Leukocyte
BCI
CD45-ECD
J.33
Pan Leukocyte CD127-PE
BCI
FOXP3-Pacific
BlueCD45-ECD
206D
Treg transcript
BioLegend
R
BCI
CD127-APC-AF700
R34.34
IL-7J.33
CD45-PC5
Pan Leukocyte
BCI
CD45-PC5
J.33
Pan Leukocyte
BCI
HLA-Dr-PacificBCI
Blue
Immu-357
Activation
BCI
CD45-PC7
J.33
Pan Leukocyte
CD45-PC7
J.33
Pan Leukocyte
BCI
J.33
Pan Leukocyte
BCI
CD45-APC
J.33
Pan Leukocyte CD45-FITCBCI
CD45-APC
J.33
Pan Leukocyte
BCI
CD45-PE
J.33 (CD152)
Pan Leukocyte
BCI
CTLA-4-PCy5 (CD152)
BNI3 SFCI12T4D11
Inhibitory signal
BD
CTLA-4-PCy5
BNI3
Inhibitory
signal BD PharMingen
CD4-PC7
MHCII PharMingen
R
BCI
CD45-ECD
J.33
Pan Leukocyte
BCI
GARP-PE
7B11
Activation
BD PharMingen GARP-PE
7B11
Activation
BD PharMingen
CD4-Krome
Orange
13B8.2
MHCII
R
BCI
CD45-PC5
Pan Leukocyte
BCI
CCR4-PE (CD194)
205410
Homing R
R&D Systems J.33
CCR4-PE (CD194)
205410
Homing R
R&D Systems
CD6-APC
6D3
TCR Costimulation
BCI
CD39-APC
A1
Ecto-nucleotidase
BioLegend
CD45-PC7
J.33
Pan Leukocyte
BCI
CD39-APC
A1
Ecto-nucleotidase
BioLegend
FOXP3-PacificCD6-FITC
Blue
206D
Treg transcript
BioLegend
CD45-APC
J.33
Pan Leukocyte
BCI
6D3
TCR Costimulation
BCI
CTLA-4-PCy5
BNI3
Inhibitory signal
BD PharMingen
CD8-APC
B9.11
MHC-I R(CD152)
BCI
+
GARP-PE
7B11
Activation
BD PharMingen
7B11
Activation
CD25-PE GARP-PE
B1.49.9
IL-2R,
activation
BCI
CCR4-PE (CD194)
205410
Homing R
R&D Systems
CD25-APC
B1.49.9
IL-2R,
activation
BCI
Table 2.
Multicolor
cytometry
panels
CCR4-PEflow
(CD194)
205410
Homing
R
CD39-APC
A1
Ecto-nucleotidase
BioLegend
FULL STAIN
FMO
Table
2.
Multicolor
flow
cytometry
CD25-PC7
B1.49.9
IL-2R,
activation
BCI
FOXP3-Pacific
Blue
206D
Treg transcript
BioLegend panels
CD39-APC
A1
Ecto-nucleotidase
BioLegend
CD45RA-ECD
2H4
Naïve/memory
BCI
FOXP3-Pacific 2H4
Blue
206D
Treg
transcript
BioLegend
CD45RA-AF750
Naïve/memory
BCI
+ cells
CD4
+
CD62L-ECD
DREG56
LN Homing R
BCI
CD4 cells
Table 2. Multicolor flow cytometry panels
CD127-PE
R34.34
IL-7 R
BCI
FULL STAIN
FMO
FULL STAIN
BCI
CD127-APC-AF700
R34.34
IL-7 R
CD4FMO
HLA-Dr-Pacific Blue
Immu-357
Activation
BCI
CD45-FITC
J.33
Pan Leukocyte
BCI
CD4+ cells
CD45-PE
J.33
Pan Leukocyte
BCI
CD45-ECD
J.33
Pan Leukocyte
BCI
+
CD45-PC5
J.33 + cells
Pan Leukocyte
BCI FULL STAIN CD4 cells
FMO
CD4
CD4
CD4
CD45-PC7
J.33
Pan Leukocyte
BCI
FULL STAIN
CD45-APC
J.33
Pan Leukocyte
BCI
+ cells
FMO
CD4
+ cells
FULL
STAIN
CD4
CTLA-4-PCy5 (CD152)
BNI3
Inhibitory
FMO signal BD PharMingen
GARP-PE
7B11
Activation
BD PharMingen
FULL STAIN
FMO
CCR4-PE (CD194) FULL STAIN
205410
Homing
R&D Systems
FMO R
CD4+ cells
CD39-APC
A1
Ecto-nucleotidase
BioLegend
CD4
CD4
FOXP3-Pacific Blue
206D
Treg transcript
BioLegend
FULL STAIN
15.0000
60.0000
15.0000
% Suppression
0
31.8% 20.8%
CD6CD127-
CD6
CD6 CD127
FOXP3
D
D
N/R
B
CD6lo/-
**
CD45RA
LN Homing R
Figure 5
70
CD127
94.9%
CD45RA
DREG56
70
1:8
1:1
% Suppression
CD127-
hi
CD6 CD127
33.8%compartments defined by CD45RA/
nTreg
functional maturation
Figure
8
I/Me
CCR4
analysis
Ma/E
Abivariate
B
N/R
CD6CD127CD6
CD6CD127CD6
15.0000
CD6-Treg
CD6-Treg
CD4+CD25hi
CD6
94.9%
lo/- lo/p<0.009
CD6CD127CD6
CD127
nTreg
p<0.009
N/RnTreg I/Me
CD127lo/nTreg
nTreg
CD45RA
CD62L-ECD
30.0000
CD6-Treg
30.0000
Figure 5
C
CD6-
CD4+CD25hi
+
FOXP3 CD6lo/-CD127lo/-
D
*
CD6lo/CD4+CD25hi
CD127lo/nTreg
nTreg
CD6CD127CD6lo/+CD25
+CD25hi
hi CD6CD4
CD127CD6lo/- lo/CD4
CD4+CD25hi
CD127
nTreg nTreg
nTreg CD127
lo/-Ma/E
nTreg
lo/lo/-
(n=5) R²
45.0000
% Suppression
% Suppression
1:1
Source
FOXP3
D
* *
C
CD45RA
Function
CD4 CD25
hi
CD4+CD25hi
CD4+CD25
+
+
subsets
1. 2.
2. CD6+CD127+
subsets
lo/lo/-
CD6
CD127
lo/-+CD25hi
CD4
22.6% CD6lo/CD127
DFOXP3
D
p<0.009
p<0.009
p<0.009
D
+
CD6+CD25
CD6+CD127+
lo/- hi
FOXP3
CD6CD127- CD4
CD6CD127
+
+
1. CD127
+CD25
hi
CD4+CD25hi
22.6% CD4 CD25
lo/-CD127
lo/lo/2.
nTregCD6
+ nTreg
++
1. CD4
94.9% lo/- 22.6%
lo/lo/- CD6 CD127
+
lo/-
lo/CD12794.9%
* *
CD45RA
CD45RA
Clone
94.9%
CD127nTreg
CD4+CD25hi cells
hi
+
CD6+CD127+
22.6%
CD6lo/-CD127lo/- FOXP3
CD6+CD127+
CD6lo/-
+
+
+
+
% positive cells
AntibodyConjugate
lo/-
lo/-
CD127 nTreg
CD6lo/-CD127lo/-
p<0.009
p<0.009
C
Figure
A 8
% positive cells
Source
CD25
Function
CD6nTreg
CD4+CD25hi
C
CD45RA
Flow cytometry analysis was
performed using a Gallios*
flow cytometer (Beckman
Coulter) equipped with 405nm,
488nm, and 633nm lasers.
Autofluorescence, isotype and
FMO controls were used to
establish negative and positive
fluorescent events (Figure1A,
B), Compensation controls were
performed using single tube
Table 1. Conjugated monoclonal antibodies
Table
2. Multicolor flow BCI
cytometry panels staining with antibodies conjuCD45-PE
J.33
Pan
Leukocyte
Table
2. Multicolor flow cytometry
panels
AntibodyCD45-ECD
J.33
Pan
Leukocyte
BCI
Clone
Function
Source
gated with the fluorochromes
Table 2.Conjugate
Multicolor
flow cytometry panels
CD45-PC5
J.33
Pan Leukocyte
BCI
Table 2. Multicolor flow cytometry panels
CD45-PC7
J.33
Pan Leukocyte
BCI
used in our staining protocols.
CD45-APC
J.33
Pan Leukocyte
BCI
Clone
Table 1. Conjugated monoclonal antibodies
lo/-
+
94.9%
C C
60.0000
CD25
AntibodyConjugate
CD4-PC7
SFCI12T4D11
MHC- II R
BCI
CD4-PC7
SFCI12T4D11
MHC- II R
BCI
CD4-Krome Orange
13B8.2
MHC- II R
BCI
CD4-Krome Orange
13B8.2
MHC- II R
BCI
CD6-APC
6D3
TCR Costimulation
BCI
Table 1. Conjugated
monoclonal antibodies
CD6-APC
6D3
TCR Costimulation
BCI
CD6-FITC
6D3
TCR Costimulation
BCI
1. Conjugated
Table monoclonal
1.AntibodyConjugatedantibodies
monoclonal antibodies
AntibodyCD8-APC
B9.11
MHC-ISource
R
BCI
CD6-FITC
6D3
TCR Costimulation TableBCI
Clone
Function
Conjugate
CD25-PE
B1.49.9
IL-2R, activation
BCI
CloneAntibodyFunction
Source
AntibodyCD8-APC
B9.11
MHC-I
R
BCI
CD25-APC
B1.49.9
activation
BCI
CD4-PC7
SFCI12T4D11 Function
MHCII R IL-2R,Source
BCI
Clone
Conjugate
Clone
Function
Source
Conjugate
Conjugate
CD25-PE
B1.49.9
IL-2R, activation
BCI
B1.49.9
BCI
CD4-Krome OrangeCD25-PC7
13B8.2
MHC- II R IL-2R, activation
BCI
CD4-PC7
SFCI12T4D11
MHCII R
BCI BCI
CD45RA-ECD
2H4
Naïve/memory
BCI
CD4-PC7 IL-2R, activation
SFCI12T4D11
MHCII
R
BCI
CD6-APC
TCR
Costimulation
CD4-PC7
SFCI12T4D11
MHC-6D3
II R
BCI
CD25-APC
B1.49.9
BCI
CD4-Krome Orange CD45RA-AF750
13B8.2
MHCII R
BCI
2H4
Naïve/memory
BCI
CD6-FITC
TCR
Costimulation
BCI
CD4-Krome
13B8.2
MHC-6D3
II R
BCI
CD25-PC7
B1.49.9 Orange
IL-2R, activation 13B8.2
BCI Orange MHCCD6-APC
6D3
TCR
Costimulation
BCI
CD4-Krome
II
R
BCI
CD62L-ECD
DREG56
R
BCI
CD8-APC
B9.11
MHC-I
CD6-APC
6D3
TCR
Costimulation
BCI R LN HomingBCI
CD6-FITC
6D3
TCR Costimulation
R BCI
BCI
CD127-PE
R34.34activation IL-7 BCI
CD45RA-ECD
2H4
Naïve/memory
BCI
CD25-PE
B1.49.9
IL-2R,
6D3
TCR Costimulation
BCI
Table
Conjugated
monoclonal
antibodies
CD6-APC
6D3 CD6-FITC
TCR1. Costimulation
BCI
CD8-APC
B9.11
MHC-I
R
R BCI
BCI
R34.34activation
IL-7 BCI
CD25-APC CD127-APC-AF700
B1.49.9
IL-2R,
CD45RA-AF750
2H4
Naïve/memory
BCI
CD8-APC
B9.11
MHC-I
R
BCI
CD25-PE HLA-Dr-Pacific
B1.49.9Blue IL-2R,
activation
BCI
Immu-357
BCI
CD25-PC7
B1.49.9
IL-2R, activation ActivationBCI
AntibodyCD6-FITC LN
6D3 BCI
Costimulation
BCI
CD25-APC
B1.49.9
activation
BCI
CD25-PETCR
B1.49.9
IL-2R,
activation IL-2R,
BCI
CD62L-ECD
DREG56
Homing R
CD45-FITC
J.33
Pan
Leukocyte
BCI
Table
1. Conjugated monoclonal
antibodies
CD45RA-ECD Clone 2H4
Naïve/memory Source BCI
Function
CD25-PC7
B1.49.9
IL-2R,J.33
activation
BCI
CD25-APC Conjugate
B1.49.9 R
IL-2R,
activation
BCI
CD45-PE
Pan Leukocyte
BCI
R
BCI
CD127-PE
R34.34
IL-7
CD45RA-AF750
2H4
Naïve/memory
BCI
CD8-APC
B9.11
MHC-I
BCI
CD45RA-ECD
2H4
Naïve/memory
BCI
AntibodyCD45-ECD
Leukocyte
BCI
CD25-PC7
B1.49.9 SFCI12T4D11
IL-2R,
activationMHCBCI RPanBCI
CD4-PC7
IIJ.33
RHoming
CD62L-ECD
DREG56
LN
BCI
Clone
Function
Source
CD127-APC-AF700
R34.34
IL-7 R
BCI
CD45RA-AF750
2H4
Naïve/memory
BCI
CD45-PC5
Leukocyte
BCI
Conjugate
CD45RA-ECD
2H4
Naïve/memory
BCIR PanBCI
CD25-PE
B1.49.9
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activation
BCI
CD127-PE
R34.34 MHCBCI
CD4-Krome
Orange
13B8.2
IIJ.33
RIL-7
CD62L-ECD
DREG56
LN Homing
R
BCI
1. Conjugated
monoclonal
HLA-Dr-Pacific
Immu-357
Activation
BCI
CD45-PC7
J.33
Leukocyte
BCI
CD4-PC7Blue
SFCI12T4D11
MHC- IITable
R
BCI
CD45RA-AF750
2H4 antibodies
Naïve/memory
BCI
R PanBCI
BCI
CD127-APC-AF700
R34.34
IL-7
CD6-APC
6D3R34.34
TCR Costimulation
R
BCI
CD127-PE
IL-7
J.33
Pan Leukocyte
BCI
CD25-APC
IL-2R,
activation
BCI
CD4-Krome
Orange
13B8.2
MHC- II R B1.49.9
BCI
HLA-Dr-Pacific
BlueCD45-APC
Immu-357
Activation
BCI
CD45-FITC
J.33
Pan Leukocyte
BCI
CD62L-ECD
DREG56
LNR34.34
Homing
R Costimulation
BCI
R
CD127-APC-AF700
IL-7
AntibodyCD6-FITC
6D3
TCR
BCI BCI
CTLA-4-PCy5
(CD152)
BNI3
Inhibitory
signal
BD PharMingen
CD6-APC
6D3
TCR
Costimulation
BCI
CD45-FITC
J.33
Pan
Leukocyte
BCI
Clone
Function
Source
R
BCI
CD127-PE
R34.34
IL-7
CD45-PE
J.33
Pan Leukocyte
BCI
HLA-Dr-Pacific
Blue
Immu-357
Activation
BCI
CD25-PC7
B1.49.9
IL-2R,
activation
BCI
CD8-APC
B9.11
MHC-I
R Leukocyte Activation
BCI
GARP-PE
7B11
BD PharMingen
ConjugateBCI
CD6-FITC
6D3
TCR Costimulation
CD45-PE
J.33
Pan
CD127-APC-AF700
R34.34
IL-7
R IL-2R,Pan
BCI
CD45-FITC
J.33
Leukocyte
BCIRBCI
CCR4-PE
(CD194)
205410
Homing
R&D
Systems
CD45-ECD
J.33
Pan
Leukocyte
BCI
CD25-PE
B1.49.9
activation
BCI
CD4-PC7 BCI
SFCI12T4D11
BCI
CD45-ECD MHC- II R
Pan
Leukocyte
CD8-APC
B9.11
MHC-I R
CD45RA-ECD
2H4
Naïve/memory
BCI
CD45-PE
J.33J.33
Pan
Leukocyte
BCI BCI
HLA-Dr-Pacific Blue
Immu-357
Activation
CD39-APC
A1 BCI Ecto-nucleotidase
BioLegend
CD25-APC
B1.49.9
IL-2R,
activation
BCI
CD45-PC5
J.33
Pan
Leukocyte
BCI
CD45-PC5
J.33
Pan
Leukocyte
BCI
CD4-Krome Orange
13B8.2
MHCII
R
BCI
CD25-PE
B1.49.9
IL-2R,
activation
BCI
CD45-ECD
J.33 Blue
Pan 206D
Leukocyte
BCI
CD45-FITC
J.33
Pan Leukocyte
BCI
FOXP3-Pacific
Treg transcript
BioLegend
CD45-PC7
Pan
Leukocyte BCI BCI BCI
CD25-PC7
B1.49.9J.33J.33 IL-2R,Pan
activation
CD45RA-AF750
Naïve/memory
BCI
CD25-APC
B1.49.9
IL-2R,
activation 2H4
BCI
CD6-APC
6D3CD45-PC5
TCR Costimulation
BCI
Leukocyte
CD45-PC7
J.33
Pan
Leukocyte
BCI
CD45-PE
J.33
Pan Leukocyte
BCI
CD45-APC
Pan
Leukocyte
CD45RA-ECD
2H4 J.33J.33 Naïve/memory
BCI BCI BCI
CD25-PC7
B1.49.9
IL-2R, activation CD6-FITCBCI
PanBCI
Leukocyte
6D3CD45-PC7
TCR Costimulation
CD45-ECD LN
J.33
Pan
Leukocyte
BCI
CD45-APC
J.33
Pan
Leukocyte
BCI
CD62L-ECD
DREG56
Homing
R
BCI
CTLA-4-PCy5
BNI3 Naïve/memory
Inhibitory
signal BCI
BDBCI
PharMingen
CD45RA-ECD
2H4
Naïve/memory CD8-APC BCI
CD45-APC (CD152)
Pan
Leukocyte
CD45RA-AF750
2H4RJ.33
B9.11
MHC-I
BCI
CD45-PC5
J.33(CD152) Pan BNI3
Leukocyte
BCI
GARP-PE
7B11
Activation
BD PharMingen
CTLA-4-PCy5
(CD152)
BNI3
Inhibitory
signal R34.34
BD PharMingen
CTLA-4-PCy5
BDBCI
PharMingen
CD45RA-AF750
2H4
Naïve/memory
BCI
α IL-2R,
CD62L-ECD
DREG56
LN Inhibitory
Homing
CD25-PE
B1.49.9
activation
BCI Rsignal
CD127-PE
IL-7
R Table
BCI
2.
Multicolor
flow
panels
CD45-PC7
J.33 (CD194)
Pan 7B11
Leukocyte
BCI
CCR4-PE
205410
Homing
R cytometry
R&D Systems
GARP-PE
Activation
BD PharMingen
CD62L-ECD
DREG56
LN Homing R CD25-APC
BCI
GARP-PE
7B11
Activation
BD PharMingen
CD127-PE
R34.34
IL-7
R
BCI
B1.49.9
IL-2R, activation
BCI
CD39-APC
A1
Ecto-nucleotidase
CD45-APC
J.33
Pan205410
Leukocyte
α
CCR4-PE
(CD194)
HomingBCI
R
R&D BioLegend
Systems
BCI
CD127-PE
R34.34
IL-7 R
R34.34
IL-7
R Blueactivation
CD25-PC7
B1.49.9
IL-2R,
BCI
RPharMingen
CD127-APC-AF700
R34.34
IL-7BCI
CCR4-PE
(CD194) CD127-APC-AF700
205410
Homing
R
R&D
Systems
Treg
transcript BCI
BioLegend
CD39-APC
A1206D
BioLegend
CTLA-4-PCy5
BNI3
Inhibitory
signalEcto-nucleotidase
BD
BCI (CD152) FOXP3-Pacific
CD127-APC-AF700
R34.34
IL-7 R
CD45RA-ECD
2H4
Naïve/memory
BCI
HLA-Dr-Pacific
Blue
Immu-357
Activation
BCI
FOXP3-Pacific
Blue
206D
Treg
transcript
BioLegend
CD39-APC
A1
Ecto-nucleotidase
BioLegend
HLA-Dr-Pacific
Blue
Immu-357
Activation
BCI
GARP-PE
7B11
Activation
BD PharMingen
HLA-Dr-Pacific Blue
Immu-357
Activation
BCI
CD45RA-AF750
2H4
Naïve/memory
BCI
J.33
CCR4-PE
205410
Homing RPan Leukocyte
R&D Systems BCI
CD45-FITC
J.33
Pantranscript
Leukocyte
BCI (CD194)CD45-FITC
FOXP3-Pacific
Blue
206D
Treg
BioLegend
CD45-FITC
J.33
Pan Leukocyte
BCI
lo/-
lo/-
+
CD6-nTreg
-
CD4 CD25
1.subsets
+CD25
hiCD127lo/CD6lo/CD4
B
+CD25hi
+CD25
hi lo/CD4
22.6%
CD6 94.9%
CD127
CD6
CD127
1.hi CD6
lo/CD4
2.CD127
CD4+CD25
cells
+CD127
+
B
+CD25
hi
CD6
CD4
+CD25
hi
CD6 nTreg
CD127
CD6 CD127 22.6%
CD127
CD4
94.9%
hi cells
subsets
1.
+CD25hi
CD4+CD25
lo/lo/2.
CD6CD6
CD127
+CD127
+
CD4
CD4+CD25hi cells
-nTreg
CD4
cells
hi
CD6
CD4+CD25
CD4+CD25hi
C
C
CD4+CD25 cells
lo/-
-nTreg
CD6-nTreg CD127
+ cells
CD4
CD4+CD25hi
subsets
+ hi
Bhi
CD25CD25
CD4+CD4
CD4+CD25hi
subsets
1.
subsets
lo/-CD127
+
hi
CD6
CD127
nTreg
CD25 lo/CD4
hi
CD6 nTreg
BCD4+CD25
CD6 CD127
CD6 CD127
% FOXP3+
cells
% FOXP3+ cells
FIG. 4
Figure
7 A
Figure
hi
lo/– 5
(n=5)
15.0000
CD4+CD25CD6-Treg
CD6
cells show
suppressive
% Suppression function Treg-suppresCD6-Treg
60.0000
= 0.9914
R² =R²0.9914
sion assay Figure 50
R² = 0.9914
45.0000
Table 1. Conjugated monoclonal antibodies
CD4+ cells
hi
+ cells
CD4
hi
CD6-nTreg CD127-nTreg
CD4+CD25hi
+
B
+
Figure
7A
CD4 CD25
lo/PBMCs. Bars represent mean ± SD from
E. FOXP3 expression60.0000
in Figure
CD6+ and
5 CD6
R² = 0.9914
% Suppression
four healthy donors; *45.0000
p<0.03, U-Mann Whitney
test.
30.0000
60.0000
15.0000
60.000045.0000
0
1:230.0000 1:4
45.0000
+
FIG. 4
Figure
7A
FIG. 4
% Suppression
Figure
30.0000
Figure
5 5
Figure
7 *A
CD4 cells
+CD25hi cells
hi cells
CD4
CD4+CD25
-nTreg
hi
FIG.
4 - CD127-nTreg
p<0.009
CD6
CD4
CD25
-nTreg
hi +CD6
CD127
nTreg
FIG. 4
CD25
Figure
7 CD4
A
C. CD4+CD25hiCD6low/- lymphocytes have significantly higher % of FOXP3 positive
cells compared
to CD4+CD25–CD6lo/– and CD4+CD25–CD6+.
Figure
5
Multicolor Flow Cytometry. Conjugated monoclonal antibodies used are summarized
in Table 1. Staining was performed according Grant et al. protocol[2]. Multicolor flow
cytometry
used to evaluate Treg markers using the panels show in Table 2.
Table 1. Conjugated
monoclonalwas
antibodies
cells
hi
+
B. MFI analysis
reveals low CD6 expression
CD6 on CD4+FOXP3+ and
CD6
CD4+CD25+FOXP3+ cells
Subjects. Heparinized blood samples were obtained with informed consent with Institutional Review Board approval at Blood Service, Beckman Coulter,
+
hi
% FOXP3+
cells
Lymphocytes
Lymphocytes
Natural T-regulatory cells, nTreg, are responsible for the maintenance of dominant self tolerFigure
4 negative expression
Low/
of CD6 in nTreg.
Figure
4
Lymphocytes
ance. nTreg cells play an important role in the prevention of autoimmune disorders, allergy,
Lymphocytes
Figure 4
and in maintenance of fetal maternal tolerance and organ graft tolerance. For these reasons
Lymphocytes
Figure 4
Lymphocytes
nTreg cells have been the subject of extensive research in the past decade[1]. While Figure
the
4
Lymphocytes
nuclear transcription factor, FOXP3, uniquely defines nTreg cells, there is a need for more
convenient surface markers that can be used for nTreg isolation. Several surface markers
have been already identified. Among them, CD25 and CD127 are the most commonly
CD6
CD6
used. We report here the identification of CD6 as a low/negative surface marker for natural
occurring regulatory T-cell (nTreg). CD6 is an important costimulatory molecule in T cell
response. Lack of CD6 expression on nTreg may have significant biological consequences
as it could explain anergy and peripheral antigen-induced tolerance, a central characteristic
of nTreg suppressor cells. The high level of FOXP3+ cells afforded by CD4+CD25+CD6lo/-CD127lo/- marker combination provides a new approach for identification, enrichment and isolation of nTreg by surface staining. In addition, CD4+CD25+CD6lo/-CD127lo/- nTreg functional
CD6
CD6
CD6
CD6
differentiation stages can be assessed based on the expression of CD45RA and CCR4
CD6
CD6
markers, where CD45RA-CCR4hiHLA-DR+ cells could represent a mature effector stage of
CD6
CD6
CD6
CD6
the nTreg population. This allows analysis of nTreg differentiation using only surface markers. Lack of or low CD6 expression on nTreg could contribute to a better understanding of
CD6
CD6
CD6
CD6
the biology of nTreg immune regulation.
MATERIAL & METHOD
CD4+CD25hi
CD6+CD127+
Highly enriched FOXP3+ population identified
by surface nTreg
FOXP3
markers.
Figure
7
Lymphocytes
Figure 4Figure 4
CD45RA
RESULTS
+ cells
% FOXP3+ cells % FOXP3
% FOXP3+ cells
Figure 4
SUMMARY
2.
22.6%
hi
CD25 hi
CCR4
CD25
CD6+CD
pure FOXP3 population
nTreg
Accurate evaluation of nTreg
Accurate
evaluation
of
Identification
and isolation
ofnTreg
high of high
Identification
and isolation
subset
subset
+
will
enrich
the
cell
preparation
with
+
will
enrich
the
cell
preparation
with
population
purepure
FOXP3
population
FOXP3
Sorted Inc.)
Populations
in
were isolated using a MoFlo* XDP (Beckman Coulter,
high speed
cell sorter.
supplemented media
untouched
nTreg
without
loosing
nTregcells
cells
without
loosing
Depletionuntouched
of
bone marrow
with
anti-CD6
Identification and isolation of high
Figure 3. Treg isolation by MoFlo XDP high speed cell sorting
lymphoid progenitors
deplet
lymphoid
progenitorsand
andcould
could
deplet
Accurate
evaluation
of nTreg
Accurate
of nTreg
Explains
anergy
in+evaluation
nTreg
cells
will enrichinflammatory/
the cell preparation
with
population
pure
FOXP3
Staining
inflammatory/
cytotoxic
T-cells
cytotoxic
T-cells
Sorting Gate strategy
subset
subset
CD4
CD4+
CD4
FSC-Gate
FSC-H
CD25
SSC-H
FSC-W
SSC-W
SSC-Gate
Lymph
FSC-A
SSC-A
PBMC
SSC-A
CD4 PC7
CD25 PE
CD6 APC
CD6
untouched nTreg cells without loosing
lymphoid progenitors and
could deplet
Thymus
Treg selection Accurate evaluation
of nTreg to induce tolerance
Mechanism
inflammatory/ cytotoxic T-cells
subset
CD4+CD25+CD6Treg cells
CD4+CD25-CD6+
Non-Treg cells
CD6
Sorted Populations in
supplemented media
In vitro suppression assay was performed based on the capacity of added nTreg to
suppress the proliferation of allogeneic CD8+ responder T-cells stimulated with CD3/CD28
soluble mAbs[3]. A carboxyfluorescein diacetate succinimidyl ester (CFSE)-based proliferation assay was used[4]. A CFSE histogram of unstimulated responder cells defined the parent population, and the proliferation of activated responders was determined by calculation
of precursor frequency (pf) using ModFit LT software (Verity Software House, v3.0; Topsham
ME). Results are expressed as % suppression of pf for each nTreg:Tresp ratio sample.
Methods of analysis. Flow cytometric analysis was performed using Kaluza* software
(v1.2; Beckman Coulter, Inc., USA). Statistical analyses included mean, standard deviation and regression testing using Excel (Microsoft Office 2003) and U-Mann Whitney non
parametric tests (GraphPad Software, Inc., USA). Proliferation of activated responders
was determined by calculation of precursor frequency (pf) using ModFit LT software (Verity
Software House, v3.0; Topsham ME).
ACKNOWLEDGEMENTS
CD6lo/nTreg
REFERENCES
We wish to acknowledge Dr Tewfik Miloud and David Bloodgood for
preparation
of anti-CD6
conjugates,
and Dianne with
Prendergast
and Joe
Depletion
of bone
marrow
anti-CD6
Gao for MoFlo* XDP assistance. We thank Dr Vincent T. Shankey for
will
enrich
theandcell
preparation
with
his
valuable
discussions
suggestions,
and Dr. William
Godfrey,
Dr
Li Yang and Dr. Mike
Reed forcells
their useful
comments.loosing
The authors
untouched
nTreg
without
are employees of Beckman Coulter, Inc., and presented research was
lymphoid progenitors and could deplet
undertaken under context of their employment.
inflammatory/ cytotoxic T-cells
1.
2.
3.
4.
Sakaguchi et al. (2004) Annu Rev Immunol 22, 531-562.
Grant, J. et. al.Cytometry B Clin Cytom. (2009) Mar;76(2):69-78).
Putnam, A. et al. (2009) Diabetes 58, 652-662.
Lyons and Parish, (1994) J Immunol Methods 171, 131-137.
Identification and isolation of high
pure FOXP3+ population
*Gallios, MoFlo XDP and Kaluza are for research use only. Not for
use in diagnostic procedures.
Accurate evaluation of nTreg
subset
Gallios, MoFlo XDP and Kaluza are trademarks of Beckman Coulter,
Inc. Biomek and the stylized logo are registered trademarks of Beckman Coulter, Inc. and are registered in the USPTO.