cd6: new low/negative surface marker for human foxp3 ......cd6: new low/negative surface marker for...

CD6: New low/negative surface marker for human FOXP3+ naturally-occurring regulatory T-cellsCarlos A. Garcia Santana M.D., Ph.D.1, James W. Tung Ph.D.2, and Sergei Gulnik Ph.D.1

1Life Science Research; 2Life Science Development, Beckman Coulter, Inc., Miami, FL., USA

SUMMARYNatural T-regulatory cells, nTreg, are responsible for the maintenance of dominant self toler-ance. nTreg cells play an important role in the prevention of autoimmune disorders, allergy, and in maintenance of fetal maternal tolerance and organ graft tolerance. For these reasons nTreg cells have been the subject of extensive research in the past decade[1]. While the 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 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/-CD-127lo/- marker combination provides a new approach for identification, enrichment and isola-tion of nTreg by surface staining. In addition, CD4+CD25+CD6lo/-CD127lo/- nTreg functional differentiation stages can be assessed based on the expression of CD45RA and CCR4 markers, where CD45RA-CCR4hiHLA-DR+ cells could represent a mature effector stage of the nTreg population. This allows analysis of nTreg differentiation using only surface mark-ers. Lack of or low CD6 expression on nTreg could contribute to a better understanding of the biology of nTreg immune regulation.

MATERIAL & METHODSubjects. Heparinized blood samples were obtained with informed consent with Institu-tional Review Board approval at Blood Service, Beckman Coulter,

Multicolor Flow Cytometry. Conjugated monoclonal antibodies used are summarized in Table 1. Staining was performed according Grant et al. protocol[2]. Multicolor flow cytometry was used to evaluate Treg markers using the panels show in Table 2.

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 staining with antibodies conju-gated with the fluorochromes used in our staining protocols.

The results were expressed as the percentage of cells above the negative region for each mAb; alternatively, results were

expressed as the mean of fluorescence intensity (MFI) of discrete populations.

Lymphocytes were gated based on forward and side light scatter, viable cells were selected as 7AAD negative cells, side and forward scatter doublets were excluded, and CD4+ population was analyzed (Figure 2)

In vitro suppression assay. Sorting regulatory T-suppressor cells. After PBMC staining, cells were pelleted and resuspended at 10 × 106 per mL cell culture media. Stained cells were isolated using a MoFlo* XDP (Beckman Coulter, Inc.) high speed cell sorter.

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 prolifera-tion assay was used[4]. A CFSE histogram of unstimulated responder cells defined the par-ent 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 devia-tion 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).

CD4-KrO

FS IN

T

FS INT FS INTSS INT

FS IN

T

SS IN

T

SS T

OF

FS T

OF

7AAD

Figure 2. Gating strategy for 10-color nTreg panel analysis.

Table 1. Conjugated monoclonal antibodiesAntibody-Conjugate Clone Function Source

CD4-PC7 SFCI12T4D11 MHC- II R BCICD4-Krome Orange 13B8.2 MHC- II R BCI

CD6-APC 6D3 TCR Costimulation BCICD6-FITC 6D3 TCR Costimulation BCICD8-APC B9.11 MHC-I R BCICD25-PE B1.49.9 IL-2R, activation BCI

CD25-APC B1.49.9 IL-2R, activation BCICD25-PC7 B1.49.9 IL-2R, activation BCI

CD45RA-ECD 2H4 Naïve/memory BCICD45RA-AF750 2H4 Naïve/memory BCI

CD62L-ECD DREG56 LN Homing R BCICD127-PE R34.34 IL-7α R BCI

CD127-APC-AF700 R34.34 IL-7α R BCIHLA-Dr-Pacific Blue Immu-357 Activation BCI

CD45-FITC J.33 Pan Leukocyte BCICD45-PE J.33 Pan Leukocyte BCI

CD45-ECD J.33 Pan Leukocyte BCICD45-PC5 J.33 Pan Leukocyte BCICD45-PC7 J.33 Pan Leukocyte BCICD45-APC J.33 Pan Leukocyte BCI

CTLA-4-PCy5 (CD152) BNI3 Inhibitory signal BD PharMingenGARP-PE 7B11 Activation BD PharMingen

CCR4-PE (CD194) 205410 Homing R R&D SystemsCD39-APC A1 Ecto-nucleotidase BioLegend

FOXP3-Pacific Blue 206D Treg transcript BioLegend

Table 2. Multicolor flow cytometry panels

A. FOXP3 FMO

FULL STAIN FMO

CD4+ cellsB. CD6 FMO

CD4

FOXP

3

CD6

CD4+ cells

CD4

FOXP

3

FULL STAIN FMO

Figure 1. Fluorescense minus one for FOXP3 and CD6 staining

ACKNOWLEDGEMENTS

We wish to acknowledge Dr Tewfik Miloud and David Bloodgood for preparation of anti-CD6 conjugates, and Dianne Prendergast and Joe Gao for MoFlo* XDP assistance. We thank Dr Vincent T. Shankey for his valuable discussions and suggestions, and Dr. William Godfrey, Dr Li Yang and Dr. Mike Reed for their useful comments. The authors are employees of Beckman Coulter, Inc., and presented research was undertaken under context of their employment.

REFERENCES 1. Sakaguchi et al. (2004) Annu Rev Immunol 22, 531-562.2. Grant, J. et. al.Cytometry B Clin Cytom. (2009) Mar;76(2):69-78).3. Putnam, A. et al. (2009) Diabetes 58, 652-662. 4. Lyons and Parish, (1994) J Immunol Methods 171, 131-137.

*Gallios, MoFlo XDP and Kaluza are for research use only. Not for use in diagnostic procedures.

Gallios, MoFlo XDP and Kaluza are trademarks of Beckman Coulter, Inc. Biomek and the stylized logo are registered trademarks of Beck-man Coulter, Inc. and are registered in the USPTO.

RESULTS

Low/ negative expression of CD6 in nTreg.

B. MFI analysis reveals low CD6 expression on CD4+FOXP3+ and CD4+CD25+FOXP3+ cells

C. CD4+CD25hiCD6low/- lymphocytes have significantly higher % of FOXP3 positive cells compared to CD4+CD25–CD6lo/– and CD4+CD25–CD6+.

E. FOXP3 expression in CD6+ and CD6lo/-PBMCs. Bars represent mean ± SD from four healthy donors; * p<0.03, U-Mann Whitney test.

CD4+CD25hiCD6lo/– cells show suppressive function Treg-suppres-sion assay

No differences in the expression of nTreg-associated markers in CD6-Treg and CD127-Treg populations

Highly enriched FOXP3+ population identified by surface nTreg markers.

nTreg functional maturation compartments defined by CD45RA/CCR4 bivariate analysis

CD45RA-CCR4hiHLA-DR+ cells could represent a mature effector stage of the nTreg population

Postulated nTreg functional maturation pathway model

DISCUSSION & CONCLUSION

CD6lo/-: New insights into nTreg cell biologyCD4+CD25+CD6-

Lymph

CD4+CD25-CD6+

FSC-A

SSC

-A

SSC

-W

SSC-H

FSC

-W

FSC-H

CD4

SSC

-A

CD6

CD

25

SSC-Gate FSC-Gate

CD4+

Sorted Populations in supplemented media

Treg cells

Non-Treg cells

Sorting Gate strategy

CD4 PC7CD25 PECD6 APC

StainingFigure 3. Treg isolation by MoFlo XDP high speed cell sorting

PBMC






CD62L-ECD DREG56 LN Homing R BCICD127-PE R34.34 IL-7 R BCI

CD127-APC-AF700 R34.34 IL-7 R BCIHLA-Dr-Pacific Blue Immu-357 Activation BCI







A. FOXP3 FMO

FULL STAIN FMO


CD4

FOXP

3

CD6

CD4+ cells

CD4

FOXP

3

FULL STAIN FMO


CD4-KrO

FS IN

T

FS INT FS INTSS INT

FS IN

T

SS IN

T

SS T

OF

FS T

OF

7AAD


A CD4+ cells

CD6-nTreg CD127-nTreg

CD6

CD

25

CD127

B CD4+CD25hi CD6-nTreg CD127-nTreg

CD39

GA

RP

CCR4

CD

45R

A

FIG. 4

CD4+ cellsACD4+CD25hi CD6-nTreg CD127-nTreg

CD4+CD25hi cells

CD6lo/-CD127lo/- CD6+CD127+

B

22.6%94.9%

FOXP3

C

p<0.009 *

% F

OXP

3+ c

ells

CD4+CD25hi CD6-nTreg

CD127-nTreg

CD6lo/-

CD127lo/-

% F

OXP

3+

cells

D

CD4+CD25+ CD6-nTreg

CD127-nTreg

CD6lo/-

CD127lo/-

1.190.5 7.9

0.4 0.566.8 31.3

1.4 1.114.0 47.1

35.8

N/R I/Me Ma/EA

2.486.1 6.7

4.8

C

CD4+CD25hi

CD6lo/-CD127lo/-

B

TDD

CD4+CD25hi

CD6+CD127+

31.8%N/R

20.8%I/Me

33.8%Ma/E

60.0%TD

CD39

HLA

-DR

CD39

HLA

-DR

B

CD

45R

A

GARP

3D: 16.8%

Pop1

Pop2

Pop3

A

CCR4

CD

45R

A

3D: 19.1%

N/R I/Me

Ma/E

-CCR4hiHLA-DR+ cells could

CD6lo/- nTreg

Depletion of bone marrow with anti-CD6 will enrich the cell preparation with untouched nTreg cells without loosing lymphoid progenitors and could deplet inflammatory/ cytotoxic T-cells

Identification and isolation of high pure FOXP3+ population

Explains anergy in nTreg cells

Thymus Treg selection Mechanism to induce tolerance

Accurate evaluation of nTreg subset

Figure 4

Figure 6

Figure 7

Figure 8

Figure 9

% Suppression

0

18

35

53

70

1:1 1:2 1:4 1:8

R² = 0.9927

nTreg: T-responder ratio

0

15.0000

30.0000

45.0000

60.0000

1:1 1:2 1:4 1:8

R² = 0.9914

CD6-Treg

CD127-Treg(PositiveControl)

Figure 5

FOXP3

HLA-Dr

CTLA-4

% p

ositi

ve c

ells

CD4+CD25hi

CD6-TregCD127-Treg

C

Note: arrows show marker expression pathway

CD4+CD25hi

CD6lo/-CD127lo/-

CD4+CD25hi

CD6+CD127+

1.

2.

CD4+CD25hi

subsets

Naïve/ Immature/ Mature/ resting memory effectors

TerminalDifferentiation?

CCR4blocking

CD45RA+ CD39+

CCR4lo

CD39lo

CD25++CD25++ CD25 hi

Regulation Treg suppression ?

CD25 +

HLA-DR

++

FOXP3lo FOXP3lo FOXP3hi FOXP3lo

CD6+CD127+CD6lo/-CD127lo/-

CCR4 hiCCR4 hi

Figure 10

(n=5)

(n=9)

Lymphocytes

CD6 CD6

CD4+CD25+CD6-

Lymph

CD4+CD25-CD6+

FSC-A

SSC-

A

SSC-

W

SSC-H

FSC-

W

FSC-H

CD4

SSC-

A

CD6

CD25

SSC-Gate FSC-Gate

CD4+


Treg cells

Non-Treg cells




PBMC














A. FOXP3 FMO

FULL STAIN FMO


CD4

FOXP

3

CD6

CD4+ cells

CD4

FOXP

3 FULL STAIN FMO


CD4-KrO

FS IN

T

FS INT FS INTSS INT

FS IN

T

SS IN

T

SS T

OF

FS T

OF

7AAD


A CD4+ cells


CD6

CD25

CD127


CD39

GARP

CCR4

CD45

RA

FIG. 4


CD4+CD25hi cells


B

22.6%94.9%

FOXP3

C

p<0.009 *

% F

OXP3

+ cel

ls


CD127-nTreg

CD6lo/-

CD127lo/-

% F

OXP3

+ ce

lls

D

CD4+CD25+ CD6-nTreg

CD127-nTreg

CD6lo/-

CD127lo/-

1.190.5 7.9

0.4 0.566.8 31.3

1.4 1.114.0 47.1

35.8

N/R I/Me Ma/EA

2.486.1 6.7

4.8

C

CD4+CD25hi

CD6lo/-CD127lo/-

B

TDD

CD4+CD25hi

CD6+CD127+

31.8%N/R

20.8%I/Me

33.8%Ma/E

60.0%TD

CD39

HLA-

DR

CD39

HLA-

DR

B

CD45

RA

GARP

3D: 16.8%

Pop1

Pop2

Pop3

A

CCR4

CD45

RA

3D: 19.1%

N/R I/Me

Ma/E


CD6lo/- nTreg






Figure 4

Figure 6

Figure 7

Figure 8

Figure 9

% Suppression

0

18

35

53

70

1:1 1:2 1:4 1:8

R² = 0.9927


0

15.0000

30.0000

45.0000

60.0000

1:1 1:2 1:4 1:8

R² = 0.9914

CD6-Treg


Figure 5

FOXP3

HLA-Dr

CTLA-4

% p

ositi

ve c

ells

CD4+CD25hi

CD6-TregCD127-Treg

C


CD4+CD25hi

CD6lo/-CD127lo/-

CD4+CD25hi

CD6+CD127+

1.

2.

CD4+CD25hi

subsets



CCR4blocking

CD45RA+ CD39+

CCR4lo

CD39lo



CD25 +

HLA-DR

++



CCR4 hiCCR4 hi

Figure 10

(n=5)

(n=9)

Lymphocytes

CD6 CD6

CD4+CD25+CD6-

Lymph

CD4+CD25-CD6+

FSC-A

SS

C-A

SS

C-W

SSC-H

FS

C-W

FSC-H

CD4

SS

C-A

CD6

CD

25

SSC-Gate FSC-Gate

CD4+


Treg cells

Non-Treg cells




PBMC














A. FOXP3 FMO

FULL STAIN FMO


CD4

FO

XP

3

CD6

CD4+ cells

CD4

FO

XP

3

FULL STAIN FMO


CD4-KrO

FS

INT

FS INT FS INTSS INT

FS

INT

SS

INT

SS

TO

F

FS

TO

F

7AAD


A CD4+ cells


CD6

CD

25

CD127


CD39

GA

RP

CCR4

CD

45R

A

FIG. 4


CD4+CD25hi cells


B

22.6%94.9%

FOXP3

C

p<0.009 *

% F

OX

P3+

cel

ls


CD127-nTreg

CD6lo/-

CD127lo/-

% F

OX

P3+

ce

lls

D

CD4+CD25+ CD6-nTreg

CD127-nTreg

CD6lo/-

CD127lo/-

1.190.5 7.9

0.4 0.566.8 31.3

1.4 1.114.0 47.1

35.8

N/R I/Me Ma/EA

2.486.1 6.7

4.8

C

CD4+CD25hi

CD6lo/-CD127lo/-

B

TDD

CD4+CD25hi

CD6+CD127+

31.8%N/R

20.8%I/Me

33.8%Ma/E

60.0%TD

CD39

HL

A-D

R

CD39

HL

A-D

R

B

CD

45R

A

GARP

3D: 16.8%

Pop1

Pop2

Pop3

A

CCR4

CD

45R

A

3D: 19.1%

N/R I/Me

Ma/E


CD6lo/- nTreg






Figure 4

Figure 6

Figure 7

Figure 8

Figure 9

% Suppression

0

18

35

53

70

1:1 1:2 1:4 1:8

R² = 0.9927


0

15.0000

30.0000

45.0000

60.0000

1:1 1:2 1:4 1:8

R² = 0.9914

CD6-Treg


Figure 5

FOXP3

HLA-Dr

CTLA-4

% p

osi

tive

cel

ls

CD4+CD25hi

CD6-TregCD127-Treg

C


CD4+CD25hi

CD6lo/-CD127lo/-

CD4+CD25hi

CD6+CD127+

1.

2.

CD4+CD25hi

subsets



CCR4blocking

CD45RA+ CD39+

CCR4lo

CD39lo



CD25 +

HLA-DR

++



CCR4 hiCCR4 hi

Figure 10

(n=5)

(n=9)

Lymphocytes

CD6 CD6

CD4+CD25+CD6-

Lymph

CD4+CD25-CD6+

FSC-A

SSC

-A

SSC

-W

SSC-H

FSC

-W

FSC-H

CD4

SSC

-A

CD6

CD

25

SSC-Gate FSC-Gate

CD4+


Treg cells

Non-Treg cells




PBMC














A. FOXP3 FMO

FULL STAIN FMO


CD4

FOXP

3

CD6

CD4+ cells

CD4

FOXP

3

FULL STAIN FMO


CD4-KrO

FS IN

T

FS INT FS INTSS INT

FS IN

T

SS IN

T

SS T

OF

FS T

OF

7AAD


A CD4+ cells


CD6

CD

25

CD127


CD39

GA

RP

CCR4

CD

45R

A

FIG. 4


CD4+CD25hi cells


B

22.6%94.9%

FOXP3

C

p<0.009 *

% F

OXP

3+ c

ells


CD127-nTreg

CD6lo/-

CD127lo/-

% F

OXP

3+

cells

D

CD4+CD25+ CD6-nTreg

CD127-nTreg

CD6lo/-

CD127lo/-

1.190.5 7.9

0.4 0.566.8 31.3

1.4 1.114.0 47.1

35.8

N/R I/Me Ma/EA

2.486.1 6.7

4.8

C

CD4+CD25hi

CD6lo/-CD127lo/-

B

TDD

CD4+CD25hi

CD6+CD127+

31.8%N/R

20.8%I/Me

33.8%Ma/E

60.0%TD

CD39

HLA

-DR

CD39

HLA

-DR

B

CD

45R

A

GARP

3D: 16.8%

Pop1

Pop2

Pop3

A

CCR4

CD

45R

A

3D: 19.1%

N/R I/Me

Ma/E


CD6lo/- nTreg






Figure 4

Figure 6

Figure 7

Figure 8

Figure 9

% Suppression

0

18

35

53

70

1:1 1:2 1:4 1:8

R² = 0.9927


0

15.0000

30.0000

45.0000

60.0000

1:1 1:2 1:4 1:8

R² = 0.9914

CD6-Treg


Figure 5

FOXP3

HLA-Dr

CTLA-4

% p

ositi

ve c

ells

CD4+CD25hi

CD6-TregCD127-Treg

C


CD4+CD25hi

CD6lo/-CD127lo/-

CD4+CD25hi

CD6+CD127+

1.

2.

CD4+CD25hi

subsets



CCR4blocking

CD45RA+ CD39+

CCR4lo

CD39lo



CD25 +

HLA-DR

++



CCR4 hiCCR4 hi

Figure 10

(n=5)

(n=9)

Lymphocytes

CD6 CD6

CD4+CD25+CD6-

Lymph

CD4+CD25-CD6+

FSC-A

SSC

-A

SSC

-W

SSC-H

FSC

-W

FSC-H

CD4

SSC

-A

CD6

CD

25

SSC-Gate FSC-Gate

CD4+


Treg cells

Non-Treg cells




PBMC














A. FOXP3 FMO

FULL STAIN FMO


CD4

FOXP

3

CD6

CD4+ cells

CD4

FOXP

3

FULL STAIN FMO


CD4-KrO

FS IN

T

FS INT FS INTSS INT

FS IN

T

SS IN

T

SS T

OF

FS T

OF

7AAD


A CD4+ cells


CD6

CD

25

CD127


CD39

GA

RP

CCR4

CD

45R

A

FIG. 4


CD4+CD25hi cells


B

22.6%94.9%

FOXP3

C

p<0.009 *

% F

OXP

3+ c

ells


CD127-nTreg

CD6lo/-

CD127lo/-

% F

OXP

3+

cells

D

CD4+CD25+ CD6-nTreg

CD127-nTreg

CD6lo/-

CD127lo/-

1.190.5 7.9

0.4 0.566.8 31.3

1.4 1.114.0 47.1

35.8

N/R I/Me Ma/EA

2.486.1 6.7

4.8

C

CD4+CD25hi

CD6lo/-CD127lo/-

B

TDD

CD4+CD25hi

CD6+CD127+

31.8%N/R

20.8%I/Me

33.8%Ma/E

60.0%TD

CD39

HLA

-DR

CD39

HLA

-DR

B

CD

45R

A

GARP

3D: 16.8%

Pop1

Pop2

Pop3

A

CCR4

CD

45R

A

3D: 19.1%

N/R I/Me

Ma/E


CD6lo/- nTreg






Figure 4

Figure 6

Figure 7

Figure 8

Figure 9

% Suppression

0

18

35

53

70

1:1 1:2 1:4 1:8

R² = 0.9927


0

15.0000

30.0000

45.0000

60.0000

1:1 1:2 1:4 1:8

R² = 0.9914

CD6-Treg


Figure 5

FOXP3

HLA-Dr

CTLA-4

% p

ositi

ve c

ells

CD4+CD25hi

CD6-TregCD127-Treg

C


CD4+CD25hi

CD6lo/-CD127lo/-

CD4+CD25hi

CD6+CD127+

1.

2.

CD4+CD25hi

subsets



CCR4blocking

CD45RA+ CD39+

CCR4lo

CD39lo



CD25 +

HLA-DR

++



CCR4 hiCCR4 hi

Figure 10

(n=5)

(n=9)

Lymphocytes

CD6 CD6

CD4+CD25+CD6-

Lymph

CD4+CD25-CD6+

FSC-A

SS

C-A

SS

C-W

SSC-H

FSC

-W

FSC-H

CD4

SS

C-A

CD6

CD

25

SSC-Gate FSC-Gate

CD4+


Treg cells

Non-Treg cells




PBMC














A. FOXP3 FMO

FULL STAIN FMO


CD4

FOX

P3

CD6

CD4+ cells

CD4

FOX

P3

FULL STAIN FMO


CD4-KrO

FS IN

T

FS INT FS INTSS INT

FS IN

T

SS

INT

SS

TO

F

FS T

OF

7AAD


A CD4+ cells


CD6

CD

25

CD127


CD39

GA

RP

CCR4

CD

45R

A

FIG. 4


CD4+CD25hi cells


B

22.6%94.9%

FOXP3

C

p<0.009 *

% F

OX

P3+

cel

ls


CD127-nTreg

CD6lo/-

CD127lo/-

% F

OX

P3+

ce

lls

D

CD4+CD25+ CD6-nTreg

CD127-nTreg

CD6lo/-

CD127lo/-

1.190.5 7.9

0.4 0.566.8 31.3

1.4 1.114.0 47.1

35.8

N/R I/Me Ma/EA

2.486.1 6.7

4.8

C

CD4+CD25hi

CD6lo/-CD127lo/-

B

TDD

CD4+CD25hi

CD6+CD127+

31.8%N/R

20.8%I/Me

33.8%Ma/E

60.0%TD

CD39

HLA

-DR

CD39

HLA

-DR

B

CD

45R

A

GARP

3D: 16.8%

Pop1

Pop2

Pop3

A

CCR4

CD

45R

A

3D: 19.1%

N/R I/Me

Ma/E


CD6lo/- nTreg






Figure 4

Figure 6

Figure 7

Figure 8

Figure 9

% Suppression

0

18

35

53

70

1:1 1:2 1:4 1:8

R² = 0.9927


0

15.0000

30.0000

45.0000

60.0000

1:1 1:2 1:4 1:8

R² = 0.9914

CD6-Treg


Figure 5

FOXP3

HLA-Dr

CTLA-4

% p

ositi

ve c

ells

CD4+CD25hi

CD6-TregCD127-Treg

C


CD4+CD25hi

CD6lo/-CD127lo/-

CD4+CD25hi

CD6+CD127+

1.

2.

CD4+CD25hi

subsets



CCR4blocking

CD45RA+ CD39+

CCR4lo

CD39lo



CD25 +

HLA-DR

++



CCR4 hiCCR4 hi

Figure 10

(n=5)

(n=9)

Lymphocytes

CD6 CD6

CD4+CD25+CD6-

Lymph

CD4+CD25-CD6+

FSC-A

SS

C-A

SS

C-W

SSC-H

FSC

-W

FSC-H

CD4

SS

C-A

CD6

CD

25

SSC-Gate FSC-Gate

CD4+


Treg cells

Non-Treg cells




PBMC














A. FOXP3 FMO

FULL STAIN FMO


CD4

FOX

P3

CD6

CD4+ cells

CD4

FOX

P3

FULL STAIN FMO


CD4-KrO

FS IN

T

FS INT FS INTSS INT

FS IN

T

SS

INT

SS

TO

F

FS T

OF

7AAD


A CD4+ cells


CD6

CD

25

CD127


CD39

GA

RP

CCR4

CD

45R

A

FIG. 4


CD4+CD25hi cells


B

22.6%94.9%

FOXP3

C

p<0.009 *

% F

OX

P3+

cel

ls


CD127-nTreg

CD6lo/-

CD127lo/-

% F

OX

P3+

ce

lls

D

CD4+CD25+ CD6-nTreg

CD127-nTreg

CD6lo/-

CD127lo/-

1.190.5 7.9

0.4 0.566.8 31.3

1.4 1.114.0 47.1

35.8

N/R I/Me Ma/EA

2.486.1 6.7

4.8

C

CD4+CD25hi

CD6lo/-CD127lo/-

B

TDD

CD4+CD25hi

CD6+CD127+

31.8%N/R

20.8%I/Me

33.8%Ma/E

60.0%TD

CD39

HLA

-DR

CD39

HLA

-DR

B

CD

45R

A

GARP

3D: 16.8%

Pop1

Pop2

Pop3

A

CCR4

CD

45R

A

3D: 19.1%

N/R I/Me

Ma/E


CD6lo/- nTreg






Figure 4

Figure 6

Figure 7

Figure 8

Figure 9

% Suppression

0

18

35

53

70

1:1 1:2 1:4 1:8

R² = 0.9927


0

15.0000

30.0000

45.0000

60.0000

1:1 1:2 1:4 1:8

R² = 0.9914

CD6-Treg


Figure 5

FOXP3

HLA-Dr

CTLA-4

% p

ositi

ve c

ells

CD4+CD25hi

CD6-TregCD127-Treg

C


CD4+CD25hi

CD6lo/-CD127lo/-

CD4+CD25hi

CD6+CD127+

1.

2.

CD4+CD25hi

subsets



CCR4blocking

CD45RA+ CD39+

CCR4lo

CD39lo



CD25 +

HLA-DR

++



CCR4 hiCCR4 hi

Figure 10

(n=5)

(n=9)

Lymphocytes

CD6 CD6

CD4+CD25+CD6-

Lymph

CD4+CD25-CD6+

FSC-A

SSC

-A

SSC

-W

SSC-H

FSC

-W

FSC-H

CD4

SSC

-A

CD6

CD

25

SSC-Gate FSC-Gate

CD4+


Treg cells

Non-Treg cells




PBMC














A. FOXP3 FMO

FULL STAIN FMO


CD4

FOXP

3

CD6

CD4+ cells

CD4

FOXP

3

FULL STAIN FMO


CD4-KrO

FS IN

T

FS INT FS INTSS INT

FS IN

T

SS IN

T

SS T

OF

FS T

OF

7AAD


A CD4+ cells


CD6

CD

25

CD127


CD39

GA

RP

CCR4

CD

45R

A

FIG. 4


CD4+CD25hi cells


B

22.6%94.9%

FOXP3

C

p<0.009 *

% F

OXP

3+ c

ells


CD127-nTreg

CD6lo/-

CD127lo/-

% F

OXP

3+

cells

D

CD4+CD25+ CD6-nTreg

CD127-nTreg

CD6lo/-

CD127lo/-

1.190.5 7.9

0.4 0.566.8 31.3

1.4 1.114.0 47.1

35.8

N/R I/Me Ma/EA

2.486.1 6.7

4.8

C

CD4+CD25hi

CD6lo/-CD127lo/-

B

TDD

CD4+CD25hi

CD6+CD127+

31.8%N/R

20.8%I/Me

33.8%Ma/E

60.0%TD

CD39

HLA

-DR

CD39

HLA

-DR

B

CD

45R

A

GARP

3D: 16.8%

Pop1

Pop2

Pop3

A

CCR4

CD

45R

A

3D: 19.1%

N/R I/Me

Ma/E


CD6lo/- nTreg






Figure 4

Figure 6

Figure 7

Figure 8

Figure 9

% Suppression

0

18

35

53

70

1:1 1:2 1:4 1:8

R² = 0.9927


0

15.0000

30.0000

45.0000

60.0000

1:1 1:2 1:4 1:8

R² = 0.9914

CD6-Treg


Figure 5

FOXP3

HLA-Dr

CTLA-4

% p

ositi

ve c

ells

CD4+CD25hi

CD6-TregCD127-Treg

C


CD4+CD25hi

CD6lo/-CD127lo/-

CD4+CD25hi

CD6+CD127+

1.

2.

CD4+CD25hi

subsets



CCR4blocking

CD45RA+ CD39+

CCR4lo

CD39lo



CD25 +

HLA-DR

++



CCR4 hiCCR4 hi

Figure 10

(n=5)

(n=9)

Lymphocytes

CD6 CD6

CD4+CD25+CD6-

Lymph

CD4+CD25-CD6+

FSC-A

SSC

-A

SSC

-W

SSC-H

FSC

-W

FSC-H

CD4

SSC

-A

CD6

CD

25

SSC-Gate FSC-Gate

CD4+


Treg cells

Non-Treg cells




PBMC














A. FOXP3 FMO

FULL STAIN FMO


CD4

FOXP

3

CD6

CD4+ cells

CD4

FOXP

3

FULL STAIN FMO


CD4-KrO

FS IN

T

FS INT FS INTSS INT

FS IN

T

SS IN

T

SS T

OF

FS T

OF

7AAD


A CD4+ cells


CD6

CD

25

CD127


CD39

GA

RP

CCR4

CD

45R

A

FIG. 4


CD4+CD25hi cells


B

22.6%94.9%

FOXP3

C

p<0.009 *

% F

OXP

3+ c

ells


CD127-nTreg

CD6lo/-

CD127lo/-

% F

OXP

3+

cells

D

CD4+CD25+ CD6-nTreg

CD127-nTreg

CD6lo/-

CD127lo/-

1.190.5 7.9

0.4 0.566.8 31.3

1.4 1.114.0 47.1

35.8

N/R I/Me Ma/EA

2.486.1 6.7

4.8

C

CD4+CD25hi

CD6lo/-CD127lo/-

B

TDD

CD4+CD25hi

CD6+CD127+

31.8%N/R

20.8%I/Me

33.8%Ma/E

60.0%TD

CD39

HLA

-DR

CD39

HLA

-DR

B

CD

45R

A

GARP

3D: 16.8%

Pop1

Pop2

Pop3

A

CCR4

CD

45R

A

3D: 19.1%

N/R I/Me

Ma/E


CD6lo/- nTreg






Figure 4

Figure 6

Figure 7

Figure 8

Figure 9

% Suppression

0

18

35

53

70

1:1 1:2 1:4 1:8

R² = 0.9927


0

15.0000

30.0000

45.0000

60.0000

1:1 1:2 1:4 1:8

R² = 0.9914

CD6-Treg


Figure 5

FOXP3

HLA-Dr

CTLA-4

% p

ositi

ve c

ells

CD4+CD25hi

CD6-TregCD127-Treg

C


CD4+CD25hi

CD6lo/-CD127lo/-

CD4+CD25hi

CD6+CD127+

1.

2.

CD4+CD25hi

subsets



CCR4blocking

CD45RA+ CD39+

CCR4lo

CD39lo



CD25 +

HLA-DR

++



CCR4 hiCCR4 hi

Figure 10

(n=5)

(n=9)

Lymphocytes

CD6 CD6

cd6: new low/negative surface marker for human foxp3 ......cd6: new low/negative surface marker for...

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