CD4+CD25highFoxp3+ T regulatory cells kill autologous CD8(+) and CD4(+) T

cells using Fas/FasL- and Granzyme B-mediated pathways

University of Pittsburgh Cancer Institute and Departments of Pathology, Immunology and

Otolaryngology, University of Pittsburgh School of Medicine,

Pittsburgh, PA 15213

FoxP3+

Laura Strauss, Christoph Bergmann, Theresa L. Whiteside

• To define mechanisms employed by Treg to mediate suppression of proliferating T responder cells (RC)

1. Cytokine- mediated death.2. Death receptor-mediated apoptosis.3. Cytolysis mediated by granzymes/perforin.

We considered 3 possible mechanisms:

Objectives of our study were:

Laser

Methods for studies of TregIsolate CD4+CD25high and CD4+CD25neg or CD8+CD25neg T cells from PBMC of NC or patients with cancer bv single-cell sorting (FACS)

+ 7-AAD

CFSE-labeled RC (+ OKT3+ IL-2) + Unlabeled S5-day co-culture

Suppression ofRC proliferation

FLOCA(Flow cytometry-based cytotoxicity assay)

Multicolor flow cytometry: phenotypeSuppressor function:

Apoptosis in fresh T cell subsets from HNSCC patient and NCApoptosis in fresh T cell subsets from HNSCC patient and NC

gated on CD3+CD8+CD25-

We have shown before that CD8We have shown before that CD8++ T T effectoreffector cells in the cells in the circulation of patients with HNSCC (but not NC) are highly circulation of patients with HNSCC (but not NC) are highly

sensitive to apoptosis sensitive to apoptosis 4,54,5

44Reichert et al., 2000 Reichert et al., 2000 55Hoffmann et al., 2002Hoffmann et al., 2002

72% 5.1%

CD8 CD4

fresh PBMC

HNSCC

0.3% 0.2%gated on CD3+CD4+CD25-

fresh PBMC

NC

Ann

ex V

CD4CD8

TregTreg mediate suppression of mediate suppression of autologousautologous CD4CD4++ or CD8or CD8++

RC proliferation via direct cellRC proliferation via direct cell--cell contactcell contact66

66Strauss et al., 2007, Strauss et al., 2007, Clinical Cancer Res, 1:13 (15):4352

0

10

20

30

40

50

60

70

80

90

non-TR TR

% S

uppr

essi

on

p≤ 0.003

S:RC ratio:1:2S:RC ratio:1:2

CD4+CD25high + CD4+CD25- or CD8+CD25- co-cultures

n = 5

1

2

3

4

5

6

7

Gated on CD4+CD25high T cells

% C

D25

high

cells

P≤0.0001

0

NC HNSCC

% Treg in PBMC

(n=15) (n=35)

Human CD4Human CD4++CD25CD25highhigh TregTreg suppress proliferation and suppress proliferation and induce apoptosis in induce apoptosis in autologousautologous CD8CD8++ responder cellsresponder cells

1200

89%

0 30 60 90 120

0%Responder cells alone

(RC) (CD8+CD25neg)

99.7%

0.3%

0%

56%

24%

20%

(RC) cells

+ CD4+CD25high(S)

7-AA

D

CD8+CFSE

CD8+ (RC) cells

60

HNSCC

30 60 90

Events

S/RC=1:1

CD8+ (RC) cells

5-day co-cultures in the presence of 150 IU/mL IL-2 + OKT3

Human CD4Human CD4++CD25CD25highhigh TregTreg suppress proliferation but do suppress proliferation but do not not mediate mediate apoptosis in apoptosis in autologousautologous CD4CD4++ responder cellsresponder cells

(RC) cells

+ CD4+CD25high (S)96%

0 30 60 90 120

0%Responder cells alone (RC)

(CD4+CD25neg)

Events

S/RC=1:1

HNSCC

0 30 120

CD4+ (RC) cells

7-AA

D

CD4+

99.8%

0%

0.2%

98.8%

0%

1.2%

CFSE

60 90

CD4+(RC) cells

77.4%

8%

18.8%

Tr cells

82%

3%

15%

Treg

CD4+

7-AA

D

5-day co-cultures in the presence of 150 IU/mL IL-2 + OKT3

Expression of Fas and FasL on CD4Expression of Fas and FasL on CD4++CD25CD25high high

T cells in NC or HNSCC patientsT cells in NC or HNSCC patients

Normal Control

0102030405060708090

100

Fas

NC (n=15)HNSCC (n=35)FRESH PBMC

% p

ositi

ve c

ells

p≤0.001 p≤0.03

HNSCC

FasL

gated on CD4+CD25high T cells95%

92%

Fas

FasL

5.5%

43%

CD4FasL

TregTreg can can ““killkill”” autologousautologous CD8CD8+CD25CD25-- RC but not RC but not CD4CD4++CD25CD25-- RC via the RC via the Fas/FasLFas/FasL pathwaypathway

30 60 90 1200

92%

CFSECD8

56% Events

+ CFSE-labeled CD8+RC

20%

0 30 60 90 120

62%

0%0.3%

99.7%

Events

0%

1%

99%

+ anti-FasL- Ab

24%

CD4+CD25high Treg (FasL+)+ CFSE-labeled CD4+RC

7-AA

D

150 IU/mL IL-2 1000 IU/mL IL-2

0 30 60 90 120

92%

Events

12%

68%

20%

7-AA

D + anti-FasL- Ab

CFSECD4

10%

72%

18%

0 30 60 90 120

97%

Events

Results of cell death obtained in FLOCA

B.

A.

0

20

40

60

80

100

120

140

160

Granzyme B Granzyme A Perforin

MFI

±SD

Gated on CD4+CD25high in fresh PBMCNC n=15HNSCC n=25

*

* *p≤0.01

0

20

40

60

80

100

120

140

160

Granzyme B Granzyme A Perforin

MFI

±SD

Gated on CD4+CD25- in fresh PBMC

Activated CD4+CD25high

0

20

40

60

80

100

120

160

Granzyme B Granzyme A Perforin

MFI

±SD *

*p≤0.03

NC n=15HNSCC n=25

*

Activated CD4+CD25high

NC n=15HNSCC n=25

0

20

40

60

80

100

120

140

160

Granzyme B Granzyme A Perforin

MFI

±SD

NC n=15HNSCC n=25

140

Expression of Expression of GranzymesGranzymes and and PerforinPerforin in fresh in fresh and activated peripheral Tand activated peripheral T--cell subsetscell subsets

+OKT3, IL-2 (150 IU/mL)

CD4+CD25high

after co-incubation with

CD4+ RC cells

1S:1 (RC)

CD4+CD25neg

after co-incubation with

Treg cells

1S:1 (RC)

(1000 IU IL-2/mL)

CD4 PerCP99%

Gra

nzym

eB

-PE

Gra

nzym

eA

-PE 92% 75%

Expression of Expression of cytotoxinscytotoxins is regulated by ILis regulated by IL--2 in 2 in the the presence presence of the of the ““partnerpartner”” T cellT cell

Gra

nzym

eB

-PE

Gra

nzym

eA

-PE

Perf

orin

-FIT

C

(150 IU IL-2/mL)

Perf

orin

-FIT

C

98% 97% 88%

0 1 2 3

3% 4% 7%

Gra

nzym

eB

-PE

Gra

nzym

eA

-PE

Perf

orin

-FIT

C

Gra

nzym

eA

-PE

Gra

nzym

eB

-PE 25% 1% 15%

Perf

orin

-FIT

C

5-day co-cultures of Treg and autologous RC

CD4

R

S

TregTreg suppress CD4+ RC at low and high dose of ILsuppress CD4+ RC at low and high dose of IL--2, but 2, but can kill RC only at high ILcan kill RC only at high IL--2 concentrations2 concentrations

96%

Events

S/RC=1:1

HNSCC

0 30 120 99.8%

0%

0.2%

CFSE

60 90

CD4+(RC) cells

CD4

(150 IU IL-2/mL)

(1000 IU IL-2/mL)0%

0%

100%

HNSCC

Treg

0 30 60 90 120

98%CD4+(RC) cells

0%

78%

22%

77.4%

8%

18.8%

Tr cells

82%

3%

15%

Treg

7-AA

D

At low IL-2 doses, Treg induce suppression of RC proliferation and then undergo apoptosis. This type of suppression does not involve death of RC.

Conclusion 1

Mechanisms responsible for RC death and Treg survival in these co-cultures are IL-2 dependent

TregTreg-- mediated killing of CD4mediated killing of CD4++ RC is RC is GranzymeBGranzymeB--dependent, but dependent, but PerforinPerforin--independentindependent

0 30 60 90 120

98%

Events

7-AA

D

Data.022

99.6%

0%

0.4%

35.5%

0.5%

64%

97%

Gra

nzym

eB

-PE

CFSE

CD412%

6%

0 30 60 90 120

+ siRNA GrB

CD4+(RC) cells Treg 1000 IU/mL IL-2

99.8%

0%

99.7%

0%

0.3%0.2%

CD4+(RC) cells Treg

CD-4 Gra

nzym

eB

-PE

CD4CD4++ RCRC-- mediated killing of mediated killing of TregTreg is is GranzymeBGranzymeB and and PerforinPerforin--dependentdependent

Events

30 60 90 1200

82%

7-AA

D0%

0%

100%

7%

0%

93%

150 IU/mL IL-2

CD4+(RC) cells Treg

+ConcanamycinA

CFSE

0 30 60 90 120

98%

35.5%

0.5%

64%

97%

Per

forin

-FIT

C

CD4+(RC) cells Treg

Data.022

99.6%

0%

0.4%

7%

CD-4

Per

forin

-FIT

C

Conclusion 2

The GranzymeB-mediated reciprocal killing mediated by RC

or Treg is IL-2-dependent

PI-9 expression in Treg and RC after co-culture + IL-2

CD4+CD25high 83 ± 5* 115 ± 10* 150* ± 12

CD4+CD25- 25 ± 12 25 ± 10 32 ± 2.5

150 IU/mL IL-2:

Expression of proteins that protect from apoptosis is Expression of proteins that protect from apoptosis is regulated by ILregulated by IL--22

CD4+CD25high 22 ± 6.7* 18 ± 4.58* 18 ± 2.5*

CD4+CD25- 85 ± 6.5 68 ± 12 58 ± 10

*p≤0.03 for differences between Treg and RC

CD8+CD25- 12 ± 1.8 28 ± 4.5 65 ± 5.8

*p≤0.001 for differences between Treg and RC

CD8+CD25- 95 ± 5.9 64 ± 8 54 ± 12

PI-9 AD NEDNC

1000 IU/mL IL-2: MFI ± SD

0

20

40

60

80

100

120

140

160

180

Foxp3high Foxp3 interm. Foxp3low

Bcl

-2 (M

FI±S

D)

NC n=15

HNSCC n=35

* p≤0.001

200Fresh PBMC

*

At low IL-2 concentrations, Treg co-cultured with RC do notup-regulate PI-9 and are sensitive to GrB-mediated apoptosis.

Conclusions

Treg can suppress RC via three different mechanismsTr1 largely use IL-10 and TGFβ1 to suppress RC proliferation (a contact independent process)CD8+RC expansion is suppressed by the Fas/FasL-mediated apoptosisCD4+RC proliferation is suppressed via a contact-dependent GrB/perforin pathway which is regulated by the IL-2 concentration in the microenvironment Resistance or sensitivity to death of Treg vs. RC is dependent on IL-2-mediated T cell activation