Costimulation and the immune response

• There are positive and negative second signals• Costimulation regulates naive and activated cells• Expanding complexity of T cell costimulation

B7:CD28 family TNF:TNFR family CD2 family

+-

The B7/CD28 superfamily provides T cell costimulatio n

The B7/CD28 superfamily provides T cell costimulatio n

Comparing PD-1 with other CD28 Family Members

late lethalityhyperactivation

early lethalityhyperactivation

↓↓↓↓T cell activation

Knockout

PD-L1/PD-L2B7-1/B7-2B7-1/B7-2Ligand

ITIM / ITSMITIM-like domainNo ITIMSignaling

inducibleinducibleconstitutiveKinetics

T, BT cellsT cellsExpression

PD-1CTLA-4CD28

PD-L1 is broadly expressed in tissues and lymphoid tissues, while PD-L2 is expressed only on APCs

PD-L1

Parenchymal

Placenta, eye, vascular endothelium,

pancreatic islets

None

Hematopoietic

B cells, T cells, DCs, MΦs

DCs, MΦsPD-L2

TCR/CD3

MY

PP

PY

YYYY

CD28M

YP

PP

YYYYY

MY

PP

PY

YYYY P

Proximal signaling kinases

Transcriptional activation

Y

Y

SHP-2PD-1

Inhibition of antigen receptor

signaling

MHC

PD-LB7

PD-1 inhibits antigen receptor signaling

PD-1 is negative regulator of T cell activation

CFSE

PD

-1

Gated on CD8+ cells

• PD-1 is upregulated upon activation of lymphocytes.

• PD-1-/- mice develop spontaneous autoimmunity.

• PD-1 signaling can affect cytokine production and cell

cycle progression.

Lymphoid organs

NaiveCD4+

NaiveCD8+

Antigen Recognition

Activation/Clonal

Expansion

IL-2

IL-2

Differentiation

Effectorfunctions

Effector CD4 +

Peripheral tissues

Activation of B cells, macrophages, etc.

Memory CD4 +

Effector CD8 +

Killing of target cells, macrophage activation

Memory CD8 +

PD-1 in the priming and effector phase of the immune response

PD-L have overlapping roles in naïve T cell priming by DCs

WT DCsPD-L1-/- DCsPD-L2-/- DCsPD-L1/L2-/- DCs

104 naïve DO11.10 T cells were stimulated 1:1 with BM derived DCs

4

8

12

16

1 2 3 4 5

ng/m

l

DAY

0.2

0.4

0.6

0.8

1

1 2 3 4 5DAY

IFN-γγγγIL-2

DCs

OT-I

RIP-ovahi pancreas

Do naïve CD8 T cells that lack PD-1 display tolerance to peripheral self-antigens?

Ovalbumin-specific CD8+ T cells

PD-1-/- OT-I T cells are increased in the pancreatic LN and traffic into the pancreas

WT OT-I

PD-1-/- OT-I

0

1

2

3

4

3 5 7 14

0

1

2

3

4

Cal

cula

ted

# O

T-I

(x1

04 )

3 5 7 14Days post-transfer

ILN

Days post-transfer

PLN

0

1

2

3

3 5 7 14

pancreas

Days post-transfer

*

*

* *

* = p<0.05

Increased effector cells and infiltration of tissue s by PD-1 -/- OT-I T cells

Pancreas

WT PD-1-/-

7 days after transfer of 5 x 106 PD-1-/-OT-I or OT-I T cells into RIP-ovahi recipient

PLN

%D

ivid

ed

ILN

WT OT-I

PD-1-/- OT-I

0

20

40

60

80

3 5 7 14

*

0

10

20

30

40

50

3 5 7 14

Lymphoid organs

NaiveCD4+

NaiveCD8+

Antigen Recognition

Activation/Clonal

Expansion

IL-2

IL-2

Differentiation

Effectorfunctions

Effector CD4 +

Peripheral tissues

Activation of B cells, macrophages, etc.

Memory CD4 +

Effector CD8 +

Killing of target cells, macrophage activation

Memory CD8 +

PD-1 in the priming and effector phase of the immune response

Naïve PD-1-/- OT-I T cells induce diabetes in RIP-ova hi mice

Adoptive transfer of of 5 x 106 purified OT-I CD8+ cells (intravenous)

0

20

40

60

80

100%

Dia

betic

3 6 9 12 15 18 21 24 27 30

OTI (n=5)

PD-1-/- OTI (n=4)

Draining Lymph Node

Target tissue

Auto-antigen

Migration to target tissue

Pancreas

T cell mediated autoimmune destruction

Pathogenic T cell survival in target tissue

Activation of autoreactive T cell

by APC

PD-1-/-

Is PD-1 exerting a cell-intrinsic effect on cross-p resentation?

WT

PD-1 exerts a cell-intrinsic effect on cell divisio n

CFSE

Thy

1.2

ILN

WT OT-I

PD-1-/- OT-I

51±3

49±3

pancreas

83±4

17±4

PLN

66±3

34±3

OT-I only

OT-I from OT-I + PD-1-/- OT-I

0

20

40

60

%D

ivid

ed

ILN PLN

NOD mouse model of autoimmune diabetes

• Non-obese diabetic (NOD) mice are an in-bred strain that develop autoimmune T-cell mediated insulin-dependent diabetes mellitus (IDDM).

• Autoimmunity in the NOD mice has been shown to be under complex heritable polygenic control.

• The development of autoimmunity in the NOD mouse is also influenced by diet and environmental factors.

• Early onset of insulitis in PD-L1/PD-L2-/- NOD mice• Increased severity of insulitis in PD-L1/PD-L2-/- NOD mice• Increased number of T cells in pancreatic LN• Increased cytokine production by T cells from pancreatic LN• Early onset diabetes phenotype is found in PD-L1-/- but not

PD-L2-/- NOD mice

Week

WTNOD maleWT NOD femalePD-L1/PD-L2-/- NOD malePD-L1/PD-L2-/- NOD female

0

25

50

75

100

% D

iabe

tic

n=9 n=16

n=12

n=13

3 6 9 12 15 18 21 24 27 30 33 36 39

Does PD-L1/PD-L2 expression protect islets from T cell mediated autoimmunity?

Week

107 T cells from non-diabetic wild type NOD mice were transferred into wild type or PD-L1/L2 -/- NOD SCID mice.

PD-L1/PD-L2 expression on non-lymphoid cells inhibits autoimmune diabetes

T cells

WT→WT NOD SCID

WT→PD-L1/L2-/- NOD SCIDn=5

n=6

%D

iabe

tic

0

20

40

60

80

100

2 4 6 8 10 12 14

p=0.0006

0

20

40

60

80

100

%D

iabe

tic

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

WT NOD SCIDPD-L1/PD-L2-/- NOD SCIDBDC2.5 CD4+

Effectors

p=0.02

PD-L1/PD-L2 expression can regulate CD4 + effector T cell function

2 x 105 BDC2.5 CD4+CD25-CD62L+ T cells from BDC2.5 WT NOD mice were transferred into WT NOD SCID or PD-L1/PD-L2-/- NOD SCID mice.

n=9n=9

Days

% o

f CD

4+ce

lls

0

4

8

12

16 PLN*

*

*

WT�

KO

WT�

WT

IFN-γ+W

T�

KO

WT�

WT

TNF-α+

IFN-γ+

WT�

KO

WT�

WT

TNF-α+

PD-L1/PD-L2 expression can limit CD4 + T celleffector cytokine production

2 x 105 BDC2.5 CD4+CD25-CD62L+ T cells from BDC2.5 WT NOD mice were transferred into WT NOD SCID or PD-L1/PD-L2-/- NOD SCID mice. Recipients were sacrificed 7 days after transfer.

*p<0.05

Can PD-L1/PD-L2 expression on antigen presenting ce lls protect against rapid induction of diabetes ?

PD-L1

PD-L1

CD11c+

CD11c+

Adoptive transfer prediabetic T cells into BM chimeras

PD-L1/PD-L2 expression on APCs, but not on parenchymal cells

No PD-L1/PD-L2 expression on APCs or parenchymal cells

A

B

Non-diabetic WT NOD

T cells

PD-L1/PD-L2-/- NOD SCID BM→PD-L1/PD-L2-/- NOD SCID

CD11c+

B

WT NOD SCID BM→PD-L1/PD-L2-/- NOD SCID

PD-L1

CD11c+A

WT→CHIMERA AWT→CHIMERA B%

Dia

betic

T cells

0

20

40

60

80

100

2 4 6 8 10 12

n=5 n=9

Week

p=0.31

PD-L1/PD-L2 expression on antigen presenting cells does not protect against rapid induction of diabetes

PD-L1 expression on islets protects against self-re active T cells

PD-L1 on islets inhibits self-reactive T cells

PancreasPancreatic

LN

Self-Ag

Self-reactive T cells

PD-L1

Draining Lymph Node

Target tissue

Auto-antigen

Migration to target tissue

Pancreas

T cell mediated autoimmune destruction

Pathogenic T cell survival in target tissue

Activation of autoreactive T cell

by APC

PD-1

PD-1 can function at two checkpoints

PD-1

Draining Lymph Node

Target tissue

Auto-antigen

Migration to target tissue

Pancreas

T cell mediated autoimmune destruction

Pathogenic T cell survival in target tissue

Activation of autoreactive T cell

by APC

PD-1

PD-L1

PD-1 delivers stronger inhibition of effector cells

A strong association between PD-1:PD-L and cancer

Breast cancerGhebeh et al, Neoplasia 8:190Ghebeh et al, Int J Canc 121:751

Esophageal cancerOhigashi et al, Clin Canc Res 11:2947

Gastric carcinoma Sun et al, Tissue Antigens 69:19Wu et al, Acta Histochem 108:19

Glioma Parsa et al, Nat Med 13:84

LymphomaDorfman et al, Am J Surg Path 30:802Yang et al, Blood 107:3639

Non-small cell lung cancerKonishi et al, Clin Canc Res 10:5094

Oral squamous cell carcinomaTsushima et al, Oral Oncol 42:268

Ovarian cancerHamanishi et al, Proc Natl Acad Sci104:3360

Pancreatic cancer Nomi et al, Clin Canc Res 13:2151

Renal cell carcinoma Thompson et al, Clin Canc Res 13:1757Krambeck et al, Clin Canc Res 13:1749Thompson et al, Canc Res 66:3381Thompson et al, Cancer 104:2084Thompson et al , Urology 66:10

Urothelial cancer Nakanishi et al, Canc Immunol Immunother 56:1173Inman et al, Cancer 109:1499

PD-1:PD-L1 and cancer

Draining Lymph Node

Tumor antigen

Auto-antigen

Migration to target tissue

Tumor

T cell mediated tumor destruction

Tumor infiltrating lymphocyte (TIL)

Activation of autoreactive T cell

by APC

PD-1

PD-1:PD-L1 and cancer

Draining Lymph Node

Tumor antigen

Auto-antigen

Migration to target tissue

T cell mediated tumor destruction

Activation of autoreactive T cell

by APC

PD-1

Tumor

Tumor infiltrating lymphocyte (TIL)

PD-1 expression on TIL

PD-L1 expression on

tumor cells

Can PD-1:PD-L1 blockade invigorate the anti-tumor immune response?

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Tumor clearance

Tumor tolerance

Tumor Ag-specific

lymphocyte

Tumor Ag

The promise of PD-1:PD-L1 blockade

• Increased anti-tumor T cell responsesBlank et al, Int J Cancer 119:317

• Increased tumor clearanceIwai et al, Proc Nat’l Acad Sci 99:12293Iwai et al, Int Immunol 17:133

• Increased efficacy of other anti-tumor therapiesHirano et al, Cancer Res 65:1089Nomi et al, Clin Cancer Res 13:2157

B7-1 also can bind and signal through PD-L1

• B7-1 interacts more strongly with PD-L1 (1.7 µM) than with CD28 (4 µM) but less strongly than with CTLA-4 (0.2 µM).

• B7-1:PD-L1 binding site overlaps with B7-1:CTLA-4 and PD-L1:PD-1 interfaces.

• Multiple classes of mAbs: single vs. dual blockers of interactions.

• B7-1:PD-L1 interaction bidirectionally inhibits T cell proliferation and cytokine production.

• Increased significance of B7-1 and PD-L1 on T cells.

Butte, Keir et al, Immunity (in press)

Lee AlbackerManish Butte

Yvette LatchmanSpencer LiangArlene Sharpe

Harvard Medical School

Gordon FreemanDana Farber Cancer Institute

Indira GuleriaMohamed Sayegh

Brigham and Women’s Hospital

Andi QipoMaria Koulmanda

Massachusetts General Hospital

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

This work was supported by the Cancer Research Institute