programmed death-1 (pd-1) in sle t cells: “that which does not kill us, makes us stronger” maida...

Programmed Death-1 (PD-1) in SLE T cells:

“That which does not kill us, makes us stronger”

Maida Wong. M.D.Assistant Clinical ProfessorDivision of Rheumatology

UCLA David Geffen School of Medicine

Systemic Lupus Erythematosus

Fate of a T cell

Von Herrath MG et al. Nature Imunology 2003

Fully activated APCCostimulation +++

Partially activated APCCostimulation +/-

Block activation

Suppression

Normal T cell response

Deletion

Naïve T cell

Why are Tregs important?

Stimulating self-Ag:

Tolerance

Immunological tolerance: Unresponsiveness to an Ag by exposing lymphocytes to that Ag (tolerogen)

• Breakdown of self-tolerance results in autoimmunity

Von Herrath MG et al. Nature Imunology 2003

Why does it matter?

Therapeutic potential Restore immune tolerance

Treat autoimmune & allergic diseases

Prevent graft rejection

PD-1 controls dysregulated T cell activation

Normal T cell activation

Bc-xLIL-2IFN-ϒ

activated T cells



Bc-xLIL-2IFN-ϒ

activated T cells

Anergy

PD-1

Functional unresponsiveness

Bc-xLIL-2IFN-ϒ



Bc-xLIL-2IFN-ϒ

activated T cells

Anergy

PD-1


Bc-xLIL-2IFN-ϒ

glucose metabolismprotein synthesisproliferationcell survival



Bc-xLIL-2IFN-ϒ

activated T cells

Anergy

PD-1


Bc-xLIL-2IFN-ϒ

glucose metabolismprotein synthesisproliferationcell survival

Abatacept (anti-CTLA-4)

What happens to a T after PD-1 is activated?

+TGF-β

What happens to a T after PD-1 is activated?

High PD-1: Cancer Infectious

disease

Low PD-1: Autoimmunity Allergy Transplant

rejection

+TGF-β

Okazaki, T et al. Int. Immunol. 2007

Anti-PD1 drugsFDA approvedNivolumab (Opdivo)Pembrolizumab (Keytruda)

In the pipelinePidilizumabREGN2810MDX1106-02

Anti-PD1 drugsFDA approvedNivolumab (Opdivo): melanoma, NSCL

Pembrolizumab (Keytruda): melanoma, NSCL

glioma, SCC (H&N)

In the pipelinePidilizumab: diffuse intrinsic pontine glioma, GBM

REGN2810MDX1106-02: Hepatitis C

Francisco LM, Sharpe A et al, Immunological Reviews, 2010.

What cells are we targeting with PD-1 & its ligand?

PD-1 & SLE

Background: PD1/PDL1 plays a role in lupus-like autoimmunity1. Normal mouse (B6) KO PD1

2. Lupus mouse (BXSB) with increased PDL1 expression

• Protects from lupus nephritis Hypercellularity & IgG deposition in glomerulio Inhibited IgG productiono Delay onset of proteinuria & anti-dsDNA

IgG

C3

Inflammatory arthritisGlomerulonephritis

Nishimura H et al, Immunity, 1999

Ding H et al; Clin Immun 2006

PD-1 and T cells in SLE

Hahn LabLupus mouse (BWF1) tolerized with pCons (suppresses SLE):

• CD8+PD1+ cells

• mRNA of PD1 in CD8+ cells

Singh RP, Hahn BH et al. JImmunol 2007

Hypothesis•Regulation of signaling through PD-1 controls Tregs and autoimmunity in BWF1 lupus mice.

Hypothesis•Regulation of signaling through PD-1 controls Tregs and autoimmunity in BWF1 lupus mice.

[PD-1]

Dis

eas

e-fr

ee

SLE SLE

No SLE

Experiment to examine PD-1 in Tregs

10 wk old BWF1

In vivo

• Apoptosis• Flow cytometry• ELISA• Microarray• Clinical data

splenocytes

PBMC

anti-PD1 mAb 100 g IP qod x3

http://www.littlebuddhababy.com/images/rat_detail.gif

http://www.health.state.mn.us/divs/idepc/dtopics/stds/images/syringe.jpg

Clinical Results in Mouse SLE

anti-dsDNA

proteinuria

survival


anti-dsDNA

proteinuria

survival

Wong M, Hahn BH et al. JImmunol 2011


• PD-1 blockade results in suppression of autoantibody production.

• Anti-PD-1 delays nephritis and prolongs survival.

anti-dsDNA

proteinuria

survival



• PD-1 blockade results in suppression of autoantibody production.• Anti-PD1 added to pCons abrogates immune tolerance.

• Anti-PD-1 delays nephritis and prolongs survival.

anti-dsDNA

proteinuria

survival


Does the timing of PD-1 blockade matter?

30 32 34 36 38 40

0

25

50

75

100

Age (weeks)

% m

ice

with

pro

tein

uri

a >2

+

35 37 39 41 43 45 47 490

25

50

75

100 IgG isotypeanti-PD-1 (early)anti-PD-1 (late)

Age (weeks)

% s

urvi

val


Does the timing of PD-1 blockade matter?

30 32 34 36 38 40

0

25

50

75

100

Age (weeks)

% m

ice

with

pro

tein

uri

a >2

+

35 37 39 41 43 45 47 490

25

50

75

100 IgG isotypeanti-PD-1 (early)anti-PD-1 (late)

Age (weeks)

% s

urvi

val

•Later in disease, PD-1 blockade can delay disease progression, but the effect is diminished.


anti-PD1 in vivoIgG isotype in vivo

What is PD-1 blockade actually doing in Tregs?

PD1hi

PD1lo

CD4+CD25+

49

16

PD

1 30

33


PD1hi

PD1lo

10 15 20 25 300

1

2

3

4

IgG isotypeAge (weeks)

%C

D4

+ CD

25+ Fo

xp3

+ cel

ls


PD-1 blockade in Tregs increases Foxp3 expression

10 15 20 25 300

1

2

3

4

IgG isotypeanti-PD1

** *

Age (weeks)

%C

D4

+ CD

25+ F

ox

p3

+ cel

ls

* p < 0.05** p < 0.01


PD-1 blockade in Tregs increases Foxp3 expression

10 wk old BWF1

Treg +/- anti-PD1

In vitro

• Apoptosis• Flow cytometry•Functional assays• ELISA

Splenocytesfor Treg, Th & B cells

Th B

Experiment to examine PD-1 in Tregs

http://www.littlebuddhababy.com/images/rat_detail.gif

In vitro blockade of PD-1 expression in Tregs

PD1hiPD1hi

CD4+Treg from IgG isotype control mice:

without anti-PD1 [anti-PD1] 75 mg/mL in vitro

PD1loPD1lo

CD4+CD25+

PD

1 64

17

19

44


0 25 50 75 1000

5

10

15

20

25

30

35

40

[anti-PD1] (g/ml)

%7

AA

D- A

nn

ex

V+ C

D4

+ CD

25+ c

ells

0 25 50 75 1000

25

50

75

[anti-PD1] (g/ml)

%7

AA

D- A

nn

ex

V+ C

D19

+ cel

ls

Does the degree of PD-1 blockade matter?


0 25 50 75 1000

5

10

15

20

25

30

35

40** *

[anti-PD1] (g/ml)

%7

AA

D- A

nn

ex

V+ C

D4

+ CD

25+ c

ells

0 25 50 75 1000

25

50

75

* *

[anti-PD1] (g/ml)%

7A

AD

- An

ne

xV

+ CD

19+ c

ells


* p < 0.05** p < 0.01


0 25 50 75 1000

5

10

15

20

25

30

35

40** *

[anti-PD1] (g/ml)

%7

AA

D- A

nn

ex

V+ C

D4

+ CD

25+ c

ells

0 25 50 75 1000

25

50

75

* *

[anti-PD1] (g/ml)%

7A

AD

- An

ne

xV

+ CD

19+ c

ells


* p < 0.05** p < 0.01


•The amount of PD-1 expression has to be finely tuned – neither absent nor high – for effective suppressive function in Tregs.

1. Attenuated PD-1 expression makes Treg more effective as suppressor cells:

survival:

• Treg

survival: • Th• B


2. Attenuated PD-1 expression alters cytokine production of Tregs that controls inflammation:

production: • TGF-β • IL-2

production: • anti-dsDNA• IgG• IFN-γ• IL-6• IL-10

1. Attenuated PD-1 expression makes Treg more effective as suppressor cells:

survival:

• Treg



Genes influenced by PD-1 that alters Treg fitness

Gene Fold up- or t-test down regulation p-value

Tcf7 7.54 0.018Bcl2l1 7.16 0.002Birc5 4.47 0.002Ccl2 -3.59 0.012Hk2 -4.2 0.028

Brca1 -4.94 0.001Cd5 -5.89 0.015

Cdkn1a -9.09 0.06Tfrc -10.41 0.0001Fasl -23.12 0.027Fn1 -23.97 0.034

Zap70/Syk

NFATNFB

Gene Up-/down- t-test regulation (fold) p-value

Prdx2 4.47 0.002Bcl2 3.84 0.002Birc5 3.36 0.05Traf2 -3.24 0.06

Tnfsf10 -3.61 0.01Card6 -3.67 0.002

Casp8 -5.43 0.002Tbfrsf10b -6.35 0.002

Traf3 -6.41 0.002Pycard -6.58 0.002Traf1 -6.6 0.002Fasl -7.39 0.002

Cd40lg -18.71 0.002

Pro-apoptotic

anti-apoptotic(TNFR)




Brca1 -4.94 0.001Cd5 -5.89 0.015


Zap70/Syk

NFATNFB



Tnfsf10 -3.61 0.01Card6 -3.67 0.002

Casp8 -5.43 0.002Tbfrsf10b -6.35 0.002


Cd40lg -18.71 0.002

Pro-apoptotic

anti-apoptotic(TNFR) - OX40




Brca1 -4.94 0.001Cd5 -5.89 0.015


Zap70/Syk

NFATNFB



Tnfsf10 -3.61 0.01Card6 -3.67 0.002

Casp8 -5.43 0.002Tbfrsf10b -6.35 0.002


Cd40lg -18.71 0.002

Pro-apoptotic

anti-apoptotic(TNFR)- OX40

Arrest: DNA damage Anti-proliferative factors UV Lack of growth factors

Cell Cycle

High PD-1 signaling arrests cell cycle progression to the G1 phase in Tregs

IgG

anti-

PD10

10

20

30

40

50

60p =0.006

%S

ph

ase

IgG

anti-

PD10

50

100

150

200p = 0.05

G1

arre

st i

nd

ex

Summary

• Regulation of PD-1 appears critical to the generation and maintenance of regulatory CD4+ T cells during immune tolerance.

• Quantitative & timing of PD-1 expression to Ag has to be finely tuned – neither absent nor high – to enable Tregs to control autoimmunity.

• PD-1 blocks cell cycle progression and proliferation in Tregs, possibly between the G1-S phase.

Human Data

Patient Demographics

Patient Control n 60 30 Age (SD) 45.2 (15.7) 43.1 (13.5) % Female 91.7 93.3 Ethnicity

Causasian 0.52 0.67Black 0.07 0.07Asian 0.17 0.20

Hispanic 0.25 0.07 SLEDAI 10.8 (4.4) 0

Higher intensity of PD-1 expression in Tregs of PBMC from SLE patients

Healthy ctrls SLE pts0.0

2.5

5.0

7.5p = 0.02

%C

D4

+C

D25

hi F

oxp

3+ c

ells



2.5

5.0

7.5p = 0.02

%C

D4

+C

D25

hi F

oxp

3+ c

ells

Healthy ctrls SLE pts0

10

20

30

40

50

p < 0.002

%C

D4

+ CD

25h

i Fo

xp

3+ c

ells

be

ing

PD

1+



2.5

5.0

7.5p = 0.02

%C

D4

+C

D25

hi F

oxp

3+ c

ells


10

20

30

40

50

p < 0.002

%C

D4

+ CD

25h

i Fo

xp

3+ c

ells

be

ing

PD

1+

SLEDAI <4 SLEDAI >40

10

20

30

40

50

p = 0.07

%C

D4+ C

D2

5hi F

ox

p3+

ce

lls b

ein

g P

D1+



2.5

5.0

7.5p = 0.02

%C

D4

+C

D25

hi F

oxp

3+ c

ells


10

20

30

40

50

p < 0.002

%C

D4

+ CD

25h

i Fo

xp

3+ c

ells

be

ing

PD

1+

SLEDAI <4 SLEDAI >40

10

20

30

40

50

p = 0.07

%C

D4+ C

D2

5hi F

ox

p3+

ce

lls b

ein

g P

D1+

•SLE patients have increased PD-1 expression in Tregs compared to healthy individuals & patients with mild disease.

Wong M, Hahn BH et al. Arthritis Rheum 2014. (Abstract)

Clinical symptoms associated with increased PD-1

Clinical manifestation %PD1hiCD4+CD25+ cells p-value

no symptoms with symptoms

Vasculitis 7.93 ± 1.59 15.54 ± 3.94 0.029

Renal 10.48± 5.26 21.45 ± 9.18 0.052

Thrombocytopenia (plt < 1K) 6.64 ± 1.03 11.55 ± 3.46 0.036

Leukopenia (WBC < 3.5) 6.61 ± 1.41 11.30 ± 2.69 0.014

Oral/Nasal ulcers 8.84 ± 1.47 11.19 ± 5.61 0.023

Rash (acute, subacute, discoid) 10.02 ± 1.93 6.79 ± 1.60 0.014

Photosensitivity 9.95 ± 1.68 3.52 ± 1.07 0.013


Low PD-1 expression induced suppressivity in Treg of SLE patients

0 2.5 10 200

5

10

15

20

25

**

p <0.05 (1-way ANOVA)

[anti-PD1] (g/ml)

%A

nn

ex

inV

+ 7A

AD

- CD

4+ C

D25

hi ce

lls


0 2.5 10 200

5

10

15

20

25

**


[anti-PD1] (g/ml)

%A

nn

ex

inV

+ 7A

AD

- CD

4+ C

D25

hi ce

lls

0 100

20

40

60

p < 0.05

[anti-PD1] to treat CD4+Treg (g/ml)

%A

nn

exin

V+7A

AD

- CD

19+ c

ells


0 2.5 10 200

5

10

15

20

25

**


[anti-PD1] (g/ml)

%A

nn

ex

inV

+ 7A

AD

- CD

4+ C

D25

hi ce

lls

0 100

20

40

60

p < 0.05


%A

nn

exin

V+7A

AD

- CD

19+ c

ells

0 100.0

0.5

1.0

1.5

2.0 p <0.005


CD

4+C

D25

- Th

cell

divi

sio

n in

dex

n = 11


0 2.5 10 200

5

10

15

20

25

**


[anti-PD1] (g/ml)

%A

nn

ex

inV

+ 7A

AD

- CD

4+ C

D25

hi ce

lls

0 100

20

40

60

p < 0.05


%A

nn

exin

V+7A

AD

- CD

19+ c

ells

0 100.0

0.5

1.0

1.5

2.0 p <0.005


CD

4+C

D25

- Th

cell

divi

sio

n in

dex

•Tolerance can be induced in Treg by PD-1 blockade at a fine-tuned concentration.


n = 11

Attenuated PD-1 expression alters cytokine production of Treg that controls inflammation:



Attenuated PD-1 expression makes Treg more effective as suppressor cells:

survival:

• Treg


Attenuated PD-1 expression alters cytokine production of Treg that controls inflammation:



Attenuated PD-1 expression makes Treg more effective as suppressor cells:

survival:

• Treg


Reprise of lupus mouse data

Future DirectionsMouse• Bone marrow chimeric model• PD-1 on/off switch

Human• PDL1 expression on target cells• TNFR: OX40• Renal disease• Clinical application

Summary

• SLE patients have aberrant, increased PD-1 expression in their circulating Tregs that may reduce the regulatory function of Foxp3+

Tregs, which are important in the suppression of autoimmunity.

• One mechanism by which PD-1 sustains these Tregs is by reducing their susceptibility to apoptosis.

Conclusion

PD-1 influences autoimmunity in part via its effect on Tregs.

PD-1 expression in Tregs must be tightly controlled in a graded fashion to maintain their numbers and suppressive functions.

PD-1 has the potential to be a target for treatment of SLE.

Acknowledgements Bevra Hahn Betty Tsao Antonio La Cava Daniel Furst Jennifer Grossman Elaine Lourenço

Grant support NIH (T32) Arthritis Foundation (Local & National Chapter) American College of Rheumatology Arthritis National Research Foundation

George Tsokos

Acute infection

Chronic infection

Revival of exhausted cells

Revival of “Exhausted” T cells

Williams MA et al. Nature Immunology, 2006.

programmed death-1 (pd-1) in sle t cells: “that which does not kill us, makes us stronger” maida...

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