Comparison of Viral Immunity in Stable Renal Allograft Recipients

The 12th Joint Annual Congress of the American Society of Transplant Surgeons and The American Society of Transplantation

Yvonne Suessmuth, PhD Postdoctoral Fellow Emory Transplant Center, Atlanta, GA I have no financial relationships to disclose within the past 12 months relevant to my presentation

My presentation does not include discussion of off-label or investigational use I do not intend to reference unlabeled/unapproved uses of drugs or products in my presentation.

Comparison of Viral Immunity in Stable Renal Allograft Recipients Treated with Belatacept or Tacrolimus Yvonne Suessmuth PhD, PW Thompson; C Breeden; B Johnson; R Jones; LA Stempora; J Cheeseman; J Joseph; B Begley; S Thomas; AD Kirk; K Newell; CP Larsen; AK Mehta Emory Transplant Center Emory Transplant Center

Belatacept • Newly approved high-affinity CTLA4Ig variant • Blocks interaction of CD28 with

•

CD80/86 Inhibits T cell proliferation and differentiation Improved GFR in belatacept groups vs. CSA Improved metabolic control

• • Increased incidence of EBV related PTLD • Very little data on impact of belatacept on protective immunity

10 9

8.8

Bela EBV (-) [n = 91] Bela EBV (+) [n = 810) CsA EBV (-) [n = 57] CsA EBV (+) [n = 399]

8 7

Patients (%)

•

X X

6

5.49

5 4

3.3

3 1.75

2 1

1.75

0.74 0.25

0 All PTLD

0.49 0 CNS PTLD

0

0.25

0.25

Non-CNS PTLD

Larsen et al. Am J Transplant 2006; 6: 876 – 883.

In vitro treatment of PBMCs with Belatacept does not inhibit EBV specific memory 1.5

ns

** ns

1.0

p = 0.0139

p = 0.0087

p = 0.0060

p =0.0056

0.5

%CD8+ T cell Double Producers

% Double Producer CD8s

1.5

ns

1.0

p = 0.1404

0.5

0.0 unstimulated

No drug

unstimulated 2 mcg Bela

unstimulated 20 mcg Bela

unstimulated 200 mcg Bela

unstimulated

TS-1

0.0 unstimulated

No drug

2 mcg Bela

20 mcg Bela 200 mcg Bela

TS-1

Figure 2: Paired analysis of the unstimulated condition by each conditions

Mehta AK, et al. ATC 2011

In vitro treatment of PBMCs with Belatacept does not inhibit EBV specific memory 1.5

ns

** ns

1.0

p = 0.0139

p = 0.0087

p = 0.0060

p =0.0056

0.5

%CD8+ T cell Double Producers

% Double Producer CD8s

1.5

ns

1.0

p = 0.1404

0.5

0.0 unstimulated

No drug

unstimulated 2 mcg Bela

unstimulated 20 mcg Bela

unstimulated 200 mcg Bela

unstimulated

TS-1

0.0 unstimulated

No drug

2 mcg Bela

20 mcg Bela 200 mcg Bela

TS-1

Figure 2: Paired analysis of the unstimulated condition by each conditions

• Lack of data on viral specific protective immunity in patients treated with belatacept Mehta AK, et al. ATC 2011

Study Design and Patient populations • Subjects were enrolled from existing immune monitoring protocols at Emory University

• Peripheral blood samples were collected at a single timepoint • Phenotypic and functional analysis of peripheral blood lymphocytes were performed using rationally-designed and validated flow cytometric panel

• 10 healthy volunteers • 10 transplant recipients (>6mos s/p renal allograft) on stable dose of Belatacept, MMF, and prednisone

• 10 transplant recipients (>6mos s/p renal allograft) on stable dose of Tacrolimus, MMF, and prednisone

Subject Characteristics Group

Treatment

Number (n)

Median Age (range)

Sex (M/F)

# months s/p txp

Seropositive (EBV/CMV)

Healthy Controls

None

10

43.8 (29- 55)

5/5

n/a

9/5

Tacrolimus + MMF + Prednisone

10

49.3 (34- 66)

6/4

51.7 (6- 120)

10/7

Belatacept + MMF +Prednisone

10

51.1 (34- 63)

4/6

92.2 (44- 128)

9/7

Tacrolimus (>6m s/p kidney txp)

Belatacept (>6m s/p kidney txp)

Subject Characteristics Group

Treatment

Number (n)

Median Age (range)

Sex (M/F)

# months s/p txp

Seropositive (EBV/CMV)

Healthy Controls

None

10

43.8 (29- 55)

5/5

n/a

9/5

Tacrolimus + MMF + Prednisone

10

49.3 (34- 66)

6/4

51.7 (6- 120)

10/7

Belatacept + MMF +Prednisone

10

51.1 (34- 63)

4/6

92.2 (44- 128)

9/7

Tacrolimus (>6m s/p kidney txp)

Belatacept (>6m s/p kidney txp)

Methods • PBMCs were rested 8h in 10% RPMI, then stimulated for 12h with either: • CMV pp65 PepMix = 15-mers overlapping by 11 aa covering the length of pp65 • EBV BZLF PepMix = 15-mers overlapping by 11 aa covering the length of BZLF • EBV Peptide pool = Peptides from several EBV proteins but restricted by HLA types • Cells were then stained for the following markers:

FITC

PE

PerCPCy5.5

APC

PE-Cy7

Alexa 700

V450

Qdot 655

APC-Cy7

Pac Orange

CD28

CD27

IFNγ

TNFα

CD4

CD8

CD3

CD45RA

CCR7

CD14/CD20 +Live/Dead

MIP1β

CD107a

IFNγ

TNFα

IL-2

CD8

CD3

CD45RA

CCR7

CD14/CD20 +Live/Dead

MIP1β

CD154

IL-17

CCR5

CD4

CD8

CD3

CD45RA

CCR7

CD14/CD20 +Live/Dead

Methods • PBMCs were rested 8h in 10% RPMI, then stimulated for 12h with either: • CMV pp65 PepMix = 15-mers overlapping by 11 aa covering the length of pp65 • EBV BZLF PepMix = 15-mers overlapping by 11 aa covering the length of BZLF • EBV Peptide pool = Peptides from several EBV proteins but restricted by HLA types • Cells were then stained for the following markers:

FITC

PE

PerCPCy5.5

APC

PE-Cy7

Alexa 700

V450

Qdot 655

APC-Cy7

Pac Orange

CD28

CD27

IFNγ

TNFα

CD4

CD8

CD3

CD45RA

CCR7

CD14/CD20 +Live/Dead

MIP1β

CD107a

IFNγ

TNFα

IL-2

CD8

CD3

CD45RA

CCR7

CD14/CD20 +Live/Dead

MIP1β

CD154

IL-17

CCR5

CD4

CD8

CD3

CD45RA

CCR7

CD14/CD20 +Live/Dead

Gating Strategy Lymphocytes

Live/Dead +CD14/CD20

FSC-H

SSC-A

97.3

62.2 FSC-A

Live CD3 cells

Singlets

75.5 FSC-A

CD3

Gating Strategy Lymphocytes

Live/Dead +CD14/CD20

FSC-H

SSC-A

Live CD3 cells

Singlets

97.3

62.2

75.5

FSC-A

FSC-A

CD3

CD4 vs CD8 cells

CD4

40

55

CD8

Gating Strategy Lymphocytes

Live CD3 cells Live/Dead +CD14/CD20

SSC-A

FSC-H

Singlets

97.3

62.2

75.5

FSC-A

FSC-A

CD3

CD4 vs CD8 cells 11.7

32.1

CD4

CD27

40

54.8

0.0395

1.33

0.655

55 TNF

CD28

CD8 99.1

0.179 IFN

Belatacept treated patients show no difference in TNFα/IFNγ production in CD4 cells compared to Tacrolimus Frequency of TNFa+/IFNg+ CD4 cells

0.3 0.2 0.1

pe V EB

V C M

pt

at

id

ed

e

0.0

ul

IFN

0.4

im

0.142

Healthy Control Belatacept Tacrolimus

st

99.4

8 6 4 2

un

0.229

TNF

0.259

% CD4 TNF+/IFN +

Belatacept patient CD4 EBV stimulated

Belatacept treated patients show no difference in TNFα/IFNγ production in CD8 cells compared to Tacrolimus Belatacept patient CD8 EBV stimulated 0.0369

Frequency of TNFα+/IFNγ+ CD8 cells

0.803

TNF

12

98.6

0.628 IFN

Belatacept patient CD8 CMV stimulated 0.0557

0.502

% CD8 TNF+/IFN +

8 4

Belatacept Tacrolimus Healthy Control

1.5

1.0

0.5

V M C

e pe V EB

0.689 IFN

un

98.8

st

im

ul

pt

at

id

ed

TNF

0.0

Belatacept treated patients show no difference in TNFα/IFNγ production in CD8 cells compared to Tacrolimus Belatacept patient CD8 EBV stimulated 0.0369

Frequency of TNFα+/IFNγ+ CD8 cells

0.803

TNF

12

98.6

0.628 IFN

Belatacept patient CD8 CMV stimulated 0.0557

0.502

% CD8 TNF+/IFN +

8 4

Belatacept Tacrolimus Healthy Control

1.5

1.0

0.5

V M C

e pe V EB

0.689 IFN

un

98.8

st

im

ul

pt

at

id

ed

TNF

0.0

Belatacept patients show more TNFα/IFNγ production in Central Memory cells but lower in Naïve CD8 cells in response to EBV stimulation Central Memory CD8

p= 0.028

2.0

% CD8 TNF+/IFN +

4

p= 0.054

HC EBV Bela EBV Tac EBV

CCR7

2

0.5

Ta c

EB

V

V EB el a B

H C

V EB c Ta

B

el a

EB

V

EB V

Stimulation Groups

EB V

0.0

0

TCM

1.5

HC EBV Bela EBV1.0 Tac EBV

H C

% CD8 TNF+/IFN +

6

Naive CD8

Naive

TEM TEMRA CD45RA

Belatacept patients show more TNFα/IFNγ production in Central Memory cells but lower in Naïve CD8 cells in response to EBV stimulation Central Memory CD8

p= 0.028

2.0

% CD8 TNF+/IFN +

4

p= 0.054

HC EBV Bela EBV Tac EBV

CCR7

2

0.5

Ta c

EB

V

V EB el a B

H C

V EB c Ta

B

el a

EB

V

EB V

Stimulation Groups

EB V

0.0

0

TCM

1.5

HC EBV Bela EBV1.0 Tac EBV

H C

% CD8 TNF+/IFN +

6

Naive CD8

Naive

TEM TEMRA CD45RA

Belatacept patients show more TNFα/IFNγ production in Central Memory cells but lower in Naïve CD8 cells in response to EBV stimulation Central Memory CD8

p= 0.028

2.0

% CD8 TNF+/IFN +

4

p= 0.054

HC EBV Bela EBV Tac EBV

CCR7

2

0.5

Ta c

EB

V

V EB el a B

H C

V EB c Ta

B

el a

EB

V

EB V

Stimulation Groups

EB V

0.0

0

TCM

1.5

HC EBV Bela EBV1.0 Tac EBV

H C

% CD8 TNF+/IFN +

6

Naive CD8

Naive

TEM TEMRA CD45RA

Belatacept patients show more TNFα/IFNγ production in Central Memory cells but lower in Naïve CD8 cells in response to EBV stimulation Central Memory CD8

p= 0.028

2.0

% CD8 TNF+/IFN +

4

HC EBV Bela EBV Tac EBV

CCR7

2

TCM

1.5

HC EBV Bela EBV1.0 Tac EBV 0.5 0.0

V Ta c

Effector Memory RA CD8

2.5 2.0

HC EBV Bela EBV Tac EBV

1.5 1.0 0.5 0.0

% CD8 TNF+/IFN +

1.5

1.0

HC EBV Bela EBV Tac EBV

0.5

V EB Ta c

el a

EB

V

EB V HC

Ta

c

EB

V

V EB el a B

H C

EB V

0.0

Stimulation Groups

Naive

TEM TEMRA CD45RA

EB

EB el a B

H C

EB c Ta

B

Stimulation Groups Effector Memory CD8

V

EB V

V

V EB el a

H C

EB V

0

% CD8 TNF+/IFN +

p= 0.054

B

% CD8 TNF+/IFN +

6

Naive CD8

In response to CMV stimulation Tacrolimus treated patients show higher production of TNFα/IFNγ in all CD8 Memory subsets Naive CD8

Central Memory CD8 % CD8 TNF+/IFN +

TCM

HC CMV

4

HC CMV

10

5

0

Tac CMV

CCR7

Tac CMV

2 1

V

V

V M

M

C c Ta

H

el

a

C

C

C

c

el B

V

V M

C

C a

M

M

M V C H C

Ta

Effector Memory RA CD8 % CD8 TNF+/IFN +

15 10 5

10

5

0

V M C c Ta

C el a B

C H

M

C M

V

V

V M C c Ta

C M a el B

C

C M

V

V

0

H

% CD8 TNF+/IFN +

15 20

TEM TEMRA CD45RA

0

Effector Memory CD8

Naive

Bela CMV

Bela CMV 3

B

% CD8 TNF+/IFN +

p=0.009

5

15

Belatacept treated patients show a robust trend towards increased CD27lo/CD28lo cells Healthy

Belatacept

Tacrolimus

Belatacept treated patients show a robust trend towards increased CD27lo/CD28lo cells Healthy

Belatacept

Tacrolimus

Belatacept treated patients show a robust trend towards increased CD27lo/CD28lo cells Healthy

Belatacept

Tacrolimus

Increased CD27lo/CD28lo cell numbers in Belatacept patients do not correlate with increased TNFα/IFNγ double producing cells in this population

0.5

0.0

Frequency of TNFa/IFNg producing CD8 cells

CMV

% CD8 TNF+/IFN +

HC EBV Bela EBV Tac EBV

1.0

HC CMV

15

Bela CMV Tac CMV

10

5

CD27/CD28 Subsets

D

27

-

27 +

-/ C

D

C

D

28

C 28 +/ D C

C

D

28

-/C

C D

D

27

27 +

-

0

D 28 +/ C

% CD8 TNF+/IFN +

Frequency of TNFa/IFNg EBVproducing CD8 cells

CD27/CD28 Subsets

Increased CD27lo/CD28lo cell numbers in Belatacept patients do not correlate with increased TNFα/IFNγ double producing cells in this population

0.5

0.0

Frequency of TNFa/IFNg producing CD8 cells

CMV

% CD8 TNF+/IFN +

HC EBV Bela EBV Tac EBV

1.0

HC CMV

15

Bela CMV Tac CMV

10

5

CD27/CD28 Subsets

D

27

-

27 +

-/ C

D

C

D

28

C 28 +/ D C

C

D

28

-/C

C D

D

27

27 +

-

0

D 28 +/ C

% CD8 TNF+/IFN +

Frequency of TNFa/IFNg EBVproducing CD8 cells

CD27/CD28 Subsets

Increased CD27lo/CD28lo cell numbers in Belatacept patients do not correlate with increased TNFα/IFNγ double producing cells in this population

0.5

0.0

Frequency of TNFa/IFNg producing CD8 cells

CMV

% CD8 TNF+/IFN +

HC EBV Bela EBV Tac EBV

1.0

HC CMV

15

Bela CMV Tac CMV

10

5

CD27/CD28 Subsets

D

27

-

27 +

-/ C

D

C

D

28

C 28 +/ D C

C

D

28

-/C

C D

D

27

27 +

-

0

D 28 +/ C

% CD8 TNF+/IFN +

Frequency of TNFa/IFNg EBVproducing CD8 cells

CD27/CD28 Subsets

Conclusions • Belatacept treatment does not appear to significantly impact virus-specific immune function as compared to Tacrolimus treatment. • Differences in TNFα/IFNγ production are possibly due to the difference in cohorts but need further investigation

• Differences observed between healthy controls and treated patients in memory subsets suggest that immunosuppressive agents influence how viral-specific memory is maintained

• Increased numbers of late differentiated cells (CD27lo/CD28lo) in Belatacept patients do not coincide with significantly decreased viral- specific immunity in these patients.

Future Plans • Enroll further 10 early post transplant Belatacept and 10 late post transplant Tacrolimus treated patients to ensure better comparison between the groups.

• Monitor patients longitudinally in the CTOT10 Trial comparing long-term treatment with Belatacept to Tacrolimus

Acknowledgments Special Thanks To: • Christian P Larsen • Aneesh K Mehta • Allan D Kirk • Kenneth Newell

• • • • •

Peter Thompson Linda Stempora Cindy Breeden Brandi Johnson He Xu

ETC Biorepository • Rachelle Jones • Stephanie Monday • Kendra Bryant • Jennifer Cheeseman ETC Clinical Research Coordinators • Elizabeth Begley • Shine Thomas • Elizabeth Ferry The Patients!

Grant support: A portion of this work was performed as part of the Clinical Trials in Organ Transplantation, supported by the National Institute of Allergy and Infectious Diseases.