Yvonne Suessmuth, PhD Postdoctoral Fellow Emory Transplant Center, Atlanta, GA

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

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.

Yvonne Suessmuth, PhDPostdoctoral Fellow

Emory Transplant Center, Atlanta, GA

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 CenterEmory Transplant

Center

8.8

5.49

3.3

0.74 0.49 0.25

1.75

0

1.75

0.25 0 0.250

1

2

3

4

5

6

7

8

9

10

All PTLD CNS PTLD Non-CNS PTLD

Patie

nts (

%)

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

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

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

XX

unstimulated No drug 2 mcg Bela 20 mcg Bela 200 mcg Bela TS-10.0

0.5

1.0

1.5

%C

D8+

T c

ell D

oubl

e Pr

oduc

ers

ns

ns ns

**

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

Mehta AK, et al. ATC 2011

% D

oubl

e Pr

oduc

er C

D8s

unstimulated No drug0.0

0.5

1.0

1.5

unstimulated TS-1unstimulated 2 mcg Bela unstimulated 20 mcg Bela unstimulated 200 mcg Bela

p = 0.1404p =0.0056p = 0.0060p = 0.0087p = 0.0139

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

unstimulated No drug 2 mcg Bela 20 mcg Bela 200 mcg Bela TS-10.0

0.5

1.0

1.5

%C

D8+

T c

ell D

oubl

e Pr

oduc

ers

ns

ns ns

**

• Lack of data on viral specific protective immunity in patients treated with belatacept

Mehta AK, et al. ATC 2011

% D

oubl

e Pr

oduc

er C

D8s

unstimulated No drug0.0

0.5

1.0

1.5

unstimulated TS-1unstimulated 2 mcg Bela unstimulated 20 mcg Bela unstimulated 200 mcg Bela

p = 0.1404p =0.0056p = 0.0060p = 0.0087p = 0.0139

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

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

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(>6m s/p kidney txp)

Tacrolimus + MMF + Prednisone 10 49.3

(34- 66) 6 / 4 51.7(6- 120) 10/7

Belatacept(>6m s/p kidney txp)

Belatacept + MMF +Prednisone 10 51.1

(34- 63) 4 / 6 92.2(44- 128) 9 / 7

• 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 PerCP-

Cy5.5APC PE-Cy7 Alexa

700V450 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:

Methods

FITC PE PerCP-Cy5.5

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 StrategyLymphocytes Singlets Live CD3 cells

Gating StrategyLymphocytes Singlets Live CD3 cells

CD4 vs CD8 cells

Belatacept treated patients show no difference in TNFα/IFNγ production in CD4 cells compared to

Tacrolimus

Belatacept patientCD4 EBV stimulated

Belatacept treated patients show no difference in TNFα/IFNγ production in CD8 cells compared

to Tacrolimus

Belatacept patientCD8 CMV stimulated


Belatacept patientCD8 CMV stimulated


Belatacept treated patients show no difference in TNFα/IFNγ production in CD8 cells compared

to Tacrolimus

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

response to EBV stimulation

NaiveTCM

TEM TEMRA

CCR7

CD45RA

p= 0.028p= 0.054

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

Memory subsets

NaiveTCM

TEM TEMRA

CCR7

CD45RA

p=0.009

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

EBV CMV

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

AcknowledgmentsSpecial 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 CheesemanETC Clinical Research Coordinators• Elizabeth Begley• Shine Thomas• Elizabeth FerryThe 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.

Yvonne Suessmuth, PhD Postdoctoral Fellow Emory Transplant Center, Atlanta, GA

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