yvonne suessmuth, phd postdoctoral fellow emory transplant center, atlanta, ga
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The 12 th 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. - PowerPoint PPT PresentationTRANSCRIPT
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
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
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 EBV stimulated
Belatacept patientCD8 CMV stimulated
Belatacept patientCD8 EBV 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
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
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
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
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
EBV CMV
Increased CD27lo/CD28lo cell numbers in Belatacept patients do not correlate with increased TNFα/IFNγ
double producing cells in this population
EBV CMV
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.