Comparison of Viral Immunity in Stable Renal Allograft Recipients

Report
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.

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