Transcript S. Typhi-specific CD8+ T EM responses

Salmonella Typhi-specific effector/memory CD8+ T cell
responses elicited in a wild-type S. Typhi controlled
human infection model
Stephanie Fresnay1, Monica A. McArthur1, Thomas C. Darton2, Claire Jones2, Claire S.
Waddington2, Christoph J. Blohmke2, Gordon Dougan3, Brian Angus2, Myron M.
Levine1, Andrew J. Pollard2, Marcelo B. Sztein1
1University
of Maryland, Baltimore, MD, United States, 2University of Oxford, Oxford,
United Kingdom, 3Wellcome Trust Sanger Institute, Hinxton, United Kingdom
Infection Prevention and Control, November 28 2016
CVD
Typhoid fever
 More than 20 millions cases of enteric fever and 200,000 death / year
 Salmonella enterica serovar Typhi: human-restricted pathogen
 Current limited knowledge on correlates of protection due to the lack
of appropriate clinical and preclinical models
 Cellular immune responses are likely to play a crucial role in protection
 Limited data on immune status before infection and its effects on
clinical outcome
http://www.infectionlandscapes.org
The Oxford challenge model
Challenge with wild-type S. Typhi to establish a 60-75% attack rate
Typhoid Diagnosis (TD), determined by fever >38C for 12 hours or bacteremia
Effector functions tested at different time points for:
- Volunteers with positive typhoid diagnosis: TD (n=13)
- Volunteers not diagnosed with typhoid: NoTD (n=7)
Experimental Design: in vitro stimulation of T
effectors with S. Typhi-infected targets
Target Cells
Autologous B-EBV cells,
HLA-E-restricted AEH cells,
autologous blasts
TD (n=13)
NoTD (n=7)
PBMC
S. Typhi infection
+
g-irradiation
14-18 hrs
Flow cytometry
Experimental Design: Flow cytometry gating
2500
R2: 95.28%
800
1000
2000
1000
500
0
200
400
600
800
104
CD62-L
CD8
TEM
IFN-g
103
104
102
CD45RA Qdot 800-A
103
101
102
TNFa Alexa700-A
103
TNF-
104
103
104
100
101
CD69 ECD-A
102
103
104
CD69 ECD-A
102
101
100
100
104
TEMRA CD45-RA
103
104
CD69 ECD-A
102
102
IFNg PE-Cy 7-A
101
104
100
104
101
101
R9: 8.56%
TCM TN
CD69 ECD-A
102
103
100
100
R7: 56.02%
101
102
CD3 BV650-A
103
104
CD69 ECD-A
102
101
100
CD107a
104
R6: 13.17%
R8: 15.53%
100
101
100
101
103
104
103
CD69 ECD-A
102
CD69
101
100
103
10 4
CD8+ subsets
CD62L APC-EF780-A
102
103
104
CD8 PerCP-Cy5-5-A
102
103
R4: 23.47%
CD3
102
CD107a/b FITC-A
10 1
10 2
10 3
YEVID + CD14/19/45 BV570-A
Yevid-CD14-CD19-CD45
CD8+ T cells
100
101
10 0
1000
FSC-H
FSC-H
100
101
102
IL-2 BV605-A
IL-2
103
104
100
600
FSC-A
FSC-A
101
400
0
0
200
R201: 75.70%
Number
1500
FSC-A
600
400
200
400
0
200
SSC-A
SSC-A
600
FSC-A
R1: 43.89%
0
100
Live T cells
800
800
1000
Singlets
1000
Lymphocytes
100
101
102
IL-17 BV421-A
103
IL-17A
104
100
101
102
MIP1b PE-A
103
MIP-1
104
S. Typhi-specific CD8+ TEM responses: Baseline
CD107a
IFN-g
TNF-
TD EBV
TD AEH
TD blast
NoTD EBV
% positive cells
NoTD AEH
NoTD blast
median
MIP-1
IL-17A
IL-2
p<0.05
p<0.01
Higher baseline responses at baseline are associated with disease development
Relationship between S. Typhi-specific baseline responses
and time to diagnosis
CD107a
IFN-g
C D 107a
2 0
TNF-
T N F -
IF N -g
p = 0.09
3
2 0
1 0
1 0
5
5
2
% positive cells
1
0
1 0 0
1 5 0
2 0 0
2 5 0
0
1 0 0
MIP-1
2 0 0
2 5 0
1 0 0
1 5 0
IL-17A
M IP -1 
1 0
2 0 0
2 5 0
IL-2
IL -2
IL -1 7 A
p = 0.09
2 0
0
1 5 0
2
0 .8
0 .6
5
1
0 .4
0 .2
0
1 0 0
0
0 .0
1 5 0
2 0 0
2 5 0
1 0 0
1 5 0
2 0 0
2 5 0
1 0 0
1 5 0
2 0 0
High S. Typhi-specific baseline responses are associated
with delayed time to diagnosis
2 5 0
Post-challenge kinetics of responses
IFN-g
TD
AEH
NoTD
5
8
MIP-1
TD
5
8
5
5
0
0
0
0
-5
-5
-1 2
-8
-4
8
4
T D
12
% positive cells
-5
-5
28
21
14
7
0
20
16
-1 2
D a y s p o s t c h a lle n g e
T im e r e la tiv e to d ia g n o s is
-8
-4
T D
4
8
12
16
D a y s p o s t c h a lle n g e
IL17A
5
5
1
5
0
0
0
0
-1 2
-8
-4
T D
4
12
8
16
-1 2
28
21
14
7
0
20
-5
-1
-5
-5
-8
-4
T D
4
8
12
16
D a y s p o s t c h a lle n g e
IL-2
TNF-
5
5
2
5
0
0
0
0
-5
-1 2
-8
-4
T D
4
8
12
16
-5
-2
28
21
14
7
0
20
-1 2
D a y s p o s t c h a lle n g e
T im e r e la tiv e to d ia g n o s is
28
21
14
7
0
20
T im e r e la tiv e to d ia g n o s is
D a y s p o s t c h a lle n g e
-5
28
21
14
7
0
20
T im e r e la tiv e to d ia g n o s is
CD107a
T im e r e la tiv e to d ia g n o s is
IFN-g+ cells
NoTD
-8
-4
T D
4
8
12
16
28
21
14
7
0
20
D a y s p o s t c h a lle n g e
T im e r e la tiv e to d ia g n o s is
Blasts
EBV
Early decreases in CD107a expression and cytokine production are associated
5
5
0
0
2
with disease development
-5
-5
-1 2
-8
- 4
T D
4
8
1 2
1 6
2 0
0
7
1 4
2 1
2 8
2
0
0
-2
-2
0
7
1 4
2 1
2 8
Multifunctional (MF) CD8+ TEM responses: Baseline
NoTD EBV
median
TD AEH
NoTD AEH
p<0.05
TD blast
NoTD blast
p<0.01
% positive cells
TD EBV
MF S. Typhi-specific responses are dominant in TD volunteers
Summary / Discussion
 Development of typhoid disease is associated with:
 Higher baseline S. Typhi-specific responses
 Early decreases in blood of S. Typhi-specific TEM cells after challenge
These results suggest that S. Typhi-specific TEM cells migrate to the site(s) of
infection (e.g., gut, RES), resulting in an inflammatory process that favors S. Typhi to
effectively infiltrate the mucosa and spread systemically.
 Delayed time to diagnosis among TD volunteers is associated with higher
baseline S. Typhi-specific responses.
These results suggest that high numbers of S. Typhi-specific T cells at baseline
are sufficient to delay, but ultimately can not prevent, disease development.
Summary / Discussion
 Protection against typhoid fever is associated with:
 Very low or no baseline S. Typhi-specific responses
 No changes observed in circulation after challenge
These results suggest that in protected volunteers innate and/or
adaptive immune responses in the gut microenvironment are able to
control S. Typhi infection, precluding systemic dissemination.
Take home messages
This study provides unique insights into the human immune
response during the development of typhoid fever
 Uncovered, for the first time, that S. Typhi-specific CD8 T cell baseline
responses correlate significantly with clinical outcome after infection
 Revealed some of the immunological mechanisms responsible for
delayed time to disease onset
 Demonstrated that multifunctional T cells appear to play a critical role in
the development of typhoid disease
 These results argue for the measurement of defined immunological
responses during the development of new generation typhoid vaccines
Acknowledgements
Center Vaccine
Development, UMB
Cellular Immunology Group
Regina Harley
Cathy Storrer
CVD
Paula Bernal
Franklin Toapanta
Rezwan Wahid
Jeffrey Floyd
Rosangela Mezghanni
Haiyan Chen
Kirsten Lyke
Jay Booth
Mark Rudolph
Sarah Boudova
UK Collaborators
Jeremy Farrar
Paul Langford
Derrick Crook
Stephen Lockhart
Statistical support
Larry Magder
Supported by grants U19 AI082655 (UM-CCHI) & R01 AI-36525
from the NIH, the Passano Foundation Clinical Investigator Award,
and by the Wellcome Trust