Download 5. Detection of Immune Responses in Mucosal T

Detection of Immune Responses in Mucosal T-cells in Kenya
Jackton Indangasi2, Hannah Cheeseman1, Onyango J. I. Obila2, Simon Ogola2, Robert Langat2,Philip J. Bergin1, Dilbinder K. Gill1, Harriet H. Park3, Jacquelyn Nyange2, Dagna Laufer3, Pat
Fast3, Bashir Farah2, Jill Gilmour1, Omu Anzala2.
2Kenya
AIDS Vaccine Initiative (KAVI), Nairobi, Kenya, 1International AIDS Vaccine Initiative Human Immunology Laboratory (IAVI HIL), Imperial College London, London, UK, 3International AIDS Vaccine Initiative (IAVI), New York, USA.
Introduction
% IFN-g Expression (CD8+ T-Cells)
The next generation of candidate HIV vaccines include replicating vectors targeted to mucosal sites, which are hoped to induce an efficacious T cell response to limit HIV-1
viral replication in the mucosa. For HIV vaccine clinical trials, it is especially important to ascertain whether there is a mucosal immune response, as this is where the bulk of
the viral reservoir is maintained. Immune responses have been shown to differ between racial and geographical populations(1), making it vital to perform comparison
studies in the country where a clinical trial will take place, to obtain the relevant background information. The aims of this study were to obtain gut biopsies from HIVseronegative individuals in Kenya, to ensure that scientists in Kenya were fully trained in the methods to be used in future clinical trials and to demonstrate comparability
between assays run in Kenya and the UK.
Methods
• A panel of 10 HIV-negative PBMC, with a range of CMV responses, were used to
demonstrate equivalence of the two LSRII cytometers, using the 10-colour
intracellular cytokine stimulation (ICS) panel (CD3 APCCy7, CD4 ECD, CD8 Pacific
Blue, CD27 PECy7, CD45RO A700, CD107a PECy5, IFN-g APC, IL-2 PE, TNF-a FITC,
DUMP [Aqua Blue Viability dye and CD19 AmCyan]) to assess responses to a 6hour stimulation with Mock, CMV and SEB. Three HIV-positive PBMC samples
were stimulated with Mock, CMV, SEB and clade A GAG and NEF pools.
• Biopsies were taken from the sigmoid colon of 31 HIV-seronegative volunteers
and mucosal mononuclear cells (MMC) isolated(2). Parallel blood samples were
also taken. MMC and PBMC were rested overnight and stimulated for 6 hours
with Mock, CMV, SEB and clade A HIV GAG and NEF pools. PBMC assessed by
IFN-g ELISpot were directly compared to pp65 CMV responses detected by flow
cytometry.
ns
> 0.9999
3
ns
0.5000
2
ns
0.2500
1
ns
0.7500
0
MOCK
CMV
GAG
NEF
Figure 3: Comparison of CD8+ T-cell IFN-g production. Graph shows 3 HIV positive donors.
Data from KAVI (o) and HIL (l) are not significantly different, using a Wilcoxon two-tailed
matched-pairs signed rank test.
b)
200K
200K
150K
2.5
100K
2.0
50K
150K
TNF-A+
0.15
150K
100K
50K
0
0
10
3
TNF-a
10
4
10
5
MMC
IL-2+
0.19
100K
IFN-G+
0.37
50K
0
1.5
SSC-A
200K
SSC-A
250K
SSC-A
250K
0
0 10
2
10
3
IFN-g
10
4
10
5
0
10
3
IL-2
10
4
10
5
1.0
250K
250K
250K
200K
200K
200K
KAVI
150K
SSC-A
0.0
SSC-A
0.5
SSC-A
Viable MMC yield x10e7
a)
250K
150K
TNF-A+
0.29
100K
150K
100K
50K
50K
0
50K
0
0
10
3
TNF-a
10
4
10
5
PBMC
IL-2+
0.074
100K
IFN-G+
0.56
0
0 10
2
10
3
IFN-g
10
4
CD8+
10
5
0
10
3
IL-2
10
4
10
5
CD4+
Figure 4: a) Viable MMC can be isolated, with an average of 1x106 cells per biopsy
collected. b) Responses of CD4+ and CD8+ T-cells to CMV stimulation in both MMC and
PBMC compartments. Mock responses were low in all cases.
Flow cytometry:
% IFN-g Expression (CD4+CD8+)
Figure 1: Gating strategy used for data analysis in FlowJo.
Results
Results from the PBMC comparison indicate that the two LSRII cytometers are
capable of producing data that is not significantly different.
We also show that is it possible to isolate MMC from patients in Kenya and that
these cells can be successfully stimulated, stained and acquired.
Of the 31 volunteers biopsied, 11 satisfied our pass/fail criteria (Viable CD3 count
of >30000 events and CD4/8 count of >10000 events.) and 8 PBMC and 5 MMC
samples demonstrated a CMV response (CD4 and/or CD8) by ICS. All 11 responded
by ELISpot (PBMC; mean 723 SFU/million; range 95-1440 SFU/million). PBMC
ELISpot CMV responses significantly correlated with ICS CMV responses; r2= 0.62.
K
A
VI
3
2
1
K
A
VI
Laboratory
1
0
-1
500
1000
1500
ELISpot:
SFU/million
Conclusions
Acknowledgements
References
0
A
VI
VI
K
A
IL
H
K
A
VI
IL
H
Laboratory
P = 0.5566
% Expression
0.0
0
Laboratory
0.5
K
0.0
2
1.0
IL
0.5
4
H
% Expression
1.0
P = 0.2324
2
CD8+
1.5
4
P = 0.1934
% Expression
P = 0.2324
Laboratory
CMV CD8+ TNF-a+
1.5
6
2.0
A
VI
IL
CMV CD8+ IL-2+
CMV CD8+ IFN-g+
2.5
Laboratory
3
This study demonstrates that it is feasible to collect biopsies and perform multiparameter ICS in Kenya and that the data generated is comparable with both data
run in London and on an extensively qualified assay (ELISpot).
These methods have been successfully transferred and may be used to assess
antigen-specific T-cell responses in future vaccine trials.
0.0
H
Laboratory
K
CMV CD8+ CD107A+
K
A
VI
IL
H
A
VI
IL
H
Laboratory
0.0
0.1
K
0.0
0.00
0.1
0.2
IL
0.2
0.3
IL
0.4
0.2
P = 0.2324
H
% Expression
% Expression
0.05
0.6
0.4
P = 0.1602
CD4+
0.10
0.3
P = 0.3223
4
Figure 5: ELIspot responses (PBMCs) correlate with flow cytometry assays. PBMC; mean
723 SFU/million; range 95-1440 SFU/million. PBMC ELISpot CMV responses significantly
correlated with ICS CMV responses; r2= 0.62.
CMV CD4+ TNF-a+
H
P = 0.9219
0.15
% Expression
CMV CD4+ IL-2+
0.8
0.20
% Expression
CMV CD4+ IFN-g+
% Expression
CMV CD4+ CD107A+
5
Laboratory
1. Reference ranges for the clinical laboratory derived from a rural population in
Kericho, Kenya. Kibaya et al. PLoS One. 2008 Oct 3;3(10)
2. Measuring human T cell responses in blood and gut samples using qualified
methods suitable for evaluation of HIV vaccine candidates in clinical trials. Kaltsidis
et al. J Immunol Methods. 2011 Jul 29;370(1-2):43-54.
Figure 2: Comparison of 10 paired HIV- samples tested at HIL and KAVI. Using a Wilcoxon
matched-pairs signed rank test to determine the P values, no significant difference is seen
between the data from samples tested at the HIL or at KAVI.