Summer Undergraduate Research Program - Fred Hutchinson ...

Profiling gene expression differences and immune cell populations in HIV exposed seronegative
individuals [HESN] and seroconverters
Augustine Ajuogu1, 2, 3 , Chloe Slichter 2,3 , Laura Pattacini, 3 and Jennifer Lund, 2,3
1Northwest University, Kirkland, WA; 2Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research
Center, Seattle, WA; 3Department of Global Health, University of Washington, Seattle, WA
2012 Best Poster Presentation Award
Methodology Results
Background
Question 1: Do gene expression levels differ between seroconverters and HESN samples?
Answer: Previous unpublished data supports our hypothesis, however we are still in the
process of analyzing the data and have yet to determine the results
Study cohorts
The peripheral blood mononuclear cells (PBMC) samples analyzed were either from exposed seronegative
subjects or from seroconverters. Seroconverters samples were obtained from the last visit before
seroconversion. All samples were obtained from two different cohorts: Partners in Prevention HSV/HIV
Transmission Study, Couples Observational Study, and Partner’s Pre-Exposure Prophylaxis study (PREP).
With more than 1.8 million deaths per year, HIV remains the most
devastating disease around the globe. Disease and death in HIV patients
can be attributed to various factors including the inability of the host
immune system to independently suppress or clear the infection.
Question 2: Are there statistical differences in immune cell populations between
seroconverters and HESN samples?
Answer: No, there are no distinct differences between frequency of immune cells between
the two cohorts.
2a. Samples shipped for RNA gene
expression analysis
Some studies show that few individuals do not become infected after
continued and prolonged exposure without the aid of antiretroviral
therapy [HESN] .
CD56-
NK Cells
CD8+ CD8+ T-Cell
CD4
CD4+ T-Cell
Serodiscordant couples in which one partner is HIV positive and the
other is HIV negative are of interest in understanding such phenomenon.
4. Immune cell frequency analysis
150
60
100
100
40
50
Phenoype Population
80
60
40
20
20
Phenotype Frequency
1. PBMC Samples processed for
gene expression studies and
cell phenotyping
0
HESN
Preinfection
0
0
HESN
Preinfection
HESN
Pre infection
P value: 0.7585
Summary: ns.
P value: 0.7390
Summary: ns.
P value: 0.5381
Summary: ns.
3. Samples stained with specific markers
and fluorochrome
2b. Samples plated
Figure 2: Simplified methodology diagram
CD19+ CD19+ B-Cells
Monocytes
50
40
100
112
80
30
20
60
Sample processing for immune cell population phenotyping and gene expression studies
Samples were processed via standard procedures and divided into two aliquots to determine the frequency
of specific lymphocyte populations and to investigate gene expression of these specific populations.
40
10
0
Phenotype Frequency
20
0
Phenotype population
HESN
Pre-infection
HESN
P value: 0.2818
Summary: ns.
Preinfection
P value: 0.4436
Summary: ns.
Lymphocyte populations were identified using flow cytometry. Phenotyping for various subsets including T
cells, B cells, Monocytes, plasmocytoid dendritic cells, myeloid dendritic cells, and natural killer cells by the
expression of various cellular markers was performed.
Figure 4: PBMC Frequencies
Conclusion & . Discussion
In order to provide confidence in our results, we performed a series of experiments to establish working
antibody concentrations (titers). Furthermore, we performed a full fluorescence minus one (FMO) panel
utilizing titers already established to draw the specific gates for each cell population. Cell populations were
analyzed using FlowJo and Prism software.
Our experiment revealed no statistically significant difference in frequency of immune cell
populations between the HESN and seroconverters at the time point prior to seroconversion.
Samples from subjects sent for gene expression studies are still being analyzed and thus may
provide unique signature of protection or susceptibility to HIV infection.
250K
200K
150K
98.6
100K
88.9
10
50K
73.9
0
0
0 50K 100K 150K 200K 250K
0 50K 100K 150K 200K 250K
FSC-A
FSC-A
PBMCs
S
211058.fcs
211058.fcs
Event Count: 57940
Event Count: 65154
2
10 3
10 4
10 5
99.4
10 5
10 5
Singlets
Lymphocyte-PBMCs
Alive
CD3+ CD3+
CD3-
CD4+/CD8+ T cells
CD19+ B lymhocytes
250K
200K
Figure 1: Seronegative vs. HESN individual
In some cases, individuals can resist infection after repeated and
prolonged exposure but eventually acquire the virus (seroconverters).
The differences between HESN and seroconverters are not fully
understood, but immune responses to the virus are well documented in
both groups.
Future Directions .
: L/D
150K
SSC-A
FSC-H
100K
50K
0
Investigate immune cell populations at different time points before and after seroconversion
o Immune cell populations may change during exposure to HIV
Evaluate cell populations for activation markers
o Though immune cell frequency may not change, activation markers in HESN
0 50K 100K 150K 200K 250K
FSC-A
Ungated
211058.fcs
Event Count: 66101
Specific Aims
CD3-
NK cells
10 5
Quantify and compare gene expression levels in HESN and
seroconverters via microarray technology.
10 4
Acknowledgements
Acknowledgments
may differ from seroconverters.
23.1
10 3
: CD16
: CD19
: CD4
: CD3
0
3 104 105
0 10
: CD56
CD3-
211058.fcs
Event Count: 8897
10
0
2
0 10 103 104 105
: CD8
CD3+
211058.fcs
Event Count: 39014
3
10 4
69.1
10
24.5
0
0 50K 100K 150K 200K 250K
FSC-A
Alive
211058.fcs
Event Count: 47922
3
10 4
10 5
81.4
10
18.6
0
0 50K 100K 150K 200K 250K
FSC-A
CD3-
211058.fcs
Event Count: 8897
3
10 4
48.5
51.5
10
0
0 50K 100K 150K 200K 250K
FSC-A
Alive
211058.fcs
Event Count: 57620
2
10 3
10 4
10 5
1.21
10
0
0 50K 100K 150K 200K 250K
FSC-A
Alive
211058.fcs
Event Count: 57620
2
10 3
10 4
10 5
14.7
10
0
2
0 10 103 104 105
: CD11c
HLA-DR+
211058.fcs
Event Count: 8465
2
10 3
10 4
10 5
CD14+ HLA-DR + HLA-DR +
CD11c+ mDCs
48.1
Figure 3: Full Panel and gating scheme for immune cell population.
Profile immune cell frequencies in both HESN and seroconverters via
flow cytometry to assist in de-convolution analysis in order to evaluate
specific expression levels within specific immune cell types of both
populations.
I am indebted to Dr. Laura Pattacini both for her faithful mentorship and her grace. I thank Dr.
Jairam Lingappa for the project, and Dr. Jennifer Lund for the opportunity to work in her lab. A
special thank you to Chloe Slichter and the entire lab for their assistance. I thank Rafick Sekaly
and his entire lab for their help with the RNA gene expression studies of samples. My work is
made possible by the Fred Hutchinson Cancer Research Center Summer Undergraduate Research
Program. The Summer Undergraduate Research Program is supported in part by the Cancer
Center Support Grant (CCSG) CURE Supplement: 5 P30 CA015704-37S1. This research is also
supported in part by NIAID/NIH 3 R01 AI047086-10S1.
HLA-DR +
CD123+ pDCs
10 5
0.732
10 4
Rationale .
10 3
: HLA-DR
: HLA-DR
: HLA-DR
: CD14
10
0
2
2
103 104 105
: CD123
0 10
HLA-DR+
211058.fcs
Event Count: 8465
Our study may identify genes and immune cell populations important to HIV
acquisition. Results will in addition enable a better understanding of host
resistance to HIV infection which is critical to vaccine and drug
development.