This study examined the effect of acrolein, a toxic chemical found in cigarettes, on IL-1β-dependent regulation of lung γδ T cells. The results showed that IL-1β signaling is important for maintaining lung Vγ6 γδ T cells and inducing their proliferation and IL-17 production. Acrolein was found to be cytotoxic to total lung cells and γδ T cells at concentrations of 5 μM or higher. Acrolein began inducing IL-1β production at 10 μM but not at lower concentrations. While IL-1β regulates Vγ6 γδ T cells, further study is needed to better understand how acrolein specifically affects IL-1β production in the lungs

1. To Induce or Not to Induce:
Acrolein’s Effect on IL-1β-
dependent Regulation of Lung γδ T
Cells
Tiger Teng
Christopher Fleming, Jun Yan
DuPont Manual High School
Louisville, KY

2. Introduction
• Lung Cancer
– 2nd most common cancer among men and women
– Majority of primary lung cancers are carcinomas
of the lung, derived from epithelial cells
– Multiple medical reports indicate the leading
causes of lung cancers are from cigarette smoking.

3. Introduction
• Acrolein
– A clear or yellow liquid that evaporates quickly
– Strong, unpleasing smell
– One of the toxic chemicals found in cigaretts (a
simple unsaturated aldehyde)

4. Introduction
• gd t-cells
– Part of the adaptive and innate immune system
– Countless direct and indirect effects on healthy
tissues and immune cells
• IL-1β
– Produced by macrophages and epithelial cells
– Important mediator of the inflammatory response
– Involved in variety of cellular activities (cell
proliferation, differentiation, and apoptosis

5. Introduction (cont.)
• The purpose of this study is to see Acrolein
induces IL-1b production and toxicity of gd t-
cells.
– Hypothetically, IL-1b will attach to gd t-cells
proliferation of gd t-cells  massive production
of IL-17 inflammation of lungs NF-kb signaling
one of the causes of COPD  increase risk of
cancer or cancer progression

6. Acrolein
Epithelial Cell
Inflammasome Activation
IL-1
Receptors
δ T-cells
V6
V4 ??
Proliferation
IL-1
Production
IL-17 Inflammation
1. Neutrophil
Infiltration
2. CD8 Infiltration
3. Increased
Chemoattractants 
Increase infiltrations
of other cells
4. NF- Signaling
 Increased cancer
risk
 Increase cancer
progression
IL-17
Production
Macrophages
Fibroblast

7. Methods
• Enzyme-Linked Immunosorbent Assay (ELISA) assay
IL-1β capture antibodies were used to coat the
bottom of a 96-well plate a day before adding the
standards and samples. Next day, IL-1β detection
antibody was added. Avidin-horesradish peroxidase
and TMB Substrate Solution were added creating a
colorimetric correlation to the concentration of IL-1β.

8. Methods
• Carboxyfluorescein Succinimidyl Ester (CFSE) Staining
Cells were resuspended in prewarmed PBS/0.1% BSA
at a final concentration of 1x107 cells/ml. Added 1μl of
1mM stock Invitrogen Cell Trace CFSE solution and
incubated the dye for 10 min at 37.5°C while shaking
after 5 min to label cells evenly. After 10 minutes, the
staining was quenched by the addition of 5 equal
volumes of ice-cold FBS to the cells and incubated for 5
minutes on ice. Cells are then washed two more times
with complete, cold RPMI.

9. Methods
• Flow Cytometry
After CFSE labeling, plate 2x106 lung cells per well of a
24-well plate. Added cytokines- IL-23 (5ng/ml) and IL-1β
(1ng/ml). After 72 hours, GolgiPlug (1μg/ml) was added
for 6 hours. The harvested cells were added into flow
tubes to be stained with flourochrome labeled,
monoclonal antibodies and read by the flow cytometer.
The cells were stained with CD3 APC-Cy7, γδTCR APC, Vγ4
PE, IL-17A PE-Cy7, CD45 PERCP, and purified anti-mouse
Vγ6 IgM with rat anti-IgM FITC secondary antibody (all
antibodies purchases from Biolegend except anti-mouse
Vγ6 IgM.

10. Results
WT IL-1r-/-
0
16
32
48
64
80 **
%ofV6Tcells
C57Bl6 WT IL-1r-/-
SSC
Vγ6
A
IL-17
Vγ6
C57Bl6 WT IL-1r-/-
B
WT IL-1r-/-
0
5
10
15
20
25
*%ofIL-17+V6Tcells
Decrease % of total
Vγ6 and IL-17+ Vγ6
γδ T-cells in the lung
of IL-1r -/- mice. (A)
Quantifying V6 γδ T-
cells and (B) IL-17+
V6 γδ T-cells in IL-
1r-/- and B6 WT single
cell lung suspension
using flow cytometry.
Bar graphs represent
duplicate
experiments. The
error bars represent
95% confidence
intervals, *P < 0.05
and **P < 0.01.

11. Results
Media alone
C57Bl6
WT
IL-1r-/-
IL-23 IL-1β IL-23+IL-1β
IL-17
CFSE
Lung Vγ6 γδ T-cell proliferation and IL-17 production are dependent on IL-1β in
vitro. Endogenous IL-1β leads to increased IL-17 production during 3-day culture.
Exogenous IL-1β induces proliferation and IL-17 production by γδ T-cells. IL-23
combined with IL-1 has a synergistic effect on proliferation and IL-17 production.

12. Results
A Lung Alone
44.4%
53.5%
0 10
2
10
3
10
4
10
5
0
50K
100K
150K
200K
250K
u alone
1
47.1%
50.5%
0 10
2
10
3
10
4
10
5
0
50K
100K
150K
200K
250K
Ac 0.01
Ac 5
Ac 10
69.3%
0 10
2
10
3
10
4
10
5
0
50K
100K
150K
200K
24.3%
73.5%
0 10
2
10
3
10
4
10
5
0
50K
100K
150K
200K
250K
Ac 0.1
Ac 1
47.1%
0 10
2
10
3
10
4
10
5
0
50K
100K
150K
200K
37.7%
60.0%
0 10
2
10
3
10
4
10
5
0
50K
100K
150K
200K
250K
7AAD
SSC
Ac 5
Ac 10
28.6%
69.3%
0 10
2
10
3
10
4
10
5
0
50K
100K
150K
200K
250K
24.3%
73.5%
0 10
2
10
3
10
4
10
5
0
50K
100K
150K
200K
250K
1μM Acro 5μM Acro 10μM Acro
B
Acrolein cytotoxicity of total lung cells.(A) Gating
on the live and dead cells, higher concentration of
Acrolein results in higher percentage of dead cells.
(B)There was no significant difference between
concentrations of Acrolein in terms of % of live
cells, however the decreasing trend of live cells is
obvious. The error bars represent 95% confidence
intervals, *P < 0.05 and **P < 0.01.

13. Results
A
Ac 0.01
u alone
1
0.427%
11.3%
0 10
2
10
3
10
4
10
5
0
10
2
10
3
10
4
10
5
0.420%
11.7%
0 10
2
10
3
10
4
10
5
0
10
2
10
3
10
4
10
5
γδTCR
CD3
Ac 5
Ac 10
0.219%
7.10%
0 10
2
10
3
10
4
10
5
0
10
2
10
3
10
4
10
5
0.101%
6.58%
0 10
2
10
3
10
4
10
5
0
10
2
10
3
10
4
10
5
Ac 1
Ac 0.1
10.2%
0 10
2
10
3
10
4
10
5
0
10
2
10
3
10
4
0.373%
8.59%
0 10
2
10
3
10
4
10
5
0
10
2
10
3
10
4
10
5
Ac 5
Ac 10
7.10%
0 10
2
10
3
10
4
10
5
0
10
2
10
3
0.101%
6.58%
0 10
2
10
3
10
4
10
5
0
10
2
10
3
10
4
10
5
Acrolein cytotoxicity to δ T-cells of the lungs (A)
Using flow cytometry to measure the percentage
of live δ and  T-cells. (B) Analyzing
representative data, lung alone vs 10 M of
Acrolein shows a significant decrease in the % of
total live δ and αβ T-cells . The error bars
represent 95% confidence intervals, *P < 0.05
and **P < 0.01.
B
Lung Alone 1μM Acro 5μM Acro 10μM Acro

14. Result
med Lu alone 0.01 0.1 1 5 10 Lu+ tu Lu+Tu+A 10
-50
0
50
100
150
200
250
300 *
*
Acrolein Concentrations (M)
Treatment
IL-1(pg/ml)
Acrolein was able to induce IL-1 production at 10μM. There is a decrease in IL-1
production between lungs only and lungs treated with 0.1 M of Acrolein. The error bars
represent 95% confidence intervals, *P < 0.05 and **P < 0.01.

15. Conclusion/Future Work
This study shows that IL-1r signaling is crucial for the maintenance and
function of lung Vγ6 γδ T cells. Comparison data between WT and IL-1r-/-
mice suggests that Vγ6 are specifically regulated by IL-1β, unlike Vγ4 and
Vγ1(data not shown). As shown in figure 3, IL-1 induces proliferation and
IL-17 production of Vγ6 T-cell and has a synergistic effect with IL-23 in IL-
17 production. However, with only 5 M of acrolein, cytotoxicity to total
lung cells and γδ T-cells is prevalent. From figure 6, it seems acrolein
begins to induce IL-1β production starting at 10 M but not at lower
concentrations. In conclusion, we used flow cytometry to demonstrate that
IL-1 does regulate Vγ6 γδ T-cells in both proliferation and IL-17
production. However, further optimization is needed in order to determine
how acrolein affects IL-1β production in the lungs.

16. Acknowledgement
• Thanks to:
– Tumor Immunobiology Program, James Graham
Brown Cancer Center, University of Louisville
– Christopher Fleming, Jun Yan, and others
Questions?