Download Cytotoxic activity of tumor-specific CD8 T cells

 Cytotoxic activity of tumor-specific CD8+ T cells
Sheila Pellegrino, Kantonsschule Wohlen
Antonio Cassarà, Martina Schmittnägel, Miki De Palma lab, ISREC, EPFL
Introduction
Cancer is one of the leading causes of mortality worldwide, with approximately 8.2 million
deaths per year. Cancer is caused by genetic mutations in normal cells, which lead to an
uncontrolled growth and, in some cases, to colonization to distant organs. Current available
treatments such as surgery, chemotherapy and radiotherapy delay the death of patients but
don’t cure the disease, sometimes due to resistance acquired by cancer cells.
Immunotherapy is currently among the most promising therapies, giving encouraging results
in the clinics. Immunotherapy aims at stimulating one’s own immune system, such as T
lymphocytes, in order to fight cancer cells.
T lymphocytes are immune cells capable of
sensing a variety of signals that can alert
them to potentially threatening pathogens
as well as cancer cells. Tumor-specific T
cells can infiltrate tumors and there
encounter
tumor-associated
antigens
presented
by
specialized
antigenpresenting
cells
(APCs).
They
subsequently activate, proliferate and
eventually kill cancer cells.
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In this project, we wanted to investigate
how CD8+ T cells get activated and
proliferate and how they can kill cancer
cells, which express their specific antigen.
Figure 1: Activation and proliferation of CD8 T cells.
Professional antigen-presenting cells (APCs), principally
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dendritic cells, present tumor antigens to CD8 T cells
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(cross-presentation) and to CD4 helper T cells. The
helper T cells then produce cytokines that stimulate the
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expansion and differentiation of the CD8 T cells.
Materials and Methods
Isolation of CD8+ T cells
We isolated CD8+ T cells from the spleen of an OT-I mouse, whose T cells have a transgenic
T-cell receptor (TCR) specific to the Ovalbumin protein (OVA), an immunodominant antigen
from chicken egg used to track antigen-specific T cells response.
We smashed the spleen, lysed the erythrocytes and magnetically labeled CD8+ T cells using
Microbeads (Miltenyi Biotec, Cat. no. 130-049-401). The cell suspension was loaded onto a
1 autoMACS Pro Separator Machine, which separates the CD8- cells fraction from the CD8+
cells fraction.
Activation and proliferation of CD8+ T cells
We seeded 200’000 CD8+ T cells/well in a round-bottom 96-well plate and added the OVA
peptide at a final concentration of 250 µg/ml. We stained some T cells using the CellTrace
Violet Cell Proliferation Kit (Invitrogen, Cat. no. C34557), to assess their proliferation. We
analyzed them by flow cytometry at day 3. The rest of T cells was used for the killing assay
at day 2.
LDH colorimetric assay
24h before the assay, we seeded 20’000 cancer cells in a flat-bottom 96-well plate and let
them adhere overnight. The day after, we added pre-activated T cells at different ratios
(20’000, 40’000 or 60’000) and co-culture them for 20 hours in a final volume of 100 µl. We
measured the level of cytotoxicity with the Pierce LDH Cytotoxicity Assay Kit (Thermo
Scientific, Cat. no. 88953). We transferred 50 µl of each sample medium to a new 96-well
plate, added 50 µl of substrate (Lactate), incubated for 30 min at room temperature and,
finally, measured the absorbance at 490 nm.
Figure 2: LDH colorimetric assay.
Time laps microscopy
We co-cultured 20’000 cancer cells with 40’000 pre-activated CD8+ T cells in phenol-free
medium. We added propidium iodide (PI) to the medium, which stains dead cells. We let the
co-culture for 4h and took an image every 5 min with a time-laps microscope.
2 Results
CD8+ T cells proliferation
Figure 2 shows how many rounds of proliferation our CD8+ T cells underwent during 3 days.
The CD8+ T cells were stained at day 0 with a dye-tracer, which dilutes at each round of cell
division. The more a cell divides, the less intense is his tracer. The x-axis indicates the
intensity of tracer, while the y-axis is a physical parameter. Our CD8+ T cells proliferated six
times, most of them being in their sixth division.
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Figure 3: Proliferation of OT-I CD8 T cells.
Killing assay
In order to check the cytotoxic effect of activated OVA-specific CD8+ T cells, we made a coculture of CD8+ T cells and OVA positive or negative cancer cells, at different ratios. As
cancer cells, we chose a murine colorectal cancer cell line (MC38, MC38-OVA) and a murine
melanoma cell line (B16, B16-OVA). We assessed the killing activity with two different
methods: a LDH colorimetric assay and time-laps microscopy.
LDH (Lactate dehydrogenase) is a cytosolic enzyme, which is released in the cell culture
medium upon plasma membrane damage. Extracellular LDH activity in the medium can be
quantified using a plate-reading spectrophotometer and is indicative of cellular cytotoxicity.
The percentage of cytotoxicity has been calculated as follows:
As expected, we observed an increased level of cytotoxicity in OVA-expressing cancer cells
(MC38-OVA and B16-OVA) compared to the respective wild-type cell lines (MC38 and B16).
Moreover the killing was more efficient with increased T cell : Cancer cell ratio in MC38-OVA
and B16-OVA, but also at a smaller extent in MC38 and B16.
3 % Cytotoxycity after 20h
80
B16
B16-OVA
60
40
20
0
1:1
1:2
1:3
Cancer cells : CD8+ T cells
% Cytotoxycity after 20h
80
MC38
MC38-OVA
60
40
20
0
1:1
1:2
1:3
Cancer cells : CD8+ T cells
Figure 4: Percentages of cellular cytotoxicity after 20h incubation with activated CD8+ T cells
We used also a time-laps microscopy to see the killing activity over a period of time (4h), to
track the dynamic movement of T cells and to see if some cancer cells were killed (became
red). We could see that the cancer cells are in general very static, while the T cells move a
lot to scan for MHC-peptide complexes specific to their TCR. We indeed observed that the
CD8+ T cells associated more with the cancer cells that express the OVA compared to the
ones without. However, we couldn’t see any cancer cells turning red, probably because the
time was not sufficient.
4 Figure 5: Time-lapse microscopy. Cancer cells were co-cultured with activated CD8+ T cells for 4h
and dead cells were stained with propidium iodide (red).
Conclusions
Our CD8+ T cells were able to get activated and proliferate 6 times after 3 days of culture
with the OVA peptide. In general, in order for a naïve T cell to become active and proliferate,
it needs an antigen-presenting cell that present its specific antigen and a T-helper cell.
However, in our experiment the CD8+ T cells proliferated just with the addition of the OVAprotein, because our isolation was not 100% pure and thus a small number of contaminants,
such as dendritic cells or T-helper cells, remained and were sufficient to activate CD8+ T
cells.
The LDH colorimetric assay worked according to our expectations. It shows that activated T
cells kill only the cancer cells that express their specific antigen. However, we also observed
a small increased cytotoxicity in the wild-type cancer cells (MC38, B16) with an increased T
cell: cancer cell ratio. This can be explained as unspecific killing.
The time-laps microscopy worked, but the protocol can be optimized. We were not able to
see any apoptotic cancer cell, for two possible reasons. The 4h time point might have been
5 too short to see the killing (indeed, in the LDH colorimetric assay the co-culture lasted 20h),
but enough to see the attacking action. Also, there might have been too many T cells,
resulting in a total coverage of the cancer cell surface, making it impossible for us to
recognize some red colored cancer cells.
Acknowledgements
I would like to thank the organization "Schweizer Jugend Forscht" and Alice Emery-Goodman
for this very special and enriching week full of new experiences and information. Also, for
giving us students the opportunity to undergo such a project. I would also thank Miki De
Palma, for having accepted me in his lab, my supervisor Antonio Cassarà for guiding me
through this project and all the interesting experiments. In addition, I would practically thank
most of the people, Martina, Jean-Baptiste, Maria, who work in that lab for their help from
any kind.
References
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International Agency for Research on Cancer; 2013. Available from:
http://globocan.iarc.fr (link is external), accessed December 2013.
2. Urry, Lisa A., et al. Campbell biology. Boston: Pearson, 2011.
3. Restifo, Nicholas P., Mark E. Dudley, and Steven A. Rosenberg. "Adoptive
immunotherapy for cancer: harnessing the T cell response." Nature Reviews
Immunology 12.4 (2012): 269-281.
4. Abbas, Abul K., Andrew HH Lichtman, and Shiv Pillai. Basic immunology: functions
and disorders of the immune system. Elsevier Health Sciences, 2012.
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