What's New in Research: Genetics & immunology of Alopecia Areata
Roswell Park Powerpoint Presentation
1. +
Effect of Inhibitory Cytokines on PD1 &
LAG3 Expression
in Activated and Rested T Cells
Research Summary
Adeiyewunmi (Ade) Osinubi, Ruea Huang, Dr. Kunle Odunsi
Department of Gynecological Oncology, Roswell Park Center Institute, Buffalo New York, 14263
Roswell Park Summer Research Program
http://www.manifestdaily.com/vitamin-helps-turn-precancerous-cells-back-healthy-ones/
4. + Multiple Co-Stimulatory Inhibitory
Interactions Regulate T cell responses
PDL1 or PDL2
CD80 or CD86
CD80 or CD86
HVEM
MHC I or II
PD1
CD28
CTLA4
ICOS
BTLA
TCR
LAG3
5. +
LAG3
Lymphocyte Activation Gene 3
Encoded by LAG 3 gene
Immune checkpoint receptors
Expressed on activated T cells, NK Cells, B Cells
Diverse biologic effects on T Cells
6. +
Programmed Cell Death protein 1
Encoded by the PDCD1 gene
Cell surface receptor
Expressed on T cells and pro-B cells
Immune checkpoint, down regulates immune system
Ligands PD-L1 & PD-L2
prevents T Cell activation
PD1
7. +
Tumor Microenvironment
Surrounding blood vessels,
immune cells, fibroblasts,
signaling molecules,
extracellular matrix
Effects the growth and
evolution of cancer cells
Hypoxia and extracellular
matrix role in metastasis
8. + Lag3 and PD-1 in Ovarian Cancer
Mastuzaki J, et. al. (2010) Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are
negatively regulated by LAG-3 and PD-1 in human ovarian caner. PNAS 107:7875-
7880
CD8 TILs and TALs taken from patients
show expression of both PD-1 and Lag3.
10. +
Tumor Microenvironment Cont…
Mastuzaki J, et. al. (2010) Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are
negatively regulated by LAG-3 and PD-1 in human ovarian caner. PNAS 107:7875-
7880
13. +
Procedure
Isolate Splenocytes from C57 Black 6 Mice
Grow Cells in 1640 Media for 72 hours in anti CD3 and B7.1
coated plates
During this time, treat the splenocytes with Cytokines
TGFβ, IL-6, IL-10
Harvest the Cells
Stain Cells with antibodies (FcBlock, CD8, CD4, PD1, LAG-3,
CD-69)
Do Flowcytometry
Assess and Analyze Data
18. + Rested Cells
Activated cells for 72 hrs
Rested Cells for 72 hrs
Treated with three cytokines for 72 hrs
Did not reactivate, Cells treated in non coated plates
21. +
Reactivated Cells
Activated cells for 72 hrs
Rested Cells for 72 hrs
Treated with three cytokines for 72 hrs
Reactivated the cells
Cells treated in coated plates with lower concentration of anti
CD3 and B7.1
24. +
Conclusions
During activation, saw increase in LAG 3 in a concentration
dependent manner
Did not see huge effect on PD1 expression
In the Rested Cells, did not see quite similar trends, might be
due to the fact that we did not reactivate
In the Reactivated Cells
Single treatment of TGFB had greatest affect on LAG3 expression in
CD8 cells
Combination treatment increased PD1 expression
In particular, combination of TGFB and IL10 had the greatest affect
25. +
Treated Splenocytes with Glucose
ELISA
Enzyme linked immunoabsorbent assay
Other Experiments
26. +
ELISA for serum sLAG3
To test whether the level of soluble LAG3 protein in the serum
samples from ovarian cancer patients correlates with clinical
outcome.
Hypothesis: more sLAG3, less full length LAG3 is a good prognostic
factor.
Method: ELISA
27. +
Detailed Methods
coat plate with capture antibody diluted in PBS, incubate overnight at room
temperature
wash 3x with Wash Buffer, block plate with Reagent wash 3x with Wash Buffer,
add 100 ul of sample/standard, incubate for 2 hours at room temperature
wash 3x with Wash Buffer, add 100 ul of detection antibody (biotin) diluted in
Reagent Diluent,
wash 3x with Wash Buffer, add 100 ul Streptavidin-HRP solution
wash 3x with Wash Buffer, add 100 ul Substrate Solution (H2O2+TMB),
add 50 ul Stop Solution (2N H2SO4)
read plate at 450 nm (correct with 540 nm)
28. y = 11251x1.3097
R² = 0.9909
0
1000
2000
3000
4000
0 0.1 0.2 0.3 0.4
Help to retrieve serum
samples from the straws,
Work out condition using
know concentration of
sLAG3 protein,
Established Standard curve
Examined a few samples
This is a diagram of some of the many co-stimulatory and inhibitory molecules
the left shows the ligands and the right shows the receptors
Interaction between the ligand and the receptor can have both a stimulatory and inhibitory effect on tcell activation and function
My focus for my project was LAG3 and PD1
Since it is an immune checkpoint receptor, many drug development programs target LAG3 as therapeutic treatments for cancers and autoimmune diseases.
Tumor microenvironment is important to consider because it greatly contributes to tumor development
There are a lot of suppressive molecules that can inhibit T Cells ability to kill tumor cells
In this diagram, it shows that two different types of tcells that overexpress PD1 and LAG3 receptors
You can also see here that there are many cytokines such TGFb and IL10 which are secreted in this microenvironment which can also inhibit T cell function
http://wirtzlab.johnshopkins.edu/research/tumor-microenvironment/
Multiple immune inhibitory and co-stimulatory pathways in the tumor microenvironment are targets of therapeutic manipulation by antibodies or drugs. Cells in the tumor microenvironment express multiple inhibitory cytokines, ligands, and cognate receptors (red) that down-modulate the antitumor activity of immune effector cells including cytotoxic T lymphocytes (CTL). Some of these inhibitory proteins are expressed by tumor cells themselves and others are expressed by tumor-infiltrating suppressive cells including T reg cells and myeloid derived suppressor cells (MDSCs). In addition to their function in dampening effector T cell responses, both CTLA-4 and PD-1 (as well as other checkpoint receptors, such as LAG-3) are highly expressed on regulatory T (T reg) cells, and in fact directly promote Treg cell–mediated suppression of effector immune responses
T reg cells inhibit antitumor CTL activity by producing soluble factors such as IL-10 and TGF-β, whereas MDSCs inhibit immunity through metabolic enzymes such as IDO and arginase. These inhibitory signals are counterbalanced by signals that enhance immune activation (green), a number of which are transduced by members of the TNFR family (CD40, CD137, OX40, and CD27). Blocking antibodies or drugs are in development or clinical testing for each of the inhibitory molecules depicted in the figure, and agonist antibodies are in development or clinical testing for each of the activating TNFR family members depicted (Table 1). DC/Mφ, DCs/macrophages.
IN Dr. Mastuzaki reported that LAG3 and PD1 are elevated in human ovarian cancer cells
In the TILs (Tumor infiltrating lymphocytes, actual tumor) and TALS (Tumor Associated Lymphocytes, in the ascites)
Dr. Huang reported that PD-1 and LAG-3 are co-expressed in mouse ovarian cancer microenvironment
Here are two examples of her results,
Rightmost- coexpression of LAG3 and PD1 receptors s
*put credit
Dr. Matusaki also showed here that IL6, IL10 anf TGFB can increase LAG-3 and PD1 expression in CD8 cells
These cells are isolated from PBMC (peripheral blood monocytes)
This graph shows LAG3 and PD1 expression over time (72 hrs), in anti CD3 and B7.1 coated plates
We wanted to observe the expression pattern over time
All three cytokines do not affect PD1 expression. However, they do show that they increase LAG3 expression in a concentration dependent manner. For the TGFB, only really increases LAG3 expression, In the cells that coexpress LAG3 and PD1
Ng/ml
Put date at the bottom of the slide
Activated cells for 72 hrs, then we rested cells 72 hrs, then treated them with the cytokines for the duration of 72 hrs
People might ask: will these cells reactivate? No, these were just treated in non coated plates
We did not really see similar trends to the activated cells, we think that maybe we should have reactivated the cells after they were rested
Here in the CD4 you can see a little bit of increase, but again, the trends were not quite the same as the activated cells
Talk about every single treatment for the first graph
Single treatment did not really have an effect on PD1 expression, however, after combination of treatments in the reactivated cells, pd1 expression increased
The combination of of TGFB and IL10 leads to the greatest effect on PD1 and LAG3 expression
The combination of TGFB and IL10 leads to the greatest effect on PD1 and LAG3 expression
In th glucose experiment, the cells did not really grow well so we stopped with that experiment
