1. The Missing Link in T-cell activation using a
Vaccine, "The Danger Signal" may be due to
an enzyme called IDO
As I research why some patients respond to therapies i.e. vaccination and other immunotherapy
and others don’t, I ask WHY? In my quest to get the answer or answers, I came across a paper
called “Marked Differences in Human Melanoma Antigen-Specific T Cell
Responsiveness after Vaccination Using a Functional Microarray”.
Daniel S. Chen1,2#, Yoav Soen3#, Tor B. Stuge4, Peter P. Lee4, Jeffrey S. Weber5, Patrick O. Brown2,3, Mark M.
Davis2,6*
1 Department of Internal Medicine/Division of Oncology, Stanford University, Stanford, California, United
States of America, 2 Howard Hughes Medical Institute, Stanford University, Stanford, California, United
States of America, 3 Department of Biochemistry, Stanford University, Stanford, California, United States
of America, 4 Department of Medicine, Stanford University, Stanford, California, United States of
America, 5 Norris Cancer Center, University of Southern California, Los Angeles, California, United States
of America, 6 Department of Microbiology and Immunology, Stanford University, Stanford, California,
United States of America
This is what I was looking for. It may hold the answer or could possibly point me in the right
direction.
In the paper I came across a diagram that peaked my interest. It was a comparison between
responders and non-responders.

3. We concluded from these studies that IL-1 and perhaps IL-6 play a critical role in the
differentiation and expansion of Th17 cells. Yoshihiro Miyahara et al
+
IL-6 controls Th17 immunity by inhibiting the conversion of naive CD4 T cells into
+
Foxp3 regulatory T cells.
Using in vitro and in vivo approaches, we determined that under neutral conditions,
simultaneous activation of Tregs and naive CD4+ conventional T cells in the presence of
APCs resulted in conversion of Tregs into IL-17–producing cells, and endogenous IL-1β
was mandatory in this process according to Vassiliki A. Boussiotis et al. “IL-1β–
Mediated Signals Preferentially Drive Conversion of Regulatory T Cells but Not
Conventional T Cells into IL-17–Producing Cells”
IL-6 protects CD4 T cells from cell death but also inhibits the suppressive effect of T
regs.
“Thus, the addition of IL-6 to the tumor microenvironment skews the balance toward
Th17 cells in a murine model of pancreatic cancer. The delayed tumor growth and
improved survival suggests that induction of Th17 in the tumor microenvironment
produces an antitumor effect.” David C. Linehan et al
They were looking at the cytokines secreted after the vaccine was given. When I saw what the
cytokines were, I knew I was on the right track. These cytokines help in the differentiation of the
CD4+ T-cells. What a find!!

4. Naïve CD4 T cells in the presence of TGF-b and IL-2 and others differentiate into
Tregs.
TGF-b accelerates the CTLA-4 expression by stimulated CD4+ CD25- T-cells
TGF-b requires CTLA-4 early after T-cell activation to induce FoxP expression
generating CD4+ CD25+ Treg Regulatory cells.
The Th-17 cells produce IL-17. .IL-17 induces the production of many other cytokines
(such as IL-6, G-CSF, GM-CSF, IL-1β, TGF-β, TNF-α)

5. So what was the non-responder missing, IL-6. With the missing IL-6, they weren’t able
to produce Th-17 that secreted IL-17.
While TGF-β is a critical differentiation factor for Treg cells, IL6 completely inhibits the
generation of Treg cells induced by TGF-β. Instead, IL6 and TGF-β together induce the
differentiation of pathogenic Th17 cells. With IL-6 missing in the microenvironment,
Treg Cells flourish.
If the CD4 + T cells differentiate into TH2 cells that produce IL-4, the other cells
inhibited to produce IL-6. IL-4 was found to inhibit TNF-α and IL-1β by activated
monocytes almost 100 %. The Secretion of IL-6 was decreased by approximatly 80 % in
the presences of IL-4 Cytokine. TE Velde et al 1990

6. They were missing “The Danger Signal”.
Friendly inflammation “The Danger Signal”
Most of the time you have no notion of the microbial life-and-death struggle being waged
within your body. At other times, though, you are acutely aware of the exact location of
the battleground, thanks to the unmistakable signs of inflammation — heat, pain, redness,
and swelling. Inflammation, the buildup of fluid and cells at the point of infection/cancer,
is put into motion by cytokines — proteins that are released into the blood by the innate
immune system when it encounters germs. Cytokines function like police dispatchers.
They signal there's a problem, which activates the immune system's highway patrol force:
the circulating lymphocytes of the adaptive immune system. These lymphocytes cruise
the highways of the blood vessels and lymphatic system. In response to the chemical
signal from the cytokines, increased blood flow rushes these circulating cells to the
trouble spot.
“The CD8+ T-cell-mediated Immune Response to Eradicate the
Tumors”
“Three major events must occur to induce CD8+ T cell–mediated, tumor-protective
immunity against syngeneic melanoma. First, the T-cell receptor must be triggered by a
(or multiple) self antigen–derived peptide MHC class I complex . Therefore, this event
depends entirely on appropriate antigen presentation, which is most efficiently provided
by mature dendritic cells. Peripherally tolerant or “ignorant” self-reactive T-cell clones,
once properly activated, may serve as tumor-specific effector T cells .Second,
simultaneously with T-cell receptor triggering, a distinct second costimulatory signal
must be delivered, mediated by IL-2, B7-1, or B7-2, which engage IL-2 receptors and
CD28 on the surface of the T cell, respectively. A source of these cofactors for effective
CD8+ T-cell stimulation can be provided by CD4+ T cells that release critical amounts of
IL-2, or by mature dendritic cells that display an increased level of B7-1/B7-2
costimulatory molecules on their cell surfaces. Third, inflammatory cytokines,
including IL-1, IL-6, IL-12, IL-17 and IFN-γ provide a third signal that acts directly
on T cells, referred to as the “danger signal”. This signal was found to optimally
activate TH1 differentiation and lead to clonal expansion of T cells.

7. Adopted from Current Opinion in Immunology

8. The responder was able to produce inflammatory cytokines, including IL-1, IL-6, IL-12,
IL-17 and IFN-γ provides a third signal that acts directly on T cells, referred to as the
“danger signal”. This signal was found to optimally activate TH1 differentiation and lead
to clonal expansion of T cells and invoke a robust immune response to the Melanoma
Cancer.

9. Conclusion: Based on my observation, the cytokine that ties this “Danger Signal” to the
immune system is IL-6.
• IL-6 protects CD4 T cells from cell death but also inhibits the suppressive
effect of Tregs.
• IL-6 controls Th17 immunity by inhibiting the conversion of naive
+ +
CD4 T cells into Foxp3 regulatory T cells.

10. So what is causing the lack of IL-6 in the non-responders? The IDO enzyme. This
enzyme catalyzes the degradation of the essential amino acid L-tryptophan to N-
formylkynurenine.
IDO enzyme degrades tryptophan and through the GCN2 kinase pathway inhibits the
transcription of IL-6. Without the transcription of IL-6, the IL-6 cytokine cannot be
produced leading to the T-cell differentialtion toward the T Regulatory cell instead of the
TH17 phenotype.
My guess is the tumor induced enzyme called IDO may the Missing Link to intiating an
immune response.IDO produced by Tumor cells significantly inhibited interleukin (IL-
2) expression and proliferative response in T-cells and increased apoptosis (death) of T-

11. cells. Tryptophan depletion is known to halt cell cycle progression by triggering the
antiproliferative GCN2 pathway in lymphocytes.
Also, IDO is upregulated in antigen-presenting dendritic cells (DC) by autocrine IFN-γ
released as a result of Treg cell–induced CTLA-4/B7-dependent cell-cell signaling.
It is well established that IDO expression by APCs or tumors can inhibit immune
responses.
Tryptophan depletion by IDO-expressing tumors is a common mechanism of
immune evasion inducing regulatory T cells and inhibiting effector T cells.
So adding IDO inhibitor to a combinatorial therapy like Yervoy for melanoma cancer
should see a syenergist response