The Missing Link in T-cell activation using a Vaccine, "The Danger Signal" may be due to an enzyme called IDOAs 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.

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!!

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-cellsTGF-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)

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

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.

Adopted from Current Opinion in Immunology

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.

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.

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-

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