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

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.

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-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)

 

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 approximately 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.

 

 

 

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 T_reg 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 synergist response.

 

 

 

 

Bristol Myer Squibb and Incyte Corporation are following this Science along
Newlink.

“It is not the strongest of the species that survives, nor the most intelligent, but the one most responsive to change.” ~Charles Darwin~

Take Care,

Jimmy B

Macrophage Activation… The Missing Link to Activation an immune response to Melanoma..Jim Breitfeller

"The development of Th1 cells producing high levels of IFN-gamma could not be induced with dendritic cells alone but required the addition of appropriately activated macrophages." Et al A. O'Gara March 23, 1993 "Dendritic cells and macrophages are required for Th1 development of CD4+ T cells from αβ TCR transgenic mice: IL-12 substitution f.or macrophages to stimulate IFN-γ production is IFN-γ-dependent"
Local IL-12 therapy stimulates Th cells to secrete Th1 cytokines. Exogenous IL-12 application creates an environment rich in IL-12 around the cancer site. In such a local environment, newly activated Th cells, responding to the presence of cancer, exit the blood and are influenced to become Th1 cells and secrete more Th1 cytokines and activate macrophages and NK cells. Activated macrophages will produce more IL-12 and via positive feedback, cell-mediated immunity can be promoted to battle the cancer thereby leading to the prevention of reoccurrence. As you can see, the activated macrophage secretes IL-12, IL-1a, IL-1b, and IL-6 along with a Chemoattractant, IP-10. Without macrophage activation, the immune response does not take place and the patient will relapse.(see graphic below)

With IL-1b and IL-6 missing, the T-Regulatory Cells (Tregs) rule the Tumor's Microenviroment. IL-6 controls Th17 immunity by inhibiting the conversion of naive CD4+ T cells into Foxp3+ regulatory T cells. IL-1β–Mediated Signals Preferentially Drive Conversion of Regulatory T Cells but Not Conventional T Cells into IL-17–Producing Cells (Danger Signal).So if the IL-1b signaling is missing,conversion of the Tregs never take place along with no Danger Signals.

Dr. Steven Rosenberg is using engineered T-cells that secrete IL-12 to stimulate Th cells to secrete Th1 cytokines. This sets in motion the positive feed back loop needed to initiate an immune response. His therapy is show great promise. “It is not the strongest of the species that survives, nor the most intelligent, but the one most responsive to change.” ~Charles Darwin~ Take Care, Jimmy B

Identifying and Over Coming Immune Barriers at the Level of the Tumor Microenvironment..Melanoma .Jim Breitfeller

Eleventh International Conference on Progress in Vaccination Against Cancer

Dr. Thomas Gajewski
University of Chicago,Chicago,IL,USA

"Immunotherapeutic approaches for the treatment of melanoma, such as tumor antigen-based vaccines, can frequently boost immune responses. However, clinical responses as measured by tumor shrinkage are seen in only a minority of patients. This observation has prompted careful analysis of the tumor microenvironment for biologic correlates to clinical response and also to identify mechanisms of tumor resistance. Patients with advanced melanoma treated with antigen-specific vaccines had pre-treatment tumor biopsies analyzed by gene expression profiling. Supervised hierarchical clustering was performed based on clinical outcome. An expanded bank of tumors was analyzed to increase the sample size and better understand gene patterns.
Two major categories of melanoma metastases have been observed.

One subgroup of patient has an inflamed phenotype that includes expression of chemokines, T-cell markers, and other immunoregulatory factors. Clinical responders to melanoma vaccines appear to fall within this subset. This group also contains the highest expression of negative regulatory factors, including PD-L1, IDO, and FoxP3, suggesting that these immunosuppressive mechanisms may dominantly inhibit anti-tumor –cell function in those patients. In addition, absence of B7 expression supports classical T-cell anergy. Preclinical experiments have confirmed a critical role for these mechanisms in limiting anti-tumor T–cell efficacy in vivo, giving candidate treatment strategies for translation back into the clinic.

A second subset of patients is represented by tumors which are non-inflamed and lack chemokines for T cell recruitment. Therefore, a major barrier in these cases appears to be failed T –cell migration into tumor sites. Experimental strategies to augment T-cell migration can have important anti-tumor effects in preclinical models. The presence of the "inflamed" gene signature was associated with a type I IFN transcriptional profile, and murine experimental models have confirmed a critical role for type I IFN signaling in promoting adaptive immunity."


So,In the first subset, tumors had a suppresive nature that may be over riddden by Anti-CTLA-4 (Yervoy) and or Anti-PD-1 Therapy

The second subset was missing the "danger signal" inflammatory cytokines and chemoattractants most likely due to STAT3 signaling from the Tumor.

Stimulation of Toll-like receptor 4 (TLR-4) activates macrophages and results in the release of TNF-alpha. It is hypothesized that melanoma inhibits macrophage activation by suppressing TLR-4 signaling.








Cytokines are small proteins which allow cells of the immune system to communicate with one another via cytokine receptors expressed at the cell surface.
Activated macrophages defend against tumors by secreting cytokines to recruit secondary immune cells, presenting antigen to T cells, and by direct tumor cytotoxicity. Peritoneal macrophages harvested from melanoma-bearing mice are less cytotoxic to melanoma cells, and produce less superoxide, nitric oxide, and tumor necrosis factor-alpha (TNF-alpha) than those from nontumor-bearing mice. Similar impairment of macrophage activation occurs in vitro using media harvested from cultured melanoma cells.

Activated Macrophages secrete the following cytokines under different conditions:
IL-1,IL-12,IL-6, IFN-gamma/alpha/beta and TNF-alpha


IL-6


Interleukin 6 is a pro-inflammatory cytokine and is produced in response to infection and tissue injury. IL-6 exerts its effects on multiple cell types and can act systemically.

IL-6 stimulates liver secretion of acute phase proteins
IL-6 stimulates B-lymphocytes to produce antibodies
IL-6 in concert with IL-1b causes T-cell activation
IL-6 induces STAT 3 Signaling
IL-6 Plus TGF-b induces the Th17 cell phenotype


IL-1 beta

Interleukin-1b is a pro-inflammatory cytokine which is secreted by macrophages activated by a number of stimuli including TNF-alpha, bacterial endotoxin and IL-1b itself.
IL-1b exerts its effects on many different cell types locally at the site of production and systemically (at a distance).


IL-12


Interleukin-12 is a heterodimer consisting of a p35 and a p40 subunit. Both subunits are required for receptor binding and biological activity.
IL-12 stimulates growth of activated Natural Killer (NK) cells, CD8+ and CD4+ T- cells.
IL-12 increases NK and T-cell g-IFN production which shifts T-cell differentiation towards a Th1-type response.
IL-12 increases NK production of TNF-alpha which can act synergistically with IFN-gamma.
IL-12 suppresses IL-4 induced IgE production.


TNF-alpha


Tumor Necrosis Factor alpha is made by many other cells as well as macrophages, which are major sources, especially after priming by Interferon gamma.
TNF-alpha initiates a cascade of cytokines which mediate an inflammatory response. TNF-alpha effects are mediated through two types of receptor, a 75kDa TNFR-a receptor and a 55kDa TNFR-b receptor.
TNF-alpha regulates the expression of many genes in many cell types important for the host response to infection.

IFN-gamma/beta

Macrophages, and many other cells produce these Type I interferons which act as immunomodulatory, as well as antiviral cytokines. Distinct receptor from interferon gamma, mediates overlapping or competing effects on macrophages. Cellular signalling pathways involve Jak/Stats, and other pathways.

So, if Melanoma suppresses Macrophage Activation, then the tumor microenviroment is missing IL-6, IL-1b and other cytokines.

If you look at the above micrographs, you will see that the two patients that had relapsed (10710 and 10737) Had IL-1b and IL-6 missing. The macrophages were not activated!!!! The "Danger Signal" known as inflammation was missing!





The missing combination of IL-1 and IL-6 meant there is no T-cell activation. And no induction of the Th17 phenotype. It is now becoming a lot more clearer based on Dr. Gajewski's findings.


Now might be the time for a critical re-evaluation of our overall approaches to targeting STAT3 and for developing new models for disrupting the protein in order to accomplish the goal of delivering clinically useful direct STAT3 inhibitors as novel anticancer agents in a timely manner.






“It is not the strongest of the species that survives, nor the most intelligent, but the one most responsive to change.”

~Charles Darwin~
Take Care,
Jimmy B

NIH Scientists Discover Secrets Of Helper T Cells Involved In Autoimmunity..Melanoma..Jim Breitfeller

NIH Scientists Discover Secrets Of Helper T Cells Involved In Autoimmunity
October 21, 2010

A race for a cure!!!!!!! Let the games begin!!!!!


WHATScientists at the National Institutes of Health have redefined the roles of several cytokines involved in the generation of immune cells implicated in severe autoimmune diseases. The study in mice showed that development of Th17 immune cells can occur without the presence of transforming growth factor (TGF)-beta, a mediator thought to be required for Th17 cell development. The study demonstrates that the interaction of three inflammatory cytokines (proteins that influence the behavior of cells) - interleukin-6 (IL-6), IL-1-beta and IL-23 - is responsible for the creation of Th17 cells that are more active in promoting autoimmunity than Th17 cells generated with IL-6, IL-1-beta and TGF-beta. These findings reemphasize the separate roles of IL-23 and TGF-beta in immunity and autoimmunity, and open up possibilities for the development of new therapies. The study appears in the current issue of the journal Nature.

The immune systems of mice and humans mainly consist of B cells and T cells. While B cells fight infections and can induce autoimmunity by producing antibodies that directly target foreign antigens or a person's own tissue, T cells are involved in overall cell-mediated immunity. Importantly, how a T helper (Th) cell differentiates (develops from an immature, unspecialized cell into a mature, specialized cell) determines how it mediates immune responses. Th17 cells produce IL-17, a powerful inflammatory cytokine, and have been implicated in multiple autoimmune diseases, including rheumatoid arthritis, psoriasis and multiple sclerosis. The established belief has been that Th17 cells initially differentiate in response to activation by IL-6 and TGF-beta. However, previous research has shown that TGF-beta is primarily associated with suppressing immune functions and promoting regulatory T cells (Treg), which can produce inhibitory cytokines that dampen inflammatory immune responses.

In the present study, the NIH scientists first looked at the conditions to differentiate Th17 cells from naïve T cells outside of the mouse (in vitro) and tried several different cocktails of cytokines to see which combinations would promote Th17 development. They found two combinations that efficiently induced Th17 differentiation. As previously described, IL-6, IL-1-beta, and TGF-beta-1 together created Th17 cells. Surprisingly, IL-6, IL-1-beta, and IL-23 without TGF-beta also created Th17 cells. Most interestingly, the action of Th17 cells generated with IL-23, designated Th17(23), was different from the action of Th17 cells generated with TGF-beta (Th17(beta)). The researchers compared transcription factors, receptors and mediators of the two Th17 subtypes and looked at the pathogenic activity of both Th17 subtypes in mice during experimental autoimmune encephalomyelitis (EAE), a common model of autoimmunity that mimics some aspects of multiple sclerosis. They found that Th17(23) cells provoked significantly more severe disease than did Th17(beta) cells.

These findings suggest a new model for Th17 generation and the existence of functionally different subtypes of Th17 cells. This study also provides a better understanding of the array of immune components involved in autoimmunity and suggests possibilities for new targeted therapies.

NIH scientists contributing to this study are affiliated with the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), the National Institute of Dental and Craniofacial Research (NIDCR), and the National Institute of Allergy and Infectious Diseases (NIAID). Additional support was provided by Merck Research Laboratories (Schering-Plough Biopharma), Palo Alto, Calif.

REFERENCE
Ghoreschi K, Laurence A, Yang XP, Tato CM, McGeachy MJ, Konkel J, Ramos HL, Wei L, Davidson T, Bouladoux N, Grainger J, Chen Q, Kanno Y, Watford WT, Sun HW, Eberl G, Shevach E, Belkaid Y, Cua DJ, Chen W, O'Shea JJ. Enhanced Pathogenicity of Th17 cells Generated in the Absence of Transforming Growth Factor-ß Signaling. Nature. 2010 October 21;467(7318): 967-971.

WHO
John J. O'Shea, M.D. Scientific Director, National Institute of Arthritis and Musculoskeletal and Skin Diseases, is available to comment on this article.

CONTACT
To schedule interviews, please contact Trish Reynolds, 301-496-8190, .

About National Institute of Arthritis and Musculoskeletal and Skin Diseases
The mission of the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), a part of the Department of Health and Human Services' National Institutes of Health (NIH), is to support research into the causes, treatment, and prevention of arthritis and musculoskeletal and skin diseases; the training of basic and clinical scientists to carry out this research; and the dissemination of information on research progress in these diseases. For more information, visit http://www.niams.nih.gov.

About The National Institute of Dental and Craniofacial Research
The National Institute of Dental and Craniofacial Research (NIDCR) is the Nation's leading funder of research on oral, dental, and craniofacial health. For more information, visit http://www.nidcr.nih.gov/.

About National Institute of Allergy and Infectious Diseases
The National Institute of Allergy and Infectious Diseases (NIAID) conducts and supports research -- at NIH, throughout the United States, and worldwide -- to study the causes of infectious and immune-mediated diseases, and to develop better means of preventing, diagnosing and treating these illnesses. For more information, visit http://www.niaid.nih.gov.

About National Institutes of Health
The National Institutes of Health (NIH) -- The Nation's Medical Research Agency -- includes 27 Institutes and Centers and is a component of the U.S. Department of Health and Human Services. It is the primary federal agency for conducting and supporting basic, clinical and translational medical research, and it investigates the causes, treatments, and cures for both common and rare diseases. For more information, visit www.nih.gov.

SOURCE: National Institutes of Health

NIH Scientists Discover Secrets Of Helper T Cells Involved In Autoimmunity
October 21, 2010




“It is not the strongest of the species that survives, nor the most intelligent, but the one most responsive to change.”
~Charles Darwin~
Take Care,

Jimmy B