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

Immunotherapeutic Barriers at the Level of the Tumor Microenvironment..Melanoma .Jim Breitfeller

Last year I Blogged about the Missing Link in T-cell activation with a vaccine. We were missing the Proflammatory Cytokines that produce the "Danger Signal" to the Immune system. Well Dr. Gajewski has come out and has backed up my theory.

Along with that, there are Two companies that are persuing this theory with Cytokine Therapy.


Thomas F. Gajewski, MD, PhD (University of Chicago) presented on the key role that the tumor microenvironment plays in determining the outcome of a tumor immune response. He noted the complexity of the tumor with respect to its structural and cellular composition and that the functional phenotypes of these cells may or may not permit an effective anti-tumor response at either the priming or effector phase. Characteristics of the tumor microenvironment may dominate during the effector phase of an anti-tumor T cell response, limiting the efficacy of current immunotherapies by inhibiting T cell trafficking into the tumor, eliciting immune suppressive mechanisms within the tumor, altering tumor cell biology and susceptibility to immune-mediated killing, or modifying the tumor stroma (i.e., vasculature, fibrosis). These features can be interrogated through pre-treatment gene expression profiling of the tumor site in individual patients; such an analysis may identify a predictive biomarker profile associated with clinical response. This strategy may also help identify biologic barriers that need to be overcome to optimize therapeutic efficacy of vaccines and other cancer immunotherapies. Mouse models have helped to define the hallmarks of an anti-tumor response, taking into account the effector phase within the tumor microenvironment. Based on these models, a DC subset (CD8α+) appears necessary for priming of host CD8+ T cells through cross-presentation of antigen within the draining lymph node. Antigen specific naïve CD8+ T cells that recognize the antigen within the lymph node and receive appropriate co-stimulatory and proliferative signals acquire their effector phenotype. In order to assert immune control over the tumor, these effector CD8+ T cells must enter the bloodstream, and via chemokine signals, traffic to the tumor site; once there, these T cells must overcome immune regulatory/suppressive mechanisms. In a small study of an IL-12-based melanoma vaccine, Dr. Gajewski and colleagues correlated pre-treatment biopsy gene expression to outcomes and noted that in responding patients, tumors expressed chemokines (e.g., CXCL9 which binds CXCR3 on activated CD8+ T cells), which in some instances were able to recruit T cells into the tumor site. A broader transcript analysis of banked melanoma tissue demonstrated a subset of tumors with T cell markers co-associated with a panel of chemokines. Among responders in the vaccine trial, there was a pattern of expression of T cell- recruiting chemokines, T cell markers, innate immune genes, and type I IFN—all of which indicate productive inflammation. These results were supported by other cancer vaccine studies that demonstrated a strong correlation between survival and the expression of T cell markers and chemokines within the tumors. The results from these gene expression studies may be useful in identifying biomarkers that could provide valuable information for selecting patients most likely to respond to immunotherapies. Additionally, these studies point toward specific strategies for overcoming immunologic barriers to immunotherapy at the level of the tumor microenvironment. Thus, based on gene expression profiling, tumors can be categorized as T cell poor tumors, which lack chemokines for recruitment and have few indicators of inflammation, and T cell rich tumors, which express T cell recruiting chemokines, contain CD8+ T cells in the tumor microenvironment, and have a broad inflammatory signature. A strong presence of T cells within the tumor is predictive of clinical benefit from vaccines.

These observations prompt several important questions:

1) What dictates recruitment of activated CD8+ T cells into the tumor?
2) Why are tumors with CD8+ T cells not spontaneously rejected?
3) What are the innate immune mechanisms that promote spontaneous T cell priming in a subset of patients?
4) What oncogenic pathways in tumor cells drive these two distinct phenotypes?

Studies of CD8+ T cell recruitment to the tumor site point to a panel of chemokines, all of which may be produced by the melanoma tumor cells themselves. These studies suggest potential strategies to promote effector T cell migration to the tumor site that may include: direct introduction of chemokines; direct induction of chemokine production from stromal cells; eliciting local inflammation that generates chemokines (e.g., via type I IFNs, TLR agonists and possibly radiation); and altering signaling pathways in melanoma cells to enable chemokines expression by the tumor cells. Studies that have been designed to evaluate why melanomas that attract CD8+ T cells are not spontaneously rejected have pointed to several mechanisms that may exert negative regulation of T cells within the tumor microenvironment, including T cell inhibition via IDO and PD-L1, extrinsic suppression via CD4+CD25+FoxP3+ Tregs, and T cell anergy due to deficiency of B7 costimulation in the tumor microenvironment. Dr. Gajewski presented data that indicate that the immune inhibitory mechanisms present in the melanoma tumor microenvironment are driven by the CD8+ T cells, not the tumor. For example, IFNγ is the major mediator for IDO and PD-L1; and CCL22 production by CD8+ T cells is the major mediator for Tregs. Thus, blockade of these mechanisms may represent attractive strategies to restore anti-tumor T cell function and promote tumor rejection in patients.
To address questions underlying the mechanisms that promote spontaneous T cell priming in a subset of melanoma patients, Dr. Gajewski and colleagues used gene array data to identify markers of innate immunity that correlated with T cell infiltration. Melanoma metastases that contained T cell transcripts also contained transcripts known to be induced by type I IFNs. A knock-out mouse model demonstrated the necessity of the type I IFN axis for effective priming of a spontaneous T cell response and tumor rejection. Additional studies in knock-out mice have demonstrated that the CD8α+ DC subset is responsible for this spontaneous T cell priming. In an effective anti-tumor response sensing of the tumor by a separate DC subset drives type I IFN production, which is required for CD8α+ DC cross-priming of T cells. This suggests additional pathways that could be altered to promote spontaneous priming and an effective tumor response (e.g., provision of exogenous IFNβ). In summary, there is heterogeneity in patient outcomes to cancer immunotherapies (e.g., melanoma vaccines). One component of that heterogeneity is derived from differences at the level of the tumor microenvironment. Key factors in the melanoma microenvironment include
chemokine-mediated recruitment of effector CD8+ T cells, local immune suppressive mechanisms, and type I IFNs/innate immunity. Understanding these aspects should improve patient selection for treatment with immunotherapies (predictive biomarker), as well as aid the development of new interventions to modify the microenvironment to better support T cell-mediated rejection of tumors.

Source: http://www.translational-medicine.com/content/pdf/1479-5876-9-18.pdf

















Induction of an immune response by antigen vaccination. Active immunization occurs following administration of tumor antigens, which are processed by antigen-presenting cells resulting in activation of immune effector cells such as T lymphocytes and B lymphocytes. Effector cells fight the tumor through several different effector pathways such as antibodies, cytokines or direct cellular interaction (Fas/Fas ligand, perforin/granzymes). Ideally this results in immunologic memory with long-lasting immunity against the tumor. Stimulation of the innate immune system is also known to have an important role in the evolution of an adaptive immune response (e.g. a rapid burst of inflammatory cytokines leads to activation of DC and macrophages). However, tumor-specific T cells also secrete chemokines (e.g. RANTES, MIP-1) that attract cells of the innate immune system to the tumor site. This mechanism may further contribute to the tumoricidal activitiy exhibited during T cell-mediated tumor regression. (TCR: T cell receptor, MHC: major histocompatibility complex, DC: dendritic cell, PMN: polymorphonuclear neutrophil, NK: natural killer cell, NKT: NK T cell, RANTES: regulated upon activation, normal T cell expressed, and secreted, (MIP-1): macrophage inflammatory protein 1)



Two companies that are pursuing Cytokine Therapies with a product of “Natural Mixtures of Cytokines.” They may provide the missing signal needed to raise the red flag, “The Danger Signal”

Multikine is in the Phase 3 and IRX-2 is in the Phase 1 clinical trial Phase
IRX Therapeutics seems to have the most appropriate mixture. Cel-SCI is missing the chemoattractants and proinflammatory cytokines.

If you combine the Cytokine therapy with anti-CTLA-4 blockage, will you get a synergistic immune response?



“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

Alarmins Initiate Host Defense against Melanoma, Let the war games begin..Melanoma ..Jim Breitfeller

The immune system has evolved to respond not only to pathogens, but also to signals released from dying cells. These signals are generated by molecules call Alarmins.
In response to infection and/or tissue injury, cells of the host innate immune system rapidly produce a variety of structurally distinct mediators that not only function as potent effectors of innate defense but also act to alarm the immune system by promoting the recruitment and activation of host leukocytes through interaction with distinct receptors. One of these Alarmins is the High mobility group box 1 (HMGB1).

(HMGB1) is a DNA-binding nuclear protein, released actively following cytokine stimulation as well as passively during cell death; it is the prototypic damage-associated molecular pattern (DAMP) molecule and has been implicated in several inflammatory disorders, but our case, it is the molecule that sends the red flag up to the immune system, stating there is a problem.

This Alarmin (HMGB1) is capable of activating antigen-presenting cells (APCs) and enhancing the development of antigen-specific immune responses.

The interaction of high mobility group box 1 protein (HMGB1) released from dying tumor cells with Toll-like receptor 4 (TLR) on dendritic cell was required for the crosspresentation of tumor antigens and the promotion of tumor specific cytotoxic T-cell responses.



Under stress conditions, such as injury or infection, HMGB1 is released and promotes inflammation. (Danger Signal) HMGB1 is passively released by Death of cells or tissues through injury or disease (necrotic) but not apoptotic death. Disintegration of cells into membrane-bound particles that are then eliminated by phagocytosis or by shedding of normal cells and actively secreted by a variety of activated immune and non-immune cells.
Through the TLR4, HMGB1 activates NFκB inducing a wide range of host changes that include activation of the innate immune system (neutrophils, NK cells, dendritic cells) and secretion of proinflammatory cytokines and mediators.

There is growing evidence that the relationship between the inflammatory process and cancer is complex. Our understanding of this relationship as it relates to Danger Signal development and or progression of malignancy is still limited. Further evaluation in patients is clearly needed if we are to truly understand whether there is therapeutic potential in targeting Pro-inflammation, “The Danger Signal”.

HMGB1 as a DAMP released into the tumor microenvironment plays a central role in the recruitment and activation of innate immune cells.

This emergent understanding of the danger signals also called alarmins or damage associated molecular patterns (DAMPs) is fueling new research that may be beneficial to finding a cure.

This ability of dendritic cells to recreate the environment associated with necrosis via the regulated secretion of HMGB1 represents a successful evolutionary strategy and places HMGB1 at the crossroads between innate and adaptive immunity.



Once T-cells are activated, RAGE and HMGB1 are upregulated during cellular activation, consistent with a role of the RAGE axis in pro-inflammatory immune responses.

So how do we cause tumor cells to die? We don’t need to kill them all at once, that would be nice, and we need to a portion so that HMGB1 is secreted. Or do we inject a mixture of naturally occurring Cytokines that act as the pro-inflammatory cytokines, (the Danger Signal)?



.
A little known company is just doing that. The company is IRX Therapeutics.

Irx Therapeutics, Inc
2350 Broadhollow Road
Farmingdale, NY 11735-1006


Dr. John W Hadden founded the company in 1994 and holds the patent on the cytokine mixture called IRX-2.

The cytokine components of IRX-2 (includes IL-1, IL-2, IL-6, IL-8, IFN-γ, TNF-α, G-CSF and GM-CSF) are known to promote both T lymphocyte and dendritic cell development and function and monocyte function.

I believe the natural cytokine mixture (NCM). The NCM includes 1L1, IL2, IL6, IL8, IL10, IL12, .delta.IFN, TNF.alpha. and G- and GM-CSF could play a pivotal role in activation of the T-cells (CD4+ and CD8+). This mixture that is in the patent (20030206885) is what I would call the Proinflammatory Cytokines, the “Danger Signal”.

The natural cytokine mixture is missing MCP-1, MIP-1 alpha and beta which is very important to attract immune cells to the tumor’s microenvironment.


CCL3/MIP-1 alpha acts as a chemoattractant to a variety of cells including monocytes, T cells, Dendritic cells, B cells and eosinophils

CCL4/MIP-1 beta is expressed primarily by T cells, B cells, and monocytes after antigen or mitogen stimulation. The functional receptor for MIP-1 beta has been identified as CCR5.

CCL4, also known as macrophage inflammatory protein 1 beta (MIP1β)
is a 7.8 kDa β chemokine that is secreted at sites of inflammation by activated leukocytes, lymphocytes, vascular endothelial cells.

CCL4 attracts a variety of immune cells to sites of microbial infection as well as to other pathologic inflammation.

Monocyte chemoattractant protein-1 (MCP-1) is important in attracting monocytes to sites of inflammation. Besides induction of monocyte recruitment, MCP-1 can also affect chemotactic response of endothelial cells.


I speculate by combining Radiation, Chemotherapy, Ipilimumab, IRX-2, and IL-2 in a systematic way, we can induce an immune response against Melanoma and win most of the Battles. Let the war games begin. Who will take on this challenge and be the first to CURE Melanoma? We shall see.

A clue: This is from a patient that is receiving Ipilimumab (Yervoy)

“The Dr. says it's possible the swelling is caused by the tumors starting to respond. My back and leg tumors did become inflamed before they receded, so it's possible the same is happening in the brain. I could also be having a delayed reaction.”




From a paper of Dr. Meenhard Herlyn.

Inflammation is the key to the Danger Signal which can be produced by dying Tumor cells or Ipilimumab differentiating T-cells into Th-17 Phenotype or by injecting a pro-flammatory cytokine mixture. Let the War on Melanoma Begin!!!!!


“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

The Missing Link in T-cell activation using a Vaccine, "The Danger Signal"Melanoma..Jim Breitfeller

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.

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 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.

The responder was able to produce 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 and invoke a robust immune response to the Melanoma Cancer.

“Our data altogether suggest the following model: Th17 cells go to the tumor site and, by secreting IL-17, activates residential cells to produce CCL2 and CCL20, which provokes the mobilization of DCs and other leukocytes to the tumor site. DCs uptake tumor antigens in the lung or tumor site and migrate to the lymph nodes where they activate CD8+ T cells against the tumor. The new wave of effector CD8+ T cells migrates back to the lung or tumor site and kills established tumors.

Therefore, our data demonstrating that Th17 cells and IL17 participate in antitumor immunity by facilitating T cell recruitment to the tumor site and CD8+ T cell priming and effector differentiation suggests a new avenue for developing Th17 cell-based therapy for tumors or chronic viral infections and as an adjuvant for vaccinations.”

(Dudley and Rosenberg, 2007; Rosenberg and Dudley, 2004)

Conclusion: We not only want to supress the Tregs, we also need to produce the "Danger Signal" to optimally activate TH1 differentiation and lead to clonal expansion of T cells to generate a robust tumor-specific immune response.






The Missing Link in T-cell activation using a Vaccine, "The Danger Signal"


“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

Dr. Polly Matzinger Danger!! Danger!! Melanoma..Jim Breitfeller

Danger signals
"In a 1994 article entitled "Tolerance, Danger and the Extended Family", Matzinger went several steps further by laying out the idea that antigen-presenting cells respond to "danger signals" - most notably from cells undergoing injury, or stress or "bad cell death" (as opposed to apoptosis, controlled cell death). The alarm signals released by these cells let the immune system know that there is a problem requiring an immune response. She argued that T-cells and the immune response they orchestrate occurs not because of a neonatal definition of "self", as in the previous model, nor because of ancient definitions of pathogens, as in Janeway's argument, but due to a dynamic and constantly-updated response to danger as defined by cellular damage."


The Danger Model

The self-non-self model, the predominant model in immunology since the 1950s, began to encounter problems in the late 1980s when immunologists began to recognize that T-cells depend on other cells to pick up and then present the things to which they will respond — and that the T-cell response depends on whether the other cell (known as antigen-presenting cells) is sending activation signals to the T-cells.

In 1989, drawing on the ideas of Thomas Kuhn, Charles Janeway proposed that the old immunological paradigm had reached the limits of its usefulness--or, as he described it, the asymptote of the increase in knowledge which it had brought. Janeway argued that the innate immune system was the real gatekeeper of whether the immune system responded or did not respond. He also argued that the innate immune system used ancient pattern-recognition receptors to make these decisions - recognizing a pathogen by its unchanging characteristics.

Source:Wikipedia

Part of my Research

Jimmy B