The Danger Theory What is it? Danger Theory

The Danger Theory What is it? Danger Theory

The Danger Theory What is it? Danger Theory What is self? Everything encoded by the genome? Everything under the skin, including structures encoded by commensal genomes. Any tissue accessible to lymphocytes (excludes privileged sites, e.g., brain, cornea, and testes)?

For T cells the set of peptides complexed with MHC molecules (that dont elicit a response)? For B cells, cell surface and soluble molecules (to which they would not respond)? The set of bodily proteins that exist at a concentration above a certain threshold? A theory put forth to address many unexplained immunological phenomenon What happens when self changes?

Puberty Lactation Aging

Why are fetuses not rejected? Why do we fail to make immune responses to vaccines composed of inert foreign proteins unless we add noxious substances (adjuvants)? Why do we fail to reject tumors even though they express new proteins (antigens)? Immune surveillance does not exist. Why do most of us harbor autoreactive lymphocytes without any sign of autoimmune disease, while a few individuals succumb (most of us also carry antibodies reactive to keratin and DNA)? Why do we respond to some pathogens and not others? Why are some transplants e.g., liver more likely to survive than other types of tissue?

What mechanism can induce tolerance to antigens found only on skin, kidney, or liver cells? How are we tolerant to the 55,000 different bodily proteins, plus 1012 potentially different B and T cell idiotypes and still be able to respond to foreign antigens. Ascaris lumbricoides Canine: heartworm Normal Gut Flora

The Evolution of the Danger Theory Immunology in the traditional sense: anything not me is non-self and will therefore be cleared. The first SNS (Self-Non-Self) model. 1959; Burnet and Medawar The Evolution of the Danger Theory

This SNS model suggested that... (i) Each lymphocyte expresses multiple copies of a single surface receptor specific for a foreign entity. (ii) Signaling through this surface Ab initiates the response. (iii) Self-reactive lymphocytes are deleted early in life (reinforced by Ray Owens observations in 1945). The Evolution of the Danger Theory SUMMARY-The antigen is in control

(Burnet): recognition of antigen (Signal 1) leads to B- and T-cell activation. Modifications to the SNS e.g., cytokines, ligands. In 1969, Bretscher and Cohn added the 2-signal hypothesis to the SNS model. New theory proposed that autoimmunity would be rare if 2 cells had to recognize different specificities

on the same antigen. Signal 1/Signal 2 This version of the SNS lasted until 1976 Modifications to the SNS e.g., cytokines, ligands. SUMMARY-The helper cell is in control (Bretscher and Cohn): Signal one leads to Bcell death, but the addition of help (Signal 2) leads to activation. Matzinger. Scand. J. Immunol. 2001.

Modifications to the SNS e.g., cytokines, ligands. Lafferty and Cunningham modified it by adding yet another new cell (the accessory cell or the APC) and a new signal (co-stimulation). This theory was largely ignored for 13 years because up until now, APCs were believed to be non specific and always active. Matzinger. Scand. J. Immunol. 2001.

Why was the co-stimulation theory ignored for so long? Co-stimulation by APCs did not fit the SNS model because... Unlike help (Signal 2), which comes from a population of T-helper cells that are antigen specific (and can be depleted if self-reactive), co-stimulation comes from APCs, which cannot distinguish self from non-self (What? Is this true?). Enter the Infectious Non-self SNS model.

Charles Janeway 1943-2003 APCs are not non-specific e.g., cytokines, ligands. Janeway, 1976. APCs are not constitutively active. APCs express pattern recognition receptors (PRRs). PRRs (e.g., Toll-like receptors) recognize bacterial

products that will lead to activation and costimulation. This means that APCs CAN recognize self from a point. TLRs and SNS TLR2

Liporoteins Hsp60 TLR4 LPS Hsp70 hyaluron TLR9

DNA -TLRs recognize both endogenous and exogenous molecules. Why? -Maybe pathogens have evolved to bind them and not the other way around. Pathogen-Ligands HIV CCR5 Toxoplasma Streptococcus &

Fc, C3 Coxsackie virus Rabies virus EBV LPS/Apoptotic cells CD4, CCR5 TCR, AbStaphylococcus ICAM-1 N-CAM CR2

CD14 The SNS and INS Models The critical event in all of these theories is the recognition of foreign antigen. What is non-self? We have spent half a century studying selfnon-self discrimination. Janeway himself pointed out that even with the addition of PRRs the SNS/INS models could not explain the immune response to transplants or tumors, nor the dysfunction(s) that lead to many autoimmune diseases.

Danger Theory Abandons the concept that the immune system is concerned with nonself. The Danger model is based on the idea that the ultimate controlling signals are endogenous, not exogenous.

Matzinger. Scand. J. Immunol. 2001. Danger Theory SUMMARY-The tissues are in control (Matzinger): APCs receive activating signals from injured cells, but not from healthy cells or from cells

dying by normal physiological death. Matzinger. Scand. J. Immunol. 2001. Matzinger. Science. 2002 The Big Picture: Danger Model A) fetuses, milk proteins, aging related changes. C) mutations INS predicts neither transplants and fetuses

would be rejected. SNS predicts both should be rejected. DM predicts that healthy fetuses should not be rejected because the do not send out an alarm. Transplants cannot be performed without surgical and or ischemic damage: danger. Matzinger. Science. 2002

Rules of the Danger Theory First Law-DIE IF YOU RECEIVE SIGNAL ONE IN THE ABSENCE OF SIGNAL TWO. Cells that receive signal one can be rescued by the addition of an appropriate Second signal. Leads to activation. Co-stimulation for T cells Help for B cells Second Law-ACCEPT SIGNAL TWO

(ACTIVATING SIGNAL) ONLY FROM APCs. Third Law-IF ACTIVATED, ONLY NEED SIGNAL 1 AND IT CAN COME FROM ANY CELL TYPE. Rules of the Danger Theory: T cells T cell differentiation state Immature

thymocyte Potentially activating (or triggering) cells NONE Tolerizing cells

ALL Resting naive T cell DC, Active M? Resting Activated experienced effector T cell

T cell DC B cells, Tissue cells ALL M B cells Tissue cells

NONE Examples of the Danger Theory Immune system should always be in off mode. Dendritic cell at work.

Mounting a Response Even though asleep, dendritic cells sample one cell volume every hour. x2 Life Changes Why is there no immune response to the many new proteins associated with lactation, puberty, aging?

First-They are not associated with tissue damage (death, destruction, distress, danger). Danger Signals? Necrotic cell death, heat-shock proteins Second-Any T or B cells that could recognize these changes will receive signal one only (which means?). Tumors Newly arising tumors may express antigens not expressed by normal

tissues but this is not enough to alert the immune system. No difference between a rapidly dividing cancer cell and a rapidly dividing hematopoietic cell, gut cell, or thymocyte. Consequently, as it grows, any tumor unable to deliver Signal 2 should induce deletion of tumor specific T

cells. Signal 1 T NO DANGER Tumor Mature Resting T cell Dendritic cell

Not active. T Anergy or Death Activated lymphocyte T

T Tumor Signal 2 Signal 1 Dendritic cell Dendritic cell Trauma e.g., -surgery, -infection,

-injection of in vitroactivated TILs. Signal 1 T Tumor Mature Activated T cell -Only needs Signal 1.

Tumor T cell Activation in the Tumor Model By trauma (as described). Vaccination* (tumor vaccine). Activation of T cell in vitro. However...the tumor size will affect the efficacy of these strategies.

Sometimes size matters not sometimes it does. T Mature Activated T cell -Activated by vaccine or in vitro by cytokines. Tumor Mature Activated T cell -Activated by vaccine or in vitro by cytokines.

T Tumor T -T cell attacks tumor. -T cell now needs to be Re-activated with Signal 1 and 2 T cell needs APC for

reactivation in the lymph node. Tumor T Without reactivation, T cell goes into resting state. -Need repeated tumor vaccination or repeated TIL treatment.

T Resting cell receiving signal one without Signal 2 will die. Tumor Tumor regenerates. Tumor Viral Infection (Skin) Sk

Epithelial cell Sk Epithelial cell Sk Epithelial cell Non infected skin cells expressing self

antigen (Sk). Viral Infection (Skin) Dendritic cell Dendritic cell Sk Sk V Sk

Infected skin cells expressing self antigen and viral antigen (V). Immune system should always be in off mode. Dendritic cell at work. Viral Infection (Skin) Sk

Dendritic cell Sk V Sk Dendritic cell Virally-infected skin cell is lysed, sending out danger signals (necrosis). Sk

Sk V Sk V Sk Dendritic cell Dendritic cell becomes activated and it picks up both Sk and V antigens and goes to the DLN.

Signal 1 and 2 Signal 1 and 2 TSk Sk V

TV Dendritic cell Activated T cells go to the site of infection. Activated T cells return to the site of infection. TS k

k TS T Sk Sk V V Sk

Infected skin cells expressing Self antigen and Viral antigen are lysed by Activated T cells. Resting T cell TSk TV Resting T cell

TSk Sk Resting T cell Sk V Sk After lysing Sk- and V-antigen expressing cells, T cells have to get recharged by Signal 1 and Signal 2.

Resting T cell TSk TV Resting T cell TSk Sk Resting T cell

Sk Sk After infection is cleared, no more dendritic cell activation and no more Signal 1 and Signal 2. T cells not active. Resting Tsk cell Receiving Signal 1 only. k

k TS TS TV Sk Resting Tv cell

Sk Sk After infection is cleared, no more dendritic cell activation and no more Signal 1 and Signal 2 in the DLN. T cells not active. TSk TSk

TV Resting Tv cell. Memory cell. VitiligoAssociated with spontaneous melanoma remission Deletion of Tsk (Signal one only) and maintenance of Tv cells. Tolerance established. Outcome dictated by tissue size. Like the tumor

model, the size of the infected tissue defines the outcome i.e., there are infinitely more Sk antigens than V antigens. Spontaneous Tumor Regression Concurrent with acute bacterial infections.

Administration of bacterial vaccines. Removal of at least some of the tumor or its metastases. Compiling 449 cases of spontaneous regression most were commonly associated with suppurative infections e.g., S. pyogenes (Nautus, 1980). Spontaneous Tumor Regression Everson and Cole (1966) reported 176 cases of spontaneous regressions.

40% of the patients had some type of operative trauma. 24% excision of the primary tumor was followed by regression of metastases. Both surgery and infection result in tissuemediated danger signals. Efficacy of Adjuvant Why so many vaccines (protein) do not work. Most do not elicit a Freunds complete

danger response, especially those approved for human use i.e., alum. Attenuated pathogens, DNA vaccines, elicit danger signals Alum without the need for adjuvants. Damage to Tissues

Danger and Tissue Transplantation Until the mid-1980s, most attempts to transplant organs were unsuccessful. Then, cyclosporine A was found to block the activation of the immune system, and soon transplant successes were occurring everywhere. Danger and Tissue Transplantation

90-95% transplants lasted longer than 1 year. However, 40-50% of kidney transplants were lost by 10 years. 30% of patients now on kidney waiting lists have already had one--and lost it. Patients need to take the drug the rest of their lives, living on the edge of immunosuppression with a constant threat of infections. Danger and Tissue Transplantation

A better approach came from: 1) Understanding how cyclosporine A works i.e., by blocking signal one. 2) The understanding that the bodys normal tolerance mechanism hinges on Signal 2, NOT Signal 1. Why you have to take CspA for life. Tx

x Activated, X-reactive T cell x CspA Tx Transplanted Tissue

S2 S1 X Dendritic cell Surgery=Danger signals Alternative strategy to CspA.

Deleted because only gets S1 Non-activated, X-reactive T cell Tx Tx x x

Transplanted Tissue S2 S1 X Dendritic cell Surgery=Danger signals

Danger signals derived from stressed and necrotic epithelial cells activate human eosinophils Stenfeldt et al., 2004. 112:605, Immunology. Background & Purpose 99% of eosinophils are found within epithelial surfaces interfacing with the environment (gastrointestinal, genitourinary and respiratory

tracts. Eosinophils increase in numbers in the intestinal mucosa during various types of inflammation (e.g., inflammatory bowel disease, celiac disease and dysentery caused by Shigella. Found in infiltrates of transplants being rejected. Background & Purpose Aim of study was to determine if blood-derived eosinophils from healthy individuals are able to recognize danger signals from damaged epithelial cells. Measured

eosinophil migration release of putative tissue-damaging factors eosinophil peroxidase (EPO) eosinophil cationic protein (ECP) secretion of tissue healing factors fibroblast growth factor (FGF) transforming growth factor (TGF-b) Generation of stressed or necrotic epithelial cells a) freeze pressed

b) freeze thawed c) heat-treated (trypan blue) d) viable HT29 Cell types tested: HEp-2: larynx carcinoma HT29:colorectal carcinoma HeLa: cervical carcinoma Chemoatxis induction 3 different concentrations of

epithelial cells were placed at the bottom of a transwell plate and 30K eosinophils were examined for migration from the top well. Eotaxin=positive control. Release of ECP ECP released by the

presence of necrotic cells. ECP implicated in immunity to parasites and pathophysiology of chronic allergic responses, Heat shock proteins from part of a danger signal cascade in response to lipopolysaccharide and GroEL

Davies et al. 2006. 145:183. Clinical and Experimental Immunology. Background & Significance Recently, heat-shock proteins (Hsp) have been implicated as members of a danger signaling cascade. Hsps are normally intracellular proteins that have functions involved in protein folding and maintenance of protein

integrity under both normal and stress conditions. Background & Significance However, bacterial Hsps (e.g., GroEL) have been found in extra cellular locations and implicated in immune reactions to infection. Human Hsps have also been shown to be present on cell surfaces, serum, and to be released from a variety of cell types (e.g., Hsp70 from lymphocytes)

Hypothesis Bacterial Hsps act as danger signals that activate the immune response. Test if Hsp GroEL and LPS up-regulate human Hsps Release of Hsp70 PBMCs (a) and Jurkat cells (b) release Hsp70 in response to LPS

and GroEL compared to control. 10x increase in the presence GroEL. Hsp60 had no effect on Hsp70 release. Summary Hsp70 was released by PBMCs, a T cell line and tumor cell line when grown at 37C for 24 h. The release of Hsp60 at these conditions was

not significantly greater than that of LDH release (control). Suggests that under normal conditions the cells are releasing low levels of Hsp70 but not Hsp60. HOWEVER, the amount of Hsp60/70 was increased after exposure to LPS or GroEL.

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