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Innate Immune system

• First found in sponges in ocean
• We became better homes for bugs:
• Temperature
• Nutrients
• Transportation/spread
• Longer life span
• Pros:
• Fast
• Recognizes pathogens based on not having ID badges
• Recognizes pathogens based on conserved danger signals
• Cons:
• Doesn’t improve
• Some pathogens have figured out ways around it
  
  
  

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 Adaptive Immune system

How does it combat genetic diversity in bacteria?

• Pros:
• Remembers old enemies
• Gets better at beating enemies with each exposure
• Can prevent infection with specific antibodies
• Recognizes pathogens based on specific proteins
• Cons:
• Takes time
• Can run amok (autoimmunity)

We develop novel antigen receptors in B cells and T cells by taking a set number of genes, and reshuffling them to create a new antigen receptor. Each of these new receptors can recognize a different antigen

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What Organs are Involved in the Immune Sytsem?

1. Lymphnodes
2. Spleen
3. Bone marrow
4. Thymus
5. Mucosal Associated Lympoid Tissue (MALT)
6. Various tissue specific cells/mechanisms

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Receptors are the immune system’s...

Innate and Adaptive receptors..

eye's for the environment

Innate immune system:
Recognizes conserved danger signals

Adaptive Immune system:
Recognizes specific proteins:

1. In native, folded 3D form (antibodies)
2. Linear peptides (T cell receptors) mounted on MHC

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Why does the adaptive immune system recognize proteins?

• The outer surface of all types of cells, self and non self, are coated in proteins
• These cell surface proteins are often specific for self and non self
• Many of these proteins are involved in how a pathogen attacks us
• So if you attack the protein, you attack the pathogen, or block its mechanism of attacking us

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What is a danger signal?

Most invasive organisims are very different than humans and thus they have metabolic and structural differences that make them clearly ‘not self’

Ex:

Bacteria: LPS, mannose, peptidoglycans, flagellin, CPG repeats
Yeast: fungal glucans (sugars)
Viruses: CGD repeats, double stranded RNA
Parasites: hemozoin (Malaria)
Damaged tissue also release DAMPS (Danger Associated Molecular Patterns ie Uric Acid)

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The situation: Small sliver in the arm, coated with a nice mixture of pathogenic bacteria

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Microbial Virulence Factors

After initial injury with sliver are colonization factors:

• Adhesion molecules
• (make them sticky)
• Ability to clump or wall of site of infection
• (Make them hard to get at and remove)
• Ability to break down CT
• (allow them to invade other tissues)

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Inflammation functions to...

• Bring cells to the site of injury
• Bring relevant serum proteins to the site of injury
• Complement
• Clotting factors
• Antibodies
• Acute phase proteins
• Filter through more extracellular fluid into lymph nodes
• Increased Antigen sampling

Or more simply:

1. Injure them
2. Tag them so they are easy to round up
3. Keep them out
4. Slow them down/surround them
5. Warn the neighborhood
6. Call for help
7. Clean up the mess
   
   

• WDMWL
• Increased Antigen sampling

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Innate Immune System Components

1. Barriers (Don’t let them in)
        Mucosal, Endothelial, Mucus, Cillia
2. Serum proteins (Injure them on the way in, slow them down)
3. Cell mediated (Surround them after beating them in hand to hand combat, then eat them)
        Neutrophils
        Macrophages
        Gamma Delta T Cells/NK T cells

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Adaptive Immune System Components

1. Cell mediated
1. T helper Cell (CD4+)
2. Cytotoxic T cell (CD8+)
   
   
2. Antibody mediated
1. B cells

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Adaptive Immune Responses

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Passive vs. Active Immunity

Example: high risk HepB exposure in unvaccinated individual

Passive:

Treat with Hep B immunoglobulin (antibodies to HepB)
Advantage: immediate protection
Disadvantage: short duration of protection (didn’t make memory cells as a result)

Active:

Treat with a Hep B vaccine that consists of killed Hep B virus
Advantage:
Develop long lived memory B and T cells that recognize Hep B and  quickly and effectively fight it off
B cells make antibodies which circulate to mop up infection
Disadvantage: takes time

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Kinetics timeline of Inflammation

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Antigens and Antibodies

An antigen (“Ag”) is any molecule that can bind specifically to an antibody.  Their name arises from their ability to cause antibodies generation.

Each Antibody (“Ab”) molecule has a unique Ag binding pocket that enables it to bind specifically to its complementary specific antigen. 

Ab are produced by B cells and plasma cells in response to infection or immunization, bind to and neutralize pathogens or prepare them for uptake and destruction by phagocytes.  They can also cause many problems in autoimmunity.

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Adjuvant
Allergen

Allergic asthma

Allergic rhinitis

Adjuvant: Any substance that enhances the immune response to an Ag with which it is mixed

Allergens: are types of Ags that elicit hypersensitivity or allergic reactions.

Most common mechanism is the binding of allergen to IgE. Ab on mast cells that causes asthma, hay fever, and other common allergic reactions. 

Allergic asthma: is constriction of the bronchial tree due to chronic airway inflammation and is an allergic reaction to inhaled Ag.

Allergic rhinitis: is an allergic reaction in the nasal mucosa, also known as hay fever, that causes runny nose, sneezing, tears and congestion.

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Allograft

Anaphylactic shock

Ab-dependent cell-mediated cytotoxity (ADCC)

Ab repertoire

Allograft: A graft of tissue from an allogeneic or non-self donor of the same species; such grafts are always rejected unless the recipient is immunosuppressed.

Anaphylactic shock (or systemic anaphylaxis): is an allergic reaction to systemically administered Ag that can result in circulatory collapse and suffocation due to tracheal swelling.

It results from binding of Ag to IgE on connective tissue mast cells throughout the body, leading to the release of inflammatory mediators (ie histamine).  Epinepherine administration is the only effective clinical response.

Ab-dependent cell-mediated cytotoxity (ADCC) is the killing of Ab-coated target cells (ie bacteria, cancer cells)  by cells with Fc receptors that recognize the bound Ab.  Most ADCC is mediated by NK cells.

Ab repertoire describes the total variety of antibodies that an individual can make, approximately 1010 different specificities.

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Ag:Ab complexes

Ag-presenting cells (APC)

Ag processing

Ag presentation

Apoptosis

Ag:Ab complexes: are non-covalently bound groups of Ag and Ab molecules which vary in size (aka immune complexes)

Ag-presenting cells (APC): are cells that activate responses of naive T cells to Ag by carrying out Ag processing then Ag presentation.  Dendritic cells, macrophages, and B cells are the main APC types.
Must be able to:

(i) display peptide fragments of Ag inside of MHC molecules
(ii) also carry co-stimulatory molecules on its surface, to activate a T cell.

Ag processing: is the enzymatic digestion of protein into peptides that can then bind into pockets of MHC molecules for presentation to T cells.  All Ags must be processed into small peptides before they can be presented by MHC molecules.

Ag presentation: is the display of Ag as peptide fragments bound in MHC molecules on the surface of an Ag-presenting cell; all T cells recognize Ag only when it is presented in this way. 

Apoptosis: form of cell death in which the cell activates an internal death program (effectively, suicide).  It is seen as:

Nuclear DNA degradation
Nuclear degeneration and condensation
Phagocytosis of cell residua. 

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Autoimmune diseases

Basophils

B lymphocyte

Bone marrow

Autoimmune diseases: are those in which the disease is caused by immune responses to self Ags

Basophils: are white blood cells containing granules that stain with basic dyes, and which are thought to have a function similar to mast cells (ie. in allergy).

B lymphocyte: The Ag receptor on B lymphocytes is a cell-surface Ab.  On activation by Ag, B cells differentiate into plasma cells which are factories producing high levels of Ab molecules of the same specificity as this receptor.

Bone marrow: is the main site of hematopoiesis, the generation of the cellular elements of blood, including red blood cells, monocytes, polymorphonuclear leukocytes, and platelets.  The bone marrow is also the site of B-cell development in mammals and the source of stem cells, some of which later give rise to T cells upon migration to the thymus.  Thus, bone marrow transplantation can restore all the cellular elements of the blood, including the cells required for adaptive immunity. 

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Cell-mediated immunity

Central (or primary) lymphoid organs

Complement proteins

Cytokines

Chemokines

Cell-mediated immunity: describes any adaptive immune response in which Ag-specific T cells have the main role

Central (or primary) lymphoid organs: are sites of lymphocyte development (B = Bone marrow, T = Thymus)

Complement proteins: are constitutively present innate immunity molecules that makeup an enzyme cascade which can be activated via several pathways.  Activation of these acute phase proteins effectively kill target cells and enhance inflammatory responses

Cytokines: is a term for about 60 , mostly soluble,  proteins that act as a communication system between cells of the immune system.  They also are produced by and interact with many non-immune cells.  Cytokines act on specific cytokine receptors expressed by the cells that they affect. 

Chemokines are a sub-family of  ~50  small cytokines involved in cell trafficking, immune regulation, angiogenesis and other areas of biology.  They have a central role both in inflammatory responses and in normal immune regulation.

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Clonal expansion

CD

Complement system

Complement receptors (CR)
Contact hypersensitivity reactions

Clonal expansion: is the selection and proliferation of Ag-specific lymphocytes upon Ag stimulation.  It precedes their differentiation into effector cells.  It is an essential step in adaptive immunity, allowing rare Ag-specific cells to multiply so that they can effectively combat the pathogen that elicited the response.

CD is the abbreviation for commonly used cell surface Ags (“cell markers” ) that are useful tools in discriminating between different cell types. The cell-surface molecule is designated CD followed by a number (e.g. CD3, CD4, CD8 etc.)

CD3 complex contains the Ag specific T cell receptor.  This receptor is responsible for Ag recognition by forming Ag binding pockets analogous to those of Ab molecules.
CD4 is a  marker expressed on helper T cells
CD8 is associated with cytotoxic T cells. 
CD19 is a B cell marker.

Complement system is a set of constitutively present plasma proteins that can be activated to act together to attack extracellular pathogens (bacteria, parasites, free virus).  Complement can be activated spontaneously on certain pathogens (alternative pathway) or by Ab attached to the pathogen (classical pathway).  The pathogen then becomes coated with complement proteins that aid pathogen removal by phagocytes or can kill the pathogen directly.

Complement receptors (CR): are cell-surface proteins on various cells that recognize and bind complement proteins that have bound an Ag. Complement receptors on phagocytes allow them to identify pathogens coated with complement proteins for uptake and destruction.

Contact hypersensitivity reactions are essentially the same as delayed-type hypersensitivity (DTH) in which T cells migrate to the site of Ags introduced by contact with skin.  This is called delayed-type hypersensitivity because the reaction usually appears 18-24 hours after Ag is introduced. Poison ivy is an example.

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Cross-reaction

Effector cells

Eosinophils

Extravasation

Fc receptors

A cross-reaction is the binding of Ab to an Ag that was not used to elicit that Ab.  Thus, if Ab raised against X also binds Ag Y, it is said to cross-react with Ag Y.  The term is used generically to describe the reactivity of antibodies or T cells with Ags other than the original eliciting Ag.  A clinical example is rheumatic fever.

Effector cells are lymphocytes that mediate pathogen removal without a need for further differentiation.  They are distinct from naive lymphocytes, which must proliferate and differentiate before they can mediate effector functions, and memory cells, which must differentiate and often proliferate before they become effector cells.  These are the end stage cells that carry out the responses themselves.

Eosinophils are white blood cells important in defense against parasitic infections; They are also very important in certain types of asthma, allergy.

Extravasation: The movement of cells or fluid from within blood vessels into the surrounding tissues is called.

Fc receptors bind the Fc (or constant) portion of antibody: the part that makes IgG distinct from IgA or IgE.

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Germinal centers

Helper T cells

HLA

Humoral immunity

Lymphatic system

Germinal centers in secondary lymphoid tissues (ie.  spleen, lymph nodes) are sites of intense B-cell proliferation, selection, maturation, and death (apoptosis) during Ab responses. 

Helper T cells are CD4 T cells that can help B cells make Ab in response to antigenic challenge

HLA, Human Leukocyte Ags, is the name for the human major histocompatibility complex (MHC), a complex found in all vertebrates

Humoral immunity is Ab-mediated specific immunity.  It can be transferred to unimmunized recipients by using immune serum containing specific Ab (ie.  IV Ig for short lived passive immunity). 

Lymphatic system is the system of lymphoid channels that drain extracellular fluid from the periphery via the thoracic duct to the blood. It connects the primary and secondary lymphoid organs.

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Lymphoid organs

Macrophages

Major Histocompatibility Complex (MHC)

Negative selection

Plasma Cells

Lymphoid organs: are organized tissues characterized by very large numbers of lymphocytes interacting with a non-lymphoid matrix which interacts with them to support, mature, activate or kill them. 

Primary (or central) lymphoid organs, where lymphocytes develop, include the thymus and bone marrow. 
Secondary (or peripheral )  lymphoid organs, in which adaptive immune responses are turned on, include lymph nodes, spleen, and mucosal-associated lymphoid tissues, such as tonsils or Peyer’s patches in the gut.

Macrophages are large mononuclear phagocytic cells important in innate immunity, in early non-adaptive phases of host defense, as Ag-presenting cells, and as effector cells in humoral and cell-mediated immunity. 

MHC is a cluster of genes encoding a set of membrane proteins called MHC molecules.  One family, MHC class I present CD8 T cells.  MHC class II molecules present to CD4 T cells.  The MHC is the most polymorphic gene cluster in the human genome, having large numbers of alleles at several different loci.

Negative Selection: During intrathymic development, thymocytes (immature T cells) that recognize self are deleted from the repertoire, a process known as negative selection.  Autoreactive B cells undergo a similar process in bone marrow to help prevent self reactive cells from escaping, something  which could cause autoimmunity.

Plasma cells: are terminally differentiated B lymphocytes.  They are the main high-production Ab-secreting cells of the body.  They are found in the medulla (centre) of lymph nodes, in splenic red pulp, and in bone marrow.

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Polymorphocuclear leukocytes

Primary immune response

Rheumatoid arthritis

Secondary Ab response

Positive selection

Polymorphocuclear leukocytes (PMN, granulocytes) are white blood cells with multi-lobed nuclei and cytoplasmic granules.
There are three main types:

Neutrophils with granules that stain with neutral dyes
eosinophils with granules that stain with eosin
basophils with granules that stain with basic dyes

All are part of the innate immune response but, because they have Fc receptors to bind different types of Ab, can participate in the Ag-specific, adaptive immune response to kill pathogens (or self cells).           

Primary immune response: is the adaptive immune response resulting from first exposure to an Ag.  Primary immunization (“priming”) generates both

(i) a primary immune response (initially IgM predominating, later IgG in serum or sIgA in secretions)
(ii) immune memory (hence the ability to mount secondary responses).

Rheumatoid arthritis: is a common inflammatory joint disease due to an autoimmune response. The disease is accompanied by the production of rheumatoid factor, an IgM anti- (self) IgG that is sometimes also produced in normal immune responses at low concentrations without clinical effect.

Secondary Ab response is the Ab response induced by a second or subsequent exposure to Ag.  Starts sooner after Ag injection, reaches higher levels, is of higher affinity than the primary response, and is dominated by a different Ab isotype: IgG antibodies in serum, IgA in secretions. For all these reasons it provides better protection than does a primary

Positive Selection: A cell is said to be selected by Ag when its receptors encounter and bind that particular Ag.  If the cell proliferates as a result, this is called positive selection (also, clonal selection) and the cell multiplies to create a clone.

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Self tolerance

Seroconversionspleen

T cells

Type I hypersensitivity reactions

Vaccination

Self Tolerence: Tolerance is the failure to respond to an Ag; when that Ag is borne by self tissues, tolerance is called self tolerance—something essential to prevent your immune system from rejecting your heart, lungs, kidney, pancreas etc. .

Seroconversion is when Ab against the infecting agent are first detectable in blood/serum.  It demonstrates that the person has previously been exposed to that pathogen (ie., HIV, hepatitis A, flu, etc).

Spleen: an organ containing a red pulp, involved in removing old blood cells, and a white pulp of lymphoid cells that respond to Ags delivered by the blood for generation of innate, primary and secondary Ag-specific immune responses.

Hypersensitivity reactions are historically (and still) classified by mechanism:
Type I hypersensitivity reactions (ie. Hayfever, asthma) involve IgE  triggering of mast cells
Type II hypersensitivity reactions (ie. penicillin allergy) involve IgG against cell surface of Ags
Type III hypersensitivity reactions involve Ag:Ab complexes;

Type IV hypersensitivity reactions delayed hypersensitivity reactions (DTH) (ie. poison ivy) are T-cell mediated.

Vaccination (immunization) is the deliberate induction of Ag-specific immunity to a pathogen by injecting a vaccine

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PAMPS DAMPS and TOLLS

Kinetics of Inflammation: There is an order to immune arrival

PAMP: Pathogen Associated Molecular Pattern
DAMP: Danger Associated Molecular Pattern
PRR: Pattern Recognition Receptor (recognizes PAMP’s and DAMP’s)
TOLL Receptors: one of the first identified classes PRR’s

Serum Proteins- seconds
Neutrophils - minutes
Macrophages - hours
T and B cells - days

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Innate Immune System Includes...

Complement Overview

1. Complement and acute phase proteins
2. Neutrophils
3. Macrophages
4. NK cells
5. Dendritic cells

"The Landmines"

Serum proteins and Enzymatic cascade

3 Functions:

1. Lysis via pore formation
2. Opsinization
3. chemotaxis

3 Pathways:

1. Alternate
2. Classical
3. Mannose/Lectin

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Complement Pathways

What happens when complement doesn’t work?

‘Classical’
Complement binds to an antibody that is bound to a bacteria/or targeted cell,  and becomes activated
Complement fixing antibody isotypes: IgM, IgG1, IgG2, IgG3

'Alternate'

Complement naturally ‘goes off’ when it touches any cell membrane unless there is an inhibitor protein (ID badge)

Mannose/Lectin

Mannose binding protein binds to bacterial mannose residues
Activates complement

Higher risk for Lupus
Nesireia meningitis infection if there is a problem with the MAC attack complex

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The Neutrophil

The Macrophage

• Short lived
• Main phagocytosing cell-oxidative killing
• May appear as large infiltrate during different disease states (Neutraphilic infiltrate)
• Dead neutrophils large component of puss
• Quickly attracted to tissues undergoing inflammation
• Tri-lobed

• Also responsible for phagocytosis in periphery-oxidative killing
• Important antigen presenting cell
• Presents Class II MHC to T cells

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Phagozome, Lysozome and Phagolysozome killing method (missing)?

What happens when we’re missing these cells?

1. Cyclic neutropenia
2. Post chemotherapy febrile neutropenia
3. Risk for sepsis
4. Stomatitis

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What happens when the oxidative burst doesn’t work?

Virulence Factors at this stage (cell killing) interfere with:

• Chronic granulotomatous disease
• TB

1. Complement pore formation
2. Antibody binding
3. Opsinization (marking cells for phago)
4. Phagocytosis
5. Digestion
        Inhibit phagosome lysosome fusion (*TB)
        Inhibit superoxides

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The Dendritic Cell (8)

1. Roam in peripheral tissues
2. Pick up antigens (not phagocytosis-pinocytosis)
3. Migrate back to lymphnodes
4. Present antigen to T helper cells via MHCII
5. Can also present MHCI to cytotoxic T cells, called ‘Cross presentation'
      The ONLY APC that can present both Class I and II
      MHC molecules to T cells.
6. These cells are ‘professional’ antigen presenting cells: give the best costimulation
7. They hold antigen’s on their surface for long periods of time
8. Stimulate memory B cells with survival signals

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The Mast Cell

• Bind IgE produced by B cells
• When something triggers the IgE: Release Histamine
• Histamine causes local swelling, immune recruitment, inflamation
• Get triggered in allergic reactions ‘Antihistamines’ –Type I immediate hypersensitivity
• Live in tissue, those in blood called basophils

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The Eosinophil

Natural Killer (NK) Cells (Innate) 

• Largely responsible for clearance of intestinal parasites/worms
• Bind IgE
• Recognizes parasite via bound IgE
• Degranulate on parasite, injuring it

• T cells, but they recognize ‘danger signals’
• Delete cells that aren’t expressing MHCI, very very important for cancer immunity
• Checking for ‘ID’
• Can delete other invaders, induce apoptosis release pore forming proteins
• Don’t mature in thymus

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MHCI and MHCII

Antigen Loading

Antigen Presentation

MHCI: the self FLAG POLE – internal (intracellular) antigens (flag) are mounted on, recognized by CD8+ T cells. Shorter aa sequence.

MHCII: the ‘what I just ate’ FLAG POLE – external (extracellular) antigens are mounted on, recognized by CD4+ T cells. Longer aa sequence.

Rule of 8: MHC 2x4 = 8, MHC 1x8 = 8

Antigen loading onto MHC:

Linear peptide sequence binds into antigen binding pocket

This is why resistance to some infections is MHC restricted
This is why some autoimmune diseases are MHC restricted

How does antigen presentation happen on MHCII?
The bacterial protein are taken in and proteases chop up the protein which is then placed onto the MHCII receptors. Then recognized by macrophages.

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Lymphocytes are the only cells with...

Two types of Antigen-Presenting cells (APCs)

What are the Effector Cells

specific receptors for antigens, therefore they are key mediators of adaptive immunity

1. Macrophages (monocyte)
   - important in innate immunity
   - phagocytose microbes, digest them, and then present the antigen for T cell activation
   - secrete proinflammatory cytokines
   - involved in initiation/coordination of cell-mediated immunity (T cell activation) and also initiation/coordination of effector cells
   
   
2. Dendritic Cell
   Capture antigens of microbes and transport them to regional lymph nodes where they display portions of the antigen for recognition by T cells
   - are phagocytic and/or pinocytic

1. neutrophil
2. eosinophil
3. basophil
4. mast cell
5. T lymphocyte
6. macrophage

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Helper T cells:

T_H0
T_H1
T_H2
T_H17
T_R1

T_H0: Naïve

T_H1: ‘Cellular Immune Response’ - Proinflammatory, macrophage and Tc activator (Autoimmunity)

T_H2: ‘Humoral Immune Response’ - Induces strong B cell response, antibody production and Eosinphils (parasites) (Allergy)

T_R1: ‘Regulatory Immune Response’ Immunosuppressive (hot topic, MS cure?), self tolerance (cancer)

T_H17: Responsible for mucosal/epi barrier immunity may play a big role in autoimmune disease

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The CD4+ T Helper Cell

Maturation of the CD4+ T cell response

• Activated by antigen presentation on MHCII molecules. It recognizes foreign antigen via T Cell Receptor (TCR).
• Once activated it in turn activates
• Macrophages
• B-cells
• CD8+ cells
• through costimulation and soluble mediators called cytokines

• T cell starts off as naïve (never seen antigen)
• After being presented with antigen, its now an Effector T cell (can be TH0, TH1, TH2, TH17 or TR1)
• After the immune response is over, some remain as memory cells
• Single cell can turn into 100s and 1000s of cells, most die from apoptosis, few memeory remain

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How do the innate and adaptive immune system assist one another?

• Antigen presenting cells that have been stimulated by PAMPs give stronger activation signals to Tcells using costimulation
• Majority of Costimulation through B7-1 or B7-2 (also called CD80/CD86) is increased on the APC which stimulates T cells through CD28
• Activated CD4+ T cells increase cell mediated killing in neutraphils and macrophages by stimulating them with cytokines (IFN-gamma)
• This stimulation increases expression of proteins involved in oxidative killing

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T Cell Selection (CD4+ and CD8+) (Diagram)

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Autoimmunity I: Transplant Rejection

Result of Self T cells that recognize the transplanted tissues MHCI as if they were a danger

Note:
HLA = MHC, same thing
MHCI  (HLA A,B,C) – is on all cells
MHCII (HLA DP,DQ,DR) –is only on APC’s
Therefore MHCI is the most important to match

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3 factors that affect the type of T cell Response

1. Antigen presenting cell
       Type: Macrophage, Dendritic cell, B cell
       Activation state (how much costimulation is it
       providing)
2. Amount of antigen
3. Previous exposure to antigen

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Cytokines you Should Know...

But why?

IL-1 –    fever
IL-2 –    T cell activation and proliferation
IL-4 –    TH2 responses
IL-10 -   TR1 Suppression of immune response
IL-17 –  Activate Th17 cells
IFN-γ -  TH1 responses
TNFα -   Inflammation and Septic/toxic Shock

Because there are drugs to block them being used on patients. These drugs are humanized antibodies against the cytokines, and are called biologicals, they are proteins

Ex:

Anikinera - blocks IL-1
Etanercept – blocks TNF-α -> juvenille rhumatoid arthritis

If someone comes in with a fever, they come in much later because cytokines that trigger us to look sick are not produced, thus these people

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Antibody Structure

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Antibody Isotypes

IgD –naïve B cells - on Bcell surface
IgM – first produced before T cell help, complement fixing (pentamer)
IgG – neutralizing or complement fixing, high affinity
IgA – mucosal surfaces (dimer), neutralizing
IgE – parasite immunity and allergy – bind to mast cells and eosinophils, cause degranulation

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 What are antibodies targeted against?

• Mostly proteins but...
• Some antibodies are targeted against sugars, but these are all IgM, and therefore not high affinity, and no memory B cells
• Some targeted against conserved pathogen sugar epitopes, these are made by B1 B cells, that are part of the innate immune system
• We can make good vaccine antibodies against sugars that are IgG and high affinity if we attach the sugar to a protein: pneuomococal congugate vaccine
• B cell recog sugar, T cell recog protein, and T cell helps create memory B cells

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CD4+ T_H2 B-Cell Help

CD4+ TH2 cell and B-cell meet leading to receptor cross linkin (recognition)

B-cell is activated and...

Isotype Switching (IgM to IgG (can't go back))
Mutation (increased affinity)
Plasma Cell Formation

Notes say "Cytokines (IL-4), Costimulation, Proliferation, Memory" beside CD4+ TH2 cell, not sure what this means

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Kinetics of the B cell response: Primary

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Kinetics of the B cell response: Secondary

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Virulence Factors at stage of CD8 Cytotoxic T cell Killing will...

• Induce inappropriate immune responses (ie superantigens)
• Induce TH1 or TH2 shifts (leshmania)
• Decrease MHC expression (most viruses)
• Induce Immune suppression (Epstein Barr virus)
• Bind to the constant region of antibodies (protein A) and neutralize them
• Target immune cells: TB, HIV, Epstein Barr

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Tolerance to Self/Non Self in the Peripery

Relies on absence of danger signals
3 Mechanisms:

- Low dose-induction of active suppresion (Tr1 and IL-10)
- Low dose-induction of anergy (not dead, but doesn’t respond)
- High dose-induction of apoptosis

All these mechanisms involve altered costimulation

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Table: Putting It Together

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General properties of all Ag receptors
 

All 4  groups (PRR, MHC, TCR, BCR, totaling billions of indiv receptors) use different terminology. However...

Each consists of  “constant” regions that define the  family it belongs to and that binds the receptor to a membrane.

- usually anchored into the cell membrane

- provide structural support

PRR (TLR, NLR, RLR)
MHC:  class I  vs class II
TcR:  alpha-beta vs gamma-delta

Ab: IgG vs IgA …

and a “variable” region at the other end of the molecule that physically captures and holds the target molecule

• The part of the molecule that forms the Ag binding
• pocket that recognizes one Ag compared to another
• Ag recognition based on physical fit into the “pocket”

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Pattern recognition receptors

What are they looking for?

Microbe selective molecules

The eyes of the innate immune response, activating your first line of immune defense

• Some are expressed by virtually every cell in the body.
• Membrane, cytoplasmic, secreted: depending on the PRR examined

Examples:

• TLR (Toll like receptors)
• NLR (NOD like receptors)
• RLR (RIG-like receptors)
• Mannose receptors
• Complement receptors
• C reactive protein

What are they looking for?

• PAMPs: pathogen-associated molecular patterns 
• Bacterial carbohydrates (e.g. LPS, mannose)
• Nucleic acids (e.g. bacterial, viral DNA / RNA)
• Bacterial peptides (flagellin)

Microbe selective molecules:

• Peptidoglycans and lipoteichoic acids (from Gram positive bacteria)
• N-formylmethionine
• lipoproteins
• fungal glucans

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What does the MHC do? (3)

1. Required for development of your T cell repertoire in thymus
2. Required for activation of mature T cells by Ag
3. Both MHC I and MHC II  collect families of digested peptides and “display” them as a necessary first step for generating any Ag-specific immune response.

Express co-dominantly -> ie alleles of both parents are expressed thus more variey (1 in 6 billion odds)

Large pool of MHC alleles in pop’n (highly polymorphic genes), but very small in individual (2x6). Think of matching kidney transplant (1-6 in 6 match for each parent)

Clinically linked to a few diseases (RA, MS, renal, neurological diseases)

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Lymphocyte Ag receptors: B and T cells

How do you generate such a vast array of specific receptors?

4 Key Points

Lymphocyte recognition of microbes is more specific
(therefore  more complex) than innate PRR:  ~ 50 PRR
(essentially identical receptors in everyone…other than SNPs) and ~ 10 7-8   different BCR, ~ 10 7-8 different TCR.  Note: 10^14 types of cells in whole body

How do you generate such a vast array of specific receptors?

By breaking all the rules of genetic inheritance. V(D)J recombination: genes encoding Ag receptors undergo a unique DNA “shuffling” process to create variability in Ag-binding receptors

The final products of V(D)J recombination = functional antigen receptors with unique “V regions” that each have differently shaped Ag-binding pockets
Note: overall structure of Ag receptor is the same no matter what Ag it binds. All contain constant “C regions” spliced onto unique “V region” combinations

Key Points:

1. Ag specific binding results from assembly of multiple exons to form an Ag binding pocket
2. Continuous generation of diversity (new Ag specificities) due to high mutation rate in receptor genes
3. Many receptors in total in person but only one receptor specificity per mature cell
4. Different families exist (IgA vs IgG,  a/b vs g/d), each with different roles in host defense                        

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Diagram comparing Ab and TCR

SF098

B cell Ag receptor exists in 2 forms...

B and T cell Ag recognition: major differences

differing only in the C-terminus  of the protein:

1. Cell surface-bound receptor (BCR)
2. Secreted form (circulating antibody, made by B cells and plasma cells)

Major Differences:

T cell:
Recognizes linear peptide sequences derived
from digested (by APC) proteins. The TCR activates the T cell to perform defense functions: 

Kill virally-infected cell

Produce cytokines to initiate local inflammation

B cell:

Recognizes intact 3D molecular structures. The Ag receptor also serves as a defense molecule (I.e. secreted antibody)

Neutralize toxins

Block viral entry

Opsonization of bacteria

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Lymphocyte activation results in Ag-specific...

Lymphocyte activation requires...(4)

Cell-mediated immunity: Ag-specific response mediated by T cells. Ag-specific T cells also activate other cell-mediated defenses (Eg. monocytes, macrophages)

Humoral Immunity = antibody = B cells
T cells also have a critical role (“helper” T cells)

1. That a lymphocyte with the right receptor for the specific Ag has been created and binds with the Ag
2. That  those cells respond by multiplying to provide enough cells to be effective
3. That that population receives an “off” signal once Ag is removed, to limit pathology
4. That a fragment of this population differentiates into long lived (~ 50y) memory cells to protect you upon re-exposure to that exact Ag (or one that looks almost identical)

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T cell Ag recognition involves..

How are Ag peptides loaded onto MHC molecules?

“presentation” of peptide fragments bound to MHC molecules on surface of “antigen presenting cells”. There are 2 types of signals:

1. TcR / MHC/ Ag complex  PLUS
2. A variety of other Ag-non specific receptor interactions between T cell and APC that are needed for activation

There are 2 main ways:

MCH Class I path: Endogenous Ags (polio, TB, parasites…). Cell ingests cytosolic microbe and breaks down its protein in the cytosol, then MHC I incorperates it, and presents on cell surface

MCH Class II path: Exogenous Ags (salmonella, toxins,
grass pollen…), Microbe ingested in vessicle. Same as above but all done in vessicles, but then presented vis MHC II

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What is costimulation of lymphocytes?

Two main arms of cell mediated immunity:

A necessary “second signal” required for T cell activation that is delivered by the APC to the Ag-stimulated T cell.

• Some act as Go signals (T cell survival, expansion, differentiation) that MAY also shape the type of response
• Some act as Stop signals (Multiple costimulation pathways exist)

Helper: CD4 effector T cells interact with antigen presented on Macrophage, then produce cytokines leading to:

1. Macrophage activation

2. Inflammation

Cytotoxic:
CD8 T cells interact with infected cell with microbes in cytoplasm. The CD8 T cells kill the infected cell.

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B cell Ag receptors (5 pts differing from TCR)

B cell receptor: Ab (also known as Ig or immunoglobulin). Unlike T cell receptors, the B cell receptor:

1. Can be produced in a secreted Ab form, often at high concentration
2. Can recognize intact 3D structures of proteins and non-protein molecules
3. Can subtly change its daughters’ Ag-binding specificity by mutations of Ab genes during an ongoing immune response
4. Is of 5 main families (IgM, IgG, IgA, IgD, IgE)
5. The isotype a B cell differentiates to is determined by locally produced cytokines
             Ex. IL-4 results in IgE

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B cell activation

Stimulation of B cells by protein Ags:
T dependent responses (3)

It is similar to T lymphocyte activation in many respects:

1. Need Ag recognition (by membrane Ab)
2. Need “help” (second signals)
3. This leads to further clonal expansion of Ag-reactive cells --> and differentiation.
4. Need to taper response once no longer required and develop memory

T dependent responses:

1. Ag binding/Ab crosslinking – initial activation and clonal expansion
2. Interaction with a pre-activated T helper cell to deliver “second signal” 
3. Collectively these interactions lead to further clonal expansion, differentiation to Ab secreting cells (IgG vs IgE) and MEMORY

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3 specific recognition events are needed to get humoral immunity (in lymph nodes)

1. T cells need to be activated by antigen presentation

2. B cells need to recognize an antigen (of a microbe)

3. T cells and B cells need to interact and T cell "helps" activate B cell

But what exactly is T cell “help”?

When B cell presents an antigen to the Tcell the Helper T cell is activated, expresses CD40L and releases cytokines. CD40L and cytokines are responsible for activating the B cell. They type of cytokine released influences which isotype the B cell will take

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Main characteristics of Ab isotypes

IgM

• First produced in primary responses and in ontogeny,  phylogeny.
• 2nd most common serum Ab
• Opsonization, activates complement, neutralizing Ab

IgG 

• Dominates memory responses in serum (Secondary responses). Makes up the majority of serum Ab
• Opsonization, activates complement, neutralizing Ab, enhances killing by NK cells (ADCC)
• Transplacental transfer: neonatal immunity

IgA

• Major Ab at mucosal surfaces, with special structure allowing transport across mucosa and stability
• In colostrum, tears, GI and respiratory secretions but also in serum
• Opsonization, activates complement, neutralizing Ab

IgD
Who knows?

IgE

• Parasite defense/arming of eosinophils
• Immediate hypersensitivity: mast/baso degranulation
• Very small amounts

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Cytokines

Key Roles (7)

the main communication system of the immune response, like “Immunological hormones” for inter-cellular communication: and not just among immune cells (ie epithelial , brain etc)

Secreted proteins “made by cells that affect the behavior of other cells”  IL-1 to IL-37, IFN-y , TNF-a...

Key roles:

1. Ontogeny of immune cells, functional maturation
2. Growth factors
3. Help activate immune responses (“go” signals)
4. Lymphocyte trafficking directional signals for cell recruitment.
5. Directing the type of immune responses that develop
6. Amplify the intensity of the immune response
7. Turn down responses (preventatively or after response no longer needed)

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Cytokine expression patterns of Th1, Th2 and Th17

Th1: IFNg , TNFa  NOT IL-4,5,13
Good: APC activation, enhanced T cell activation
Bad: Autoimmune inflammatory diseases

Th2: IL-4,5,13,  NOT IFNg, TNFa
Good: Enhanced Ab production, parasite resistance
Bad: IgE and allergic diseases

Th17… IL-17
Good: importance in host defense, inflammation often with Th1
Bad: autoimmune disease, chronic regional inflammation

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The Allergic March

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Diseases in which immediate hypersensitivity plays a role (8)

1. asthma
2. allergic rhinitis
3. atopic dermatitis/eczema
4. anaphylaxis
5. acute urticaria 
6. food allergy
7. insect allergy
8. drug allergy

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Tests for investigation of Immediate Hypersensitivity

Note:

General tests for evidence of atopy

• eosinophil counts in blood and secretions
• total serum IgE

Tests for evidence of sensitization to specific allergen(s)

• Skin tests for immediate hypersensitivity
• ELISAs for allergen-specific IgE
• Allergen-specific challenge tests (in hospital)

 Note:

• the history is the most critically important investigation
• skin tests/allergen specific IgE levels confirm sensitization
• 50% of the population are sensitized to one or more allergens
• sensitization is a risk factor, not a disease
• asthma, rhinitis, and anaphylaxis may have non-allergic triggers

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Treatment options for Allergic Diseases (5)

• Avoidance of environmental triggers (except exercise)
• Medications
• Regular anti-inflammatory meds; “reliever” meds as needed
• Preventer (controller) versus reliever
• topical (inhaled, intranasal, skin) versus systemic
• Education
• Allergen-specific immunotherapy
• New immunomodulators

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Glucocorticoid (GC) efficacy in allergy treatment

WHO says

Has proven anti-inflammatory activity: down-regulate eosinophils, lymphocytes, macrophages, monocytes, dendritic cells. Highly effective for asthma, allergic rhinitis, atopic dermatitis -> the “gold standard” with good benefit to risk ratio by inhaled, intranasal, or topical routes

Systemic GC rarely used in outpatients with allergic diseases, instead non-systemic b/c of dec side FX. Topical is 1st line.

WHO says Inhaled beclomethasone is an essential drug for asthma

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2 types of immunotherapy for Allergy control

Allergen-Specific Immunotherapy

• Traditional route: subcutaneous injections over many years
• ↓ allergen-specific IgE and ↑ allergen-specific IgG4
• downregulates the Th2 response (↓ IL-4, IL-13, eosinophils, etc)
• used in allergic rhinitis and in insect venom anaphylaxis

Allergen Non-Specific Immunotherapy

• anti-IgE antibody
• binds to circulating IgE
• used as adjunctive treatment in severe asthma
• symptoms despite inhaled glucocorticoid use

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10 warning signs of

Primary Immunodeficiency

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Congenital (Primary) Immune Deficiency

Severe Combined Immunodeficiency (SCID):

• Defects in maturation of both B and T cells (steps prior to Pre B and Pre T cell)
• Several genetic abnormalities identified (50% x-linked)
• Decreased T cells. B cells can be normal, increased or decreased
• Present in first few weeks of life with fungal infections such as Candida, PCP, viral (eg. CMV pneumonia), atypical Mycobacteria
• ie opportunistic infections
• Fatal early in life unless bone marrow transplant
• Blood products must be irradiated to prevent Graft vs Host Disease
• No live vaccines (measles, rubella, mumps, BCG)

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B Cell Immunodeficiencies

Example

• Recurrent bacterial infections affecting lungs, sinuses
• Present > 4 months of age, corresponding to waning of maternal IgG (can cross placenta)
  
  Eg. X-linked agammaglobulinemia: decrease in all Ig isotypes, reduced B cell numbers. Block in maturation beyond pre B cells due to mutation in B cell tyrosine kinase
• Treatment: Lifelong IgG replacement (IVIG or SCIG)
  No replacement for IgM, IgA.
  
  

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T Cell Immunodeficiency

Di George Syndrome: incomplete development of thymus due to anomalous development of 3rd and 4th branchial pouch, failure of T cell maturation.

• Decreased T cells, normal B cells, normal or decreased Ig
• May have abnormal facial development, heart defects, hypocalcemia (incomplete development of parathyroid glands)

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Common Variable Immunodeficiency

• Recurrent sinusitis and pneumonia in adolescence and adulthood
• Reduced levels of IgG, IgA, and often IgM
• T lymphocytes may be increased, decreased or normal. T cell function may be normal or abnormal
• Likely defects in B cell maturation and activation as well as defects in helper T cell function.
• Treatment: IgG replacement
• Increased incidence of autoimmune disorder and lymphoma

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Defects in Innate Immunity (3 manifestations)

1. Chronic granulomatous disease

• mutations in phagocyte oxidase.
• Neutrophils and macophages which phagocytose microbes are unable to kill the microbes.

2. Leukocyte Adhesion Defects

3. Deficiencies in complement components

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Acquired (Secondary) Immunodeficiency

Manifestations of Humoral Immunodeficiency

Acquired (Secondary) Immunodeficiency:

1. HIV
2. Leukemia or cancer metasteses to bone marrow
3. Chemotherapy and irradiation
4. Immunosuppressant medication
5. Protein calorie malnutrition
6. Removal of spleen

Manifestations of Humoral Immunodeficiency:

Increased susceptibility to infection:   

1. Recurrent otitis media, chronic sinusitis, pneumonia.
2. Increased severity of infection (Sepsis, meningitis, osteomyelitis, cellulitis)
3. Prolonged duration of infection
4. Repeated infection without a symptom free period.
5. Increased dependency on antibiotics (Need for IV antibiotics to clear infection)
6. Unexplained or severe complications of infection.
7. Infection with an unusual organism.
   
   

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Diagnosis of Immune comprimised patients

1. High index of suspicion
2. CBC to determine lymphocyte count (75% T cells)
3. Quantitative serum IgA, IgG, IgM
4. IgG subclasses (if IgG normal)
5. IgG function: specific IgG titers to protein antigens (tetanus, diphtheria), and polysaccharides (pneumococcus)
6. Quantification of B cells (CD19 and CD20) and T cells (CD3, CD4, CD8)
7. CXR (thymic shadow, signs of infection or bronchiectasis)
8. Tcell function:
   intradermal injections of Candida, mumps, Trichophyton. Swelling of site at 48 hours indicates presence of sensitized competent T cells

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What is tolerance?

• Specific immunological non-reactivity to an antigen resulting from a previous exposure to the same antigen
• Active ongoing process, not passive!
• It is different from non-specific immunosuppression and immunodeficiency. It is an active antigen-dependent process in response to the antigen.
• Like immune response, tolerance is specific and like immunological memory, it can exist in T-cells, B cells or both and like immunological memory, tolerance at the T cell level is longer lasting than tolerance at the B cell level.

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Autoimmunity Examples

Can be classified into clusters that are either organ-specific or systemic

Organ Specific Ex: Diabetes, MS, Hashimotos Thyroiditis, Vitiligo

Hashimotos Thyroiditis: antibodies against thyroglobulin or thyroid peroxidase cause destruction of thyroid follicles

Vitiligo: autoimmune destruction of melanocytes

Systemic: Rheumatoid arthritis, Sclerodoma, Systemic Lupus erythematosus, Goodpasture’s Disease...

Goodpasture’s Disease: Antibodies to Vascular Basement Membranes affects Renal Glomeruli and Lung Alveoli

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When lymphocytes encounter antigen there are

3 outcomes possible

Tolerance and Autoimmunity: Central Tolerance

1. Lymphocyte Activation (immune response)
2. Functional Inactivation/killing of lymphocyte (Tolerance)
3. No response (Ignorance)

Central Tolerance:

Central tolerance is a consequence of immature lymphocytes recognizing self-antigens.

During maturation, all lymphocytes pass through a stage where encounter with antigen leads to tolerance rather than activation. This process occurs in the generative lymphoid organs:

Thymus (T cells)
Bone Marrow (B cells)

Only self-antigens are normally present at high concentrations in these lymphoid organs, thus only self-antigens are encountered at high concentrations by immature lymphocytes.

Lymphocyte clones whose receptors recognize these self antigens with high affinity are killed (negative selection)

Some T cells that encounter self-antigen may develop into regulatory cells, which inhibit immune responses.

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Tolerance and Autoimmunity: Peripheral Tolerance

Occurs as a result of mature self-reactive lymphocytes encountering self-antigens under particular conditions.

Induce by:
1. Peripheral tolerance is induced when mature lymphocytes recognize antigens without adequate levels of co-stimulators that are required for activation
OR
2. as a result of persistent and repeated stimulation by self-antigens in peripheral tissues.

These act as a second level of control to restrain potentially auto-reactive T cells, which escape from the thymus.

Peripheral tolerance is important for maintaining unresponsiveness to self antigens that are expressed in peripheral tissues and not in the central lymphoid organs.
         
To maintain self-tolerance, responses to these antigens by mature lymphocytes are either not initiated or tightly regulated.

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Principal mechanisms of lymphocyte tolerance (3)

1. Clonal deletion = Apoptotic cell death

• Two main factors determining whether a particular self antigen will induce apoptosis are:
• a. The concentration of that antigen in the thymus
• b. The affinity of the thymocyte T-cell receptors (TCRs) that recognize the antigens.
• Self-antigens are presented in association with MHC molecules on thymic antigen presenting cells (APCs).
• Some thymic immature lymphocytes specifically recognize self-peptide-MHC complexes with high affinity, resulting in apoptotic cell death.

2. Clonal anergy = functional inactivation without cell death

• T cells recognize self-antigens presented by APCs deficient in co-stimulators
• These T cells survive but are rendered incapable of responding to the antigen even if it is later presented by competent APCs

3. Clonal Suppression = the functional inactivation of self-reactive clones induced by Regulatory T cells.

• Precise mechanism of T regs is unknown
• Regulatory T cells are defined by expression of transcription factor Foxp3, and are induced by self-antigen.
• Regulatory T cells secret immunosuppressive cytokines:
• TGF-β - inhibits T and B Cell proliferation
• IL-10 - inhibits macrophage activation and antagonizes IFN-gamma
• Regulatory T cells may directly interfere with APCs or responding T cells

Central tolerance is mainly due to deletion, all 3 mechanisms contribute to peripheral tolerance.

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Clonal Anergy Further Explained

What does Abatacept do?

Some Antigen presenting cells that recognize self-reactive lymphocytes are:

1. deficient in costimulators (B7 or CD 80/86) or

2. the self-reactive cell expresses CTLA-4

both result in functional inacitvation of the lymphocyte, even if lymphocyte is later restimulated with an APC with costimulators.

CTLA-4 is part of a feedback loop, following t cell activation, it is upregulated. Binding of CTLA4 to CD80/CD86 provides a control signal that suppresses ongoing T-cell activation

Abatacept:

Abatacept (CTLA4Ig) interrupts the interaction between CD80/CD86 and CD28 or CTLA4, thereby disrupting the second signal to the T cell. Binding of abatacept to CD80/CD86 on antigen-presenting cells might also affect the antigen-presenting cell through induction of indoleamine 2,3-dioxygenase.

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What are CD25+ CD4+ cells?

They are regulatory T cells which are naturally anergic and suppressive, appear to be produced by the normal thymus as a functionally distinct subpopulation of T cells.

They play critical roles in:

1. preventing autoimmunity (main purpose)

2. controlling tumor immunity

3. transplantation tolerance

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Possible mechanisms for breaking tolerance (4)

1. Molecular Mimicry:
Infectious microbes may contain antigens that are structurally similar to, and cross react with self antigens.
Immune responses to the microbial antigen then also result in reactions against self-antigen.
E.g. Rheumatic fever (classical) and reactive arthritis

2. Polyclonal B cell and T cell Activation:
Microorganism can induce polyclonal B cell or T cell activation

E.g. cytomegalovirus and Epstein-Barr virus are polyclonal B cell activators. EBV imptnt with lupus, but we are all exposed, need combo of genes and env.
E.g. Staphylococcal and streptococcal toxins are superantigens. Superantigens bind to and activate all T cells with specific receptor types.

3. Disturbance of Th1/Th2 balance:
CD4 helper T cells functionally develop into two functional subsets

• Th1 (cellular immunity) responses promote release of pro-inflammatory/and cytodestructive cytokines
• Th2 (humoral immunity) responses promote B cell activation.

A shift in the balance leads to autoimmune disease

4. Epitope Spreading:
Autoimmune reactions initiated against 1 self-antigen may cause tissue injury. Tissue injury results in the release or alteration of other tissue antigens, and activation of lymphocytes specific for these antigens, with disease exacerbation.
May explain why autoimmune disease tends to be chronic and progressive.

SF118

Mechanisms of Autoimmune Damage (3)

1. Circulating autoantibodies

Complement lysis
E.g. Hemolytic diseases

Direct binding of antibodies to red cells induces cell lysis

Interaction with cell receptors

E.g. Myasthenia gravis

Autoantibodies to the acetylcholine receptor block neuromuscular transmission from cholinergic neurons.

E.g. Grave’s Disease

Stimulatory antibodies dysregulate thyroid function.

2. Toxic immune complexes
Circulating complexes of autoantibody and self-antigen may deposit in tissues (as in lupus nephritis), where they induce complement mediated inflammation and tissue damage.
= Antibody dependant cellular cytotoxicity

3. T cell mediated damage:
This term implies that the recognition of autoantigen by T cells leads to tissue destruction without requiring the production of autoantibody:

• CD4 cells polarized to TH1 response via cytokines
• E.g. Rheumatoid arthritis, Multiple sclerosis,
      Diabetes Type I
• Direct T cell cytotoxicity via CD8+ CTL
• Recruitment and activation of macrophages leading to bystander tissue destruction

SF129

Immunization with Vaccines

• Induce antigen-specific lymphocyte memory without making the person or animal ill
• the most effective public health intervention
• No vaccine developed to date actually totally prevents infection by the pathogen

SF129

Vaccines: classification

Subunit vaccines

1. Polysaccharide based = Polysacc antigen -> B-cell

• Produce primary antibody responses
• Short lasting immunity
• Poor response among young children
• Ex. 23-valent Pneumococcal polysaccharide vaccine, 4-valent meningococcal polysaccharide vaccine

2. Protein based

APC ->Tcells (cell mediated immunity) -> Bcells (ABs)

• Longer lasting immunity
• Boosting may be necessary
• Examples include: hepatitis b, tetanus toxoid, diphtheria toxoid, acellular pertussis, HPV

3. Combination

• Chemically attaches a polysaccharide to a carrier protein
• polysaccharide then presented as T-cell dependent -> memory cells
• Examples: conjugated H. flu; pneumococcal, meningococcal C vaccines

Whole cell vaccines

1. Inactivated

APC ->Tcells (cell mediated immunity) -> Bcells (ABs)
attenuated

• Grow infected cells or bacterial culture, then inactivate with formaldehyde or other chemical
• Examples include: Hepatitis A, Influenza, “old” whole cell pertussis vaccine; IPV

2. Attenuated

• Passage pathogens through repeated cell cultures or animal cultures until non-pathogenic but still "alive"
• Examples include: BCG, measles, mumps, rubella, varicella, smallpox, yellow fever, oral polio vaccines

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How is Influenza vaccine made?

Vaccine Development

• Influenza strains grown in embryonated eggs
• Inactivated with formalin
• Preserved with thimerosal
• Process takes ~12-16 weeks (including testing)

Phase 1 studies

• <100 subjects
• Immunogenicity
• preliminary safety studies

Phase 2 studies (Liscensure)

• <1000 subjects
• Safety
• Dosage

Phase 3 studies

• 10’s thousands
• Safety
• Efficacy

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National Immunization Strategy:

Five Major Themes

Five Cross Cutting Themes

Five Major Themes:

1. National Goals and Objectives for Immunization
2. Immunization Program Planning
3. Immunization Registries
4. Immunization Safety
5. Vaccine Procurement

Five Cross Cutting Themes

1. Research
2. Professional Education
3. Public Education
4. Vaccine Preventable Disease Surveillance
5. Needs of Special Populations

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Define Herd Immunity

Which vaccines are presently available for Canadians in which herd immunity is relevant

• This is when a sufficiently large percentage of the population is immunized so that non immunized person is protected.
• must prevent transmission as well as disease
• amount of people that need to be immunized depends on how infectious the agent is i.e. how many people one person can infect (this number is called “Ro”)
• many diseases such as encephalopathy, autism, MS are diagnosed at times around when vaccines are give
• parents may then associate vaccine with disease, leading to belief that vaccine causes the disease

Disease         Ro
Diphtheria     6-7
Measles       12-18
Mumps         4-7
Pertussis     12-17
Polio             5-7
Rubella         5-7

When R0 is larger, this results in a higher herd immunity threshold

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The mucosal system Generally

Found in....(7)

MALT= mucosa-associated lymphoid tissue -> mouth to rectum
It is the largest mammalian lymphoid organ system and in an adult it comprises approximately 80% of all lymphocytes.

Found in:

1. Bronchial tree (BALT)
2. Gastrointestinal tract (GALT)
3. Nasopharyngeal (NALT)
4. Mammary gland
5. Salivary and lacrimal glands
6. Urogenital organs
7. Inner ear

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Physical organization of the mucosal immune system

Innate defenses at the mucosal surfaces, 4 cell types

Top down:

1. Mucous layer

mucosal secretion, mucins, defensins, IgA

2. Epithelial cells
3. Immune cells

A: All epithelial cells (called enterocytes here, 80%)

Tight junctions:

Mechanical barrier, cross talk between epi cells

Cell surface receptor:

Innate recognition of pathogens

B: Paneth cells

Defensin, lysozyme
Have an anti-bacterial effect

C: Goblet Cells

Mucins secretion

Gel formation, physical barrier

Trefoil factor protein

Epithelial defense & reconstitution

D: Follicle-associated Epithelium (FAE): consist of enterocytes and M cells

In the small intestine, these macroscopic structures consist of aggregates of 5 - 10 lymphoid follicles known as Peyer’s patches

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What are M-cells?

Are specialized epithelial cells which overlie lymphoid follicules domes along the length of the small & large intestine

Differ from epithelial cells (enterocyte):

• Few or no microvilli, do not secrete enzyme, mucus
  
  
• Transport organism via distinct features from gut to immune cells (lymphoid follicles) across the epithelia barrier
  
  
• Express innate immunity receptors (TLR or PRR), may facilitate Ag recognition and facilitate endocytosis and phagocytosis.

Attachment of particulate antigens or pathogens to the apical surfaces of M cells results in their transepithelial transport and delivery into intraepithelial pocket or SED (Sub-epithelial dome)  populated by B cells, T cells and occasional DCs.

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What are Peyer’s patches?

• Organized lymphoid follicles that contain germinal centers which represent the primary sites of mucosal B cell differentiation and somatic cell hypermutation
  
  
• MALT lack afferent lymphatics; germinal center activity is driven exclusively in response to antigens present in the intestinal lumen -> don’t sample systemic pathogen
  
  
• Uptake and transepithelial transport of macro-molecular antigens from the intestinal lumen to organized lymphoid follicles is achieved by FAE (Includes M cells)

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What immune cells are within the mucosa? (6)

1. T-cells - mostly CD4+ - LP layer
   
   
2. Intraepithelial lymphocytes (IELs): 90% are T cells; 80% CD8+
   
   
3. B Cells - secretes IgA - secreted in the form of a dimer held together by a J chain
1. IgA does not activate complement pathway
2. Does not trigger inflammatory response
3. Restricts commensal flora to the lumen
4. Provide efficient microbial agglutination and virus neutralization
5. Inhibits epithelial adherence and invasion
6. Exhibit extensive cross-reactive (‘innate-like’) activity which provides cross-immunity and herd protection
4. Dendritic cells (DC) - recruited to mucosa in response to chemokines
   Extend processes across epithelium to capture Ag in lumen. Suck them into Peyer's Patch and present them to T cells.
5. Granulocytes
6. Macrophages
7. Mast Cells
   
   
   
   
   

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IBS

Celiac Disease Generally

Irritable Bowel Syndrome:

Steps:

Genetic Susceptibility

Barrier defect: Too little mucus -> constant inflam when pathoges reach epi

Infection

Sustained innate immunity = IBS

• An autoimmune disease caused by exposure to gluten
• Results in villous atrophy and crypt hyperplasia of the SI
• Symptoms included bloating, cramping abdominal pain, diarrhea, malabsorption.
• Grains that are bad: Wheat, Barley, Rye
• ~1:150 in N. America

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Celiac's Disease: Pathogenesis

• 33 AA (proline rich) peptide Gliadin passes across epithelium
• Intestinal permeability regulated by a protein “zonulin” -> possible drug actions for cure
• Increase in intestinal permeability allows peptide across mucosa and into lamina propria
• The enzyme Tissue transglutaminase deamidates glutamic acid residues in gliadin peptide
• Deamidated peptide (negatively charged) increases the affinity for APC
• APC express HLA DQ2 or DQ8 receptors
• APC binds to CD4+ T lymphocyte and activates it
• Inflamm cytokines produced – TNF alpha, IFN gamma etc. -> in tun causes tisssue damage and it is even easier for the gliadin to get it
• Also IL-15 produced by enterocytes stimulates T cells to migrate to eptithelium and results in activation of T-NK cells and enterocyte damage
  
  
  
  
  

SF140

Cyclosporin

Autograft

Isograft

Xenograft

Composite tissue transplants

Cyclosporin: Imptnt in transplant surgery!

Allograft: organ/tissue transplanted between genetically different (allogeneic) individuals of the same species (i.e. vast majority of solid organ transplants)

Autograft: (autologous graft) organ/tissue transplanted from one site to another in an individual (eg. skin grafts from one site to another in a burn victim)

Isograft: (Syngeneic graft) organ/tissue transplanted between genetically identical individuals

Xenograft: (Xenogeneic graft) organ/tissue transplanted between genetically different individuals (eg. a porcine heart valve)

Composite tissue transplant: Experimental - face, limbs

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3 antigen systems that determine Histocompatibility

ABO blood group antigens

• These antibodies can cause early and severe rejection of a transplant
• Note: Rh doesn’t matter for solid organ transplants!

The MHC (major histocompatibility complex)

• The major antigen system that determines histocompatibility. Polyallelic and highly polymorphic. Commonly referred to as the HLA system among humans.

Minor histocompatibility antigens (sometimes acronym is mHags)

• These are a diverse population of polymorphic antigens – mostly proteins bound to the MHC antigens. They are called minor because although they trigger rejection, it is typically not as quick or severe. Nevertheless, even perfectly MHC matched transplants can be rejected due to mismatches at mHags

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The MHC

• Located on chromosome 6
• Includes HLA Class I  (HLA-A, -B, -C) and Class II (HLA-DR, -DP, -DQ) antigens
• HLA Class I antigens are membrane proteins expressed on all nucleated cells and platelets
• HLA Class II antigens are membrane proteins expressed on APC’s (dendritic cells, B lymphocytes, macrophages/monocytes), and some T cells.
• HLA class I present intracellular peptides to T cells
• HLA Class II present peptides taken up by endocytosis (from outside the cell)
• The HLA genes are polyallelic (as above – HLA A,  B, DR etc.) and highly polymorphic (there are many different alleles at each locus among different individuals within a population)
• Inherited in a co-dominant fashion (ie. one set from mom’s chromosome, other set from dad)
• Haplotype – refers to the combination of alleles on the same chromosome inherited together
• In transplantation - typically haplotype refers to combination of HLA Class I A, B loci and Class II DR locus as these are the major determinants of transplant rejection

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Allorecognition

• The high degree of polymorphism at HLA loci (and to a lesser degree due to mHags) means that each transplant recipient is likely to recognize some foreign (allogeneic) peptides and mount an immune response to them.
• T cells must first be activated prior to rejection pathways being initiated and the first step in this alloimmune response is allorecognition. Importantly, in transplantation foreign peptides can be recognized by T cells in two distinct allorecognition pathways, the standard process (indirect) and one unique to allogeneic transplantation (direct).

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Indirect allorecognition

Direct allorecognition

But...

Indirect allorecognition: Allogeneic proteins (e.g. donor MHC) are taken up by recipient APC’s, processed, and presented on self MHC (i.e. class II HLA) to T cells leading to activation and proliferation

Direct allorecognition: Donor antigen presenting cells expressing their (foreign) peptides loaded onto their own foreign MHC migrate out from the graft and bind to recipient T cells leading to activation and proliferation.

Allorecognition involves more than just MHC-peptide complexes binding to lymphocyte T cell receptors. The latter is often referred to as signal 1. Several other molecular interactions are required for T cell activation.

Signal 2 refers to a number of other APC-T cell interactions that serve to:

1) strengthen the bond between cells for signalling (eg. adhesion molecules binding between cells and

2) enhancing intracellular signalling in lymphocytes leading to activation.

Allorecognition leads to T cell activation, then proliferation primarily driven by the activated T cell cytokine IL-2, and then activation of effector pathways that mediate rejection.

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Effector mechanisms of rejection

Why is the alloimmune response so strong?

Involves both cell-mediated and antibody mediated (humoral) processes.

Cell mediated rejection
1. CTL’s (cytotoxic T lymphocytes)

• after being activated by direct allorecognition CTL’s traffic to the transplanted organ and mediate damage by ether perforin-granzyme (pokes holes)or Fas-Fas ligand dependent killing

2. Delayed-type hypersensitivity

• Activated T cells -> IFNγ -> macrophages -> release enzymes/toxic products and TNF that causes endothelial changes like increased vascular permeability enhancing immune cell trafficking into transplanted organs

Antibody mediated (humoral) rejection

• With help from activated T helper cells, B cells with specificity for foreign HLA antigens (as well as other alloantigens) generate antibodies
• This can occur immediately if the antibodies are present at the time of transplantation but may also develop long after transplantation

Two general reasons:

1. Lots of potential alloantigens – So lots of antigens to catch the immune system’s attention
2. The potency of direct allorecognition b/c a very high frequency of recipient T cells are capable of reacting with donor MHC-peptide complexes (e.g. 1 in 104). This is in sharp contrast to recognition of self MHC-peptide complexes where only a tiny fraction of T cells may respond to a given MHC-peptide complex (<<<1 in 104). This is likely due to the fact that T cells binding too strongly to self MHC-peptide complex were deleted during lymphoid development leading to a very flexible ‘loose-fitting’ TCR structure capable of recognizing a multitude of allo-MHC-complexes.

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 3 Timeframes of Clinical Transplant Rejection

Hyperacute (within minutes to hours of transplantation):

• Due to preformed antibodies in the recipient’s circulation (Anti-ABO or anti-HLA antibodies -> via pregnancy, transfusions, prior transplantation)

Acute rejection (within the first few weeks to months):

• May occur at any time point and presents as a sudden deterioration in organ function
• More commonly, T cell mediated but can also be due to HLA antibodies
• Management is prevention with adequate immunosuppression

Chronic rejection (develops gradually over months to years):

• Remains a major cause of transplant failure
• Due to slow and continuous immune injury to the allograft
• Involves both cell mediated and antibody mediated rejection
• Prevention is immunosuppression
• Gradual ‘smoldering’rejection

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4 Commonly Used Immunosuppressant Medications

Note: twins, avascular tissues such as corneas, heart valves, or connective tissue are an exception and their recipients do not need immunosuppression. No tolerance to an allogeneic graft is thought to develop over time. Exceptions to this need for lifelong immunosuppression are bone marrow transplant recipients, some liver transplant recipients, and the very rare nonadherent patient who stops taking their medication.

1. Calcineurin inhibitors

Eg. Cyclosporine

Bind to calcineurin inside T cell cytoplasm and prevent this enzyme from activating a nuclear transcription factor which triggers the production of key cytokines driving lymphocyte activation and proliferation (e.g. interleukin-2)

2. Antimetabolites
    Eg. Azathioprine
Both are purine analogues which interfere with the production of purines and DNA synthesis, limiting T cell proliferation.

3.    Corticosteroids

Eg. prednisone (orally)

Major FX is blockade of transcription of numerous cytokine genes (e.g. IL-1, IL-2, TNFα, IFNγ). Steroids can bind to GRE (glucocorticoid receptor elements) sites on DNA preventing gene transcription and affect the nuclear translocation of other transcription factors.

4. Biologic agents (polyclonal or monoclonal antibody preparations)

Ex Thymoglobulin (rabbit)

Antibodies produced either by inoculating animals or using engineered cell lines. They are typically given at the time of transplantation to prevent rejection when the risk is highest in the initial weeks to months.