Immunology

Exam 2 guide

Antibody Structure and Generation of Diversity

General Features Ig
- Glycoprotiens
- 4 chains, 2 H and 2 L (kappa or lambda)
Papsin cleavage
- Fab fragment: 2 identical, antigen specific
- Fc fragment: interact with receptor Fc cells (macrophages)
- Cut at hinge regions
Pepsin cleavage
- F(ab’)2: 2 antigen binding sites linked
- Unable to react with any effector molecule

Globular motif: diff. folds that make up Ig domain
- Initial IgM; if receive help: diff isotypes

Complementarity-Determining Regions (Cdr)
- 3 regions in V region of both heavy and light chains to make up antigen binding site
- Combinatorial Diversity: Cdr’s from both Vh and Vl determine final ant. Specificity
Antigen binding: Electostatic, H-bonds, Van der Waals, Hydrophobic (reversible non-covalent)

Affinity: Strength of binding 1 antigenic determinant to single binding site
Avidity: overall binding energy b/t antigen with many determinants and multivalent antibodies
- Multimeric antibodies may have low affinity but hi avidity
Valency: total # of interactions

Constant Regions
- Diff Fc receptors can bind to diff. subtypes of isotypes
- IgM, IgD, IgG, IgA and IgE
- Humans: IgA: IgA1 and IgA2; IgG: IgG1, IgG2, IgG3 and IgG4
- All with diff. effector functions

Antibody Flexibility
- @ hinge region, also between V and C domains
- Allow for better orientations (ball and socket joints)

Kappa vs. Lambda Light chains
- No known differences in function, determined by the carboxyl-terminal constant region
- Humans typically have 60% kappa, 40% lambda
- Can be used to diagnose B-cell lymphomas (one B-cell clone produces only one species of antibody,
therefore skewing the ratio)

When antibodies of another species are introduced to humans, they are seen as foreign
A response is induced mainly against the C region of these antibodies

Isotypes: Diff b/t H chain C regions
Allotypes: diff in diff alleles of the same C regions (mom vs dad)
Idiotype: Diff in rearranged Vl and Vh genes with same C region

Cross-reaction: antibody binds to diff but structurally similar antigen (m-protiens/S. pyogenes)
-can lead to immunologic diseases

 Serum: antibodies remain in fluid after blood clot
Antiserum: any serum sample that contains antibodies that bind to particular antigen
Serology: study of antibodies RXN with antigens
High titer: hi conc. of particular antibody in serum

Polyclonal antibodies: produced by more than one clone of B-cell (produce more than one type of antibody for
antigen with multiple epitopes)

Monoclonal antibody: produced by a single clone of B-cells

To make antiserum:

Hybridomas: Take antigen and inject in animal. Isolate spleen cells from animal (activated B-cells). These B-cells
recognize the epitopes on the antigen. Fuse these cells with immortal myeloma cell line. Then the antibody is
selected with the desired specificity: MONOCLONAL ANTIBODIES.
Applications: Used for diagnostic to detect specific antigens associated with particular microbe
Biopsy from tumor (protiens from cancer cells)
Therapeutically (name always ends in ___mab)

Generation of Diversity in Ig’s
Somatic recombination: site-specific rearrangement (VDJ)
Functional antigen receptor genes are created ONLY in DEVELOPING LYMPHOCYTES after DNA rearrangement
- 3 different Loci:
Encode light chain (kappa or lambda) with V, J and constant
Encode heavy chain with V, D, J and constant
Limited constants (5 in heavy), limited in light

Humans:
- Kappa light chain: 1 C gene
- Lambda light chain: 4 C genes
- Heavy chain: 9 C genes
Light chain, variable region encoded by V and J
Heavy chain, variable region encoded by V, D and J

Heavy Chain
- Select single D and J segment first, then V segment (makes the “fingers”)
- Rearrangement puts the VDJ unit next to the constant region genes Cm(IgM) and Cd(IgD)
- Single resting B-cell may express both IgM and IgD with same antigen specificity

Light Chain
- Only difference is no D segment
- Either Kappa or Lambda (2 separate loci)

Random selection of gene segments generates great diversity!

V(D)J Recombination
- Involves a number of sequential steps
- Chromatin opened at specific regions of chromosome to make Ag receptor segments accessible to enzymes
that mediate recombination
- 2 selected segments must be brought next to each other across a lot of chromosomal dist.
- Double stranded breaks at coding ends, nucleotides added or removed at broken ends (div)
- Ends are ligated to produce clonally unique (diverse) Ag receptor genes for transcription
- Subsequent splicing brings together V(D)J exon and C region exons

RAG- recombination- activating genes: only in developing B and T cells at certain times
- RAG-1 and RAG-2 recognize recombination signal sequences adjacent to V, D, J segments
- RAG cuts the DNA!
- TdT mediates addition or removal of nucleotides at broken ends of DNA

Steps for Ig Gene rearrangement

Early pro-B cell
- H-chain rearrangement: D, J gene on both alleles
Late pro-B cell
- H-chain rearrangement: V-DJ rearrangement on one allele (if no good than other allele)
o Only one allele goes through process (allelic exclusion)
Pre-B cell
- Rearrangement of Kappa light chain OR Lambda light chain
Immature B cell
- Rearrangement ceases

Generation of diversity in B and T cells
- Somatic recombination: diff combinations of gene segments used in diff rearrangement events
- Junctional Diversity: TdT mediates addition/removal of nucleotides at joints GREATEST CONTRIBUTION TO
DIVERSITY OF ANTIGEN RECEPTORS
- Combinatorial Diversity: diff combination of heavy and light chain V regions can pair to form antigen binding
site

Mature B cell
- Only when cell expresses both IgM and IgD
- Can splice our of M to make D with same antigen specificity4


Secondary Lymphoid Organs (after antigen recognition and cell activation)

Affinity Maturation:
- With repeated exposure to the same antigen, host will produce antibodies with greater affinities
- Somatic mutation followed by selective survival
- Helper T-cells required for somatic mutation: T-dependent protein antigens
Somatic hypermutation
- Occurs in 2ndary lymph organs after func. Ig genes have been assembled
- Point mutations in V regions of H and L chains lead to mutated B cell receptors on surface
- Many mutations result in decline or loss of Ag binding, so selection of best B cells is crucial
- Migrate to follicular dendritic cell-rich areas, or sticky magnets to present Ag in germinal centers
- Die unless recognize Ag and receive T cell help
- Hi BCR affinity results in more MHC on cell surface
- Greatest share of T-cell help-> drives positive selection

H chain Isotype Switching
- T-dependent responses
- Regulated by CD40L signals and cytokines from helper T cells
- At 5’ end: switch region
- The intervening C region DNA is “looped out”, but can switch again if other switch regions are still in “tail”
DNA
- AID is enzyme needed for stimulation

Differences in Isotype Monomers
- Diff location of interchain disulfide bonds, # attached oligosaccharide moieties, # C domains, and length of
hinge region (M and E have less flexibility)
Multimers
- IgM and IgA have C regions with tailpiece of 18 AA contain Cisteine residu for polymerization
- Additional polypeptide chain called J chain promotes by linking these Cisteine res together
- IgM: PENTAMERS
- IgA: DIMERS, required for transport through epithelia
- Imp. In binding of repetitive epitopes
- J chain interacts with transporter receptor- NOT J-SEGMENT!

Humoral Immune Response Part 1- Thymus dep and indep Mechanisms
**Type and Amt of Ab vary dep on:
- Type of Ag driving response
- Prior history of Ag exposure
- T cell help
- Anatomic site of activation
Memory cells most likely have undergone Isotype Switching

B cell Subsets
FOLLICULAR B CELLS: Located in lymphoid follicles of spleen and lymph nodes, T-Dependent isotype switched,
long lived plasma cells
MARGINAL ZONE B CELLS: Abut marginal sinus of spleen, T-independent, mainly IgM, short lived
B-1 CELLS: mucosal tissues, peritoneal/pleural cavities, T-independent, mainly IgM, short lived

B-1 Cell Subset
- Limited Ag receptor diversity
- Low-affinity IgM Ab often rx with microbial polysaccharides and lipids and oxidized lipids
- Ab often called NATURAL ANTIBODIES
- Unknown source of Ag
- Respond to commonly encountered Ag in epithelial mucosal interfaces (not necessarily pathogenic foreign)

Thymus Dependent vs Thymus Independent
TD antigens: Ag unable to induce Ab response in animals who lack T cells (protiens)
- Able to undergo isotype switching, affinity maturation and Secondary response
TI antigens: Ag able to induce Ab production in absence of helper T cells (bacterial polysaccharides or polymeric
protiens)
- Little to no isotype switching (some IgG), little or no affinity maturation, secondary response only to some
antigens (polysaccharides)
- 2 types
o TI-1 antigens
o TI-2 antigens
TI-1 Activation
- LPS can activate mature/immature B cells
- Hi conc: Signal sufficient for antibody secretion in absence of specific antigen binding to surface Ig
(Polyclonal Activation-all B cells respond
- Low conc: B cells specific for TI-1 antigen bind enough to focus its B cell act. Properties onto the B cell
(Specific Antibody Response)
o TI antibody= IgM
TI-2 Activation
- Bacterial Capsular Polysaccharides activate mature B cells
 - Extensive cross-linking of B cell receptors specific for the Ag (repeating epitopes) can signal B cells to
produce Ab
- Not require peptide-specific T cell help; BUT cytokines produced by helper T cells greatly augment response,
can lead to isotype switching-> unique type of T cell

Antigen and Follicular B cell interaction
- Ag enter in afferent lymphatic vessels
- Mature recirculating naïve B cells enter via HEV’s (T cell zone) then move to B cell zone
- Ag then drain to subcapsular sinus (SCS)
- May reach B cell zone through fibroblastic reticular cell(FRC)-lined collagenous conduits b/t SCS and follicle
- SCS macrophages capture large microbes and Ag-Ab complexes and deliver to follicles under sinus
- Larger Ag not captured are captured in Medullary region by resident dendritic cells and sent to follicle

SCS Macrophages
- Extend processes through sinus
- Limited phagocytic activity to present INTACT Ag on surface (coat with Ag)

Follicular B cells
- Bind to particular Ag on SCS macrophage surface
- Does NOT depend on specificity of BCR; mediated by Compliment Receptors (CR’s) and Fc receptors
- Mediate transport of Ag to follicular dendritic cells which express higher levels of complement receptors

Sequence of Events During T Cell-Dependent Ab Responses
- Naïve CD4 T cells activated in T cell zone by Ag from dendritic cell (processed peptides)
- Naïve B cell activated in follicles by same Ag (diff epitope)=linked recognition
- Th cells and activated B cells move toward each other, interact at edges of follicles, initial Ab response
develops, short lived plasma cells
- Some cells migrate back to follicles (germinal centers) memory cells, more specialized Ab responses induced

B cell Activation
Occur in Follicles of Lymph Node
- Protein Ag endocytosed upon being recognized intact, broken down into peptides that bind to MHC II
- Protein that elicit T cell dependent B cell response makes use of at least 2 epitopes ( 1 for BCR, 1 for Th cell)
- Internalized linear peptide presented on MHC II, recognized by T cells
- Single B cell may present multiple diff. peptides, however the antibody response remains specific for native
protein (Epitope that B cell recognizes is intact peptide from B cell)

Activation by Thymus-Dependent Ag
Signal 1: Bind to Ag, process peptide, present on MHC II
Signal 2: Peptide-MHC II recognized by Effector T cell, Requires cell bound (CD40L-CD40) and released
(cytokines) molecules. (Effector T cell expresses CD40L)

CD40L-CD40 interaction stimulates B cell proliferation

T helper Cells
- Addition to CD40L, cytokines released at point of contact -> IL-4, 5, 6
- Th2 release IL-4

REMEMBER
- T-cell dependent Ab response requires activation of B cells by Th cells that respond to SAME Ag, not epitope
(linked recognition)
- B/f B cell can be induced to make Ab for this pathogen, a CD4 T cell must be act. Specific for peptides from
this pathogen to produce effector Th cells.

How do these two cells find each other?
B and T cells up/down regulate chemokine receptors oppositely.
- T cell act. In T cell zone, B cell act. In B cell zone change Chemokine receptors
- Come towards eachother
- T cells: CCR7 decrease, CCR5 increase
- B cells: CCR7 increase, CCR5 decrease

Different Locations of B cell Reponse
Primary Foci:
- medullary cords/ junction between T cell zone and redpulp
- Extrafollicular helper T cells
- Limited class switching
- Low rate somatic hypermutation
- Low Ab affinity
- Short lived Ab
Secondary Foci:
- Germinal centers
- T follicular helper cells help in germinal centers
- Extensive class switching
- High rate somatic hypermutation
- High Ab affinity
- Long lived Ab migrate to MALT or bone marrow, and memory cells
- T fh cells help B cells undergo Affinity Maturation in germinal centers

Induction of T follicular helper Cells
- 2 Steps
o Initial activation by APC dendritic cells
o Activation by B cells
§ ICOS on T cells and ICOS ligand on act. B cells promote differentiation of T cells in to T fh
cells
- T fh cells express hi CXCR5, drawn into Lymphoid follicles by CXCL13 (ligand fro CXCR5)
- Defining cytokine of Tfh cells = IL-21, required for Germinal center development

Germinal Center
- Proliferating B cells accumulate in Dark zone (no FDC’s or T cells)
- Progeny migrate to light zone, come into contact with FDC and Tfh, selection event occur
- Germinal center formation is dependent on CD40L (Tfh) interacting with CD40 (B cells)
- Higher affinity receptors are selected in dark zone (specificity also selected against)

Follicular Dendritic Cells
- Only found in lymphoid follicles, express compliment receptors and Fc receptors
- Recognize opsonins
- Display antigen intact
- NO MHC II
- Sticky magnets

Isotype Switching Directed by Cytokines
- Cytokines induce isotype switching
- Stimulate mRNA transcribed from switch recomb sites
- Lie 5’ to each C gene
- Th regulate both production of Ab and isotype that determines effector function of Ab
- CD40 engagement induces AID enzyme, required for isotype switching and affinity maturation

X-linked Hyper-IgM Syndrome
- Mutation in CD40L, defects in Ab production, isotype switching, affinity maturation, and memory B cell
generation

B Cell Differentiation into Antibody Secreting Cells
- Short lived: T-independent responses/early T-dependent responses in Primary foci
- Long lived: T-dependent responses to protein antigens in Germinal centers
- Signals from BCR and IL-21 cooperate in generation of plasma cells and their precursors: PLASMABLASTS

Long Lived Antibody Secreting Cells
- Plasmablasts generated in germinal centers, home to the bone marrow: differentiate into long lived plasma
cells
- Chemokine ligand 12 (CXCL 12) and receptor (CXCR4) imp. In recruitment of ASC to bone marrow and
retention here
- Plasma cells maintained by cytokines of the BAFF family
- Live for months-years, continue produce Ab even after Ag is eliminated

Humoral Memory
- Constitutive Humoral Memory: Pre-existing Ab from long lived plasma cells are first line of defense
- Reactive Humoral Memory: Pathogen-experienced memory B cells are rapidly reactivated to produce Ab

REMEMBER:
Late B cell differentiation is a prominent source of human lymphomas owing to the HIGH PROLIFERATIVE RATE
of germinal center B cells combined with the expression fo AID, responsible for class switch recombination and
somatic hypermutation (DNA ALTERING PROCESSES)
THINGS CAN GO WRONG

Humoral Response Part 2: Function and Properties of Ig Isotypes

Main function of Ab: neutralize and eliminate infectious microbes and microbial toxins
o Toxoid: inactive toxin, generates Ab against toxin
o Capsular Polysaccharide attached to carrier protein: Hapten, opsonized/phagocytozed
Short lived: IgM
Long lived: other isotypes

Effector Functions of Ab
- Neutralization of microbes/toxins
- Opsonization/phagocytosis of microbes
- Compliment activation

NEUTRALIZATION
- Ab block binding to cellular receptors
- Hi affinity IgG/IgA Ab can inhibit viruses
o Block viral surface receptors to prevent viral fusion in membrane of host cell
- IgA in mucosal surfaces important at blocking adhesins that enable microbes to bind to host surface
- IgA and IgG inhibit toxin binding, IgG=Tissues, IgA=Mucosal surfaces
Tetanus Toxin Example
- A/B toxin
- B subunit binds to ganglioside receptor on nerve cell
- Internalized A subunit carried to axonal transport to CNS
- Blocks neurotransmitter release
- Constantly stimulate motor neurons/ Spastic Paralysis

OPSONIZATION
- Mononuclear phagocytes and neutrophils express receptors for Fc portion of IgG Ab
- Microbes are coated with IgG or Compliment, enhances phagocytosis
- Deliver signals to regulate the activity of leukocytes
- Fcγ receptors IMPORTANT in recognizing Heavy Chain of IgG

FcγRI= on Phagocytes, high affinity receptor
FcγRIII-A= low affinity, Natural Killer cell
FcRN= Transport mother/fetus, internalizes IgG, protect from ex. Environment, longest half lfe

Phagocytes distinguish Ab bound to pathogen from Free Ab through Ab Aggregation State
o Hi avidity->receptor crosslinking->sends activation signal

Role of OPSONINs
- Binding of Fc and compliment signals increased rate of phagocytosis, fuse w/lysosomes, increase
antimicrobial activity
- pH changes when fusing lysosome with phagosome, activates enzymes

Ab dependent Cell-mediated Cytotoxicity (ADCC)
- Ab bound to microbe can interact with Natural Killer Fc Receptors
- FcγRIII-A on NK cell recognizes IgG1 and IgG3, triggers attack

Three Pathways of Compliment Activation
1- Classical
2- Lectin
3- Alternative

Activation of Classical Pathway
- C1: complex of C1q, C1r and C1s
- C1q has 6 globular heads, bind to Fc domains, binding 2 or more activates complex
- Triggers inflammation, opsonization, formation of membrane attack complex
- IgM is good because it form a multimeric Ig, more Fc available
- IgG must be close together

Important Clearance Mechanism
- Complement activation leads to covalent binding of C4b and C3b to immune complex
- Erythrocytes have CR1 on surface, C4b and C3b bind to it
- Complex is transported to spleen/liver via erythrocyte
- Macrophages here with CR1 and Fc receptors remove w/o destroying erythrocyte and degrade

Ig Distributed in Different Location in the Body
- IgM/IgG predominate in plasma, IgG and monomeric IgA major isotypes in extracellular fluid
- Dimeric IgA predominate in secretions across epithelia (inc. breast milk) MUST BE DIMERIC
- Maternal IgG transported across placenta directly into fetal blood stream, FcRN transports
- IgE found associated with Mast Cells beneath epithelial surfaces

Funtional Activity of Isotype Classes

IgA: Neutralization
IgG: Neutralization and Compliment activation
IgG1 & IgG3: Opsonization and Sensitization for killing by NK cells
IgE: Sensitization of Mast Cells
 IgM: Activate compliment

Distribution of Isotype Classes

IgA (dimeric): transport across epithelium and diffusion into extravascular sites
IgG: Transport across placenta, Diffusion into extravascular sites, mean serum level (longest half life)

Antibody Isotype of Mammals
IgA- monomer or dimer, mucosal surfaces
IgD- B cell receptors on naïve B cells
IgE- worms, allergic rxns
IgG- serum, FcRN, principle isotype in blood
IgM- on B cells as receptor, low affinity, hi avidity (pentamer), eliminates pathogen in early humoral immunity
b/f sufficient IgG.

Key Concepts of Ab
- Ab produced in lymph nodes, spleen and bone marrow may enter blood and circulate
- Ab produced in MALT are transported across epithelial barriers into lumens of mucosal organs, block entry
of inhaled/ingested microbes
- Ab actively transported across placenta into circulation of fetus

Polymeric Ig Receptor helps transport dimeric IgA across epithelia
- Most IgA ab synthesized in plasma cells just beneath basement membranes
- IgA dimer bound to J-chain is bound to polymeric Ig Receptor as it diffuses across membrane
- At luminal surface, receptor is cleaved, fragment is called secretory component

INNATE IMMUNITY
Innate immune effector cells are fully functional even b/f infection or rapidly activated by microbes

- Recognition of Microbes and Damaged self
o Relatively limited set of molecular structures
o About 1000 products of microbes and damaged host cells
o MAMPS: Products shared by microbial associated molecular patterns
o PAMPS: Products shared by pathogen associated molecular patterns
o DAMPS: components released from damaged host= damage associated molecular patters (alarmins,
danger signals)

- Examples of PAMPS
o Nucleic acids, proteins (pili, flagella), Cell wall lipids (LPS), Carbohydrates
- Examples of DAMPS
o Stress induced protiens, Crystals (gout), Nuclear proteins

Receptors for these
- Patter Recognition Receptors (PRR’s)
- Found in areas that are more likely to find a ligand
- Phagocytes, dendritic cells, barrier cells, phagocytic vesicles etc.
- Activate signal transduction pathways promoting antimicrobial and pro-inflammatory functions

TOLL-like Receptors and NOD-like Receptors

Toll-like Receptors
- Vertebrates and Invertebrates
- Recognize wide variety of microbes/molecules released from stressed or dying cells
 - Structurally conserved
- Leucine rich repeats in extracellular domain
- TIR intracellular domain for signaling

Different TLR’s recognize Different Components
TLR4: LPS
TLR5 and heterodimers TLR2/1 and TLR2/6: diff components of wall
- Localized in plasma
TLR3, 7, 8, 9: recognize nucleic acids
- Contained in intracellular membranes (endosomal compartments)

LPS is recognized by TLR4=GRAM – bacteria
Lipoteichoic Acid recognized by TLR2=GRAM + bacteria

INTRACELLULAR COMPONENT
- Interact with adapter molecule
- Ex: TLR4 requires MD-2 for interaction with LPS

Ligand Binding to TLR
- Signals cascade to transcription factors which express genes important for inflammatory/antiviral responses
- Gene activators
o NF-kB and MAP kinases are transcription factors for pro-inflammatory mediators

COMBINED TLR Activation Produce Specific Responses
- TLR 4 and 5 activated: likely a flagellated Gram – bacteria
- TLR 5 and 2/6 activated: likely a flagellated Gram + bacteria
- More specific towards the pathogenic microbe with combined responses

CYTOSOLIC RECEPTORS: linked to signal trans pathways promoting inflamm or IF-1 production
(usually viral infection)
3 classes
1) NOD-like receptors
2) RIG-like receptors
3) Cytosolic DNA sensor

NOD-like receptors
- 3 families, focus on NOD1 and NOD2
- Both expressed in cytosol,
- Respond to bacterial cell wall peptidoglycans
- NOD2 highly expressed in intestinal Paneth cells, stimulates antimicrobial DEFENSINS
- NOD1 detects iE-DAP, primarily found in GRAM – bacteria
- NOD2 detects MDP, present ubiquitously in bacterial peptidoglycan (BOTH – and +)

NODs detect intracellular and extracellular pathogens
- Intracellular pathogens interact w/tight junctions->induced membrane ruffles

Peptides released from extracellular bacteria require transport into host cell Cytosol
- Taken up by endocytosis
- Transferred via gap junctions
- Directly transported into cytosol by bacterial secretion systems
- Directly imported into cytosol by host proteins

NLRP-Inflammasome Subfamily of NOD-like receptors
 - Respond to cytosolic PAMPS and DAMPS by forming INFLAMMASOMES
- INFLAMMASOMES: generate active forms of IL-1β and IL-18
- Induced by:
o Microbial products
o Environmentally or endogenously derived crystals
o Extracellular ATP, reactive oxygen species, reduction in cytosolic K
- After binding to ligand, multiple NLRP3 proteins interact to form oligomer
- Bind to adaptor protein (ASC)
- This binds to inactive form of enzyme caspase-1
- Caspase-1 becomes active after recruitment of inflammasome complex
- Cleaves inactive precursor of cytokines IL-1β and IL-18
- Active forms of these IL’s leave cell and perform pro-inflammatory functions


COMPONENTS OF INNATE IMMUNITY

Epithelial Barriers
- Physical, produce defensins, maintain integrity
- Cilia in airways
- Normal microbiota in gut outcompeting invaders
- Enzymes degrade

Cellular Components
- Neutrophils :#1, Macrophages, Dendritic Cells, NK cells
- Monocytes->macrophages
o Release mediators
- Tissue macrophages
o Osteoclasts (bone)
o Microglia (brain)
o Alveolar Macrophages (lung)
o Histiocytes (interstitial connective tissue)
o Kupffer Cells (liver)
- Macrophage Activation phenotypes
- M1: Inflammatory, classically activated, IFN-γ
- M2: Repair, alternatively activated, IL-4 and IL-13, suppress T cell response, Anti-inflammatory

Phagocytic Cells
- Facilitate removal of RBC, necrotic tissue and foreign substances
- Homeostatic functions increased after injury or during infection
- Express pattern recognition receptors for PAMP’s and DAMPS
- These receptors instruct to engulf/destroy via respiratory burst (promote inflammation)

Macrophages
- Long lived
- In tissues, can migrate back into lymph nodes
- O2 dependent killing not vigorous
- Virus can “hide” in macrophage

Neutrophils
- Short lived, die (PUS)
- Circulating
- Respond to inflammation, die there
- Production in bone marrow INCREASES upon infectious response
 - O2 dependent killing lethal and vigorous
- Hostile environment for intracellular pathogen

Receptors Include
- PRR(pattern recognition receptors), Fc or Compliment Receptors

Phagocytosis
- Activation
o Enhance rate of phagocytosis
o Enhance production toxic reactive O2 species(superoxide, H2O2, Hydroxyl radical)
o Enhance nitric oxide production (iNOS)
o Enhance phagosome-lysosome fusion
o Increase MHCII
o Secrete IL-12 (Th1)

Enzymes trigger initiation cascade
- NADPH oxidase: makes reactive oxide species (superoxide, H2O2, hydroxyl radicals)
- iNOS: makes nitric oxide
- Neutrophils contain MPO to make choramines, macrophages can aquire by consuming neutrophils

Host Microbicidal Factors
- V-ATPase: produces acidic environment, allows H+ inside
- NADPH oxidase: ROS production
- iNOS: RNS production
- Antimicrobial proteins/peptides: Lactoferrin (compete for Fe), defensins, lipases, lysozymes disrupt integrity,
hydrolases and peptidases, general enzymes that degrade

Microbe Response to evade Factors
- Interfere with phagocytic engulfment: degrade/inhibit opsonins
- Modify surface to degrade against antimicrobial factors
- Counteract Acid accumulation
- Produce neutralizing enzymes
- Prevent synthesis of ROS/RNS via enzymes
- Overcome reduced Fe
- Dispose/replace damaged proteins

Natural Killer Cells
- Innate lymphoid cell
- Acts on tumor cells, viral infections, intracellular bacteria, stressed cells
- Kill other cells, produce cytokines
- Main antiviral cytokine=IFNγ
- Kills the same way cytotoxic T-cells Kill (CD8+)
- Respond to IL-12, IFNγ activate macrophages to kill phagocytosed microbes

NK balance of Receptors
- Activating receptors AND inhibitory receptors
- If viral infected cell does not express MHC I, then inhibitory cell receptor will notice and attack
- Activating receptors stimulate protein kinases that phosphorylate downstream signals
- Signals cancel each other out
- Activating receptors recognize ligands on infected/injured cells (eliminate), inhibitory receptors recognize
healthy normal cells (preserve)
- FcγRIIIA: low affinity receptor for IgG Ab; imp for ADCC
o Cell is opsonized with IgG

Early Viral Infection
- NK cells activated by IL-12 and IL-15, kill b/f Ag specific CTL’s can become active

Later Viral Infection
- NK cells kill infected cells that have escaped CTL-mediated immune attack by reducing MHC I on surface

Antiviral Response
- Type I IF
o IFN-α and IFN-β important
o Viral nucleic acids stimulate
o PRR’s in cytosol/endosomal vesicles recognize (TLR3,7,8,9)
o DNA is recognized as WRONG TYPE and in the WRONG PLACE
o IF signaling activate genes that confer antiviral state
o Act on neighboring cells to protect them too (PARACRINE)
o Inhibit replication of the infected cell (AUTOCRINE)

Cells that don’t belong to INNATE or ADAPTIVE
- Natural Killer T Cells
o 2 types
o Invariant NKT and diverse NKT
o Able to respond in innate-like manner to danger signals and proinflammatory cytokines
o Preferentially use TCRα gene (not randomly selected): Vα24-Jα18 in humans
o Use restricted Vβ gene segments: Vβ11 in humans
o Do not recognize MHC I or II, but recognize LIPID bound to CD1d molecules
o CD1d is a MHC I-like molecule, expressed by many cells of hemopoetic origin
- 2 Signals Involved in Activating NKT
o First, TCR mediated: activated by strong foreign Ag
o Second, cytokine signal: Ag can be low affinity lipid, but APC generates proinflammatory cytokines
like IL-12
- Different Subpopulations of NKT exist
o Key job is to produce cytokines
o Diff. cytokines are produced depend on what type and where it is found

Innate immune response provides signal for Adaptive Immunity
- Activation of lymphocytes requires:
1) Signal from Ag
2) Signal from molecule produced by innate immune response to microbe or injured cell
a. Examples: Constimulators (T-cells), Cytokines, Compliment breakdown products)

COMPLIMENT SYSTEM
- 30 membrane bound and fluid phase proteins
- Mostly synthesixed in liver
- Zymogen: inactive precursor, is activated in proteolytic cascades to become active protease
- Do 3 things:
o Opsonize microbes, induce inflammation and lyse cells in SOME cases

Sequential Proteolysis
- Each step can amplify in cascade because multiple enzyme molecules can be generated
- Some products become covalently attached to the cell surface (C3b, C4b)
- Inhibited by regulatory proteins

3 major Pathways of Compliment Activation
 - Classical Pathway
- Lectin Pathway
- Alternative Pathway

Classical Pathway
- C1 complex (C1q, C1r, C1s)
- Can only bind to Ab bound to Ag
- When 2 or more globular heads of C1q interact with ligand (typically IgM or IgG), C1r is activated, which
then cleaves and activates C1s
- C1s then cleaves C4 into C4a and C4b
- C4b binds to cell surface (C4a floats away/inflammation)
- C2 complexes with cell bound C4b and cleaved by C1s
- C2a remains associated and C2b leaves
- This forms C3 Convertase: C4bC2a
- C3 Convertase can then bind to C3, C2a cleaves into C3a and C3b
- Some C3b binds to cell surface (opsonize), but some binds to C3 Convertase, forming C5 Convertase
- C5 Convertase: C4bC2aC3b

Lectin Pathway is the Same exept for starting Components
- Mannose Binding Lectin (MBL) forms complexes with proteases MASP-1 and MASP-2 (similar to C1 complex)
- MASP is activated by MBL binding to pathogen carbohydrates
- Activated MASP cleaves C4 and continues down pathway

Alternative Pathway
- Spontaneous hydrolysis “tickover”
- Don’t need recognition of anything on microbial surface
- Host cells have regulators to prevent opsonizing host, microbes do not
- Spontaneous hydrolysis of C3, making C3(H2O)
- This can bind Factor B, which then is cleaved by Factor D, producing short lived, fluid phase C3 Convertase:
C3(H2O)Bb
- This can turn to membrane bound C3 Convertase: C3bBb, can cleave many molecules of C3 into a and b.
- C3b can bind to more Factor B (making more convertase), or bind to the cell surface or to C3 convertase
itself, generating C5 Convertase
- C5 Convertase: C3bBbC3b


Key Point to Remember
- Alternative Pathway C3 Convertase can use C3b generated by all 3 pathways
- Amplified

Another point
- The thioester bonds from C3b and C4b are hydrolyzed(inactivated) if not quickly formed

What happens after C5 Convertase is Formed?
- Culminate in the formation of Membrane Attack Complex (MAC)
- Up to 1000 molecules of C3b can bind near a C3 Convertase (highly opsonized)
- After C5 convertase is made, C5 binds to C3b and is cleaved by C2a or Bb
- C5b had no thioester bond, C5a is most potent inflammatory inducer
- No more cleavage from this point (C6,7,8,9)
- C7 is Hydrophobic and inserts into lipid bilayer, which has high affinity for C8
- C9 then can polymerize at this site to form pore, but more than one are needed (C9n)
- 10-16 C9 are needed to form pore

Compliment Receptors
- CR1: promote phagocytosis
o Hi affinity for C3b and C4b
o Use receptor to bind/internalize foreign particles
o When Fcγ also engaged, antimicrobial mechanisms are activated
o CR1 on erythrocytes bind to circulating immune complexes and transport to liver and spleen

- CR2: enhance B cell Activation
o CR2 expressed on B cells
o Binds cleavage products of C3b (C3d, C3dg and iC3b)
o This leads to the signaling cascades that enhance B cell response to Ag
o Functions as second signal, no T cell help

- Other CR out there
o Mainly all help Phagocytosis, inflammation and antimicrobial properties

Compliment Regulation
- Low level of compliment activation goes on spontaneously and can damage normal cells
- Degradation products need to be controlled

Host cells protected
- Activation is triggered by signals not normally found on host cells
- Thioester bonds on C3b and C4b are labile
- Multiple steps needed
- Host regulatory Proteins
o Fluid phase
o Membrane bound
- Try to inhibit before formation of C3 convertase to prevent amplification

C1 INH: serine protease inhibitor that mimics C1r and C1s
- Becomes cleaved and results in C1r/s dissociating from C1q

If compliment not Controlled
- Rapid inflammation (C3a and C5a)
- Increased breakdown of C4 and C2
- Must be treated with an inhibitor

Assembly of C3/C5 Convertase Inhibited By:
- C3b/C4b may be bound to several membrane bound proteins
o Both can bind to MCP, CR1, DAF and C3b to Factor H, C4b to C4BP
o Competitively inhibit the binding of other components for C3 Convertase
o Factor H only regulator for Alternative, not Classical

- Broken down C3 still works as opsonin

MAC is inhibited by CD59 Membrane Protein
- Inhibits addition of C9 molecules
- Linked to cell surface by GPI tail (anchor)
- If this gene is mutated, results in PNH: RBC lysis

FACTOR I
- Can cleave C3b and C4b opsonins when bound to a cofactor (compliment regulators)

Paroxysmal Nocturnal Hemoglobinuria (PNH)
- Regulators CD55 and CD59 are missing allowing MAC to form and lyse RBC’s

Hereditary Angioedema
- Deficiency in C1INH

Inflammation
- C3a and C5a are potent inflammatory inducers (C5a is most, C4a least)
- When produced in large amts, can induce shock-like syndrome: Generalized Circulatory Collapse

Local Compliment Activation and T Cell Activation
- Directly through stimulating Complement mediated signaling events
- Indirectly through modulating Ag presenting cell function (up regulate costimulatory act.)

Compliment Deficiencies
- More than 50% with deficient C1q, C2 and C4 develop Lupus
- Important role in cleaning up apoptotic bodies that contain DNA that may trigger autoantibody responses

Carboxypeptidase N
- Cleave free floating C3a and C5a to reduce inflammation