Immunology: Antigen Presentation

Pharmaceutical Immunology: Basic Concepts

• Vaccination involves inoculating healthy individuals with inactivated or weakened pathogens/constituents.
• This process induces protective immunity, as demonstrated by Edward Jenner and the eradication of smallpox in 1979.
• The main function of the immune system is to protect against infection.
• Leukocytes such as T cells, B cells, NK cells, eosinophils, basophils, neutrophils, immature dendritic cells, and monocytes are key components.
• Primary lymphoid organs like the bone marrow and thymus generate immune cells.
• Secondary lymphoid organs such as Peyer's patches, spleen, tonsils, appendix, and lymph nodes induce adaptive immune responses.

Bone Marrow and Pathogen Categories

• Bone marrow contains pluripotent hematopoietic stem cells.
• These cells give rise to leukocytes, erythrocytes, and platelets through common lymphoid and myeloid progenitors.
• Pathogens can be categorized as viruses, intracellular bacteria, extracellular bacteria, archaea, protozoa, fungi, or parasites.
• Pathogens differ in sizes and reside in different places within the body.

Levels of Defense

• Anatomic barriers, such as skin, oral mucosa, respiratory epithelium, and the intestine, provide the first line of defense.
• Complement/antimicrobial proteins, like C3, defensins, and RegIIIγ, form part of the innate immune response.
• Innate immune cells, including macrophages, granulocytes, and natural killer cells, offer rapid, non-specific defense.
• Adaptive immunity, involving B cells, antibodies, and T cells, provides a targeted response.
• Innate immunity is fast-acting but less specific, whereas adaptive immunity takes longer to establish, but is highly specific.

Innate Immune System: Recognition and Response

• Microbes possess pathogen-associated molecular patterns (PAMPs).
• PAMPs are detected by specific receptors on sensor cells in tissues or blood, triggering the production of inflammatory mediators like cytokines and chemokines.
• This detection leads to amplification by inducing antimicrobial/antiviral factors, and recruitment/activation of other leukocytes.
• Pattern recognition receptors, such as Toll-like receptors (TLRs) on macrophages in the skin, enable initial discrimination between self and nonself.

Adaptive Immune System: Lymphocyte Activation and Function

• During infection, lymphocytes are activated in adaptive immunity.
• T cells recognize and destroy infected cells and activate other leukocytes.
• B cells, activated by pathogen-specific T cells, secrete antibodies.
• Antibodies bind specifically to foreign structures = antigens & make inactive.
• Dendritic cells (DCs) are major antigen-presenting cells in the body.
• DCs capture antigens through micropinocytosis, and present them to T cells, initiating a T cells antigen-specific response.
• The communication is Key link between innate & adaptive immune system.

Lymphocyte Recirculation and Clonal Selection

• Most T lymphocytes constantly recirculate between blood and lymph nodes via efferent lymphatic vessels and the thoracic duct.
• This increases the chance of encountering a cognate antigen.
• Upon recognizing a foreign antigen on an antigen-presenting DC, a naïve lymphocyte is activated and undergoes clonal selection.
• Clonal selection leads to proliferation and differentiation into an army of identical, antigen-specific T cells that acquire effector functions.

Antibody Structure and Function

• An antibody is Y-shaped, about 150 kD, and consists of 2 heavy and 2 light chains linked by disulfide bridges.
• Antibodies can also exist as transmembrane proteins on original B cells.
• The variable region is the site of antigen-binding and has a different amino acid sequence in different antibodies.
• The constant region is identical in antibodies of the same subtype.
• Fc part interacts with phagocytes and NK cells (Fc receptors).
• Antibodies in plasma and extracellular fluids mediate humoral immunity.

Epitopes and Antibody Function

• The epitope/antigenic determinant is a small portion of the antigen's molecular structure recognized by an antibody.
• Antigens may be proteins, glycoproteins, polysaccharides of pathogens, or self-antigens.
• Neutralization prevents viruses from binding to receptors.
• Antibodies neutralize bacterial toxins and bacteria in extracellular space or plasma by blocking their activity or access to cells.
• Opsonization enhances ingestion by macrophages.
• Complement activation leads to lysis and ingestion of pathogens by macrophages.

Antibody Titer and TCR Antigen Recognition

• Antibody titer is the concentration of antibody in the blood, and decreases over time after vaccination.
• Antibodies bind directly to native antigens, whereas T cell receptors (TCRs) only recognize peptide fragments of antigens presented on MHC molecules.
• For TCR recognition, antigens must first be broken down into peptide fragments and bound to MHC molecules.

TCR Structure and Antigen Recognition

• The T-cell receptor (TCR) consists of α and β chains linked by a disulfide bridge.
• The variable region of the TCR binds to peptide-MHC molecules, with different amino acid sequences in different T cell clones.
• The constant region of the TCR is identical in most T cells.

Major Histocompatibility Complex (MHC) Molecules - MHC Class I

• MHC class I molecules are present on all cells.
• MHC present fragments of proteins expressed by the cell itself.
• MHC class I molecules are recognized by cytotoxic CD8+ T cells, leading to cell death.

Major Histocompatibility Complex (MHC) Molecules - MHC Class II

• MHC class II molecules are expressed by antigen-presenting cells (APCs), B cells, macrophages and DCs
• MHC present fragments of proteins taken up into the APC from outside
• MHC class II molecules are recognized by CD4+ T cells

Lymph Node Structure

• Outermost cortex contains B cell follicles and T cell zones
• Inner medulla contains macrophages and antibody-secreting plasma cells (medullary cords)
• Afferent lymphatic vessels drain fluid from tissues & carry antigens & antigen-presenting DCs from infected tissues
• High endothelial venules (HEV) in paracortical area serve as entry portals for lymphocytes into lymph node

Lymph Node Dynamics

• DCs & T cells meet & interact in paracortical area
• DCs are short-lived & die there.
• Efferent lymphatics are exit routes for all lymphocytes from lymph node.
• Spleen's red pulp is for red blood cell destruction/disposal.
• White pulp is immune compartment, lymphocytes around arterioles.

Spleen's White Pulp Organization

• Periarteriolar lymphoid sheath is the T cell zone. This is the site of DC-T cell interactions.
• Follicles are B cell zones, germinal center surrounded by B cell corona & marginal zone.
• Peyer's patch includes subepithelial dome with DCs, T cells & B cells
• It lacks afferent lymphatics
• Antigen enters directly from gut across specialized epithelium made up of microfold (M) cells,
• Lymphocytes enter across walls of HEVs & leave via efferent lymphatic

Macrophages

• Long-lived, present in all tissues
• Perform phagocytosis & destroy bacteria or dead cells
• In spleen, macrophages of red pulp help degrade old red blood cells or immune complexes

Granulocytes

• Neutrophils contain characteristic intracellular granules, are short-lived
• Produced in bone marrow
• Rapidly recruited to sites of infection/inflammation, take up & kill pathogens
• Eosinophils & basophils are less abundant
• Granules contain many enzyme & toxic proteins
• Released when cells activated, defense against parasites & allergic response

Mast cells

• Begin development in bone marrow
• Migrate as immature precursors that mature in peripheral tissues (skin, intestines, airway mucosa)
• Granules contain inflammatory mediators (histamine, proteases protect internal surfaces from pathogens like parasitic worms)
• Dendritic cells are phagocytic when immature acttivate T lymphocytes after maturation
• NK cells Of innate immune system lack antigen-specific receptors

NK Cells

• Express Fc receptors = if Ab binds to virally infected cell/tumor cell, NK destroys it
• Have similarities with lymphoid lineages of adaptive immune system

Chapter 2: First Lines of Defense

• Extracellular bacteria is mostly cleared by phagocytes.
• Extracellular viruses are prevented to adhere or enter host cells by antibodies.
• Intracellular viruses are infected cells attacked by NK cells/cytotoxic T cells.
• Intracellular bacteria/protozoa cleared by macrophages with help from T cells.

Mechanisms of Tissue Damage

• Direct damage from the passage of pathogens.
• Damage caused by the host defense.
• Endotoxins are intrinsic components of microbes that trigger pathogen recognition receptors (PRRs).
• The most famous is Lipopolysaccharide (LPS) of the outer cell membrane of Gram-negative bacteria: Fever, rashes, pain, septic shock.
• Exotoxins are secreted toxins released by microorganisms & act on host cell surfaces.

Epithelial surfaces

• First barrier against infection.
• Epidermis has multiple layers of keratinocytes in different stages of differentiation from basal layer of stem cells.
• Differentiated in stratum spinosum: ẞ-defensins and cathelicidins in secretory organelles (lamellar bodies) into intercellular space to form waterproof lipid layer (stratum corneum) with antimicrobial activity

Lung

• Ciliated epithelium beats & moves mucus secreted by goblet cells outward. Traps & ejects potential pathogens.
• Type II pneumocytes in lung alveoli produce & secrete antimicrobial defensins.
• Mucins do not have antimicrobial activity; only produce mucus.

Intestine

• Goblet cells produce thick layer of mucus.
• Paneth cells in epithelial crypts produce antimicrobial proteins (a-defensins (cryptdins), antimicrobial lectin RegIIla).
• Morbus Crohn is a common inflammatory disease of intestines.

Lysozyme

• Saliva, tears, also produced by phagocytes.
• Digests cell walls of Gram-positive/negative bacteria (cleaves B-(1,4) linkage between N-acetylglucosamine and N-acetylmuramic acid).
• More effective in Gram-positive due to lack of LPS outer layer Lysozyme is short cationic
• Defensins peptides that are amphipathic. Disrupts cell membrane of microbes, produced in inactive form, activated by proteolytic cleavage
• RegIIla: family of bactericidal proteins produced by Paneth cells in intestine, C-type lectins, kills bacteria directly by forming hexameric pore in bacterial membrane, preferentially Gram-positive

Complement system

• System of soluble pattern recognition receptors & effector molecules detecting & destroying microorganisms.
• The similarity to blood coagulation system (enzymatic cascade of protein activation, rapid amplification).
• Activators & inhibitors of complement activation, several proteases (synthesized as zymogens = inactive pro-enzymes).
• Products of cleavage reaction designated by adding lowercase letter as suffix (a=smaller, b=bigger).

Activation Pathways

• Three activation pathways: lectin, classical & alternative
• Lectin: recognition of carbohydrate motifs
• Classical: recognition of Abs bound to pathogen
• Alternative: spontaneous activation

Outcomes of complement activation

• Production of C3a (& C5a = anaphylatoxins) inflammation & leukocytes recruitment
• Phagocytosis of C3b-tagged microorganisms by complemetn receptor expressing phagocyte
• Lysis of microbes/cells on which complement activation (C5b deposition) took place

Lectin Pathway

• Terminal mannose only in bacteria/yeast recognised by lectins.
• Triggered by binding of mannose-binding lectin (MBL, synthesised in liver, binds to mannose or fructose)/ficolins (synthesised in liver, lung & red blood cells, binds to oligosaccharide containing acetylated sugars) to microbial surfaces. ### Classical Pathway
• Lectin production is homologous
• Initiated by binding of C1 complex (C1q = pathogen/Ab sensor, 6 identical subunits with globular head & long collagen-like tails; 2 C1r & 2 C1s = serine proteases.
• Cinding to C1q cleavage of C2 & C4 C4b2a = C3 convertase) to surface-bound Abs

Alternative Pathway

• Happens all the time at spontaneously activated.
• Deposition of C3b on cell/pathogen surface.
• Amplification loop for C3b formation: alternative C3 convertase = C3bBb complex, with help of factor D.

Complement System

• The spontaneous hydrolysis of thioester bond in C3 in blood C3(H2O) short-lived fluid-phase C3 convertase by Factor B & D further increase in chances of C3b deposition on cell surfaces & generation of alternative convertases by Factor B & D.
• C3b on surface + C3 convertase C5 convertase: cleaves C5 into C5a & C5b C5b initiates assembly of terminal complement components membrane-attack complex (MAC) generates pore in membranes.

Cell-Surface Complement Receptors

• CR1 phagocytosis.
• C5a receptor G-protein-coupled receptors and enhance phagocytosis.
• Opsonisation is the decoration of pathogen surface with Abs/complement protein.

Regulation of Complement

• Anaphylatoxins (C5a & C3a) cause local inflammatory responses by acting on blood vessels: vascular permeability, upregulation of adhesion molecules; also activate mast cells release inflammatory mediators
• Regulatory proteins help protect host cells from unwanted complement activation in plasma & host-cell membrane, typically inhibit either activation C1q, C3 or C5 convertase activity or formation of MAC

Decay-Accelerating

• Factors present on host cells & displaces C2a from C4b2a to block convertase activity; inhibition of activation of C1 complex; inhibition of C5 convertase activity (CR1 &H displace C3b, cofactors for cleavage of C3b by I); inhibiting assembly of MAC.Pathogens produce inhibitors of complement activation:

Staphylococcus Aureus

• Protein A binds to Fc portion of Abs & inhibits complement activation & opsonisation; immune evasion & industrially exploited for affinity chromatography of therapeutic Abs.PRRs (pattern recognition receptors):

Receptors:

• Free receptors in serum (MBL or ficolin) or membrane.
• Bound phagocytic receptors that trigger immediate responses & recognize broad classes of pathogens

PRRs (Pattern Recognition Receptors)

• Interates with a range of molecular structures of a given type, and can be cytoplasmic with signaling receptors like NLRs

Chapter 3: The Induced Responses of Innate Immunity

• Microbes recognised, ingested & killed by resident phagocytic cells (macrophages, dendritic cells), recruited phagocytes (granulocytes, inflammatory monocytes);phagocytes & monocytes express high levels of PRRs.
• Receptor interacts with microbial surface being internalised in phagosomes being fused with lysosomes to form phagolysosome.
• The pathogen destroyed by this in turn causes bactericidal agents upon uptake of microorganisms: acidification, toxic oxygen-derived products (ROS), toxic nitrogen oxides (NO), antimicrobial peptides, enzymes (lysozyme), competitors microbicidal respiratory burst.
• Initiated by activation-induced assembly of phagocyte NADPH oxidase, bacterial fMLF & C5a receptors involved in generating reactive oxygen species (ROS).

Neutrophils

• non-tissue resident cells = first recruited to site of inflammation from bloodstream, short-lived, high phagocytic capacity.
• Dead & dying neutrophils = pus (Eiter), generate neutrophil extracellular traps (NETs, expelled chromatin) capturing microorganisms more efficient phagocytosis.
• Tolllike receptors:

Human TLR Genes

• 10 expressed recognizes distinct PAMPs
• Sensors for microbes in extracellular spaces (cell surface receptors or intracellular in membrane of endosomes phagocytosis)
• TLR Structure: single-pass transmembrane proteins, extracellular region leucine-rich repeats (LRR), multiple LRR ligand binding & Formation of dimer or conformational changes in preformed TLR dimer

TLR Activation

• TLR-3/7/8/9 viral RNA or bacterial DNA with unmethylated CpG motifs (9) only released when pathogen taken up by cell & broken down->TLRs in intracellular compartments of phagocytes or B cells or TLR-4: On cell surface & ligand

Gram -Negative Bacteria

• Already low levels in humans lead to septic shock due to overwhelming secretion of cytokines TLR signalling: =Dimerisation of 2 TLR ectodomains brings cytoplasmic TIR domains together
• Cytoplasmic adaptor molecules interact with it & start signalling cascade: MyD88 (most important activates links to NF*B which can then occur
• Results: in production of inflammatory cytokines, chemokines/chemokine receptors, antimicrobial peptides, Type I interferons NOD-like receptors (NLRs): in cytosol, contain nucleotide-binding oligomerisation domain (NOD), detect microbial products or cellular damage.

The Cytokine Production

• Cytokine production is via NF*B activation, mainly expressed in epithelial cells (barriers), macrophages, dendritic cells.

Cytokine Activation

• NLRP3 activated by: ROS, reduced intracellular K+, high ATP, disruption of lysosomes, uric acid.
• Crystals are released- associated molecular patterns -Activation-> formation of inflammasome, production of pro-inflammatory cytokines IL-1b & IL-18 & cell death through pyroptosis Sensors of intracellular infection & of cellular damage.RIG-I-like receptors (RLRs):
• These detect viral RNA produced - within infected cell, in many tissues, induce production of type I interferon inflammatory cytokines (via NF*B & IRF3), discrimination for example by capping .
• Viral DNA in cytoplasma

Co-Stimulatory Molecules

• Activation of innate sensors in macrophages & DCs trigger expression, C08 cells & C086 are most important & induced

Endothelial Activation

• Activation needs high levels for proper T cell activation & induction of adaptive immunity Adjuvants in vaccines as PAMPS & DAMPs to induce MHC & costimulatory molecule expression in antigen-presenting DCs Tissue inflammation.

Endothelial Activation & Leukocytes

• Cytokines stimulate blood vessels causing leukocytes increase the quantity of expression of these adhesion molecules - Leukocytes to extravasate at the site of infection -Blood clotting occurs in the microvessels- Inflammation to deliver - effector molecules & recruitment of leukocytes to sites of infection, induce - local blood clotting physical barrier, promote repair of injured tissue -Endothelial activation: increase is diameter, expression adhesion molecules, increase increase vascular permeability, clotting microvessels; due to pathogen recognition by macrophages -endotoxins, released due to damaged cells-lipid -Leukocytes: FlowingRolling-Integrin activation-Tight binding-Transmigration

Adhesion Molecules

• The tight-binding enables selectins by inducing activated endothelium that initiates rolling Cell adhesion molecules are small proteins, such as cytokines that directs the trafficking.
• The chemokine function involves: extravasation, chemotaxis attraction of leukocytes, in migration . It can be small proteins involved(immune cell communication

Tissue Inflammation (Cytokines)

• Activation of stimulus, autocrine/paracrine/endocrine Families: interleukins(T cell activation)
• Tumor necrosis tumor/chemokines
• Common gamma: for receptors & ligands: homotrimers signaling that activates TNF,some activate STAT pathway that activates/inhibits the arthitus,

Macrophages and Cytokines

• Activated macrophages produce inflammatory cytokines to recruit more cells. This leads to adaptive immunity. Produced by activated macrophages, local inflammation-> leukocyte recruitment & containment of infection. Tumor Necrosis factors. Activation happens due to activated macrophages as TN Fa in the tissue which leads to local & systemic infection of gram - negative bacteria.

Inactivated BActeria & Cytokines

_B cells stimulate the liver through C- reactive protein which binds to bacteria and aids in binding with opsonization with MBL as well through acute phase proteins C- reactive proteins .This results in type 1 interferons ==END.==

Explore antigen presentation: immunoproteasome's role, TAP1/2 mutations, invariant chain (CD74) significance, cross-presentation, MHC gene characteristics, TCR interaction region, unconventional T cell antigen recognition, HLA-DM function, cytosolic antigen delivery for MHC class II, MHC restriction, and TH1 immunity mechanisms.