Immunology Midterm

Cytokine-Tumor necrosis factor-alpha

• TNF-alpha

• cytokine that plays major role in cancer and inflammatory disorders of the gut, joint, and skin

Psoriasis

immune-mediated chronic inflammatory and hyper proliferative skin disease

NSAID Immunosuppressants

Cyclosporine, tacrolimanalgesic, anti-inflammatory, antipyretic, antirheumatic

Immunization

Vaccines, toxoids

Monoclonal Antibody drug examples

• rituximab (Rituxan)

• Aducanumab (Aduhelm)

• Lecanemab (Lequembi)

Main function of immune system

• eliminate infectious agents, toxins, and malignancies

• protect against pathogenic invaders

• distinguish between self and non-self

Immunity

the ability to respond to foreign substances, including molecules and microbes

Immune System

the specific molecules, cells, tissues, and organs that function to provide protection from foreign substances

Immune Response

the body’s response to a foreign substance involving cells and molecules of the immune system reacting with the substance to render it harmless

2 components of immune system

1. Innate: 1st line of defense, present from birth, non-specific

2. Adaptive: specific, slower

Pluripotent Hematopeietic Stem Cells

• cells in adult bone marrow and fetal liver that all immune system cells derive from

Hematopoiesis

• generation of blood cells

• main sites: sternum, vertebrae, iliac bones, ribs

Leukocyte

• white blood cell

• involved in innate and/or adaptive immunity

Neutrophils

• lobed nucleus and granulocytes

• most abundant leukocyte

• first cells to arrive at inflammation

• phagocytes

• granules contain antimicrobial agents

Eosinophils

• polymorphonuclear granulocytes

• main defensive job: parasites, allergic reactions

• eosinophil increase in blood associated with allergy, parasitic infection

  • surface receptors for IgE antibodies responsible for immediate hypersensitivity (ex. hay fever)

• Extrude granules into external environment, including

  • major basic function (MBP)

  • eosinophilic cationic protein (ECP)

  • Eosinophil peroxidase (EPO)

  • Eosinophil-derived neurotoxin (EDN)

• extruded granules kill large extracellular pathogens that cannot be ingested by phagocytes

MBP

• major basic protein

• cytotoxic to helminth larvae

• causes release of histamine and other preformed mediators from basophils

ECP

• eosinophilic cationic protein

• cytotoxic by pore formation in cell walls

EPO

• eosinophil peroxidase

• neuronal and axonal damage in the cerebellum and spinal cord

Basophils

• polymorphonuclear granulocytes

• have surface receptors for IgE antibodies responsible for immediate hypersensitivity (allergies)

• found in BLOOD

Mast cells

• polymorphonuclear granulocytes

• have surface receptors for antibodies (IgE) responsible. for immediate hypersensitivity (allergies)

• found in soft tissue: distributed beneath epithelial linings of skin and respiratory, intestinal, and genitourinary tracts

Monocytes/Macrophages

• circulate in blood

• phagocytes

• mature into macrophages when they enter tissue

• arrive at inflammation after PMNs

• effectors in both innate and adaptive immunity

• recruit other cells to inflammation

• present antigens to T cells for cellular immune response

• have surface receptors for antibodies that are attached to pathogens enhancing phagocytosis

• tissue macrophages produce chemokines as one of first steps in inflammatory process

Histocytes

Macrophage in connective tissue

Osteoclast

Macrophage in bone

Kupffer cells

macrophage in liver

Microglial cells

macrophage in neural tissues/brain

Alveolar macrophages

macrophages in lung

Dendritic cells

• large macrophages found in most soft tissue

• especially found in lymph nodes, spleen

• important as antigen presenting cells to initiate T cell response

• bridge innate and adaptive immune systems by acting as messengers between two systems

Cells important in immediate hypersensitivity

• eosinophils, basophils, mast cells

Phagocytic cells

neutrophil, monocytes, macrophages, dendritic cells

Lymphocytes

• respond to antigens in adaptive immune response

• make up 20-45% of circulating white blood cells

• 3 different types: T cells, B cells, Natural Killer T cells

B lymphocytes

• develop in bone marrow independent of antigens and due to DNA rearrangement

• immature B cells in bone marrow undergo apoptosis upon recognition of self antigens

• B cell activation into antibody producing plasma cells is antigen dependent

• each B cell can have 1000s of surface receptors that are specific for the same antigen

• can interact with T cells to switch from IgM to other class

• can interact with T cells to develop into antibody secreting plasma cells or B memory cells

• responsible for humoral immune response

T lymphocytes

• immature T cells (thymocytes) develop antigen specific receptors in thymus independent of antigenic stimulation via DNA rearrangement

• selected for maturation only if T cell receptors do not react with self

• regulate the adaptive immune response (cell mediated immunity)

• play part in humoral immunity by helping b cells to produce antibodies and mature into plasma cells

• receive processed antigens from antigen-presenting cells

• stimulate other T cells and APCs

• assist in B cell activation, isotype switching, development into plasma cells, production of memory cells

Clusters of Differentiation (CDs)

• surface molecules on leukocytes with specific functions

• used as identifying markers for subpopulation of cells

• proteins with specific functions

• cells often referred by CD designation

• mature T cell will always be CD3 positive and either CD4 or CD8 positive in addition

T Helper Cells

• express CD4

• regulate antigen-directed effector functions in cell mediated immunity

• stimulate B cells to proliferate, undergo isotype switching, and differentiate into antibody producing cells

• recognize antigen complexed with MHC class II molecules on dendritic cells, macrophages, monocytes, and B cells

Cytotoxic T lymphocytes

• express CD8

• cytotoxic to host cells infected with intracellular pathogens and tumor cells

• cytotoxic in antigen specific manner dependent upon expression of MHC class I molecules expressed on all nucleated cells

Natural Killer Cells

• large granular lymphocytes that express markers found on both T. and B cells

• comprise about 10% of lymphocytes in blood

• secrete cytokines (ex. interferon-gamma)

• distinguishes stressed cells from healthy

• neither T or B cell

• may represent 3rd cellular linage that expresses CD16 and CD56

• play role in apoptosis of virus-infected cells and tumor cells (same killing system as cytotoxic T cell)

• Have FC receptors on surface

• kill by antibody dependent cell mediated cytotoxicity

• can be used as immunotherapy for cancer treatment

Lymphoid Organs

• where leukocytes originate, mature, differentiate, and proliferate

• divided into primary and secondary organs based on processes that occur within organs

Primary Lymphoid Organs

• where stem cells develop into leukocytes

• liver in fetus

• bone marrow in adults

• thymus reaches maturity prior to puberty and slowly loses function

  • site of T cell maturation

  • educate T cells to differentiate between self and non-self antigens

Secondary Lymphoid Organs

• where lymphocytes come in contact with antigens

• lymph nodes

• spleen

• MALTS (mucosa associated lymphoid tissues found lining digestive and respiratory tracts)

• bone marrow can also function as secondary lymphoid organ because of recirculation

Spleen

• left upper quadrant of abdomen

• filters blood

• major site of immune responses to antigens in blood

• contain areas for T and B cells

• APCs found in germinal centers and present antigens to lymphocytes in those areas

• acts like large lymph node in circulation

Lymph Nodes

• network of thin tubes that drain interstitial fluid from body and eventually return to circulatory system

  • fluids/cells enter through afferent lymphatic vessels, percolate through node, exit through efferent vessels

  • naive B/T cells enter node through artery and enter stroma of node through high endothelial venules inside node and exit through efferent lymph vessels

  • lymph enters back into blood through superior vena cava via thoracic duct (about 2 L a day returned)

• many lymph nodes in body where filtration of lymph takes place

• APCs reside in nodes, collect antigens brought to nodes from tissues, and present to T and B cells

• disruption of draining system leads to swelling

• lymph nodes provide enclosed system with all cells necessary to carry out adaptive immune response and return antibodies and lymphocytes to circulation

MALT

• aggregates of lymphoid tissue that reside along the respiratory and digestive tracts

• same function as lymph node

• active in delivering antigens in respiratory and digestive tracts to lymphocytes

Thymus

• bi-lobed organ in anterior mediastinum

• primary site of T cell development

• base of thymus on heart

• grows until puberty, undergoes progressive involution, and hardly functional by adulthood

• extensive network of epithelial cells and APCs that is involved in selection process leading to the development of an appropriate T cell receptor repertoire

Casirivimab plus imdevimab (REGEN-COV)

recombinant human monoclonal antibodies that bind to non-overlapping epitopes of the RBD spike protien of SARS-CoV-2

Sotrovimab

Monoclonal antibody that targets an epitope in the receptor binding domain of the spike protein conserved between SARS-CoV and SARS CoV-2; originally identified in 2003 from SARS-CoV survivor

Major structural proteins of SARS-CoV-2 genome

Spike, Envelope, Membrane, Nucleocapsid

Spike protein (divided into 2 subunits, S1 and S2) targeted by monoclonal antibodies

ACE2

• Angiotensin-Converting enzyme 2

• receptor through which SARS-CoV-2 enters host cells

IVIg or SCIg

• immunoglobulin replacement therapy

• treatment for patients with immunodeficiency diseases involving poor IgG levels/function

Humoral Immunity

• humoral = blood

• antibodies secreted by plasma cells float in blood

• B cells stimulated by specific antigens develop into plasma cells, which produce and secrete thousands of specific antibody molecules (immunoglobulins) that react with the antigen

Parts of blood serum

• found by electrophoresis (applying electricity)

• serum separated into 4 parts: albumin, alpha, beta, and gamma globulins

• antibodies are in gammaglobulin portion

Antibody structure and function

• proteins with biological activity at each end of molecule

• comprised of 2 identical polypeptide arms, each arm having one heavy and one light chain

• heavy chains are connected to each other by disulfide bonds and a hinge that allows flexibility of molecule

• antibody reacts with antigen between variable regions of heavy and light chains

  • F(ab)2 fragment antibody binding site

  • 2 antigen molecules can be captured by each Ab molecule

  • variable regions allow reaction with the thousands of antigens that may be encountered

  • amino terminal end

  • different B cells produce antibody molecules with different antigenic specificity via DNA rearrangement before antigen contact

• Constant region: Fc or fragment, crystalline region, interacts with host cell surface receptors

  • carboxy terminal end reacts with host cell surface receptors, gives important biological properties to molecule

  • must do biological work after antigen has been captured, so much remain constant

Antibody Isotypes

• IgM, IgG, IgD, IgA, IgE

• all have two heavy chains, two light chains, and hinge

• each isotype based on slightly different amino acid sequences in constant region of heavy chain

• IgG has 4 subclasses: IgG1, IgG2, IgG3, IgG4

• IgA has 2 subclasses: IgA1, IgA2

Light chain types

• kappa

• lambda

• differ in amino acids of constant region

• one antibody molecule has either kappa or lambda light chains (never one of each)

IgG

• most abundant isotype

• longest-lasting

• antibody-dependent cell cytotoxicity

• monomer

• 4 subclasses: IgG1, IgG2, IgG3, IgG4

  • IgG3 is strong binder of complement

• all 4 subclasses cross the placenta to provide antibodies to fetus from mother

• neonate begins to make IgG at birth to replace maternal IgG

IgE

• mediates hypersensitivity (allergy) and reacts with parasites

• monomer

IgM

• largest isotype molecule

• first antibody produced in response to antigen

• strongest activator of complement

• pentamer

• effective agglutinator of particulate antigens, bacterial opsonization

• only antibody that a fetus can make

IgA

• found as monomer or dimer

  • dimer has 2 molecules hooked together by J chain and secretory piece

  • dimer can combine with four epitopes

• 2 subclasses: IgA2, IgA2

• dimer found on surfaces of epithelial cells and in secretions of respiratory and digestive system

• main immunoglobulin in saliva, tears, milk

• secretory IgA (dimeric) protects molecules from proteolytic attack and facilitates its transfer across epithelial cells into secretions

Idiotype

• portion of antibody molecule between amino terminal heavy and light chains in variable region that confers specificity

• allows particular antibody molecule to react with particular epitope

Epitope

portion of antigen to which antibody binds

Antibody valency

number of binding sites on antibody molecule

IgG valency

2

IgM valency

10

Affinity

strength of a single binding site between antibody and antigen

Avidity

overall strength of interaction between antibody and antigen

depends on affinity of each binding site multiplied by valency

Immunogen

• any substance that can induce an immune response

• not all antigens are immunogens

• requirements: large size, complexity, non-self, degradability

Haptens

• molecules with low molecular weight that are not large or complex enough to evoke an immune response or are non-immunogenic

• must combine with larger carrier molecule (ex. protein) to act as epitope and induce immune response

• after specific antibody is made, the hapten can combine with the antibody even if no longer attached to carrier

• haptens are antigenic (combine with specific antibody) but not immunogenic (induce immune respone) because it can’t induce immune response by itself

Binding forces between antigens and antibodies

non-covalent bonds

Antigens

• any substance that can be recognized by the immune system

• proteins are best antigens because they are generally large, complex molecules

• immunoglobulins can be antigens (if rabbit antibodies injected into humans, humans will produce antibodies to it)

• all immunogens are antigens, but not all antigens are immunogens

Cross Reactivity

• specific antibody can combine with antigen epitopes that are very similar

• reaction to a cross-reacting antigen has less affinity

• in certain diseases, a reaction to a pathogen can produce antibody that reacts with self (ex. antibodies to certain streptococcal substances cross react with heart tissue = rheumatic fever)

V(D)F recombinase

• enzyme complex responsible for recombination of genes in both heavy and light chains

• mediates joining of variable (VH), diversity (DH), and joining (JH) heavy chain gene segments in variable region of germline DNA through mechanisms by which intervening segments are spliced out of the genome in that particular B cells

• same for light chain genes, except there are no D regions in light chains

Antibody Genetic Events

• many VH, DH, and JH genes present in germline DNA are eliminated

• recombination of remaining genes code for immunoglobulin

• over 6000 possible heavy chain variable regions

  • 50 V gene segments in heavy chain

• 200 different VJ combinations in kappa light chains; 160 in lambda light chains

  • 40 V gene segments in both

• independently recombined heavy chains combine with independently recombined light chain, resulting in many more possibilities for variable region of immunoglobulin

Junctional diversity

recombination between heavy chain VDJ segments is not precise, often resulting in slight location differences resulting in amino acid sequences differences

Insertional diversity

TdT (terminal deoxynucleotide transferase) adds nucleotides randonly at the VDJ junctions of the heavy chain genes

Terminal deoxynucleotide transferase

Tdt

enzyme that adds nucleotides randomly at the VDJ junctions of the heavy chain genes

Antibody diversity have the capability of recognizing how many different epitopes?

10¹⁴

Isotype Swtiching

• changing of surface B cell IgM antibodies to another isotype

• occurs when antigen stimulated B cells receive cytokine signals from helper T cells

• variable region remains constant and is recombined with a different constant region gene sequence

• specificity remains the same (will react with same epitope) , but isotype and bio-function of antibody will change

• switch is irreversible

• B cell will always express IgM in early immune response, than switch to IgG, IgA, or IgE

• IgD is found with IgM but is not involved with isotype switching

Affinity Maturation

• occurs after antigen stimulation of B cell

• selectively stimulates b cells with higher affinity for a specific epitope

• results from hypermutation of V regions of heavy and light chains that increase the affinity of antibody for antigen (somatic mutation) which result in a better fit for the antigen

• as more b cells of this specificity are produced, those with the higher affinity are selectively stimulated resulting in more cells with higher affinity as the immune response progresses.

Trastuzumab

• Brand name: Herceptin

• monoclonal antibody drugs used in treatment of breast and stomach cancer

Antibody Drug Conjugates

• Brentuximab vedotin (brand name: Adcetris), which treats Hodgkin’s lymphoma

• anti-CD30 antibody conjugated via protease-cleavable linker to anti-microtubule agent monomethyl auristatin E (MMAE)

• binds to CD30, brentuximab vedotin internalized and transported to lysosomes where MMAE is released and binds to tubulin, leading to cell cycle arrest and apoptosis

TCR complex

• T cell receptor

• most comprised of alpha and beta chain (5% are gamma+delta)

• surface molecules are necessary for TCR to perform

• CD3 complex helps with signal transduction (nucleus receives info that results in cell response when activated by joining of TCR with its antigen)

• members of immunoglobulin superfamily

• constant region accounts for biologic function, variable region responsible for reacting with antigenic molecule presented by APC

• 10¹⁸ possible antigen receptor for T cells

• no somatic mutation, no change in affinity for antigen, no isotype switching

Complementarity Determining region

• CDR

• region of the TCR that MHC II molecule presents peptide to

• surface molecules of both APC and T helper cells transmit signaling through protein kinases and protein tyrosine phosphatases forming a link between receptor activation and activation of internal pathways leading to cellular activation and proliferation

• stimulation of both APC and TH cells occurs, clones produced, and immune response attacks

CD4 molecule

• stabilizes interaction between CDR on TCR and MHC II on APC

• helper T cell

CD3

• responsible for signal transduction of messages into the T cell

• present on all T cells, used as identification marker for T cells

CAR-T cell

• chimeric antigen receptor T-cell immunotherapy

• Tisagenlecleucel (Kymriah; Novartis) and Axicabtagene cileucel (Yescarta; Kite Pharma)

• modify patient’s cells so they attack specific antigens on tumor cells and destroy them

Common Properties of TCR and Antibody

• integral membrane proteins

• thousands of identical copies at cell surface

• made before cell encounters antigen

• gene assembled by DNA recombination

• allelic exclusion for single antigenic specificity

• N region addition during gene rearrangement

• Antigen binding by noncovalent forces

• binding signals replication and activation

Properties Unique to TCR

• structure and encoding genes

• binding presented epitopes

• never secreted from T cell

• No somatic mutation

• No isotype switching

• High junctional diversity

Development of T Cells

• immature thymocytes rearrange genes (DNA rearrangment) to generate specific TCRs

• Undergo positive and negative selection

• 98% of developing T cells die in thymus via apoptosis

• Mature T cells exit thymus with CD3 and CD4 or CD8 marker and specific TCR and enter the circulation

• T cells can also become natural Tregs (nTregs) or NKT cells

Positive selection (T cell development)

• thymocytes that do not recognize self MHC molecules are programmed to die by apoptosis

• thymocytes with affinities for self-MHC molecules develop further in thymus (cortex)

Negative selection (T cell development)

• thymocytes that do not react with self-antigens are allowed to further develop (in medulla)

Cytokines

• low molecular weight, short-lived molecules secreted by cells in response to an external stimulus, often another cytokine

• principal mediators of communication between cells of the immune system

TH1 cells

• T helper cells that aid the cytotoxic T cells in cell-mediated immunity

• activate APCs to kill intracellular pathogens

• APCs present to TH1 via MHC II, stimulates to produce/secrete cytokines that stimulate APC to be more aggressive in destroying intracellular pathogens

TH2 cells

• T helper cells that aid B cells in humoral immunity

• APC (can be B cell itself) presents antigenic peptide via MHC II to TH2 cell, stimulating TH2 to produce cytokines that stimulate B cell to proliferate, undergo isotype switching, and develop into plasma cell that secretes antibodies

TH3 cells

T helper cell subset that little is known about

Effector Function of Cytotoxic T Cells

• virus infected cells presents antigenic molecule via MHC I to TCR of CTL, which produces cytotoxic effector molecules which initiate apoptosis in infected cell

• 2 methods:

  • CTL secretes performin molecules to generate transmembrane pores in infected cell; CTL enzymes enter infected cell and induce apoptosis

  • CTL utilizes Fas molecules of infected cell that react with corresponding ligand on CTL, result in apoptosis

• Remember: Antigen presented via MHC I!

MHC I

• major histocompatibility complex I

• 1000s expressed on each nucleated cell of body

• NOT on red blood cells (do not have nucleus)

• contains HLA-A, HLA-B, HLA-C

MHC II

• major histocompatibility complex II

• only on select antigen presenting cells

• Monocytes, Macrophages, Dendritic cells, B cells

• contains HLA-DP, HLA-DQ, HLA-DR

MHC Gene

• polymorphic locus

  • large number of allelic determinants (MHC molecules are different from one person to another)

• co-dominant expression

  • both parent-derived allelic forms expressed as cell surface proteins

MHC I Antigen Presentation

• present to CD8 cell

• always associated with invariant (always same) β2M molecule next to APC surface, member of immunoglobulin superfamily

  • not good antigen because same in everyone

• α3 molecule anchored in APC

• α1 and α2 hold processed peptide for presentation to CDR of CD8 cell

• present antigen fragments that are 8-10 amino acids long

MHC II antigen presentation

• present to CDR of TCR of CD4 cell

• 2 transmembrane alpha2 and beta2 polypeptide chains anchored in APC

• presents antigen fragments up to 13-25 amino acids long

Endogenous antigenic fragments

• fragments from virus-infected or tumor cells that have developed antigens not recognized as self (from within own cells)

• presented by MHC I molecules to cytotoxic T cells