MICB 212 Immunology Notes

Levels of Defense Against Infection

1. Anatomical and Physical Barriers (External Defense)

-intact skin provides a physical barrier

-mucosal surfaces of gut, respiratory urogenital and conjunctivae surfaces

-barrier pathogens encounter

• intact skin

• lysozyme in tears/saliva

• stomach acid

• mucus

-functions to keep pathogens from entering the body

-pathogens enter body via

• wound/bites

• inhalation/air breathed in

• ingesting infected food

  • meat, dairy, etc

2. Innate Immune Response (Initial Internal Defense)

-quick and non-specific

-phagocytosis and activation of complement proteins

-limits spread of infection

-often resoles infection

-starts in minutes post-infection

• peaks in 2-3 days

3. Adaptive Immune Response (Last Line of Defense)

-slow to respond but specific to particular pathogen

-involves antibody-mediated responses and cell-mediated responses

-reaches full activity in 7-10 days

Active Immunity

-immune system makes antibodies

-B cells, T cells activating, proliferating, differentiating

-develops a memory response

• excepts T-independent B cell activation

-natural — no intervention by medical personnel (infection)

-artificial — intervention by medical personnel (vaccine)

Passive Immunity

-getting antibodies made by someone else transferred from one individual to another

-immune system doesn’t make antibodies

-no memory responses developed

-temporary — introduced antibodies degrade over time

-natural — antibodies crossing pregnant women’s placenta into baby — no intervention by medical personnel

• breastfed antibodies

-artificial — convalescent plasma to SARS-Cov-2 antibodies after a snake bite to neutralize venom — intervention by medical personnel

• used in life-threatening emergencies

Pathogen

-a microorganism that causes disease

Inflammation

-caused by physical or chemical insults or invasion by microorganisms

Infectious Inflammation

Acute Inflammation

• short duration

• initial response to infectious agent

• causes very little tissue damage to host

Chronic Inflammation

• occurs for months to years

• persistence of infectious agent

• due to microorganisms involving special pathogens that have extraordinary abilities to evade host’s immune response

• causes tissue destruction

  • due to release of oxygen metabolites, nitric oxide (NO), proteases by inflammatory cells

Acute Inflammatory Response to Infection

-begins after damage to tissue

1. pathogen recognition by tissue-resident macrophages

   • bacteria enters wound and detected by tissue-resident macrophages

   • PRRs on macrophages bind to specific structures on bacteria → phagocytosis

   • TLRs recognize PAMPs → signaling cascade initiated

2. cytokine release and inflammatory response

   • signaling cascade activates transcription factors → production of pro-inflammatory (alarm) cytokines

     • TNF-α

     • IL-6

       • produces fever

     • IL-1

       • increases production of neutrophils in bone marrow

   • promotes inflammation → recruit immune cells to infection site

3. vascular changes induced by mast cells & macrophages

   • tissue-resident mast cells release histamine

     • vasodilation → increased blood flow → redness & heat

     • increased vascular permeability (cells, proteins, fluid leak into tissues) → swelling & pain

       • immune cells & proteins enter tissues

4. leukocyte recruitment & migration to infection site

   • IL-8 creates gradient that guides neutrophils, monocytes, dendritic cells to bacteria

   • adhesion molecules on blood vessel walls allow leukocytes to stick and squeeze through into infected tissue

   • neutrophils and monocytes arrive → mature to macrophages → phagocytic

5. phagocytosis of bacteria

   • neutrophils and macrophages engulf/phagocytose bacteria → phagosome forms

   • bacteria broken down and destroyed

6. clotting & tissue repair

   • clotting mechanisms activate wall off the site of infection and immobilize bacteria

   • tissue repair mechanisms begin to heal wound

7. pus formation

   • pus accumulates; consists of

     • dead and dying neutrophils and macrophages

     • live and dead skin cells

     • dead bacteria

     • plasma (fluid from blood)

Extracellular Bacterial Infections

-live outside of host cells

• blood, tissue, mucosal surfaces

-releases toxins

-bacteria doesn’t invade cells of host

Appropriate Response

-antibody production most appropriate response

-antibodies would neutralize or opsonize pathogen to make phagocytosis more efficient

• or activate complement to kill pathogen

Innate Immune Response

• neutrophils & macrophages recognize and phagocytose bacteria

• complement system (MAC) lyses bacterial membranes

• inflammatory cytokines (IL-1, IL-7, TNF-α) recruit immune cells

Adaptive Immune Response

• B cells produce antibodies that neutralize bacteria

• help phagocytes clear infection

• CD4 T helper cells bost neutrophil activity & enhance phagocytosis

Intracellular Bacterial Infections

-live inside of host cells

• macrophages, epithelial cells

-evades immune detection by hiding in cells

-able to reproduce inside macrophages

-macrophage eventually dies and bacteria released from dead macrophage an invade other macrophages and further spread infection

-resists phagocytosis and survives inside macrophages

-antibodies not successful in eliminating infection

• infection is inside cell and antibodies can’t enter

-evades antibiotics if host is taking them

• antibiotic has to enter host cell and get to critical concentration

Appropriate Response

-cell-mediated immune response needed to combat infection

• T cells and macrophages to kill pathogen compared to soluble proteins in blood

Super-Activated Macrophages

• strong macrophages that can phagocytose and kill bacteria and viruses

• super-activated by 2 signals

  1. regular activation

     • macrophages detect bacteria or viruses → activate and try to destroy pathogens

     • phagocytose pathogens and release some toxic molecules but not at full power

     • binding of CD40L on T helper cell to CD40 on macrophage

  2. super-activation by T cells

     • CD4 T helper cell detects macrophage needs more power

       • releases IFN-γ cytokine that super activates macrophages

       • IFN-γ + TNF-α = super-activate macrophage

• more effective at fusing lysosomes with phagosomes

• increase production of antimicrobial products potent enough to kill intracellular pathogens

  • reactive nitrogen oxide (NO)

  • oxygen radicals

  • proteases

  → reactive compounds may leak outside macrophage and damage healthy cells and tissues of host → inflammation

• attracts neutrophils and macrophages that release bactericidal substances and phagocytose bacteria that have escaped from lysed cell

• activates transcription of different genes

Tuberculosis (TB)

-infectious disease caused by Mycobacterium tuberculosis (MTB) bacteria

-MTB spread from one person to another via tiny droplets released into air

• coughing & sneezing

-generally affects lungs

• can affect other parts of body (kidney, spine, brain)

Latent Tuberculosis Infection

• bacteria present in body

• inactive site

• doesn’t cause symptoms

Tuberculosis Infection

• patient ill with symptoms

• can spread bacteria to others

• may occur in first few weeks after initial infection with TB bacteria or occur years later

• if not treated properly → can be fatal

  • antibiotic therapy for ~6-24 months

Symptoms

• coughing that lasts 3+ weeks

• coughing up blood

• chest pain, pain with breathing or coughing

• unintentional weight loss

• fatigue

• fever

• night sweats

• chills

• loss of appetite

• tuberculosis of spine — back pain

• tuberculosis in kidney — bloody urine

Progression of Infection

1. inhalation of bacteria

   • bacteria enters lungs

2. bacteria engulfed by macrophages in alveoli

   • some killed by macrophages

   • some survive & proliferate

   • macrophage may burst and bacteria spreads to infect more macrophages

3. dendritic cells in area engulfs bacteria and moves to lymph nodes to activate CD4 T cells

   • tells T cells to move to tissues to aid macrophages

4. CD4 T cells activated differentiate to effector T helper cells

5. effector T helper cells arrive at infection site

   • try to super-activate macrophages

   • formation of tubercule or granuloma

6. bacteria inside granuloma → inactive → latent TB

   • if immune system weakened → outer layer of granuloma may break → bacteria released & TB reactivated

Vaccination

• BCG vaccines for TB disease

• most asians have administered

• scar develops

• doesn’t protect children from pulmonary disease caused by TB bacteria

• doesn’t prevent spread of disease or latent TB infection from progressing to active disease

• prevents serious TB complications in children (TB meningitis)

• ~7-10 days to fully develop

• generates memory T helper cells that activate macrophages and not an antibody or CTL response

• secondary response to vaccine ~2 days to develop

Tuberculin Skin Test

• test to identify if a person might be infected with MTB

• small amount of tuberculin protein injected

• positive tuberculin test

  • secondary reaction to a protein made by bacilli

  • indicates presence of effector & memory T helper cells

  • X-ray to confirm

  • person vaccinated with BCG may also test positive

    • reactivation of memory T helper cells — doesn’t indicate disease/infection

Location of Immune System

-distributed throughout the body

-cells of immune system found in

1. blood circulatory system

   • moves blood throughout body (including spleen)

2. lymphatic circulatory system

   • moves lymph fluid throughout lymph nodes

   • enables lymphocytes and proteins to move around body

   • lymph fluid similar to blood but lacks RBCs and platelets

   • connects lymph nodes together

3. lymphoid organs

   • primary lymphoid organs

   • secondary lymphoid organs

-lymphatic circulatory system and blood circulatory system are connected

• fluids in tissues drain into lymphatic capillaries then into lymph nodes

• lymphatic fluid returns to circulatory system via thoracic duct near heart

-spleen and lymph nodes act as filters

• traps pathogens so immune system responses can develop

Lymph Fluid

• contains WBCs and plasma

• returned to blood circulatory system at thoracic duct

Primary Lymphoid Organs

-sites where lymphocytes develop and mature

• bone marrow

• thymus

-all HSC (blood & immune cells) complete developmentation & maturation process in bone marrow

• except T cells → begins in bone marrow → completes in thymus

Secondary Lymphoid Organs

-sites where mature lymphocytes encounters pathogens/foreign molecules and begins adaptive immune system

• spleen

• lymph nodes

-specialized structures that allow lymphocytes to scan for antigens

• spleen and lymph nodes

• gut-associated lymphoid tissues (tonsils, adenoids, appendix, Peyer’s patches)

• bronchial-associated lymphoid tissues (BALT)

• other mucosal surfaces

Cells of Immune System

Hematopoietic Stem Cells

-all blood cells from hematopoietic stem cells (HSC)

• common lymphoid progenitor cells (CLP)

  • lymphoid cell types

• common myeloid progenitor cells (CMP)

  • myeloid cell types

-HSCs divide to replenish HSC pool and to provide progenitor cell types

-HSC and progenitor cells know what type of cells to develop into various chemical and environmental signals

• soluble chemicals, receptor/ligand binding interactions

-HSCs are self-renewing

• found in bone marrow, umbilical cords, in blood

-HSCs, CMPs, CLPs are sensitive to radiation

• divide frequently

• radiation damages the DNA

  • cells die when they divide

-bone marrow cells contains a mixture of cells — some HSCs but some cells that are in the process of maturing into lymphocytes, neutrophils

• HSCs account for less than 1% of total bone marrow population

Blood

-3 major groups of cells in blood

1. erythrocytes (RBC)

2. platelets

3. leukocytes (WBC)

Erythrocytes (RBCs)

-carries oxygen to tissues

Leukocytes (WBCs)

-cells of immune system

-separated into

• myeloid cells

  • granulocytes

    • neutrophils

    • basophils

    • eosinophils

  • monocytes

    • macrophages

• lymphoid cells/lymphocytes

• dendritic cells

Myeloid Cells

-participate in adaptive immune response

-includes

• monocytes (mature into macrophages)

• mast cells

• granulocytes (neutrophils, basophils, eosinophils)

Mast Cells

• live in tissues

• found in skin, lungs, gut, around blood vessels

• associated with allergic reactions/anaphylaxis

• detects invaders → release signals by exploding and releasing tiny granules filled with histamine that triggers immune response/causes inflammation → blood vessels dilate and leak fluid to allow immune cells to rush to site of infection → release cytokines to attract macrophages, neutrophils, T cells to fight infection

• fast immune response (first line of defense)

Neutrophils

• phagocytose & kills bacteria

Macrophages

• phagocytose and kill bacteria

• alert immune system when presence of infection

• tissue repairing

• wound healing

• not motile

• serious pathogens can prevent macrophages from killing them after phagocytosis

  • macrophages recruit T helper cells to overcome time

• presents antigen to T cells that had been activated before

Resident Macrophages

• resides in tissues throughout the body

• involved in early detection against invading pathogens

Lymphoid Cells (Lymphocytes)

-participate in adaptive immune response

-most are present in specialized lymphoid tissues

-develop and mature into primary lymphoid organs

-3 groups of lymphocytes

1. CTL (cytotoxic/killer T cells)

   • kill viral infected cells

   • prevents spread of viral infection

   • Th cells (T helper cells)

     • provide cytokines and other stimulatory signals to B cells, CTLs and macrophages

   • Treg

   • development occurs begins in bone marrow → completes in thymus

2. B Cells

   • secrete antibodies into body fluids

   • entire development occurs in bone marrow

   • presents antigen to T helper cells to receive T cell help

     • T cell help needed to get B cells to make high affinity, class-switched antibodies and develop memory B cells

3. NK (natural killer) Cells

Dendritic Cells

-phagocytose and kill bacteria/viruses

-migrates from site of infection to lymph node to activate adaptive immune response

-bridges innate and adaptive responses

-important cells for

• activating T cells

• initiating adaptive immune response

-derived from lymphoid cells or myeloid cells

Innate Immune Response

-first line of defense internally

-acts immediately to remove pathogen without development of disease in host

• fast, non-specific, triggered by components of pathogens

-includes both cellular component and protein component

-if innate immune response is unsuccessful or gets bypassed by pathogen → adaptive immune response is required

-innate immune response is able to distinguish dangerous things from non dangerous things

• pathogens are recognized as foreign by PAMPs

• recognized via pattern recognition receptors (PRRs)

-occurs in 2 phases

1. Immediate/Early Innate Response

• preformed proteins already in blood/tissues and phagocytic cells already in tissues

  ex: resident macrophages, complement in blood — when C3 breaks down to C3b → alternate pathway starts

• starts within a few minutes after infection

  1. activation of complement via alternative pathway

  • C3b — opsonin

  • C3a, C5a — enhances developing inflammatory response

  • membrane attack complex (MAC)

  2. mast cells release histamine, dilation of blood vessels

  • brings more blood to injury site

  • more complement and cells, antibodies if preset

  3. phagocytosis of bacteria by resident macrophages

  • C3b enhances phagocytosis

  • macrophage recognizes danger

  4. production of alarm cytokines by resident macrophages → induced innate response

2. Induced Innate Response

• recruitment of phagocytic cells from blood stream and into tissue (site of infection)

• resident macrophages responsible for setting induced phase in motion (sending out alarm when pathogens are detected)

• starts within a few hours after infection

1. resident macrophages recognizes bacteria and send out alarm to recruit phagocytic cells (neutrophils, monocytes, dendritic cells) to site of infection

2. change in blood vessel wall of vein so that neutrophils & monocytes can get to site of infection

3. phagocytic cells migrate towards bacteria in tissue → engulf & kill bacteria

4. leads to inflammatory response (complement activation)

Pathogen-Associated Molecular Patterns (PAMPs)

-pathogens (virus & bacteria) have PAMPs

-molecular structures that are

• not found in multicellular hosts

• present on numerous groups of pathogens

• essential for pathogen’s survival, don’t mutate frequently

ex:

• LPS; gram (-) bacteria

• peptidoglycan; gram (-) & gram (+) bacteria

• LTA; gram (+) bacteria

• dsRNA; virus

• single stranded DNA; virus

-recognized by PRRs in innate response and binds to phagocytic PRRs

• results in

  • phagocytosis of pathogen

  • cytokine production by immune cell

Pattern Recognition Receptors (PRRs)

-receptors on neutrophil, macrophages, dendritic cells that bind PAMPs

-different PRRs have different functions

-some pathogens recognized outside of host cell via cell surface PRRs

-other pathogens recognized once inside host cell

• recognized by PRRs in membranes of intracellular vesicles

Phagocytosis (Endocytotic) Receptors

-type of PRR

-on innate immune cells

-engulfed by cell when pathogen binds to this receptor

-used to bring particle inside phagocyte

Toll-Like Receptors (TLRs)

-type of PRR

-allow phagocyte to determine if particle is dangerous

-binding of PAMPs initiates intracellular signal

-upon binding of an infectious organism to toll

• antifungal or antimicrobial peptides are released

-signal results in

• release of cytokine

• up-regulation of MHC class II proteins

• up-regulation of B7 co-stimulatory molecule

-13 different TLRs in mammals

• 11 functional in humans

-every TLR recognizes a set of molecular patterns not found in host

-functions as homodimers or heterodimers

Complement

- > 20 plasma proteins in blood that work in a cascade process

-able to work alone or with other proteins (antibodies or soluble PRRs)

Function

-to attack extracellular pathogens

-form membrane attack complex on pathogen → causes death of pathogen

-some can work as opsonins to enhance phagocytosis of pathogen by phagocyte

-some can work to promote development of cellular inflammatory response

Complement Activation

-complement proteins are activated when antibodies made during a previous adaptive immune response binds to pathogen surface

-involves cleavage of protein to make 2 smaller proteins

-works as a cascade of reactions → one reaction is followed by another

• product of one reaction catalyzes next reaction which catalyzes next reaction and so on

1. component A is cleaved

2. fragment of A acts by cleaving component B

3. fragment of B acts by cleaving component C

4. fragment of C acts by cleaving component D etc

-occurs almost immediately after infection

Alternative C Pathway

-spontaneous breakdown of component C3 into

• C3a (attracts & activates)

• C3b (binding)

-C3b is deposited on membrane of pathogen and recruits rest of components leading to formation of pore in membrane of pathogen

-in absence of infection → C3b broken down further → no harm to host

-in presence of infection → C3b binds to bacteria

-complement protein C3 (blood) → cleaves → C3a & C3b form → C3b binds to pathogen surface & recruits other complement proteins to form C3 convertase → additional C3b protein & C3 convertase forms → some C3b used as opsonin, some C3b used to form C5 convertase → C5 convertase cleaves component protein C5 → C5a & C5b forms → C5b used to form membrane attack complex

Activation of Inflammatory Response

-complement activation contributes to inflammatory response activation

-C3 convertase cleaves additional C3 → C3a & C3b → some C3b forms C5 convertase → C5 convertase cleaves C5 → C5a & C5b forms

C3a & C5a

• affect blood vessel permeability when produced in large amounts

  • induces a generalized circulatory collapse → anaphylactic shock

• small fragments that bind to

  • specific receptors on blood vessels

    • results in increased permeability of blood vessels

  • receptors on resident mast cells resident macrophages

    • release additional histamine & TNF-α

• induces expression of adhesion molecules

  • allow leukocytes (neutrophils & monocytes) to attach to blood vessels

• bind to recep

• C5a is a power chemoattractant for neutrophils & monocytes

Opsonization & Enhancement of Phagocytosis

-opsonization is alteration of pathogen surface/particle surface so phagocytic cells can engulf efficiently

-C3b binds to pathogen surface can function as an opsonin

• binds to receptor on surface of phagocytic cell

• results in increase number of contact points between pathogen & phagocytic cell

-phagocytic cells have receptors that bind to C3b to pathogen surface

-coating pathogen with C3b enhances phagocytosis by neutrophils & macrophages

Formation of Membrane Attack Complex & Bacterial Cell Lysis

-when C5b is deposited on pathogen surface

• assembly of late/terminal complement components into membrane attack complex (MAC) is initiated

  • punches holes in pathogen surface → pathogen cell lysis → death

Adaptive Immune Response

-2 main weapons

• antibodies

  • produced by B cells

  • main defense against extracellular bacteria

  • protects against infection

• T cells

-activated when innate immune response can’t eliminate infection

• last line of defence

Primary Immune Response

-first exposure body has to pathogen

-takes longer to develop a defense

-5-7 days to develop

• time lag due to differentiation and proliferation of naive T helper cells & B cells

1. recognition & activation

   • APCs detect pathogen, processes its antigens and presents them to naive T cells & naive B cells

   • few days for naive T cells and naive B cells to activate

2. clonal expansion & differentiation

   • activated B cells multiply and differentiate into plasma cells

     • produces antibodies (mostly IgM at first)

   • activated T cells expand and carry out immune functions

   • symptoms develop

3. memory B cells and memory T cells formed

   • remain in body for future protection

Secondary Immune Response

-re-exposure of pathogen in body

-2-3 days to develop

-faster and stronger than primary response

1. activation of memory cells

   • memory B cells & memory T cells recognize pathogen and activates immune response

2. strong antibody production

   • memory B cells → plasma cells

   • high affinity antibodies produced (mostly IgG)

   • antibodies produce much faster

3. fast elimination of pathogen

   • infection often cleared before symptoms develop

T Cells

-WBC (lymphocyte/leukocyte) that plays a role in adaptive immune response

-from HSC produced in bone marrow → matures in thymus

-responsible for recognizing specific antigens on surface of infected cells

• recognizes and responds to antigens via T Cell receptor (TCR) complex

-co-receptors CD4 & CD8 defines function of T cell

CD4 T Cells (T Helper Cells)

• recognizes peptides present on MHC class II proteins

• help coordinate immune response by activating other immune cells

  • B cells, CD8 T cells

CD8 T Cells (Cytotoxic/Killer T Cells, CTLs)

• recognizes peptides present on MHC class I proteins

• directly attack and kills infected or cancerous cells

4 (CD4) x 2 (MHC II) = 8 (CD8) x (MHC I)

-every T cell has its own TCR and antigen specificity

-single mature T cells have ~30 000 identical copies of TCR on its cell surface

T Cell Receptor (TCR)

-structure on surface of T cells that recognize and bind to antigens presented by MHC molecules

• allows T cells to recognize pathogens and trigger immune response

-membrane-bound protein made of 2 polypeptides (α & β chains) joined via disulfide bond

• 1 variable (V) region

  • Vα and Vβ chain regions combine to form antigen-binding site

  • one antigen-binding site per TCR molecule

• 1 constant (C) regon

-TCR antigen-binding subunit recognizes peptides bound to MHC proteins

• doesn’t recognize native, intact antigens

-T cells respond to peptide antigens on MHC class I/II proteins

• if host is healthy → peptide is from self protein

• if host has infection → peptide is from pathogen protein

-CD3 is the signalling component of the T cell receptor complex

Regulatory T Cells (Treg)

-type of CD4 cell that help controls immune system and prevents autoimmune diseases

• thymocytes that are positively selected with MHC class II

-self-reactive & secretes IL-10

• calming, anti-inflammatory cytokine

-Treg deficiency → increased chances of autoimmune disease

-prevent autoimmunity

• stops immune system from attacking body’s own tissues

-control inflammation

• suppress overactive immune responses and limit tissue damage

-regulate other immune cells

• keep T cells, B cells from attacking harmless molecules

-maintains immune balance

• ensure immune system reacts properly

T Cell Development

Thymocytes

• immature T cells

• develop in thymus before becoming fully functional T cells

• T cell precursors (thymocytes) starts in bone marrow and travels to thymus

• don’t have TCR

• functionally distinct from T cell populations

• developing thymocytes tracked by changes in expression of TCR, CD4, CD8

-T cells don’t know to be CD4 or CD8 T cell → undergoes thymocyte development

• undergoes negative & positive selection

Double Negative Stage (Early Development)

-thymocytes don’t have CD4 and CD8 markers

-rearrange TCR genes to form unique TCR

Double Positive Stage (Intermediate Development)

-thymocytes express CD4 and CD8

-undergo positive selection to ensure TCR can recognize MHC molecules

• T cells with TCR unable to recognize MHC molecules undergoes apoptosis (dies)

  • rendered useless

Single Positive Stage (Final Development)

-thymocytes that recognize MHC class I → CD8 T cells (CTL)

• with high enough affinity to activate cell

-thymocytes that recognize MHC class II → CD4 T cells (T helper cell)

• with high enough affinity to activate cell

-undergo negative selection to eliminate cells that might attack body

• self-reactive T cells

• identifies thymocytes that bind MHC and self-peptide too tightly

  • bind too tight → may be activated in lymph node/spleen → autoimmune disease

-surviving thymocytes mature into functional T cells and leave thymus

T Cell Activation

-T cell gets activated to fight infections

• when antigen presenting cells present pathogens to naive T cells

Naive T Cell

• T cells that haven’t been activated before

-activated only when it needs to be → T cell activation can be dangerous

• requires 3 signals to activate

  • 2 signals come from antigen-presenting cell

Signal 1 — Antigen Recognition (TCR-MHC Interaction)

-APC presents peptide on MHC class I/II molecule

• CD8 T cell → MHC class I molecule

• CD4 T cell → MHC class II molecule

-TCR on T cell binds to MHC antigen complex

Signal 2 — Co-stimulation

-makes sure immune system doesn’t attack by mistake

-prevents self-reactive T cells from attacking body’s own cells

-co-stimulatory receptor CD28 protein of T cell binds to B7 protein of APC

• T cell gets activated

-signal 1 & 2 together allows T cell to start making IL-2 (cytokine)

-if no co-stimulation occurs → T cell becomes living but non-responsive (anergic)

Signal 3 — Cytokine Signalling

-cytokines directs T cell response and differentiation so T cells

• know what type of response to generate

  • attack viruses/bacteria/parasite

• multiply into

  • CD8 effector T cells

    • kills virus infected cells

  • CD8 memory T cells

    • reactivated if future infection with pathogen occurs

-T helper cells (CD4) make enough of their own IL-2 to proliferate but CTLs (CD8) don’t make enough

• depend on IL-2 made by T helper cells

Dendritic Cells

-best antigen presenting cell (APC) for activation of naive T cell

• presens MHC class molecules to naive T cell to activate it

-expresses

• MHC class I

• MHC class II

• co-stimulatory molecule B7

-able to undergo endogenous, exogenous, cross-presentation pathways of antigen processing and processing

-bridge between innate and adaptive immune response

Dendritic Cell Functions

-phagocytose and break up pathogens

-transport pathogen remains to local lymph node

-presents peptides from pathogens to bind to MHC class molecules for activation of T cells

Dendritic Cells in Innate Immune Sytem

-has PRRs that detect PAMPs

-detect pathogen → engulfs & digests pathogen (phagocytosis) → break down into antigens

B Cell

-secrete antibodies into body fluids

-entire development occurs in bone marrow

-presents antigen to T helper cells to receive T cell help

• T cell help needed to get B cells to make high affinity, class-switched antibodies and develop memory B cells

-becomes plasma cell after activation by Igα/Igβ signal

B Cell Development

-from common lymphoid progenitors in bone marrow

• remains in bone marrow for whole development process

-begins to express B cell receptor

-immature B cells with functional BCR are screened for self-reactivity in bone marrow

• high affinity BCRs → deleted

B Cell Receptor (BCR)

-major developmental stages of developing cells can be tracked by changes in expression of B cell receptor (BCR)

-protein complex on B cell surface

-every B cell has a unique BCR

-allows B cells to recognize and bind to antigens

-helps B cells detect infections/invaders and start an immune response

• when BCR binds to matching antigen → triggers signal inside B cell → antibody production

-provides long term immunity

• helps immune system remember past infections

-BCR of mature naive B cell is

• membrane bound immunoglobulin M (mlgM)

  or

• membrane bound immunoglobulin D (mlgD)

B Cell Receptor Structure

-quaternary protein structure

• made of 4 polypeptides

-2 antigen binding sites per BCR

Antibody Component

• binds with antigen

• made of 2 heavy (H) and 2 light (L) chains → forms Y-shape

  • joined by disulfide bonds

• each chain has a variable region (V_L V_H) located at tips of Y

  • determines antigen specificity

  • hypervariable region within variable region

    • makes direct contact with antigen

• each chain has a constant region (C_L C_H) located at base of Y

Signaling Component (Igα/Igβ)

• BCR alone can’t send signals to B cell

• Igα/Igβ proteins transmits signal inside cell after antigen has bound

B Cell Receptor Process

1. Antigen recognition

   • B cell with correct BCR encounters antigen

2. BCR — Antigen binding

   • BCR binds directly with antigen

   • doesn’t require APC (like T cells)

3. Signal transmission

   • binding of antigen triggers Igα/Igβ → sends signals inside B cell

4. B cell Activation

   • activate and prepare for immune response

   • engulf antigen for further processing

   • communicate with helper T cells

   • differentiate into plasma cells → mass produce antibodies that match antigen

   • become memory B cells → long-lived cells that remember antigen for a faster response on the next encounter

B Cell Activation

-so B cells synthesizes and secretes antibodies

-requires multiple signals to differentiate antibody secreting cell

-responds to antigen in

• T-independent manner

• T-dependent manner

T-Independent Activation

-T helper cells not involved

-fast antibody response but low affinity IgM produced, no memory cells

• few days

-short lived plasma B cells

-antigen has many epitopes

-repetitive, multivalent antigens that bind to multiple BCRs on a single B cell

-antigen binds directly to BCRs on B cell → Igα/Igβ signal inside cell → activates B cell → IgM antibodies produced

• 1 signal for activation

  • binding of antigen to 2 BCRs

-weaker immune response

• no class switching to IgG, IgA, IgE

• no memory B cell production → no long lasting immunity

Ex: Pneumococcal Vaccines

• capsular material (polysaccharide)

• 13 strains of pneumococcus bacteria (> 90 strains)

• promotes IgM antibody response → no memory cells

• antibody neutralizes bacteria

• for conjugated vaccines to activate T cells & B cells

  1. covalently couple non-protein part (polysaccharide) to a protein molecule

     • dendritic cells can use protein part to generate peptides to activate T cells

     • one set of B cells recognizes protein part

     • one set of B cells recognizes non-protein part

     • T helper cells activated by peptide from protein part

       • helps both sets of B cells

  2. add something to antigen to trick dendritic cells to think it’s dangerous

     • adjuvant → causes arm to be sore/hurt

       • stimulates danger → dendritic cells start immune system

T-Dependent Activation

-T helper cells involved

-slower, high affinity, class switched antibody response with memory cells

• 7-10 days

-long lived plasma B cells

-antigens have few epitopes

-T helper cells produce cytokines (signals) required for class switching and memory cell formation

-3 signals needed for activation

1. binding of antigens to 2 BCRs

   • cross-linking of BCRs

   • B cells identifies itself to T helper cell that it needs help binding to antigen

     • antigen-BCR complex into cell via endosome → displays peptide on MHC class II → moves to lymph node or spleen → TCR of T helper cell (CD4) recognizes peptide displayed on MHC class II → CD28 (T helper) binds to B7 (B cell)

       *B cell acts as APC so T helper cell will help

2. binding of CD40 on B cell to CD40L on T helper cell

   • T helper cells provide CD40 ligand (CD40L) binding to CD40 on B cell via cytokines

3. cytokines secreted by T helper cell supports B cells to turn into

   • memory B cells

   • plasma cells

   • secreted antibodies

     • IgG, IgA, IgM, IgE antibodies that target specific pathogens

-T helper cells signals can promote class switching in B cell

• can produce IgG, IgA , IgE from IgM

Antibodies (Abs)/Immunoglobulins (Igs)

-essentially the same as BCRs but BCR is attached to B cell while antibodies are secreted by activated B cells (plasma cells) into blood or tissues

-proteins made and secreted by B cells

-circulates in blood and other body fluids (mucus)

-antibodies bind to molecules that are foreign to individual

-kills bacteria (in presence of complement)

-opsonize pathogens (make phagocytosis more efficient)

-neutralize pathogens (prevent pathogens from binding/infecting cells)

Membrane Bound Immunoglobulins (mIg)

• activates B cells

• stays attached to B cell surface

• part of BCR

• amino acids are hydrophobic

• IgM, IgD

Secreted Immunoglobulins (sIg)

• destroys pathogens

• free floating antibodies produced by B cell after it is activated and becomes a plasma cell

• circulates in blood, lymph and tissues to attack pathogens

• amino acids are hydrophilic

• IgG, IgA, IgM, IgE

Antibody/Immunoglobulin Binding

-bind to epitopes (antigenic determinants)

• distinct 3D shape

• ~4-5 amino acids

  • can be carbohydrates, lipids, synthetic chemicals etc

• antigens have more than one kind of epitopes

-bivalent → 2 binding sites

Antibody/Immunoglobulin Classes

-IgM, IgG, IgA, IgE, IgD

-all have different functions in immune system

-can be

• membrane bound (mIg) (IgD only)

• secreted (sIg)

IgD

• not secreted

• only found on surface of naive B cells

IgM

• secreted

• low binding affinity

• neutralizes pathogen

• first antibodies produced in immune response

IgG

• secreted

• found in blood

• neutralizes pathogen

• kills bacteria in 2 ways

  1. complement proteins kill bacteria coated with IgA

     • amino acid sequence in γ chain constant region binds to complement proteins

  2. phagocytes ingest bacteria coated with IgG antibodies better than bacteria without IgG

     • amino acid sequence in γ chain constant region facilitates IgG binding with specific receptors on phagocytic cells

IgA

• secreted

• in serum → monomer

• in bodily secretions → dimer

• main antibody class in bodily secretions

  • binds to and neutralizes pathogens

• prevents attachment of pathogens to host surfaces

IgE

• secreted into serum

  • binds immediately to mast cells and basophils

• not available in serum to neutralize pathogens

• triggers allergic reactions

  • when IgE on mast cell/basophil binds an antigen

    • cell degranulates & releases large amounts of histamine

• amino acid sequence in ε chain constant region binds to receptors

  • only antibody class that can bind to receptor (FCεRI)

• main antibody class induced in response to infection by parasites

  • ex: intestinal worms → too big to be killed via phagocytosis

    • mast cell degranulation → diarrhea & vomiting to expel worms

Structure of Antibodies/Immunoglobulins

-Y shaped proteins made of 2 binding sites

-4 polypeptide chains

• 2 identical H chains

• 2 identical L chains

-variable region at tips of Y shaped antibody

• binds specifically to antigen (hypervariable region)

• hypervariable regions (HV_H HV_L)

  • makes direct contact with antigen

  • 3 short stretches of amino acids

-constant region at base of Y shaped antibody

• determines antibody type

• interacts with immune cells

• difference in amino acid sequence of C regions of heavy chains is significant

L Chain Types

• kappa (κ) chain

• lambda (λ) chain

• immunoglobulin may have κ chain or λ chain L chain, but never both

H Chain Types

• gamma (γ) chain

• mu (μ) chain

• delta (δ) chain

• epsilon (ε) chain

• alpha (α) chain

• type of H chain defines immunoglobulin class or isotype

Function of Antibodies/Immunoglobulins

-different antibodies have different roles in immune system

• determined by H chain of antibody

Neutralization

-antibodies neutralize pathogens by binding to them → prevents them from binding to surface

-reliez on variable region of antibody

Antibody/Immunoglobulin Class Switching

-best method for adaptive response to match antibody to antigen

-B cells can class switch more than once

• switching is unidirectional

  • can’t switch back to original antibody after splicing → DNA is lost

-B cells always secrete IgM antibodies first (sometimes IgD)

• fast first response but not good enough

• switches to make a different class of antibody → IgM, IgA

-only C_H regions change; V_H/V_L and C_L regions don’t change

• cutting out DNA of heavy chain genes (base of Y shape)

• only constant region of heavy chain

-T helper cells induces class switching via different combinations of cytokines

• antibody constant region is spliced out and gets replaced with a different constant region

• ex: IgM made initially → switch to IgE BUT wants to switch to IgG afterwards

  • T cell sends signals from cytokines to B cells → IgM constant region spliced out → IgE constant region inserted to antibody

  • B cell now produces IgE → switch to IgG later on

  • T cell sends signals from cytokines to B cells → IgE constant region spliced out → IgG constant region inserted

  • B cell now produces IgG

Antibodies as Tools in Research and Medicine

-clinical uses, research laboratories, diagnostic services

-antibodies can be produced by

• immunizing animals (rabbits, mice, goats)

• cells (hybridomas) that grow in tissue culture

-antibodies can be purified and used for detecting and quantifying antigens that they recognize

ex:

• detection of pathogens (bacteria, viruses, toxins) in. patient samples

• detection of antibodies in blood that indicate exposure to antigen (covid test)

• detection and measurement of hormone levels (thyroid hormones, pregnancy tests)

• analysis of blood cells and immune cells (blood and tissue typing, enumeration of cell types)

• therapeutic medication for medical conditions (cancer, psoriasis, Crohn’s disease)

-amino acid sequence of variable regions of antibodies different for constant regions of dogs & humans

• species difference

Polyclonal Antiserum

-mix of different antibodies in serum (anti-serum)

-animals can be immunized with antigen (virus, snake venom, etc) and antibodies can be isolated from animal’s serum

• contains mixture of antibodies that recognize different epitopes on antigen that was injected

  • antigens have many epitopes that activate B cells

  • each B cell recognizes a different epitope

• antibodies with different specificities and affinities

-blood serum that contains polyclonal antibodies specific to particular antigen

-after immunizing animal with specific antigen

• created by immunizing an animal with specific antigen

-constant regions of heavy chains of antibodies from different species are immunogenics

• antibodies from another species can be antigens → antibodies against antibodies are made

  ex: inject dog anti-rabies antibodies into human → patient’s immune system produces human anti-dog antibody antibodies

Polyclonal Serum

-blood serum containing mixture of antibodies against multiple antigens or epitopes

• broad mix of antibodies, not specific to one antigen

-normal blood serum of organism after exposure to infections

Monoclonal Antibodies

-antibodies produced by descendants of one clone of B cells

• maintained in cell culture (in vitro)

1. repeatedly immunize mouse with antigen to get secondary immune response

   • B cells specific for epitopes on antigen get activated → plasma cells secreting antibodies

2. remove mouse’s spleen → antibody secreting plasma cells immortalized by fusing them with myeloma cell that grows in culture

   • myeloma — plasma cell tumour that can’t make antibodies

3. B cells fuse to myeloma cells via polyethylene glycol → hybridoma

   • hybridoma — characteristics of B cell (make antibody) & myeloma (immortality)

     • grow and divide indefinitely in tissue culture dishes

     • mixture of immortal hybridomas make antibodies against different epitopes

4. clones of hybridoma making antibody made

   • single cell placed in each well of 96 well tissue culture plate

   • cells in each well divide and form clones of identical cells

5. hybridoma clones making antibodies against desired epitope are selected

   • small amount of culture medium from each well

   • using ELISA assay to see if antibodies secreted into medium by hybridoma cells bind to epitope of interest

Antigens

-markers found on surface of viruses, bacteria, blood that immune system recognizes

-can be foreign or self

-if immune system recognizes antigen as foreign → triggers immune response

-if antigen from body → immune system ignores

• sometimes makes mistakes → autoimmune diseases

Antigen Processing

-degradation of protein into peptide fragments

Antigen Presentation

-displaying peptides derived from pathogens or other protein on dendritic cell surface that T cells can see

-MHC proteins on cell surface provides physical structure to display antigenic peptides to T cells

-binding and display of antigen as peptide fragment bound to MHC proteins on surface of a cell

MHC Class I

• displays peptides derived from proteins in cytoplasm of cell

• proteins coming from inside of cell

• proteins synthesized by ribosomes

MHC Class II

• displays peptide derived from soluble proteins taken up by cell via endocytosis/phagocytosis into an endosome/phagosome

• proteins coming from outside of cell

• proteins include

  • normal blood proteins

  • toxins

  • bacteria

  • virus particles

Antigen Presenting Cells (APC)

-immune cell that activates immune system (tells immune systems about infections)

• shows pieces of pathogens to immune cells

1. capture & engulf (phagocytosis)

2. processing antigens

   • pathogens broken down into peptides (antigens)

3. displaying antigens

   • APC places antigen on MHC molecules (class I/class II)

     • MHC class I presents antigens to cytotoxic T cells (CD8 T cells) → destroys infected cells

     • MHC class II presents antigens to helper T cells (CD4 T cells) → coordinates immune response

4. activation of T cells

   • T cells recognize antigen

     • activates to kill infected cells

     • help other immune cells fight infection

Types of Antigen Presenting Cells

• dendritic cells

• macrophages

• B cells

Major Histocompatibility Complex (MHC) Proteins

-region of chromosome containing genes that encode proteins that have a role in immune response

-proteins that have grooves so peptides can bind

-allow dendritic cells to communicate to T cells that an infection is occurring so adaptive immune response can be initiated

-MHC is polygenic

• several genes that encode proteins of similar function

-MHC is polymorphic

• different alleles in human population

-HLA = human leukocyte antigen

• codominantly expressed

• cells transcribe both alleles and make protein products of both genes

MHC Class I Proteins

-expressed on all nucleated cells

-transmembrane α chain non-covalently bonded with β2-microglobulin

• peptide bonding groove on α chain

• ~8-10 amino acids long

-3 different MHC class I proteins

• HLA-A

• HLA-B

• HLA-C

-50% of MHC class I expressed on each cell from mom, other 50% from dad

-cells have ability to make 6 different variants of MHC class I proteins

• 3 (1 HLA-A, 1 HLA-B, 1 HLA-C) from mom

• 3 (1 HLA-A, 1 HLA-B, 1 HLA-C) from dad

MHC Class II Proteins

-expressed only on antigen-presenting cells (dendritic cells, macrophages, thymic epithelial cells)

-2 transmembrane polypeptide chains

• α & β chains

• peptide bonding groove between α & β chains

• at least 13 amino acids ong

-3 different MHC class II proteins

• HLA-DP

• HLA-DR

• HLA-DQ

-all proteins only expressed on antigen presenting cells

MHC Polymorphism

-different alleles in human population

• different alleles for each MHC gene

-allows cells to present as many types of peptides as possible

-protects against wide range of pathogens

-recognition by immune system wouldn’t work properly without MHC polymorphism

Disadvantages to MHC Polymorphism

-hard to find matching donor for someone that needs an organ/bone marrow transplant

• same versions of MHC molecules

MHC Diversity

-goal of MHC protein is to be able to bind and present peptide derived from any protein

• especially foreign proteins from a pathogen

-if unable to present peptides from a pathogen → adaptive immune response not activated

-single MHC proteins can bind many but not all peptides

Pathways for Processing and Presentation of Protein into Peptide Fragments

MHC Class I Peptide Loading (Endogenous Pathway)

-MHC class I displays peptides from inside of the cell to cytotoxic T cells (CD8 T cells)

• proteins coming from inside cell

• helps immune system detect infected or cancerous cells

-occurs 24/7

1. proteins inside cytoplasm are broken down into peptides via proteasomes

2. peptides moved to lumen of endoplasmic reticulum (ER)

   • TAP (transporter protein) moves peptides from cytoplasm to ER

3. MHC class I molecule made in ER

   • newly made MHC I associates with β2-microglobulin

4. peptide binds (loaded) to MHC class I molecule

   • only peptides of right length and quality are able to bind

   • MHC class I becomes stable and ready to leave ER after binding

5. MHC class I peptide complex moves from ER to Golgi apparatus

   • MHC class I peptide complex gets packed up in Golgi

6. MHC class I peptide complex sent to cell surface via vesicles

   • displayed on cell surface for ~24 hours before internalized and replaced

7. CD8 T cells inspect MHC class I peptide complex

   • peptide is normal (self-protein) → T cell ignores it

   • peptide is from a virus/cancer → T cell recognizes cell as threat and kills it

MHC Class II Peptide Loading (Exogenous Pathway)

-MHC class II displays peptides from outside of cell to helper T cells (CD4 T cells)

1. external proteins are taken into cell

   • cell takes in bacteria, viruses, blood from surroundings via endocytosis

     • cell becomes phagosome

2. proteins are broken down into peptides via phagosome — lysosome fusing

3. proteins are broken down into peptides via lysosomal proteases

4. MHC class II molecule made in ER

   • invariant chain (li) blocks peptide binding groove

     • prevents molecule from binding to wrong peptide

5. MHC class II-invariant chain complex travels from ER to phagosome via vesicle

   • enzymes cut invariant chain

     • leaves small piece (CLIP - class II associated invariant chain peptide) in binding groove

6. peptide exchange

   • CLIP replaced by peptide from degraded pathogen (antigenic peptide)

7. MHC class II peptide complex sent to cell surface

   • displayed for immune system inspection for ~48 hours before internalized and replaced

8. CD4 T cells inspect MHC class II peptide complex

   • scan MHC class II molecules on antigen presenting cells (macrophages, dendritic cells, B cells)

   • peptide is from a pathogen → CD4 T cell activates → coordinate immune response → release cytokines

MHC Class I & MHC Class II Peptide Loading (Cross-Presentation Pathway)

-mix of exogenous and endogenous pathways aided by dendritic cells

-different subsets of dendritic cells used

• from common myeloid precursors

• from common lymph precursors

-presents peptides on

• MHC class I via endogenous pathway

• MHC class II via exogenous pathway

-extracellular antigens (virus/blood/bacteria proteins → peptides) presented on MHC class I molecule to CD8 T cells

Cytosolic Pathway (Endogenous Pathway)

-proteins from extracellular antigens escape into cytoplasm → broken down into peptides via proteasome in cytoplasm → peptides travels to ER via TAP → peptides binds to MHC class I

1. dendritic cell engulfs extracellular antigens

   • cell takes in bacteria, viruses, blood from surroundings via endocytosis

     • cell becomes phagosome

2. some proteins escape from phagosome into cytoplasm

3. proteins are broken down into peptides via proteasome in cytoplasm

4. peptides in cytoplasm transported to ER via TAP

5. peptides bind to MHC class I molecule in ER

6. MHC class I peptide complex sent to cell surface

7. CD8 T cell inspect MHC class I peptide complex

Vacuolar Pathway (Exogenous Pathway)

-proteins from extracellular antigens broken down into peptides via lysosomal proteases in phagosome → MHC class I travels to phagosome → peptides bind to MHC class I

1. dendritic cell engulfs extracellular antigens

   • cell takes in bacteria, viruses, blood from surroundings via endocytosis

     • cell becomes phagosome

2. proteins broken down into peptides via lysosomal proteases in phagosome

3. MHC class I molecules made in ER travels to phagosome

4. peptide binds to MHC class I in phagosome

5. MHC class I peptide complex sent to cell surface

6. CD8 T cells inspect MHC class I peptide complex

Virus

-must infect cells in order to replicate

• viruses are not living/cells

• can’t synthesize their own ATP or amino acids or nucleotides or proteins

-binds to a structure on surface of cell

-takes over host cell’s metabolic resources and uses cell’s machinery in order to replicate genomes and assemble new virus particles

Innate Immunity Response

-less effective than innate responses to bacterial pathogens

-TLRs and PRRs to recognize virus-specific molecules

• double-stranded RNA

• double-stranded DNA

• uncapped single-stranded RNA

-PRRs initiate signal transduction cascade that results in expression of Type I interferon (IFN-α/β) genes and secretion of IFN-α/β

-IFN-α/β cytokines

• bind to a common receptor found on neighbouring uninfected cells

• triggers a signalling cascade that induces anti-viral response

-uninfected cell shuts off ability to synthesize protein

• virus infected cell → virus can’t replicate since protein synthesis is essential in virus replication cycle

Adaptive Immunity Response

-involves antibodies and cytotoxic T cell responses

• prevents infection of more cells by virus

-antibody production against viruses can be induced via

• natural infection with virus

• use of vaccine

-antibodies secreted by B cells bind to and neutralize viruses

• prevents them from infecting host cells

Intracellular Virus

-protein coat is shed

-CTL response

• sacrifice infected cell so that virus replication cycle can stop and not other cells can be infected

-APC picks up viral peptides and loads them onto MHC class I molecule

• MHC class II peptide complex is delivered to naive CD8 T cell in lymph node

-CD8 T cell makes CTLs that spread through body to find infected cells

-CTL-P activated to CTL

• CTL-precursor (naive T cell) in lymph nodes or spleen

• T helper cells provide extra IL-2 for CTL proliferation

• dendritic cells to activate T helper cells and CTL-P

-CTLS recognize infected cells by binding to MHC-class I viral peptide

• CTL releases toxic molecules

-memory CTLs remain for future infections

• some CTLs turn into memory T cells

Extracellular Virus

-antibody response

• neutralize virus so it can’t bind and infect cells

-B cells to synthesize & secrete antibodies

-T helper cell to provide signal to B cells to fully activate them

-dendritic cells to activate T helper cells

Autoimmunity

-immune system attacks body’s own healthy cells

-when immune system responds against normal tissues/organs

• results in damage to or impaired function of tissue

-clonal deletion failure causes autoimmune diseases

• clonal deletion ensures that the T cell & B cells that recognize own cells are eliminated during development in thymus/bone marrow

-antibody-mediated autoimmune disease

• making antibodies against self-components causes autoimmune diseases

• block function of tissue/organ

• activation of complement by antibodies → death of tissue/organ

-cell-mediated autoimmune disease

• activated T cells

  • damage to tissues/organs via cytokine secretion

  • activate macrophages that destroy tissues/organs

-T cells activated against normal components of an individual

• normal components reacting with BCR or TCR → auto-antigens or self-antigens

-self-reactive B cells & self-reactive T cells

-everyone has self-reactive antibodies & self-reactive T cells

• go through negative selection but process isn’t perfect

Autoimmune Diseases

Autoimmune Hemolytic Anemia

-RBCs removed from circulation at end of normal life span (~120 days)

-hemolysis

• premature destruction of RBCs

• shortened RBC life span (< 120 days)

• caused by antibodies against RBCs

-anemia results when bone marrow production can’t compensate for shortened RBC survival

Myasthenia Gravis

-severe muscle weakness

-nerve impulses don’t reach muscle

• weakness and fatigue in affected muscles

-antibodies binding to acetylcholine receptor on muscle cell

• blocks receptor and prevents muscle cells from responding to neurotransmitters (acetylcholine) released by nerve cells

• acetylcholine released by nerve cells unable to bind to receptor because antibodies are already bound to it

-T-dependent B cell response

• patient has memory B cells and long-lived plasma cells

• repeated relapses and remissions

Type I Diabetes

-insulin dependent diabetes

-self-reactive T helper cells against antigen on pancreatic islet cells

-T helper cells cause inflammatory response similar to DTH response

-activated macrophages infiltrate tissue containing self-antigen

-insulin-producing cells in pancreas are destroyed as result of inflammatory response

Multiple Sclerosis

-immune system mistakenly attacks protective nerve of brain & spinal cord

-protective coating around nerve fibers attacked

• damages myelin sheath → inflammation

-without myelin → nerve signals slow down or get disrupted

• muscle weakness, vision problems, trouble with movement/coordination

Celiac Disease

-genetic autoimmune disease that results from breakdown of tolerance of foods containing gluten

• gluten — protein in grains; gives dough its elastic feature

-in healthy systems → soluble proteins from food don’t stimulate an immune system

-oral tolerance of food breaks down → loss of villi in epithelial cells

• dendritic cells are activated → CD4 T cells activated → secrete cytokines that damage epithelium of GI tract and activate CD8 T cells → antibodies

Treatment of Autoimmune Diseases

-non-specific immunosuppression with steroid hormones

• suppresses immune system

-avoidance of trigger (gluten in celiac diseases)

-some conditions — removal of spleen may improve patient’s conditions

Cytokine Release

-cytokines are important for recruiting and activation of cells but they can cause other symptoms

• fever, muscle aches, headaches, fatigue, nausea

• symptoms result from immune response to a pathogen

Cytokine Release Syndrome

• too much cytokine is released

• potentially fatal

Medical Complications from Immune Responses

-sometimes it’s better not to have an immune response

• allergies

• delayed type hypersensitivity (DTH)

• transplant rejection

• contact dermatitis

• serum sickness

• autoimmune diseases

-body’s response involves the same events that occured in localized inflammatory response

• but on a systemic scale

• results in septic shock

  • low BP & organ failure

Septicemia

• bacteria entering bloodstream and multiplying

• very serious

• difficult to treat

• antibiotics unhelpful

Snake Venom

-some snake venom are toxic and potentially lethal

• neurotoxins, cytotoxins, cardiotoxins, hemotoxins

• rapid neutralization of venom is required → anti-venoms

  • preformed antibodies to venom administered as passive immunization

Anti-Venom

• mostly protein; adjuvant is added so dendritic cells recognize it as dangerous

• often produced in horses (large blood volume)

• milk snake → get venom → add adjuvant to venom → inject small amount to horse (small enough not to harm animal) → few months later antibodies are extracted via blood draw → formulation of snake antivenom

• binds to and neutralizes venom → resolves emergency

  • stops further damage

  • doesn’t reverse damage already done

  • effective at preventing death from bites if enough is administered in time

• people given large amounts of anti-venom

  • leftover anti-venom → production of human anti-horse antibody antibodies

    • results in rashes

Poison Ivy/Poison Oak/Poison Sumac

-produce urushiol (type of oil)

• penetrates skin & covalently binds to self-proteins

• can penetrate cell lipid bilayers and bind to intracellular proteins

• can be active for ~1 year

  1. sensitization on first time exposure

  2. delayed type reaction if previously exposed

First Exposure

• dendritic cells pick up bound self-proteins and present to MHC class II molecules to activate T helper cells (CD4)

• proteins can be processed by proteasome and presented to MHC class I molecules to activate cytotoxic T cells (CTLs/CD8)

Second Exposure

• if re-exposure → memory T cells & memory CTLs are reactivated

• macrophages will pick up urushiol bound to self-proteins and present to memory T cells

• macrophages and T helper cells secrete cytokines to activate B cells → antibodies

Contact Dermatitis

• inflammation of skin

• if allergic to something that causes dermatitis as a response upon skin contact→ allergic contact dermatitis

  • delayed type hypersensitivity (DTH)

  • rash develops ~2-3 days upon re-exposure

• hypersensitivity because urushiol in absence of immune attack is harmless

Allergies

-person has primary antibody response to antigen causing the allergy

• T helper cells somehow convinces B sells to secrete IgE antibodies

-IgE antibodies bound to FcR of mast cells

• burst of mast cells triggers release of histamine → causes allergy symptoms within minutes of exposure to antigen

-mast cell gets primed

• primary & secondary response, class switching of antibodies to IgE

-cross-linking of IgE

• allergen needs to enter tissue to bind to IgE

• IgE bound by same antigen (cross-linking)

  • triggers signal that causes explosion of mast cells (release histamine into tissus) → dilation of blood vessels

Respiratory System

• excess mucous secretion → sneezing & coughing

Skin

• hives/welts

Gut

• diarrhea/vomiting

Hay Fever

• localized/seasonal allergy

• allergic rhinitis

• allergic reaction to airborne particles inhaled through nose or mouth

  • pollen → trapped in upper airway leading to degranulation of mast cells

• symptoms localized to upper respiratory tract

Mosquito Bites — Skeeter Syndrome

• localized allergy

• 2 types of responses

  1. small round (2-10 mm) & surrounding edema that peaks 20-30 minutes after bite

  2. pruritic papules

     • IgE mediated allergic response to proteins in mosquito saliva

Penicillin Allergy

• systemic allergy

• reactive B lactam ring reacts with amino groups on host proteins to form conjugates

• naive CD4 T cells recognize penicillin modified peptides and present to MHC class II molecules

  • B cells get activated in T-dependent manner

• memory B cells express IgE BCRs and plasma cells secrete IgE antibodies

  • IgE antibodies bind to FcR of mast cell → degranulation of mast cell → release of histamine

Anaphylactic Shock

• severe and life threatening allergic reaction

• within minutes of exposure to allergen

  • peanuts, penicillin, bee venom may induce anaphylactic shock

• severe over reaction of immune system

• symptoms

  • swelling, wheezing, shortness of breath, difficulty swallowing, weak pulse, drop in BP

Allergy Treatments

Localized Allergic Reaction

• anti-histamines

  • block histamine receptor on smooth muscle cells and endothelium that binds histamine

  • doesn’t prevent release of histamine; just blocks histamine from binding to cells

Systemic

• epinephrine (epipen)

  • reverses symptoms of anaphylactic shock

  • relaxes airways, constricts blood vessels

• lie person on floor if epipen unavailable

Allergy Shots

• desensitization

  • deliberate exposure to small amounts of allergen

  • since new B cells constantly made

    • new B cells could be encouraged to switch to IgA or IgG type antibodies and out-compete IgE for binding to allergen

• ex: grass pollen injected under skin trigger B cells to switch to IgG/IgA

Hypersensitivity

-substance not dangerous but immune response to substance is dangerous

-inappropriate immune response to substance that isn’t dangerous

-can be

• CD4 T cell mediated

• CD8 T cell mediated

• B cell mediated (antibody)

Ex: Tuberculin Test

• tuberculin isn’t harmful → test to see if person has tuberculosis

• immune response to it is harmful

Immune Deficiency

-immune system is too weak or missing key components

• unable to fight infections properly

• frequent/severed illnesses

-can be genetic (primary) or acquired (secondary)

Primary Immunodeficiency

-person born with immunodeficiency

Severe Combined Immunodeficiency (SCID)

-mutation in HSC that impacts development of all mature lymphocytes

-lack of T cells

• life isn’t sustainable without T cells (medical intervention required)

-some cases where B cells present but not fully functional

• life is sustainable (continued medical assistance)

-T^-B^-

• no T cells

• no B cells

• most severe form of SCID

• no adaptive immune response

• no antibodies

-T^-B⁺

• no T cells

• has B cells

  • not functioning properly because some plasma cells are T-dependent to make antibodies

• no functional antibodies

Treatment

• HSC transplant/bone marrow transplant

  • within first 2 years of life

• in future → CRISPR-Cas system may be an option

  • gene editing that can be used on living cells

  • HSCs could be removed and isolated from patient

• bubble boy syndrome → extreme isolation to protect against infections

SCID-Like/Bare Lymphocyte Syndrome Type II (BLS II)

-absence of functional CD4 T cells but different reasons than SCID

• immune cells don’t express MHC class II molecules → CD4 T cells not activated

• MHC class I expression is normal → CD8 T cells activated

-normal amounts of B cells, neutrophils, monocytes/macrophages, dendritic cells

-weakened antibody production

• plasma cells that rely on T cells for secretion of antibodies and class switching

Treatment

• HSC transplant within first 2 years of life

  • not a complete cure

• new macrophages, B cells, dendritic cells able to express MHC class II but epithelial cells don’t

• patients do better → gain ~20% of CD4 T cell population

Neutropenia

-too little neutrophils produced

• weak immune system → high chance of infection

-neutrophils essential in controlling infections from extracellular bacteria

Treatment

• HSC transplant

• daily injection of recombinant human granulocyte colony-stimulating factor (rhG-CSF)

Secondary Immunodeficiency

-acquired immune deficiency

• not inherited or congenital

-result of infection or injury to immune system

AIDS

-acquired immunodeficiency syndrome

-untreated HIV infection can lead to AIDS

-T helper cell (CD4) count is < 20% of normal level

-people with AIDS usually die to opportunistic infections

• can’t mount an adequate immune response against infections

Treatment

• anti-retroviral medications

• medications that control virus load in patient → maintenance of CD4 T cell population

Graft Rejection and Transplantation

-removal of organ, tissue or hematopoietic stem cell from a donor and placed into body of recipient

-replacement of solid organs/tissues or bone marrow

• due to congenital defects or infectious disease that has damaged the organ

Transplant Rejection

-after transplant, graft initially survives and appears healthy

• due to slow activation of T cells

-CD8, CD4 T cells in charge of destroying grafted organs/tissues

• antibodies play a minor role in graft rejection

-when organ, tissue, or bone marrow is transplanted (unless from identical twin)

• there will be foreign proteins

• major mismatch → differences in MHC (tissue type)

• minor mismatch → differences outside MHC

-transplantation rejection caused by strong immune response to non-self MHC

Allo-Antigen

• MHC on transplanted tissue

Self-Antigen

• MHC on recipient’s cells

Indirect Allorecognition

-APC from recipient take up and process MHC proteins from transplanted organ/tissue

-recipient T cells recognize presented peptide as foreign and mount a response

-proteins released from cells of grafted issue (surgery process may damage cells)

• DAMPS — damaged associated molecular patterns

• activate signaling PRRs (TLRs)

Direct Allorecognition

-T cells become activated against graft via donor-derived APC

-transplanted into recipient with donated organ

-once in recipient’s body → donor-derived APC able to move out of transplanted organ and interact with recipient’s T cells

-recipient T cells recognize MHC proteins on donor derived APCs as foreign

• react and triggers immune response

-acute graft rejection

Solid Organ/Tissue Transplant

-transplanting

• organ

  • heart

  • kidney

  • lungs

  • liver

  • pancreas

• tissue

  • bones

  • tendons

  • cornea

  • skin

  • heart valves

-concerns

• donor and recipient may have different MHC class I molecules on their cells

• immune responses are directed against foreign MHC molecules

• transplanted organs/tissues from donor may have different proteins than those of recipient

Host vs Graft Disease

-recipient’s immune system attacks transplanted organ/tissue

-immune system sees transplanted organ/tissue as foreign and attacks it

-T cells of recipient activate and attack graft due to differences in MHC class I molecules

• inflammation & organ failure

-transplanted organ may need to be removed → second transplant

-improve chances of successful transplant

• donors HLA matched as closely as possible to recipient

• identical twin is the best donor → all genes identical

• close relative is next best choice → high chance of sharing MHC alleles

ex: kidney rejection after kidney transplant

Bone Marrow or HSC Transplant

-transplanting an immune system

-concerns

• donor and recipient may have different MHC class molecules on their cells

• immune responses are directed against foreign MHC molecules

Graft vs Host Disease

-donor’s immune cells attack recipient’s body

• transplanted system attacks body it goes into

-donor’s immune cells recognize recipient’s body as foreign and starts attacking it

-immunosuppressant may help with problem

ex: bone marrow transplant rejection leading to skin, gut, liver damage

Autograft

-organ, tissue, HSC cells transplanted within same person’s body

ex: veins from leg in a heart bypass surgery

Allograft

-organ, tissue, HSC cells harvested from one individual and placed into body of a different person

-alleles of MHC proteins on donor graft not normally present in recipient’s body (non-self antigens)

-recipient may have T cells that bind strongly to foreign MHC proteins on transplanted person

Increase Successful Surgeries

-better surgical procedure & healthier organs = less damage to graft

-matching MHC molecules

• donors that are related to patient

-immunosuppressive drugs to stop T cells from proliferation

• anti IL-2 to block T cell proliferation

• stop T cell activation (cyclosporin A)

• disadvantage → person’s susceptibility to infection increases

ELISA

-enzyme-linked immunosorbent assay

-useful for measuring molecules in solution

1. detection of soluble antibodies in solution

2. detection of any other type of soluble molecules

Detection of Soluble Antibodies in Solution

-useful for measuring hormones or any type of molecule in solution

-need 2 antibodies

• bind to different epitopes of antigen

• 1 of 2 antibodies tagged with enzyme

-determine if a soluble antigen is present in sample

-antigen x is coated to well of microtitre plate

• unbound antigen washed away

-sample added to well

• if antibody present → binds to antigen (primary antibody)

• excess antibody washed away

-secondary antibody (with enzyme bonded to it) is added

• if antibody bound to antigen → antibody will bind to it

-colourless substrate added and converted to coloured product by enzyme on secondary antibody

• concentration can be measured

  • proportional to amount of secondary antibody present (proportional to amount of primary antibody)

Detection of any Other Type of Soluble Molecules

-detection of presence of antigens

-antibody specific for the antigen is coated to well of microtitre plate (primary antibody)

• excess antibody is washed away

-sample added to well → if soluble antigen is present → binds to primary antibody

• excess antigen is washed away

-secondary antibody (with enzyme covalently bonded to it) is added

• recognizes different epitope

• doesn’t bind primary antibody

• excess antibody washed away

-colourless substrate added and converted to coloured product by enzyme on secondary antibody

• concentration of product measured

  • proportional to amount of antigen captured by first antibody

Lateral Flow Devices — ELISA Variation

-used to detect pathogens (virus, bacteria)

-has a control line to confirm test is working properly

• along with one or more target/test lines

-time to complete: 5-10 minutes (not including sample preparation)

-uses immunoassay technology using nitrocellulose membrane coloured nanoparticles and antibodies to produce results

-when sample added → flows along test device passing through conjugate pad into nitrocellulose membrane then onto absorbent pad

-conjugate pad storing conjugated labels and antibodies receive sample

• target present → immobilized conjugated antibodies and labels bind to target and continue to migrate along test

-as sample moves along device → binding reagents situated on nitrocellulose membrane will bind to target at test line → coloured line forms

Fluorescence Microscopy

-small fluorescent dyes (fluorochromes) covalently attached to antibody

• antibody becomes useful molecular probes

-different dyes excited by different light wavelengths

• allows to stain sample with several antibodies at once

-fluorescent labeled antibodies used to detect

• antibodies covalently linked to fluorescent molecules

• expression of protein

• change in expression

• distribution (location) of a cell

• movement of protein in cell

-cell needs to be fixed to microscopic slide

-if target on cell surface → labeled antibodies can be incubated with samples directly

-if target inside cell → cells treated with weak detergent solution to permeabilize cells

Fluorescent Activated Cell Sorting (FACS)

-machine that counts and sorts cells

• antibodies with different fluorescent molecules attached or cells without any fluorescence are sorted and category counted

• sort cells based on expression of particular protein

  • on or inside cell surface

• count cells and see if protein is expressed on cell

-cells sorted with laser that detects colour and counts them

-after sorting & counting cells

• computer generates 4 quadrant cell

• experiment thresholds indicated by green and red dashed line

-if amount of fluorescence detected exceeds threshold → cell considered positive

-if amount of fluorescence detected doesn’t exceed threshold → cell considered negative

-A^-B^-

• B cells and dendritic cells

-A⁺B^-

• T helper cells

-A^-B⁺

• cytotoxic T cells

-no cells in A⁺B⁺

• cells aren’t stained with either antibody

• non-T cells (eg. B cells, lymph node stromal (structural) cells)

ABO Blood Groups

A Blood Group

-A antigens on surface of RBCs

-anti-A cells removed by negative selection

-anti-B antibodies in plasma

-can donate to A, AB

-can receive from A, O

Type A Blood (Type A Rh-)

-RBCs have A type polysaccharide and B cells that make anti-A antibodies are negatively selected

-type A RBCs should never be given to

• type O blood (anti-A & anti-B antibodies)

• type B blood (anti-A antibodies)

-B cells that make anti-B antibodies aren’t negatively selected

• plasma contains anti-B antibodies

• person with type O blood can receive plasma (already have anti-B antibodies)

• person with type B blood can’t receive plasma

B Blood Group

-B antigens on surface of RBCs

-anti-A antibodies in plasma

-can donate to B, AB

-can receive from B, O

AB Blood Group

-A & B antigens on surface of RBCs

-no antibodies in plasma

-can donate to AB only

can receive from A, B, AB, O

O Type Blood

-no A or B antigens on surface of RBCs

-anti-A & anti-B antibodies in plasma

-can donate to A, B, AB, O

-can receive from O only

Type O Blood (Type O Rh-)

-universal donor

• only for RBCs, not whole blood

-RBCs lack A and B type polysaccharide

-someone with type A or B blood should never receive plasma from person with type O blood type

• type O plasma only safe for type O blood type

Rh Blood Groups

-protein found on surface of RBCs

-D antigen/Rh factor

• Rh⁺ → have D antigen

• Rh^- → don’t have D antigen

-Rh⁺ can receive Rh⁺ or Rh^- blood

-Rh^- can only receive Rh^- blood

-IgM antibodies produced by B cells that respond in T-independent manner

Blood Donation

-whole blood donations are spun in centrifuges to separate it into transferable components

• red blood cells

• platelets

• plasma

  • may be processed into components (cryoprecipitate)

    • controls risk of bleeding by helping blood to clot

• allows for flexibility when it comes to matching donors and recipients

-blood type of donor and recipient must be known

-patient with iron deficiency or anemia may receive RBCs to increase hemoglobin & iron levels → improving oxygen in body

-patients unable to make enough platelets due to illness or chemotherapy may receive platelet transfusions to stay healthy

-plasma transfusions used for patients with liver failure, severe infections, serious burns

-whole blood can be used for transfusions

• matching of donor and recipient has to be precise

Scenarios

1. Person with O Rh- blood receives a transfusion of B Rh- blood

   • antibodies that recognize the B antigen bind to transfused cells

     • IgM can activate the complement

     • transfused RBCs are lysed

   • contents of RBCs leak out

     • unpleasant symptoms

   • complicate person’s medical treatment

2. Person with O Rh- blood receives transfusion of O Rh+ blood

   • person is sensitized to Rh antigen & will produce IgG antibodies

   • depending on how much transfused blood was received

     • person may make anti-Rh IgG antibodies ~10 days later

       • these antibodies activate complement

   • if there are any O Rh+ blood cells present → content of RBCs leak out

     • unpleasant symptoms

   • person is okay as long as they don’t receive O Rh+ blood again

3. Mother with O Rh- blood carries fetus with O Rh+ blood

   • mother is sensitized to Rh antigen after childbirth

   • mother makes anti-Rh IgG antibodies ~10 days later

   • mother & baby is okay

   • Problem: future pregnancies, if 2nd fetus also has O Rh+ blood → pregnancy complications

     • IgG antibodies can cross placenta and enter fetus’ circulation

     • problem occurs because mother has memory B cells, long-lived plasma cells producing anti-Rh IgG type antibodies

     • severe anemia in fetus → prenatals important

4. Mother with O Rh- blood carries a fetus with B Rh- blood

   • mother & baby is okay

   • IgM antibodies are too big to cross placenta → don’t enter fetus’ circulation

5. Person with Rh- blood accidentally receives a transfusion Rh+ blood

   • make anti-Rh antibodies of IgG class

   • these antibodies are important medically to treat expecting mothers with Rh- blood carrying a fetus with Rh+ blood

   • mothers are passively immunized with some anti-Rh+ antibodies during late stage of pregnancy and after childbirth

   • prevents mother’s immune system from being sensitized to Rh antigen and no anti-Rh antibodies are made

     • protects fetus in subsequent pregnancies

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