Lecture Notes - Immuno

B Cell Development & Activation (3/4) - Wk 5

Case Study

• Complement system and kinins

• Reading up to the case was harder-

B Cell Development and Activation

• Immunoglobulin gene rearrangements

• Negative selection and central tolerance

• somatic hypermutation

• antibody isotypes and class switching

• development of plasma and memory B cell

• T-B Cell interactions

• B cell migration

• Intracellular B Cell signaling

• B Cell (AKA B Lymphocytes)

  • Develop and mature inside the bone marrow

  • Responsible for Humoral Immunity

  • Have B-Cell receptors (BCR) (antibodies stuck to the surface of a cell) that are specific for antigens

  • Make and secrete antibodies - ONLY CELL THAT DOES THIS

  • Name from bursa from chicken

Genetic Recombination

• How do you get such a large diversity of receptors on the surface of lymphocytes?

  • A human can generate trillions of different B cell receptors (which are proteins)

  • A human only has ~21,000 protein-coding genes

• Genetic recombination is responsible for the large repertoire of B cell receptors an individual person has

  • Repertoire - all the different lymphocyte receptors a single individual has in their body

• Why so many BCRs?

  • Recognize as many antigens as possible

B-Cell Development

• The process if B cell development occurs continuously throughout your life

• Stem cell = hematopoietic stem cell (HSC, found in Bone Marrow)

• Development of B Cells from HSC to Mature B cell occurs in the Bone Marrow

• Pro B Cell

  • earliest recognizable

  • No longer a HSC

  • also called B progenitor cells

  • Initial genetic recombination occurs at this stage

Genetic Recombination in B Cells

• B cells experience genetic recombination in order to produce a unique B Cell receptor, or antibody = more BCRs = more antibodies; THEY ARE THE SAME THING

• BCR (antibody) structure

  • Composed of 4 proteins

    • 2 identical heavy chains - bottom portion

    • 2 identical light chains - "top arm portion”

  • Chains are held together with disulfide bonds

• FC = constant fragment - “main body”

  • This region is the same for all BCRs of a particular isotype

• Fab = antigen binding fragment - the “arms”

  • F = fragment ab = antigen binding

    • This is what is different on each newly developed B cell

• All 4 chains have a unique region

  • These are proteins which are encoded by genes

B Cell Receptor Structure

• One region encodes for heavy chains in humans

  • H chain locus (specific region) on chromosome 14

• Two different gene regions encode for light chains in humans

  • kappa locus on Chromosome 2

  • lambda locus on Chromosome 22

B Cell Development

• During Pro B Cell

  • during the Pro B cell stage, the H chain locus begins to undergo recombination

Genetic Recombination

• Top portion - Germline

  • Each H chain locus is made up of

    • ~50 versions of V_H gene (V = variable)

    • ~20 versions of D_H gene (D = diversity)

    • ^ versions of J_H gene (J = joining)

    • 9 options for a C region (C = constant)

  • The VDJ genes will recombine to make the variable region of the heavy chain (composing Fab)

  • One C region gene will be selected to make the constant region of the heavy chain (composing the Fc) determining the isotype

• In a pro B cell, one D and J region are first cut out and recombined

• Important enzymes

  • VDJ recombinase

  • RAG 1

  • RAG 2

• In a pre B cell, a second rearrangement occurs in H chain Locus

• One V gene is recombined with DJ recombination made in the pro B stage

• VDJ unit is transcribed along with the first C genes: mu and delta

• Notice that there are two J gene options and 2 C gene options

• The primary RNA transcript is alternatively spliced so that mature mRNA ends up with

  • 1 V, 1 D, 1 J, 1 C

• These mature mRNA are translated to produce heavy chain proteins with:

  • A unique combination of VDJ to make up the variable region

  • Either C(mu) or C(delta) to make up the constant region

  • BCRs with C(mu) are known as IgM isotype and C (delta) are IgD isotype

• This is an error prone process!

  • If the H chain rearrangement does not work (does not produce a viable protein product)

    • The cell will try again using H chain locus on the matching homologous chromosome

  • If the H chain rearrangement try 2 does not work:

    • The pre B cell will undergo apoptosis (programmed cell death)

• If the H chain rearrangement is productive

  • The pre B cell sends the heavy chains to the cell surface forming a pre B cell receptor

• Once the pre BCR is on the surface of the cell, intracellular signaling occurs from the pre-BCR and tells the cell to rearrange its light chain genes

• Light chain recombination is very similar to heavy chain recombination

• Only consist of V and J (no D)

• Only one option for C region

• Cell tries to rearrange kappa locus first

  • if rearrangement is productive, stops there

• Cell tries. to rearrange kappa locus first

  • if second kappa locus try is not productive, tries to rearragne the genes on the lambda loci

• If no productive rearrangements are made from any kappa loci or lambda loci, the cell will die

• If productive rearrangements are made

  • light chains are sent to the surface

  • from. afunctional BCR

  • leads to the Immature B cell stage

B Cell Activation II (3/6-3/10) - Wk 5 & 6

Immunoglobulin Gene Rearrangements

• Takeaway: The genome of developing B cell is rearranged to form novel combinations of DNA to produce heavy and light protein chains to form antibodies/BCRs

Negative Selection and Central Tolerance

• The immunoglobulin gene rearrangements are random

  • There is nothing that stops the production of a BCR that can recognize something belonging to the self

    • How does the immune system solve the problem?

      • Negative Selection

• Negative selection - The elimination of self-reactive B cells during development

• Before leaving the bone marrow, the immature B cell is exposed to self-antigens from the stroma of the bone marrow

• If an immature B cell BCR binds to a self-antigen in the bone marrow

  • Receptor editing OR

  • Apoptosis

• Receptor Editing

  • BCR + self-antigen interaction in the bone marrow reactivates VDJ recombinase

  • V and J genes that were not deleted in the first rearrangement can. be edited out so that the cell can try again to make a different receptor

  • Originally in this example, V1 and J5 were not edited out of the genome

  • They either were not transcribe (V1) or alternatively spliced out (J5)

  • They reactivated VDJ recombinase can edit V2 and/or J4 out of the genomes to try make a different light chain

  • If receptor editing does not work OR

  • receptor editing produces another , different self-reactive BCR

  • then the cell undergoes apoptosis

• Negative selection leads to deletion of self-reactive immature B cells in the bone marrow

• This contributes to the development of central tolerance

  • The self tolerance that is developed in the primary lymphoid organs

  • peripheral tolerance later!

• Immature B cells that survive negative selection leave the bone marrow and travel to the spleen

• Once in the spleen, they become fully mature, naive B cells (hasn’t seen antigen yet)

B Cell Activation and Somatic Hypermutation

• A B cells job is to become activated when it sees its appropriate antigen

• Once activated it should

  • Clonally expand (make a bunch of copies of itself)

  • Differentiate into either

    • A plasma cell (to pump out antibodies for a short amount of time)

    • A memory cell (long-lived, non-proliferating cells that live in tissues and wait for future antigen exposure)

• A mature B cell migrates btw secondary lymphoid organs

  • spleen, lymph nodes, tonsils,

• Antigens arrive in secondary lymphoid organs

  • lymph nodes drain all body tissues, including foreign antigens

  • APCs bring processed antigen from anywhere in the body to secondary lymphoid organs

• Some antigens require T-cell help to fully activate their B cells

  • These are called Thymus Dependent (TD) antigens

    • If someone doesn’t have a thymus, they won’t respond to TD antigens bc they won’t have mature T cells to provide help to B cells

• B Cell Activation by TD antigens

  • B cells and CD4+ T cell that both recognize the same antigen are required

    • They can recognize different epitopes (parts) of the same antigen

  • These are cognate B and T cells

    • They recognize the same antigen

  • B cells spend time in the follicle area of secondary lymphoid organs

  • If a B cell recognizes a TD antigen in the follicle, its BCR signaling will cause it to move toward the T cell region of that organ

  • Simultaneously, a T cell in the same organ has recognized the same antigen (multiple copies of the antigen are present simultaneously) and its TCR signaling tells it to move toward the follicle

B Cell Activation

• B Cell activation by TD antigens - THE B CELL IS NOT ALWAYS PRODUCING ANTIBODIES

  • The B cell presents the antigen T_helper Cell

  • The cognate B and T cell interact for several hours

  • The B cell presents the antigen to the T_helper Cell

  • The cognate B and T cell interact

  • This interaction causes some proliferation of B cells

  • Some of these cells are short-lived plasma cells that start making antibody of the IgM isotype

  • The cognate T and B cells develop a germinal center in the follicle ~48 hours after they see each other

  • In the Germinal Center

    • Somatic Hypermutation

    • Isotype Switching

B Cell Activation and Somatic Hypermutation

• B Cell Activation by TD antigens

  • In the Germinal Center, B cells begin proliferating in what is called the ‘dark zone’

  • These B cells turn on expression of any enzyme called AID

  • AID is important in Somatic Hypermutation

• Somatic Hypermutation

  • AID = Activation Induced Cytidine Deaminase

  • AID deaminates (removes an amine group from) Cytosine, converting it to Uracil, specifically in the immunoglobulin gene loci

  • When the DNA replicates next, the Uracil base is complementary base paired with A instead of the G that would have been present if AID hadn’t acted

  • This generates mutations at 100,000 times the expected rate in immunoglobulin gene loci

  • The mutant offspring cells have BCRs that are altered by a few random amino acids bc of these mutations

  • These altered BCRs may be better, worse, or the same at recognizing their antigen

  • The pool of B cells with slightly altered BCRs migrate to the light zone of the germinal center

  • They have a competition to try to recognize their antigen, which is presented by T_helper and follicular DCs

  • There is a limited amount of antigen

  • Only the best BCRs with the highest affinity will get it

  • B cells with BCRs that don’t manage to compete for antigen will die

  • Macrophages will eat up and dispose of dead B cells

  • The B cells that leave this process are better at binding their antigen than the original B cells

  • This is called affinity maturation and is due to somatic hypermutation

B Cell Activation and

• B cell activation by TD antigens

  • In the Germinal Center

    • Somatic Hypermutation

    • Isotype switching

Antibody Isotypes

• there are 5 different classes of heavy chains

• Each class is known as an isotype

  • IgG, IgM, IgA, IgD, IgE

  • IgM and IgD are the most common

• There are 5 different classes of heavy chains

• each class is known as an isotype

• Each isotype has different properties

• IgG: Most prevalent isotype in serum, critical roles in pathogen defense

• IgM: First isotype produced after B cell activation

• IgA: isotype especially important in secretions (including breastmilk)

• IgD: Mystery isotype - what is its function?

• IgE: Isotype especially important in responses to parasites, worms, and during allergies

Isotype Switching

• Recombination of heavy chain initially does not recombine the constant region genes that determine isotype

• IgM and IgD are produced initially bc they are the first TWO constant region genes next to the VDJ region

• Therefore, mature naive B cells produce IgM and/or IgD BCRs and antibodies

• In the Germinal Center after a B cell has interacted with…:

  • its antigen AND

  • its cognate T cell

• …it will undergo istoype switching

• Isotype switching rearranges the H chain DNA to choose a different C gene for transcription and translation with VDJ unit

• The VDJ unit does not change!

• Allows plasma cells to make different isotypes of antibody with the same specificity

• Which C gene is ‘switched’ to will depend o the cytokines in the environment

B Cell Development & Activation III (3/11) - Wk 6

Effector Function

• In the Germinal Center after a B cell has undergone (found in 2ndary lymphoid organs)

  • Somatic Hypermutation

  • Isotype Switching

• it is ready to become either a Plasma Cell or a memory cell

• The plasma cells that are produced from a germinal center

  • Are much longer lived than the ones produced before the germinal center

  • Migrate to lymphoid organs (especially bone marrow) to produce antibody

• The memory B cells that are produced from germinal center

  • are very long lived and non proliferating

  • Migrate into various body tissues

  • Reactivate to produce more plasma cells upon second antigen exposure without germinal center formation

B Cell Migration

• How do all these B cells get to where they are going

  • They use the leukocyte homing system

• Important molecules in leukocyte homing

  • chemokine receptors

  • integrins

  • selectins

• All produced on the surface of the B cell

• Chemokine receptors

  • Allow B cells to sense and respond to chemical gradients in the body

    • Respiratory burst, histamines, etc.

• Integrins & Selectins

  • These molecules adhere to other molecules expressed in various tissue types

  • B cells alter which types of these are expressed based on development, and this helps them stick to different places

Intracellular B-Cell Signaling

• Intracellular B cell signaling: What happens inside a cell when its BCR binds its antigen?

• BCRs do not function alone

• BCRs are associated with two molecules called Ig(alpha) and Igß (called together CD79)

  • sends signal to nucleus to tell what is happening on the outside of the cell

  • w/o CD79 singaling will not work and receptors will not react to antigen binding

• When BCR binds its antigen more than one BCR complex gets brought together in the membrane

• IgAlpha /Igß become phosphorylated, starting a signaling cascade on the inside of the cell

• Co-Receptors also enhance B cell signaling

• BCR co-receptors are a complex of CD19/21/81/225

  • both bind to the antigen

• When complement activated (C3dg) and the BCR is signaling, the signal is enhanced through the co-receptor

T Cell Development & Maturation (3/13) - Wk 6

BE ABLE TO COMPARE AND CONTRAST B & T CELLS FOR EXAM (Both, Similar, Diff)

T Cell Receptors

• T Cell Receptors (TCRs) are specific for a particular antigen

• TCRs are found on the surface of all T cells

• TCRs are generated using genetic recombination

  • This process is very similar to that for BCRs/Antibodies

• TCRs are made of two protein chains

  • Most T cells have 1 alpha and 1 beta chain

• Like BCRs, there is a variable region (Valpha & Vbeta) and a constant region (Calpha & Cbeta) for each chain

• BCRs could bind to lots of different kinds of biological molecules

• TCRs mostly bind to peptides - bind btw alpha & beta chains

• BCRs bind to antigens free in solution - body fluids, blood

• TCRs bind of antigens that are presented on molecules called MHCs on the surface of another cell

• TCRs DO NOT undergo processes that are analogous to somatic hypermutation or isotype switching

• REFER TO COMPARE AND CONTRAST SLIDE

Molecules on the T Cell Surface

• All T cells express CD3

  • This marker can be used in Flow Cytometry assays to identify T Cells

T cells Development and Activation II (3/17) - Wk 7

T Cell Development

• A CLP from the bine marrow enters the thymus at the cortico-medullary jucntion

• when entering the thymus, the CLP has its T cell receptor genes in the ‘germline configuration’

  • same way they were at birth

• CLPs move into the cortex and genetic recombination begins

Double Negative Cells (DN)

• Do not express CD4

• Do not express CD8

• A DN cell will receive signals to begin to rearrange TCR B locus that codes for the beta chain of the BCR gene rearangment

• RAG ½ VDJ recombinase
  

Double Positive

• both T cell receptors on the outside

  • leads to selection processes - either positive or negative

Thymic Selection

• TCRs have to interact with MHC molecules (host cell - self cell or non self pathogen)

• allows the t cell to know what is going on in another cell

• selects the best cells

postive slection is to ensure that TCRS can successfully interact with self MHC I and MHC II moleucles

• body turns on own body cells if this process does not work successfully

90% of DP T cells die at this stage

Make either CD4 or CD8, NOT BOTH DOUBLE POSITIVE

Molecules on T cell surface

• CD8 interacts with MHC I (found on all nucleated bodies)

• CD4 interacts with MHC II - MHC ii found on APCs

T Cell Development

• Positive selection in cortex of thymus with DP cells

• Negative selection in medulla of thymus with SP cells

• AIRE transcription factor - causes cells to produce proteins not usually seen in thymus

• Understand the difference btw negative selection & positive selection

• Understand diff btw the cells

T Cells III (3/18) - Wk 7

Resulting cells are

• Self MHC restricted

• Self tolerant

CLP to SP takes about 3 wks

Two activation signals

• Signal 1: MHC + Peptide + TCR - antigen specific

• Signal 2: Co stimulator pairs

• Causes changes with the cell

Co receptor - CD8 or CD4

Co stimulator molecules

• CD28, CTLA-4, CD40L, ICOS

  • interact with a partner on an APC/B cell to help stimulate the T cell

TCR complex v. CD3 complex

T Cell Activation

• DCs are the most imporant APCs for T cell activation

• DCs phagocytose antigen from body tissues and carry that antigen to secondary lymphoid organ

• DCs that recognize PAMPs with their PRRs become mature

  • Upregulate MHC II expression - APCs generate MHC

  • Upregulate Co stimulator ligands

  • Upregulates CCR7

• CCR7 is a molecule that binds to high endothelial venules at the entrance to the lymph nodes

  • Causes DCs to go into the lymph nodes when they’ve seen a PAMP

• The mature DC with high MHC II and Co stimulatory molecules present antigen to T cells in the T cell area of the Lymph Node

• DCs that recognize PAMPs with their PRRs become mature

  • Upregulate MHC II expression

  • Upregulate Co stimulator Ligands

  • Upregulate CCR7

• How would the requirement of DC maturation prevent autoimmunity

  • recognize MHC I and MHC II properly - recognize a foreign antigen rather than own self cells

TH1: IF(lambda), IL-2, kill intracellular pathogens (viruses & bacteria), activates macrophages & Killer T cells

TH2: IL-4, IL-5, IL-13 - fight against extracellular parasites and allergens

TH17: IL 17(maintain mucosal barriers), IL 22, IL 21, fight against bacteria & fungi (extracellular)

Treg: IL 10, IL 35, & TGF-B - suppress excessive immune response

Effector cells affect another function in the body

Know each breifly

T Cells IV (3/19) - Wk 7

Activation of CD8+

• CD4+ requires 2 signals similar to CD8+

  • antigen presentation by APC
    Co-stimulatory molecules from APC

• happens in secondary lymphoid organs

• results in generation of effector CD8+ T cells

• CD8+ are very powerful and very effective

  • kind of like suicide bombers - destroys infected cell along with antigen and at times, itself.

  • Therefore, they are not good at defending against cancer cells because they can kill off 2-3 immune cells to kill only one antigen

    • body cant keep up

• Mechanisms of killing

  • FasL-Fas interactions

  • FasL binds to Fas receptor

  • expressed on mnay body cells

  • can cause apoptosis

Memory T Cells

• analogous to Memory B cells

  • formed by both CD4+ & CD8+

  • can make t cell clones for an antigen seen before

• Two types

  • Effector: migrate to peripheral tissues, particularly skin and mucosa

  • Central: circulate through secondary lymphoid organs like naive cells - keep an eye out for antigens that are filtering through the 2ndary organs

CD8+ - MHC I

CD4+ - MHC II (primarily)

Peripheral Tolerance (3/25) - Wk 8

Peripheral Tolerance

• Self-tolerance: The state of specific unresponsiveness to self-antigen

• Central tolerance: B and T cells that are self reactive are eliminated during development

What is self-reactive B and T cells are not eliminated?

• Affinity is low for self- antigen

  • can later cause problems if a self-antigen increases for some reason

  • self-antigen is not expressed in primary lymphoid organ

• Peripheral tolerance

  • takes place after T and B cells leave primary lymphoid organ

  • prevents auto-immune disease from self-reactive T & B cells that escape central tolerance

  • prevents immune responses to food & other harmless antigens

Anergy

• a state where an immune cell is present but will bot initiate a response against its antigen

• B cell sees antigen with not T cell help

  • outcome is anergy

  • no activation

• an anergic B cell will not get into the B cell follicle very well

  • soon undergoes apoptosis

• If a T cell sees its antigen w/o an macrophage or immature APC no second signal

  • outcome is anergy

  • no activation

Regulatory T cells (T_regS)

• subset of CD4+

• some Tregs develop in the thymus during T cell development

• during negative selection some self-reactive T cells are not deleted, but instead become Tregs

• Tregs produced during T cell development are called natural Tregs (nTreg)

• Some Tregs develop in the periphery after T cell development

  • called induced Tregs (iTreg)

• All Tregs will

  • upregulate expression of a transcription factor of FoxP3

  • inhibit activation of T cells by dendtrictic cells

  • produce anti-inflammatory cytokines

    • IL 10

    • TGFbeta

Pathogens: Resistance to Infectious Diseases (3/27) - Wk 8

Immunity to Viruses

• Viruses - obligate intracellular parasites

  • invade host cells and replicate within them

• Important elements

  • cytokine production

  • innate immune cells

  • complement

  • humoral immunity

  • cellular immunity

• Cytokine production

  • interferons are critical antiviral cytokines

  • Interferons alter transcriptional profiles of immune and non immune cells

  • interferons increase antigen processing and MHC I expression

• Innate Immune Cells

  • NK cells can directly kill virally infected cells

  • antibodies coat the surface of cell with viral antigens and these called in the NK cells

  • Called antibody dependent cellular cytotoxicity

Mucosal Immunity (4/7) - Wk 9

Mucosal Immunity

• Mucosal Immune System

  • Part of the immune system that is located near the mucosal surfaces of the body

  • Mucosal surfaces - lines with mucosa or mucous membranes (as opposed to surfaces lined with skin)

• Mucosa is found lining:

  • Respiratory, digestive, and reproductive systems

  • Sensory organs

• Mucosa is made up of:

  • Epithelium

  • Lamina propria

  • muscularis mucosae

• Mucosal surfaces are semi-permeable to the outside world by design

• this makes them vulnerable to pathogens

• Mucosal surfaces also need to be able to tolerate

  • non-harmful, non-living foreign antigens

  • commensal microbes

• Mucosal Immune System

  • Tissues are known as MALT (Mucosa-Associated Lymphoid Tissue)

• Tonsils and adenoids are part of MALT

  • frequently become enlarged during childhood infections

  • individuals with XLA often have tiny or non existent tonsils!

• MALT in the gut are known as GALT (Gut-associated lymphoid tissue)

  • Important GALT include:

    • Appendix

    • Peyer’s Patches

    • Lymphoid follicles (in the large intestine and rectum)

• The epithelial layer overlying GALT contains specialized epithelial cells called M cells

  • M cells contain microfolds on their surface instead of the usual microvilli for mucosal epithelial cells

  • M cells do not secrete mucus, which allows them to physcially interact closely with antigen present in their environment

• M cell take up an antigen from the lumen of the GI tract

• They transport the antigen to lymphoid cells that are present on the other side of the M cells

• Lymphocytes are restricted to the mucosal compartment through expression of these homing molecules

• Other lymphocytes are restricted to the peripheral compartment through expression of different homing molecules

• T and B cells circulate among peripheral secondary lymphoid organs after maturing

• Some instead circulate among mucosal secondary lymphoid organs after maturing go btw various MALT tissues

• Peripheral lymphocytes generally remain peripheral

• mucosal lymphocytes generally remain mucosal

• Activation of these lymphocytes occurs directly in the secondary lymphoid organ that is part of the MALT

• For EX:

  • tonsils and peyer’s patch

• If these lymphocytes are activated to become effector cells in any MALT tissue they will:

  • drain to the lymph node that drains that tissue

  • then travel thru the thoracic duct back to the circulatory system

  • use their specific homing receptors to end up in MALT tissues throughout the body

• Outcome: detection of foreign antigen in one mucosal region of the body leads to effector lymphocytes against that antigen in all mucosal regions

• T cells in the mucosal compartment of the immune system are more likely to have weird, non-conventional TCRS

  • conventional TCRS = 1 alpha chain + 1 Beta chain

  • Mucosal TCRS more likely to be I gamma chain and 1 lambda chain

• Non-conventional TCRS do no tbind to conventional MHC+Peptide antigens

• Instead bind to MHC Class IB molecules, or can bind to antigen directly

• IgA is the dominant antibody isotype produced by plasma B cells in mucosal tissues

• The mucosal immune system has to be unique bc its job is unique

• Many foreign antigens are routinely found at mucosal surfaces that we do not want an immune response against

  • food (in oral and gut mucosa)

  • Environmental airborne antigens

  • Non-self antigens from sexual activity

  • commensal microbes (at all mucosal surfaces)

• At the same time, many pathogens are introduced at mucosal surfaces that we do want an immune response against

• Oral Tolerance - the phenomenon that antigens are less immunogenic when delivered orally

  • APCs that find antigen in the context of the gut tend NOT to mature and up-regulate co-stimulatory molecules T cells for these molecules but instead induce anergy

  • Naive T cells circulating through the GALT that see their antigen are more likely to become iTregs due to cytokine milieu

  • Compounds in breastmilk can further promote oral tolerance of food antigens in human infants

Immunization I (4/8) - Wk 9

Immunizations

• Process of rendering an organism resistant to a pathogen

Introduction

• natural immune memory

  • natural infection with pathogen which you can build a memory response

• Ex: Smallpox

  • viral illness

  • eradicated in 1970s

  • 20-30% mortality rate

• Goal of vaccination - induce immune memory more rapidly and with less harm than natural infection

Smallpox

• rashes on eqyptian mummies - existed for at least 3,000 years

• Japan - smallpox demons

Varioloation

• a process of grinding up dried smallpox scabs from a smallpox patient and inhaling them or scratching them into an arm of an uninfected person

• used in China (inhalation technique) and India (cutaneous technique) to control smallpox

Edward Jenner

• English Physician

• realized that dairymaids rarely contracted small pox after bout of cowpox

• infects a milkmaid’s boy with cowpox

  • boy did not get sick

  • vacc - cow - vaccine

  • why cant we do this today?

    • ethics!

Introduction to Vaccines

• how do you induce immune memory more rapidly and less harm than natural infection

  • vaccines deliver something that causes an immune response that will be protective against later infection

Epitopes

• Antigens

  • name orginall comes from antibody generating molecules

  • also stimulates cellular immunity

• Antigens - any molecule that stimulates an immune response

  • proteins make great antigens

  • any type of biological molecule can be an antigen (or can be part of an antigen)

• Epitopes - small exposed regions on surface of antigen that T cell/B-cell receptors or antibodies interact with

Requirements fro Immunogenicity

• immunogens or antigens must be/have:

  • foreignness - have to be foreign

  • high molecular weight

  • chemical complexity

  • degradability and compatibility with host MHC molecules

Primary & Secondary Responses

• Primary Immune Response is critical in generating memory

• During the primary immune response, some immune cells are programmed to remember a specific antigen

• these cells differentiate into memory cells

• when an antigen is seen a second time you get a secondary response

Cross Reactivity

• An immunological reaction in which the immune components, either cells or antibodies, react with two molecules that share epitopes but are otherwise dissimilar

  • Ex.: Toxin v. toxoid - binds to epitopes

  • Ex.: Scarlet fever

Adjuvants

• Adjuvant - any substance that enhances an immune response against an immunogen

• Mechanism

  • increasing half life of antigens

  • increasing production of local inflammatory cytokines

  • improving antigen delivery and processing APCs

Vaccine Types

• attenuated (“live”) - contain live viral particles

  • EX. chicken pox

  • best immune response

• deactivated vaccines - contain dead viral particles

  • deactivated by heat/chemical methods

  • EX: flu, pneumonia

  • needs boosters

• conjugate (mixed)

  • EX: MMR

Danger of Adjuvents

• Allergic reactions

• Egg (flu vaccine)

• aluminum salts (DTAP, HPV, Hep B, pneumonia vaccines)

• formaldehyde (polio vaccine)

• Vitamine E, squalene (flu vaccines)

Polio Disease

• caused by poliovirus

  • spreads person to person via fecal-oral transmission

  • leads to seasonal epidemics of poliomyelitis

• Poliovirus disease

  • most cases asymptomatic

  • 25% cases minor viral illness

  • 0.5% cases acute flaccid paralysis

Polio Vaccine

• development of polio vaccine

  • first vaccine licensed in US in 1955 (inactivated)

  • live oral polio vaccine in 1960s (attenuated)

• Global Polio Eradication Initiative launched in 1988

  • India - attenuated vaccine given out

  • vaccine reverted to wild form which caused the disease

• Inactivated vaccine

• Delivered in an injection

• used in US exclusively since 2000

• Live Oral Vaccine

  • delivered orally as a solution on the tongue

  • used worldwide

Immunizations II (4/14) - Wk 10

Immunizations

• Immunization is the process of rendering an organism resistant to a pathogen

Epidemiology of Immunizations

• epidemiology - the study of disease at a population level

• Herd Immunity - a state achieved whereby transmission of an infectious disease is unlikely w/in a population bc a certain proportion of the population is immune to that disease

• R₀ - the average number of people one person will infect over the course of their disease

• R₀ - when R₀ is higher, the proportion of individuals who need to be immune to achieve herd immunity is also higher

• Herd Immunity = 1 - 1/R₀ × 100

• Measles is a childhood illness

  • caused by measles morbillivirus

  • leads to characteristic rash and high fevers

• Pre-vaccine, in the US, measles caused

  • 3 to 4 million infections

  • 400 to 500 deaths

  • 48,000 hospitalizations each year

• Measles was eliminated in the US in 2000

  • no continuous disease transmission for greater than 12 months

• Measles cases are climbing and we are at risk of losing elimination

• Measles has a high R₀ and requires high vaccincation numbers for herd immunity (~93%)

Active v. Passive

• Natural immunity - not induced by medical procedures

  • active (developed by the person themselves)

    • Ex. getting chicken pox → make own antibodies

  • Passive (transferred from another person)

    • Ex. breastfeeding → baby gets mom’s antibodies

• Artificial immunity - induced by medical procedures

  • Active (developed by the person themselves)

    • Ex. vaccine → produce own

  • Passive (transferred from another person)

    • Ex. serum/plasma transfusion

Vaccine Types

• Killed whole organisms

  • bacteria or parasites or fungi that have been killed after grown in culture

  • whooping cough vaccine

• Inactivated virus

  • doses of viruses that have lost disease causing capabilities

  • rabies virus vaccine

• Attenuated Bacteria

  • weakened versions of the true pathogen

  • strong immune response bc they can still often replicated

  • TB vaccine (BCG)

• Live attenuated Virus

  • weakened form of the virus that doesn’t’ cause disease (often genetically modified)

  • still causes immune response

  • EX. MMR

• Toxoids

  • inactivated toxin with suppressed toxicity (chemical or heat treatment)

  • antigens are maintained

  • toxoids mimic toxins secreted by bacteria

  • EX. tetanus vaccine

• Recombinant viral Proteins

  • subunits of virus or bacteria are extracted to be antigens

  • genetic code is manufactured in yeast or other cells

  • EX. Hep. B vaccine

• Nucleic Acid Vaccines

  • DNA or RNA directly given to humans

  • Instructs human cells to produce the antigen

  • COVID mRNA vaccines

• Viral Vector Vaccines

  • Harmless virus delivers genetic code to human cells

  • No licensed viral vector vaccines yet

Hazards of Vaccine Development

• Ex: RSV Vaccine

  • RSV is a respiratory disease

  • 100 to 300 children under 5 die annually in the US

• RSV Vaccine Trial in 1967

  • Vaccine mediated enhanced disease

  • 2 immunized children died of RSV

  • pathogenic Th2 memory response

• Influenza and antigen change

  • A,B, & C types

  • HxNx

  • New combinations with recombination

• HIV

  • gets into body fairly easily

  • mutates easily - high mutation lvl of surface proteins of virus

• Societal and Political Difficulties

  • lack of funding

  • misinformation

  • skepticism - fear

  • aversions - need for natural immunity

Tumor Immunology (4/15) - Wk 10

Intro to Cancer

• Cancer - a group of related diseases

  • Hallmarks

    • cells divide w/o stopping

    • cells invade surrounding tissues

• Tumor - a growth of cells from inappropriate cell division

• Malignancy - the ability to invade other tissues

• Why do tumor cells divide w/o stopping?

  • ignore cell death signals

  • inappropriate expression of telomerase

  • broken cell cycle checkpoints

  • cause blood vessel growth to feed themselves

  • evade the immune system

Immune Surveillance

• Immune Surveillance Hypothesis

  • proposed in the 1950s

    • immune system patrols body looking for aberrant, tumor-related cells and eliminates them

  • evidence for immune surveillance

    • you can successfully transplant tissue btw genetically identical hosts

    • you cannot successfully transplant tumor tissue btw genetically identical hosts, bc the host will reject the tumor

    • you can induce a cancer in a mouse mode with a carcinogen, then find tumor-specific immune memory against tumor tissue

    • individuals with immunosuppression (ie AIDS) develop higher rates of cancer than normal

      • Caveat: many of these cancers are known to be driven by oncoviruses

        • therefore, the immunosuppression could indirectly be leading to cancer formation by failure to protect against viral infection

      • failure to find ‘tumor-specific transplantation antigens’ TSTA”

        • TSTA = strongly immunogenic and considered foreign by the immune system

  • Updated Hypothesis

    • tumors likely expression aberrant antigens, not neoantigens

    • tumor associated antigens - often re-expression or tissue inappropriate expression of fetal antigens

Immunosuppression by Tumors

• Tumor associated antigens

  • most ‘driver’ mutations of cancer are invisible to the immune system

  • some tumors re-express fetal antigens

  • some tumors express antigens from other tissues

  • these antigens aren’t very immunogenic

  • they are self

  • but they can be used as targets in immunotherapy

• Immune system is involved in the desmoplastic response in tumor environment

  • formation of dense fibrous tissue around nests of tumor cells

  • could be an attempt to ‘wall off’ the tumor from the body

  • is the tumor hiding itself?

• innate immune system is called in by the destruction of normal tissue caused by invasive tumors

• Immune surveillance hypothesis has been updated to the Cancer Immunoediting Hypothesis

  • there is some recognition of tumors as aberrant, during which the immune system may act to eliminate some tumor cells

  • some tumor cells are unrecognizable to the immune system, they survive and the tumor grows

  • the tumor escapes immune control, leading to symptoms that cause patient to present to medical system

• Direct cell killing by NK cells and CD8+ T cells seems to be most important in early killing of tumor cells

  • some tumor cells downregulate MHC-1

    • can no longer identify self from non self

  • This males the NK cells think they are ‘foreign” and kill them

• Direct killing by NK cells seems to be the most important in early killing of tumor cells

  • some cells downregulate MHC-1

  • this. makes the NK cells think they are ‘foreign” and kill them

• Tumor cell have many ways of turning off the immune response

  • macrophages polarization

    • M2 macrophages are anti-inflammatory type of macrophages

    • normally used to turn off immune response once threat has passed

    • tumors can produce an enviroment that skews macrophages toward M2 phenotype, which supresses immune activity

• Tumor cells have many ways of turning off the immune response

  • PDL1/PD1 interactions with T cells turn them off

Tumor Microenviroment

• Hot v. Cold tumors

Immunotherapy

• classes of cancer immunotherapy

  • therapeutic antibodies

  • t cell checkpoint inhibitors

  • CAR-T therapy

  • Vaccination

BE ABLE TO WRITE OUT HOW TUMOR CELLS HIDE FROM IMMUNE SYSTEM

Tumor Immunology II (4/17) - Wk 10

Immunotherapy

• Cancer immunotherapy - modulating the patient’s own immune system treat cancer

• Classes of cancer immunotherapy

  • Therapeutic antibodies

  • T cell checkpoint inhibitors

  • CAR T therapy

  • vaccination

Therapeutic Antibodies

• some tumors express specific antigen at higher levels than others cells

• delivery of antibodies against these antigens can lead to antibody-dependent cellular cytotoxicity by NK cells

• Antibodies bind antigens on the surface of target cells → NK cell CD16 Fc receptors recognize cell bound antibodies → cross-linking of CD16 triggers degranulation into lytic synapse → tumor cells die by apoptosis

T-Cell Checkpoint Inhibitors

• Recall: Many tumors evade immunity by turning off T cell that approach them

• T Cell Checkpoint Inhibitors prevent T cells from being turned off

  • Many T Cell checkpoint inhibitors are therapeutic antibodies

• Some patients exhibit ‘autoimmune’ like phenomena during treatment with checkpoint inhibitors

• T cells all over the body become easier to activate

  • some patients have developed permanent autoimmune disease after conclusion of these therapies

CAR-T Therapy

• Chimeric Antigen Receptor T cells (CAR - T) Cells

  • made in the laboratory by genetically manipulating the pateints own t cells

• Scientists and clinicians design a T cell receptor that would be specific for patient’s tumor

• T cells are removed from the patient

• DNA encoding the engineered TCR is transfected into the cells

• The cells are returned to the patient

Vaccination

• Vaccination is possible against cancer causing viruses (oncoviruses)

  • HPV

• Sipuleucel-T is the only FDA approved direct cancer vaccine

  • for prostate cancer

  • educated immune system as to a prostate cancer antigen

Allergies - Intro (4/21) - Wk 11

Overview

• Exam 5 - May 13th

Allergies

• Belong to a group of immune responses called hypersensitivity reactions

• these are just regular immune responses, but too much

• lead to immune-mediated damage to the host

Hypersensitivity Reactions

• Four categories of hypersensitivity reactions

  • Type I: what we traditionally call allergies, depends on IgE

  • Types II + III: Inappropriate activation of complement cascade

  • Types IV: dependent on T cell, sometimes called delayed-typed hypersensitivity (occur days or weeks after antigen challenge)

Phases of an Allergic Reaction

• Sensitization phase

• activation phase

• effector phase

• Sensitization phase

  • IgE is produced in response to an antigen

  • the Fc region of IgE binds to receptors on mast cells and basophils

• Activation phase

  • re-exposure to antigen causes mast cells and basophils to degranulate (spill their granules)

• Effector phase

  • systemic and local responses to degranulation of mast cells and basophils

  • can include

    • rhinitis

    • asthma

    • anaphylaxis

Exam Review (April 22)

• Share what anergy is and an example using B and T cells

Allergies - Hypersensitivity Reaction Types (4/24) - WK 11

• Refer to slides bc I was lazy

Types II-IV Reactions (4/28) - WK 12

• Type II and III are immediate reactions

  • this is similar to type I

  • they can be activated within 24 hours of exposure to antigen

• Type II and III are grouped togther bc they share common features

  • mediated by anibodies (usually IgG)

    • this is in contrast to IgE mediated Type I reactions

  • Often result in activation of complement

    • some type II/III hypersensitivity reactions are examples of autoimmune diseases

      • this is in contrast to the foreign antigens implicated in Type I reactions

Genetic & Chemical Causes of Autoimmunity (4/29) - WK 12

Autoimmunity

• Failure of tolerance leads to auto immunity

• Example

  • Myasthenia Gravis

    • MG is an autoimmune disease

    • Caused by a type II hypersensitivity

    • antibodies block ACTH receptors causing muscle weakness and no muscle contraction

Genetic Factors

• Autoimmune diseases are complex in etiology

  • genetic factors

    • rarely have a single gene cause

  • environmental factors

• Twins - monozygotic (one egg) - dizygotic (two eggs)

  • mono: same genetics

  • di: diff genes, one could have autoimmune, one couldnt

• Role of MHC

Autoimmune Diseases (5/1) - WK 12

Autoimmunity - Enviromental Factors

• The mircrobiome

  • the commensal microbes that live in and on humans have an important role in educating the immune system

  • Individuals with dysbiotic microbiomes are at higher risk of autoimmune disease

  • the microbiome can mediate the risk that is observed from diet, etc.

• Sex hormones

  • many autoimmune disorder are more common in women

  • female sex hormones may contribute to this phenomenon

  • mechanism unclear

• drug exposure

  • like the Type II Hypersensitivity example from Monday

    • drug molecules bind to surface of host cells and result in formation of new epitopes that a host can raise an immune response against

    • this autoimmunity is usually transient and resolves when the drug is removed

• Abiotic environmental factors

  • environmental exposure to heavy metals increases autoimmune risk

    • possibly through formation of new epitopes, like with drug exposure

  • cigarette smoking increases risk of some autoimmune diseases

Autoimmunity

• Autoimmune diseases are complex in etiology

  • genetic factors

    • rarely have a single gene cause

  • Environmental factors

• Autoimmune hemolytic anemia (AIHA) (Type II)

  • results in immune mediated destruction of red blood cells

  • leads to anemia

    • fatigue

    • SOB

  • RBCs normally live 3-4 months

  • RBCs in someone with AIHA last a few days

  • Antibodies are produced against a person’s own RBCs antigens

  • Antibodies

    • activate complement and lyse RBCs and/or

    • opsonize the RBCs

    • induce antibody-dependent cellular cytotoxicity mediated by NK cells

• Myasthenia Gravis (Type II)

  • antibodies are produced against ACTH

  • leads to muscle weakness

    • eventual death from resp. failure

Antibody-Mediated Autoimmune Diseases

• Myasthenia Gravis (Type II)

• Graves Disease

  • results in hyperactive thyroid gland (hyperthyroidism)

  • production of too much thyroid hormones leads to

    • increased metabolism/weight loss

    • insomnia

    • hair loss

    • heat tolerance

    • anxiety/panic

    • bulging eyes

  • antibodies are produced against the TSH receptors

  • antibodies act as agonists and turn ON the TSH receptors

  • TSH receptor - put out more T3 and T4

  • (explain diff btw MG and GD)

• Hashimoto’s Thyroiditis

  • damage of the thyroid eventually results in hypothyroidism

    • production of insufficient lvls of TH

  • production of too little thyroid hormone leads to

    • weight gain

    • fatigue

    • cold tolerance

    • depression

  • antibodies are produced against two specific thyroid proteins

    • thyroid peroxidase

    • thryoglobulin

  • antibodies lead to ADCC

  • cellular responses are also impacted, with direct killing by T cells

  • when the thyroid is damaged, it will attempt to regenerate

  • this can lead to formation of a characteristic goiter

• Lupus (systemic lupus erythematosus) (Type III)

  • Immune-mediated destruction of multiple tissues/organs leads to

    • muscle and joint pain

    • rashes - butterfly rash on face

    • fevers

    • organ dysfunction (especially kidney, heart, lungs, and CNS)

  • Autoantibodies are present against a number of antigens in lupus pts

    • a lot of nuclear components

      • dsDNA

      • ribonucleoproteins

      • histones

    • why would these nuclear components be exposed?

      • during apoptosis, internal cellular components can be exposed

      • apoptosis is a normal part of tissue/organ management

      • usually, phagocytic cells efficiently clear those components

      • lupus pts often display defects in apoptotic clearance

    • development of lupus thus depends on multiple issues

      • presence of autoantibodies

      • impaired apoptotic cell clearance

      • other genetic/environmental factors that can lead to the above, as well as exacerbate them

    • antibody-antigen interactions result in deposition of antibody antigen complexes in various tissues

    • consequent inflammation leads to tissue damage

T Cell Mediated Autoimmune Diseases

• Multiple Sclerosis (MS)

  • results in destruction of the myelin sheaths that surround the CNS axons

  • symptoms

    • muscle weakness/spasms

    • difficulties with coordinates and balance

    • vision/speech/swallowing problems

  • T cells directly destroy cells in the myelin sheath

  • the antigens they are attacking are unclear/not well defined

    • they likely vary widely from person to person

  • good recent evidence that the autoimmune damage that leads to MS triggered by post-infection sequelae from EBV infections

• Type I Diabetes

  • immune mediated secretion of insulin producing beta cells in the pancreas

  • lack of insulin producing capacity leads to

    • high BS

    • frequent urination/excessive thirst

    • lack of glucose inside cells leads to breakdown of other cellular for energy → ketoacidosis

  • cytotoxic T lymphocytes are implicated in the destruction of pancreatic Beta cells in T1D

• Rheumatoid Arthitis

  • Chronic inflammation in joints leads to stiffness, swelling, and deformity

  • the inflammation occurs in the synovium - soft tissue that lines the joints

  • t cell activity is required for RA pathogenesis

Therapeutic Strategies for Autoimmune Diseases

• general suppression of the immune system

  • pharmacological

  • splenectomy

• Anti-cytokine therapies

• targeting cytokine signaling pathways

• B cell depletion

• Treg promotion - regulate the immune response

Overview of Transplants (5/5) - WK 13

Slides on canvas

did not feel like taking notes

Why so many transplants (2022)

• Reached 1 million transplants

• Rate of diseases increased

Ethics and morals in 1950s

• giving healthy organ to sick person

• questions on if this makes the healthy person sick

• questions on why you would take a healthy organ out of an individual

Why Identical twins

• identical MHC

• less chance of rejection

What did you hear Dr. Murrary share about immunosuppression and rejection

• Did not want surgeon to take the lead on

Transplant Immunology II (5/8) - WK 13

Organ Donor Matching

• Tissue typing - determining which HLA alleles are possessed by potential donor and recipients

• usually based on genetic sequencing of recipient and donor

• Functional Testing

  • incubate recipient’s serum sample with the donor’s cells to see whether an immune response is initiated

  • test for preformed antibodies in the recipient that are specific for donor antigen

Immunosuppressive Therapy

• A good match btw donor and recipient can reduce the immune response

• but immunosuppressive therapy is still generally necessary to maintain allograft survival in the recipient

• Induction therapy - starts at or before transplant, continues for abt two weeks after transplant

• Maintenance therapy - a reduced dose of immunosuppressive therapy that a pt will be on long term

• Specific treatments - used to manage episodes of acute rejection

• Inhibitors of lymphocyte gene expression

  • corticosteriods

  • cyclosporine

  • FK-506

• Inhibitors of cytokine signal transduction

  • rapamycin

  • lefluomide

• inhibitors of nucleotide synthesis

  • azathioprine

  • mercatopurine

  • chloambucil

  • cyclophosphamide

• New frontiers

  • antibody based therapies

  • Induction of Tregs

  • changes in transplant

    • co-transplant thymus/ bone marrow

HSC Transplants

• Hematopoietic Stem Cell (HSC) Transplants

  • sometimes called bone marrow transplant

• Required for pts with cancerous and non-cancerous issues with their blood that stem from their HSCs

  • Leukemias

  • Anemias

• Goal of HSC Transplant

  • restore normal hematopoiesis in the recipient

• HSC transplant pts usually have their own HSCs destroyed through radiation or chemo before the transplant

• HSC transplants can be analogous or allogeneic

  • autologous come from the pt themselves

  • for ex., a pt who has cancer and needs high dose chemo for a non-blood cancer

  • their own HSCs are taken and stored then re-transplanted after chemo

  • Allogeneic come from someone else

  • donor stem cells should be closely HLA matched

• HSC transplants are different than solid organ transplants before you are essentially transplanting the immune system itself

• recipient immune system has to be ‘removed’ through radiation or chemo

• This means less risk of rejection by the host

• instead: major risk in the other direction

  • the ‘donated’ immune system will try to ‘reject’ the whole recipeint

  • this is know as GVHD (graft v. host disease)

Being a donor for HSC transplants

• be the match

  • US based non profit

• 70% of pts who need HSC transplant don’t have a fully matched donor in their family

• be the match maintains a registry of willing donors

• peripheral blood stem cells can be used for donation

• sometimes bone marrow extraction

Xenogenic Transplantation

• 50k people who need organ transplants die annually while waiting for compatible donor

Pregnancy

• Women do NOT typically mount immune responses against their fetuses

  • why not? they contain foreign antigen

• pregnancy globally alters immune system in ways we don’t entirely understand

• placenta that contacts mom does not express MHC I or MHC II