Unit 2 Immunology - Adaptive Immunity

How did Elie Metchnikoff’s studies on starfish larvae and rabbits lead to the discovery of phagocytosis?

Metchnikoff observed starfish cells engulf tangerine tree thorns, noted as defense mechanism

he observed WBCs in rabbits phagocytose the bacterium B. anthracis

Kitasato and von Behring used serum from which animal to confer immunity against which infection? What in the serum mediates immunity?

Scientists used the serum of immunized horses. The serum contained antibodies against tetanus toxin

What was correct about Ehrlich’s side-chain theory? What was he wrong about?

Ehrlich proposed that cells have nutrireceptors (nutrient uptake) - like side chains on cell surface. Toxins would match side chain and bind, blocking the receptor function → cell breaks off and releases receptor (incorrect) → cells produce and release more receptors to bind toxins → receptors in blood called antitoxins

Helper T cells recognize antigens presented on which type of surface glycoprotein? How?

APCs like dendritic cells have MHC II on their cell surfaces, which associate with pathogen antigens and present them on the cell surface. The dendritic cells present antigens to helper T cells (CD4+): MDC MHCII + antigen → CD4+ T cell TCR → T cell recognizes antigen

CD proteins (cluster of differentiation) are cell surface proteins that function like cellular ID badges. What do CD3 proteins do?

Multi-protein complex that conducts intracellular signaling after the TCR recognizes an antigen

Follicle vs. Paracortex: Where are they located, and what cells are located there?

In lymph nodes, the follicle is the B-cell zone, and the paracortex is the T-cell zone

Where do APCs encounter naïve B cells and T cells and present foreign antigens to them?

Lymph nodes

What are the zones of the spleen that house B cells and T cells? What is the difference between white and red pulp?

Follicles - B cells, Periarteriolar lymphoid sheath (PALS) - T cells. White pulp is rich in lymphocytes (including PALS), while red pulp filters blood and removes damaged red blood cells (contains RBCs, WBCs, platelets, etc.)

Where do immature B cells undergo maturation into functional B cells?

Spleen - for b cells to → antibody-producing plasma B cell or memory B cell, must meet antigen and help (?) from helper T cell

• naïve B cell BCR + antigen → internalize, display peptide → MHC II + peptide antigen → matching TCR binds → T cell CD40 ligand binds to B cell CD40 receptor → T cell releases cytokines → B cell activated

Which receptors on NK cells help recognize vs. kill healthy or infected cells? Which glycoproteins do NK cells recognize?

NK cells recognize MHC I on healthy cells when their inhibitory receptors bind to the MHC I. Oppositely, infected/cancer cells stop expressing MHC I - NK cells’ activating receptors bind to ligands on infected cells that signify stress → NK triggers response to kill cell

MHC I is typically for ____ or ________ antigens. MHC II is for ________ antigens acquired by ___________

MHC I - self or internal antigens (on healthy cells)

MHC II - external antigens acquired by phagocytosis (APCs), used to present antigens to adaptive immune cells

Which genes encode MHC proteins?

Human leukocyte antigen (HLA) genes

Add NK card from last deck

MHC I is a protein complex made up of two proteins: what are they called, which is larger, and between which chain’s domains is the peptide-binding groove?

The two main proteins are the ⍺ heavy chain protein, which is made up of five domains (⍺1, ⍺2, ⍺3, transmembrane domain and cytoplasmic domain), and the β-2 microglobulin protein.

• The peptide-binding groove is between ⍺1 and ⍺2

• the β-2 microglobulin protein stabilizes the peptide binding groove

MHC II is also made up of two proteins, ⍺ and β. What are the main differences between MHC II and MHC I?

MHC I is made up of an ⍺ heavy chain protein (⍺1-⍺3), while MHC II is composed of an ⍺ chain and a β chain, each made up of 4 domains (⍺1, ⍺2), (β1, β2), a transmembrane domain, and a cytoplasmic domain.

Where is the peptide-binding groove in MHC I? MHC II?

Between ⍺1 and ⍺2 in MHC I, between ⍺1 and β1 in MHC II

Of the MHC II domains, which two have more conserved amino acid sequences? Which are more variable?

The AA sequences of the ⍺1 and β1 domains are more variable than the conserved, constant sequence of the ⍺2 and β2 domains

What is the purpose of a chaperone protein? Which chaperones bind to MHC I vs. MHC II?

Chaperones stabilize the MHC’s, holding them together and helping them sample peptides. Tapasin stabilizes empty MHC I and selects a high-affinity peptide that binds tightly to MHC I.

HLA-DM does the same but for MHC II.

MHC’s will degrade without chaperones.

What is peptide editing, and what do MHC’s require to conduct peptide editing?

Chaperones are required for peptide loading and editing, or selecting a high-affinity peptide that fits the binding groove

Once it’s fully assembled, the DM chaperone removes what from the MHC II binding groove so an antigenic peptide derived from a phagocytosed and digested microbe can replace it?

CLIP - class II-associated invariant chain peptide

What role does the DM protein play in MHC II presenting antigens to immune cells?

DM matches a specific peptide antigen with a compatible MHC II molecule → transported and placed on APC surface

What are activating molecules/ligands?

Self-proteins that get upregulated during stress, infection, or cancer cell formation. These ligands are upregulated on the cell surface → trigger NK cell activation

What are the major types of mature B and T cells? How many of each?

B cells: Memory B and plasma B cells

T cells: Helper T cells (CD4+, subtypes Th1, Th2, and Th17), Cytotoxic T cells (CD8+), NK T cells, 𝛄δ T cells, Regulatory T cells, memory T cells

Describe thymic selection, including positive and negative selection

CLP cells in bone marrow → thymus, differentiate into ɑβ or 𝛄δ. the latter migrate to the skin or mucous membranes AFTER negative selection, while ⍺β differentiate into CD4 and CD8 cells

• ⍺β thymocytes can also differentiate into pre-NKT cells- they don’t interact with MHC- double pos → double negative, don’t express CD4 or CD8 once they recognize CD1 → activated NKT cell

Positive selection:

doubly positive thymocyte → TCR matches an antigen from MHC II → receive signals to shut off CD8 expression → CD4+ helper T cell

doubly positive thymocyte → TCR matches an antigen from MHC I → receive signals to shut off CD4 expression → CD8+ cytotoxic T cell

Negative selection:

T cell screening: cells that recognize self-antigens → apoptosis, while those that don’t migrate to the spleen and lymph nodes

T cell receptors are made up of which 2 pairs of proteins? These define the two types of ________ T cells (based on their TCR structure)

⍺β or 𝛄δ proteins, immature T cells (thymocytes)

thymocytes are temporarily double-positive

APCs are required for T cell activation of ____ T cells. How do helper T cells and cytotoxic T cells use CD4 and CD8 to bind antigens?

Helper T cells and cytotoxic T cells use CD4 and CD8 as co-receptors that grab onto regions of MHC II and MHC I, respectively.

Describe T cell activation, including the difference between helper T and cytotoxic T cell activations

T cell activation: Helper T cells use TCR and CD4 to recognize and bind peptide antigens displayed by MHC II on APCs. Cytotoxic T cells use CD8 instead and bind antigens displayed by MHC on abnormal host cells

Major families of cells surface CD proteins*

Complement control proteins i.e. DAF, CD55: degrade C3 convertases/block MAC assembly on host cells

C-type lectin: PRRs that detect carbohydrates like NAG and mannose (pathogens)

TM4 and TM7: number of membrane-spanning alpha helices in transmembrane proteins

What are the two arms of adaptive immunity?

antibody-mediated (humoral) and cell-mediated

How does CD28 on helper T cells binding to CD80/86 (B7) on APCs result in a co-stimulatory signal for full T-cell activation?

Helper TCR binds to a MHC-peptide + C28 binds to B7 → cytokine production → full T-cell activation

What’s the main difference between ⍺β TCRs and 𝛄δ TCRs?

⍺β TCRs - more specialized binding capacity, MHC-restricted (recognize antigens presented by)

𝛄δ TCRs - flexible and MHC-independent; can bind free-floating, nonpeptide antigens i.e. lipids

𝛄δ T cells recognize lipids, bacteria, and other pathogens. What is the main cytokine the T cells produce, and which CD protein does it recognize on stressed cells?

IL-17: pro-inflammatory cytokine, can sometimes help tumors and trigger autoimmune diseases

BTN3A1 (CD277): marker of stress on cell, presented → 𝛄δT cells flood with granzyme → destroyed

𝛄δ T cells can act as secondary APCs → present antigens to ⍺β T cells - link adaptive and innate immunity

Which cells display peptides to thymocytes during thymic selection, and in which area of the thymus?

Cortical thymic epithelial cells (cTECs) present MHC I and MHC II to display self-peptides to thymocytes (positive selection) in the CORTEX, while medullary APCs eliminate self-reactive T cells by triggering apoptosis (negative selection) or conversion into regulatory T cells in the MEDULLA

Goldilocks model: many lymphocytes die by neglect → rest undergo positive selection → last step: undergo negative selection (2-5% of all thymocytes pass)

Regulatory T cell functions

High affinity of CD4+ regulatory TCR for self → Tregs release IL-10 and IL-35 (anti-inflammatory cytokines) → inhibit T cell function

Release granzymes and perforin to lyse rogue T cells

• if a treg and an effector T cell recognize same antigen, Treg inhibits or destroys that T cell

Express high levels of CD25 (IL-2 receptor) → deprive T cells of IL-2 → cytokine deprivation → decreased inflammation due to T cell death

Targeting dendritic cells- Treg receptors bind to DC, inhibit DC function (displaying unhelpful peptides)

S epidermidids card

What are the main differences between CD4 and CD8 T cell activation?

CD8 T cells: cytosolic protein antigen uptake by APC → processing (cleaving into smaller peptides) through proteasome → peptides associated with MHC I (aided by chaperone Tapasin) → vesicle transports MHC I to cell surface → CD8 protein and TCR attach to MHC I expressed on surface

CD4 T cells: extracellular protein antigen uptake by APC → processing (cleaving into smaller peptides) through endosome → peptides associated with MHC II (lysosomal proteases cleave antigen and invariant chain) → vesicle transports MHC II + invariant chain to cell surface → CD4 protein and TCR attach to MHC II expressed on surface

How do cTECs and medullary APCs pick antigens for T cell training?

cTECs: limited pool of peptides from many housekeeping proteins

Medullary APCs: large, diverse pool of peptides from tissue-specific proteins

What transcription factor enables medullary APCs to sample tissue-specific proteins and present them to T cells for negative selection?

Autoimmune regulator (AIRE) turns on the expression of tissue-specific genes.

Less than 1% of peptides generated by proteosomes are loaded onto MHCs- need to be right size and charge

THE CORN DOG MODEL: what’s the corn dog, and what’s the hot dog- which one corresponds to intracellular tumors or viral antigens? Bacterial, fungal, or parasitic antigens?

The MHC I binding groove attaches to intracellular tumors and viral antigens- includes only HYDROPHOBIC amino acids. The peptide backbone and terminal amino acids are enclosed within the MHC binding pockets- CORN DOG!

The MHC II binding groove attaches to bac/fungi/parasitic antigens- includes a terminal aromatic amino acid and 3 other amino acids (hydrophobic/polar/charged and one characteristic to MHC II type) - peptide backbone extends past MHC binding pockets- HOT DOG!

MHC I and CD1 are structurally similar. What’s the main difference between the two proteins?

CD1 displays fragments from LIPIDS, not peptides (contains ⍺1, ⍺2, ⍺3, and β2m chains)

What about NKT cell receptors allows them to recognize lipids presented by CD1?

NKT cells have semi-invariant ⍺β TCRs that recognize lipids (mostly hydrophilic heads that protrude from CD1) presented by CD1 better than antigens presented by MHCs

Pathogens are often coated in lipids and produce non-protein molecules, so CD receptors on APCs allow the immune system to recognize them

How are NKT cells activated after interacting with APCs?

Foreign lipids with unique hydrophilic heads or orientations w/in CD1 → NKT cell activation

Detection of MAMPs by TLRs → cytokine production → increased display of abnormal self-lipids on CD1

Perforin and granzymes

Released by cytotoxic T cells and NKT cells. Perforin makes pores in cell membranes → granzyme enters, triggers apoptosis

Innate NK cells vs. NKT cells

NK cells look for activating surface ligands and lack of MHC I - NKT cells ignore MHC I, look for lipids on CD1, can be stimulated to attack by IL-12 signaling

What are the main types and functions of helper T cells?

Th1: activates cytotoxic T cells, recruits macrophages, and targets viruses and intracellular bacteria

Th2: activates B cells and recruits eosinophils, targets parasites

Th17: recruits neutrophils; targets extracellular bacteria and fungi

Cross-presentation of exogenous antigens by dendritic cells allows for exogenous antigens to be displayed where?

Normally MHC II, but cross-presentation allows them to be displayed on both MHC I and MHC II- trains CD8+ t cells to be on the lookout for extracellular antigens

BCRs bind __ segments of antigens (__) while TCRs bind ______ peptides displayed on MHCs

3D segments from MHC II on APCs, linear peptides that come from denaturing/cleaving proteins

BCRs and antibodies bind epitopes with specific geometric structures

T/F: B cells are APCs

True! B cells internalize and denature protein antigens → display LINEAR peptides on their MHCs → train T cells

What are the main 3 signals to activate B cells?

1. BCR + antigen

2. B cell CD40 binds to CD40L on Th cell

3. B cell recognizes cytokines secreted by helper T cell

*Membrane-bound BCR vs. antibody

BCR includes light chain, heavy chain, spacer, hydrophobic segment, cytosolic segment, and Ig⍺ + Igβ - secreted form has light + heavy chains + hydrophilic segment

Central tolerance selects against _________ immature B cells through clonal deletion (apoptosis)

high-affinity autoreactive BCRs, or autoreactive immature B cells. if they pass, immature B cells migrate from the bone marrow to the spleen via blood (transitional B cell) → mature naïve B cell

What are the gene segments that get recombined to code for BCRs and antibodies?

Variable, diversity, and joining gene segments at the heavy chain locus, V and J segments at light chain loci. RAG recombinase recognizes recombination signal sequences by the V, D, + J and randomly recombine the sequences to generate unique antibodies → V(D)J recombination

Which transitional type immature B cells in the spleen express B cell-activating factor? How does this affect negative selection?

Unlike transitional type 1 (T1) cells, which are vulnerable to apoptosis if they have autoreactive BCRs, T2 cells are in the follicles of the spleen and express B cell-activating factor receptor (if non-autoreactive) → survival signal

*Ig⍺/Igβ function

Transmembrane proteins bound to BCRs - BCR signaling: lipid rafts btwn adjacent BCRs where Lyn kinases are concentrated. BCR binds antigen → BCR moves to lipid rafts rich in lipids → kinase phosphorylates Ig⍺/Igβ → signal transduction pathway

C3d is a product of complement protein C3b, which is an opsonin that breaks down microbes. How does C3d contribute to the lowering of the amount of antigen needed to activate a B cell?

C3d binds an antigen and surface receptor CD21

C3d + antigen + CD21 → increased phosphorylation of CD19 → lowers B cell activation threshold → B cell activation → cytokine production and kinase activation → B cell survival, proliferation, and differentiation

B cell internalization process

antigen + BCR → endocytosis → proteolysis → MHC II + peptide in MHC II-enriched compartment (MIIC) once CLIP is replaced by antigen → MHC II + antigen expressed on surface

Kitasato and Behring discovered antibodies that protected against tetanus and diphtheria in what of animals?

Blood serum of animals injected with the diphtheria or tetanus toxins

Ehrlich’s side-chain theory: nutrireceptors, side chains on the cell surface, would bind to foreign toxins based on matching structures. What happened to receptors once toxins were bound?

Toxins would block receptor functions, so the cell would release the receptor → produce and release more receptors to bind toxins → into bloodstream, antitoxins

Antibodies are made up of the variable and constant regions on both the heavy (inner layer) and light (smaller, outer layer) chains. What regions are located at each end of the antibody?

The fragment of antigen-binding (Fab) region is located at the top (Y-opening, hinge) of the antibody, while the fragment crystallizable (Fc) region is at the end of the antibody where the heavy chain extends- effector activity.

• Fab region diversit in binding site shape, specifically at the V_H and V_I regions

Which antibody is most common in serum and is involved in placental transfer? Which antibody activates the classical complement pathway?

1. IgG- gamma/y shaped

2. IgM (and IgG, but less)

IgM first responder post-infection, can bind to multiple antigens due to pentameter shape. IgG more specialized function, released second.

Trimming and insertion of nucleotides in what region of which type of gene segment creates more antibody diversity?

CDR3 region of diversity (D) gene segment - the size of the spacer differentiates Ig configurations

Central vs. peripheral tolerance

central tolerance: bone marrow → spleen, where peripheral tolerance occurs: more negative selection, T1 → T2 if non-autoreactive BCRs → T2 + BAFF → survival signals → mature B cell

Fibroblastic reticular cells, subcapsular sinus macrophages, and follicular dendritic cells

Germinal centers form 4-7 days post-antigen exposure, where B cells migrate to the follicle center. Why are mutations intentionally introduced in the GC (to produce what?), and what is the process called?

B cells rapidly grow and divide in the GC → expression of activation-induced cytidine deaminase (AID) enzyme → switches C to U in DNA → U:G mismatches → U’s removed → new mutations → called somatic hypermutation (SHM)

• AID enzyme performs SHM to introduce mutations in antibody-coding genes- variable (V) gene segments (CDR1 and CDR2 hotspots vs. CDR3 in D segment)

What are the main downsides of T-cell-independent activation of naive B cells?

The TI activated plasma B cells are short-lived, they produce lower-affinity antibodies (not via SHM), doesn’t promote long-term memory