Immunology Exam 2

What are the three functions of the innate immune system?

1. Physical and chemical barriers (skin, mucus, stomach acid)

2. Cellular responses to infection (macrophages, neutrophils, NK cells)

3. Activation of the adaptive immune system (through antigen presentation via MHC)

Why is the MHC necessary?

T cells cannot recognize free antigen.

They only recognize: Peptide + Self MHC

Without MHC:

• No T cell recognition

• No T cell activation

• No adaptive immune response

What are the similarities between the BCR and TCR (particularly as they related to the MHC?)

Similarities:

• Both are antigen-specific receptors

• Both are part of adaptive immunity

• Both recognize peptides derived from proteins

What are the differences between the BCR and TCR (particularly as they related to the MHC?)

 Major Difference:

• BCR → Recognizes free antigen directly

• TCR → Only recognizes antigen when presented on MHC

👉 TCR is MHC-dependent
👉 BCR is NOT MHC-dependent

What is (self) MHC-restriction?

T cells will only recognize: Foreign peptide presented by self MHC molecules

They will NOT respond to:

• Free antigen

• Peptide on non-self MHC

On which cell types (in general) are MHC molecules expressed? On which cell types are TCRs expressed? (this isn’t a trick question)

MHC Expression:

• MHC Class I → All nucleated cells

• MHC Class II → Antigen Presenting Cells (APCs)

TCR Expression:

• Only on T cells

What are the components (including subunits) of the MHC proteins? How are the similar to each other? How are they different?

MHC Class I:

• 1 α (alpha) chain

• 1 β2-microglobulin (β2m)

MHC Class II:

• 1 α (alpha) chain

• 1 β (beta) chain

How are the MHC proteins similar to each other? How are they different?

Similarities:

• Both have peptide-binding grooves

• Both present peptide to T cells

• Both contain alpha chains

Differences:

• Class I has β2-microglobulin (non-MHC encoded)

• Class II has both α and β chains encoded in MHC region

• Class I presents to CD8

• Class II presents to CD4

What is the cell surface expression profile for each MHC protein? (i.e. on which cells are MHC class I expressed? MHC class II?)

MHC Class I → All nucleated cells

MHC Class II → APCs (dendritic cells, macrophages, B cells)

 What is the binding groove? Why is it important?

• Region of MHC molecule where peptide sits

It Binds peptide backbone and Binds amino acid side chains (R groups)

importance:

• Determines which peptides can bind

• Determines what T cells can recognize

 In general, what is the process of antigen presentation?

1. Protein is made (intracellular) or taken up (extracellular)

2. Protein is degraded into peptides

3. Peptide binds MHC in binding groove

4. MHC-peptide complex moves to surface

5. TCR recognizes complex

6. T cell becomes activated

What is the central dogma of biology?

• DNA → RNA → Protein (Replication → Transcription → Translation)

Allele

Different versions of a gene

Allotype

Protein product of an allele

SNP

Single nucleotide polymorphism (one base change)

Co-dominance

Both maternal and paternal alleles are expressed

Haplotype (including syngeneic and congenic)

Set of linked genes inherited together

• Syngeneic → genetically identical

• Congenic → identical except for one locus

Polygeny

Protein encoded by multiple genes

Polymorphic/Polymorphism

Many different alleles exist in the population

How are the MHC genes organized? What does HLA mean?

Located on: Chromosome 6 (short arm)

In humans called: HLA (Human Leukocyte Antigen)

MHC Complex 1

Encodes:

• HLA-A

• HLA-B

• HLA-C
  (These are MHC Class I molecules)

MHC Complex 2

Encodes:

• HLA-DP

• HLA-DQ

• HLA-DR
  (These are MHC Class II molecules; each has α and β chains)

MHC Complex 3

Encodes complement proteins and other immune proteins (not antigen-presenting molecules)

 You should understand the HLA designations

• HLA-A → Class I

• HLA-DP, DQ, DR → Class II

• Class I → one gene, one chain

• Class II → two genes (α and β)

Why is it important that MHC genes are co-dominant?

• You express MHC from BOTH parents

• This increases peptide presentation diversity

• Improves immune protection

How are MHC genes inherited?

• Inherited as haplotypes

• One from mother

• One from father

• Expressed co-dominantly

What is meant by MHC diversity? Why is it important? What is it and why is it important? 

Large variation in MHC genes within a population. Allows population to:

• Present many different pathogens

• Avoid extinction from one infection

What is MHC promiscuity? (How are diversity and promiscuity related?) 

• Ability of MHC to bind many different peptides.

• Class II = more promiscuous

• Class I = more specific

• Diversity + promiscuity = broader immune protection.

What is the heterozygote advantage? 

Having two different MHC haplotypes;

• Increases peptide presentation range

• Improves survival

What is balancing selection? 

Maintains multiple alleles in population. (MHC genes are under balancing selection.)

What is directional selection?

• One allele becomes dominant over time.

What are MHC anchor residues? Why are they important? Are they more important to MHC class I or MHC class II? How is that related to promiscuity?

Specific amino acids in peptide that:

• Fit into pockets of MHC binding groove

• More important for:
  👉 MHC Class

• Why:

• Class I binding is more specific

• Class II is more flexible (more promiscuous)

How does the MHC help activate T cells?

The MHC:

• Displays peptide to TCR

• Allows T cell to recognize infection

• Initiates T cell activation

• Triggers adaptive immune response

No MHC → No T cell activation.

What are the two major compartments of the cell?

• Cytosol + Nucleus

• Vesicular System

  • ER

  • Golgi

  • Endosomes

  • Lysosomes

  • Exocytic vesicles

  • Contiguous with extracellular environment

Why are the two major components of the cell kept separate?

• They perform different biochemical functions

• Separation maintains proper protein folding, degradation, trafficking

• Prevents inappropriate mixing of proteins/enzymes

• Energy is required to move material between them

How is material passed from one compartment to another?

• Via membrane transporters

• Via vesicular trafficking

• Via ATP-dependent transport (e.g., TAP)

What is the relationship between MHC class I and endogenous antigens?

• MHC Class I presents endogenous (intracellular) peptides

• Found on all nucleated cells

• Presents to CD8⁺ cytotoxic T cells

Endogenous = proteins made inside the cell
• Self proteins
• Viral proteins
• Tumor proteins

How does antigen presentation work under healthy circumstances?

Healthy circumstances: 

• Many proteins misfold (~30%)

• Degraded via ERAD (Endoplasmic Reticulum-Associated Degradation)

• Broken down by proteasomes

• Self peptides loaded onto MHC I

• Displayed on surface

• Do NOT activate CD8 T cells

This is normal immune surveillance

 How does antigen presentation work under under pathogenic circumstances?

Pathogenic Circumstances 

• Viral or abnormal proteins made in cytoplasm

• Degraded into peptides

• Loaded onto MHC I

• Presented to CD8 T cells

• DO activate cytotoxic T cells

What is the difference between self antigen and non-self antigen?

Self Antigen

• Normal host proteins 

• Presented regularly 

• Does not activate T cells 

Non-self Antigen

• Pathogen-derived proteins 

• Presented during infection

• Activates T cells 

What is presentation of self-antigen? 

Normal display of host peptides on MHC

Why does it happen? Is it bad? Or just a normal process?  

• Continuous protein turnover

• Immune surveillance

• Not bad!- completely normal but only problematic if tolerance fails (autoimmunity)

What is ERAD?

ERAD = Endoplasmic Reticulum-Associated Degradation

• Misfolded proteins in ER

• Sent back to cytosol

• Degraded by proteasome

• DO NOT activate CD8+ cytotoxic T cells

What is the proteasome/immunoproteasome?

• Proteasome: Degrades intracellular proteins into peptides

• Immunoproteasome: 

• Specialized form

• Produces peptides better suited for MHC I binding

What causes the switch between the two?

• IFN-γ (Interferon gamma)

• Cytokines can have multiple effects → pleiotropy

What is a constitutive proteasome?

The normal, baseline proteasome present in healthy cells.

In what cellular compartments do various stages of MHC class I presentation take place?

Step	Location
Protein degradation	Cytosol
Peptide transport	Across RER membrane
MHC assembly	Rough ER
Peptide loading	ER
Surface expression	Plasma membrane

What is the role of TAP (1/2)?

Tap= Transporter associated with antigen processing

• ATP-dependent heterodimer

• Transports peptides from cytosol into ER

• Essential for MHC I loading

Deficiency → Bare Lymphocyte Syndrome (very low MHC I expression)

What is the function of calnexin?

• ER chaperone protein

• Stabilizes MHC I α-chain

• Ensures proper folding

• Holds MHC I until β2-microglobulin binds

What are the components of the peptide loading complex (PLC)? What are their functions?

Tapasin; what is peptide editing?

• Links MHC I to TAP

• Performs peptide editing

• Ensures only high-affinity peptides bind

ERp57- PLC

• Chaperone

• Assists in folding and disulfide bond formation

Calreticulin- PLC

• Stabilizes MHC I complex

• Part of chaperone system

(MHC Class I and β2M)- PLC

Structural components 

What is ERAP( endoplasmic reticulum aminopeptidase 1)? What is its function?

• Trims peptides inside ER

• Adjusts peptide length (8–10 aa optimal)

• Ensures proper MHC I binding

What is immunodominance?

• Not all peptides are presented equally.

• The immune system preferentially responds to a small number of dominant peptides, even though many are generated

What is cross presentation of self-antigen? Why does it happen? Is it bad? Or just a normal process? (In general), what’s the purpose of cross-presentation?

• Antigen presenting cells present exogenous antigens on MHC Class I

• Purpose:
  ➡ Activate CD8 T cells against pathogens that do NOT infect APCs directly
  ➡ Critical for antiviral and anti-tumor responses

What are the three major antigen presenting cells? Which one initiates adaptive immunity?

1. Dendritic Cells: initiates adaptive immunity 

2. Macrophages 

3. B cells 

What is the role of the dendritic cell in antigen presentation?

• Capture antigen

• Activated via PRRs

• Phagocytose pathogen

• Migrate to lymph node

• Present antigen to naïve T cells

• Initiate adaptive immunity

What is the relationship between MHC class II and exogenous antigen?

• MHC II presents exogenous (extracellular) peptides to CD4⁺ helper T cells

• Found on professional APCs

How does exogenous antigen enter cells?

• Phagocytosis 

• Pinocytosis 

• Receptor-mediated endocytosis 

Processed in endocytic/lysosomal pathway

What are the purposes of the invariant chain? (Which two problems does it solve?)

•  Prevents MHC II from binding endogenous peptides

• Directs MHC II to endosomal compartment

What is CLIP? 

CLIP = Fragment of invariant chain

• Temporarily occupies peptide groove

• Prevents premature peptide binding

What is HLA-DM? What are their purposes/functions?What is HLA-DM? What are their purposes/functions?

• Removes CLIP

• Facilitates loading of exogenous peptide

• Acts like peptide editor for MHC II

Which family of molecules are responsible for lipid antigen presentation? How are those molecules similar to MHC class I? MHC class II? To what cell type do those molecules present?

Family: CD1 molecules

Similar to:

• Structurally like MHC Class I

• Processing resembles MHC Class II pathway

Presents to:
➡ NKT (Natural Killer T) cells

What are the ‘important’ T cell responses?

CD8+ Cytotoxic T cells 

• Kill infected cells

• Release perforin & granzymes

CD4+ Helper T cells 

• Activate macrophages

• Help B cells make antibodies

• Coordinate immune response

Be familiar/comfortable with the general biology review content

DNA → RNA → Protein

• Replication = DNA → DNA

• Transcription = DNA → RNA

• Translation = RNA → Protein

Antigen receptors follow this general process after recombination occurs.

What are the general characteristics of Antigen Receptor Diversity (The How’s and Why’s)?

• Immune system must recognize pathogens it has never encountered

• Up to 100 million different receptors

• Each lymphocyte has one unique specificity

• Antigen selects the lymphocyte → Clonal selection theory

Adaptive receptors are NOT directly germline encoded.

What are the potential dangers in receptor diversity?

1. Autoimmunity – self-reactive receptors may form

2. Nonproductive rearrangements – out-of-frame joins

3. Chromosomal translocations

4. Severe defects → SCID (if RAG defective)

What are the four (three) types of receptor diversification?Note: I realize that the title of the slide says four, but there are only three discussed in this lecture. The fourth (somatic hypermutation) does not occur during development, but during the B cell response. It will be discussed in a forthcoming lecture

Occur during development:

1. Somatic Recombination (VDJ recombination)

2. Combinatorial Diversity

3. Junctional Diversity
   

Occurs later (B cell response):

4. Somatic Hypermutation (not during development)

 How is expression of antigen receptor genes similar to ‘typical’ gene expression? How are the two different?

Similar:

• Transcription

• RNA splicing

• Translation
  Different:

• Requires DNA recombination first

• Functional gene does not exist until rearranged

• Only lymphocytes express recombination enzyme

What region of the immunoglobulin protein (or T cell receptor) is influenced by antigen receptor genetics?▫ For the sake of clarity, the answer is the variable regions of both heavy and light chains (of the BCR) and of both α and β (of the TCR). Specifically it’s the hypervariable regions, or CDRs (in both the BCR and TCR).

The Variable Regions:

• Heavy chain (VH)

• Light chain (VL)

• TCR α and β chains

Specifically:
➡ Hypervariable regions (CDRs)

These form the antigen-binding site.

 make sure you’re familiar with the general structure of the antibody

Antibody = 2 Heavy Chains + 2 Light Chains

Each chain contains:

• Variable region (V)

• Constant region (C)

Heavy chain also contains:

• Diversity (D) segment

What are the differences between membrane-bound and soluble immunoglobulin?

Membrane-bound	Soluble
BCR	Antibody
Has transmembrane region	No membrane anchor
Involved in signaling	Effector function

Difference is due to alternative RNA splicing, NOT recombination.

 What is the purpose of Igα and Igβ?

Purpose:

• Signal transduction

• Contain ITAMs

• Required because antibody itself cannot signal

Equivalent concept to CD3 in T cells.

What regions do the light chains contain? What regions do the heavy chains contain? What is the purpose of the leader (L) sequence?

Light Chains (κ or λ):

• V

• J

• C

• Leader (L)

Heavy Chains:

• V

• D

• J

• C

• Leader (L)

Leader (L) Sequence:

• Directs protein to ER

• Required for secretion or membrane expression

What is the RAG1/2 complex?

Recombination Activating Genes

• Enzyme complex

• Mediates V(D)J recombination

• Recognizes RSS

• Randomly selects segments

Defect → SCID

What are recognition signal sequences? (noncoding sections of DNA recognized by RAG1/2)? What are its components?

Non-coding DNA sequences flanking gene segments.

Contain:

• Heptamer (7 bp)

• Spacer (12 or 23 bp)

• Nonamer (9 bp)

 What is the 12/23 rule? What is its purpose?

Recombination only occurs between:

• One 12 bp spacer RSS

• One 23 bp spacer RSS

Purpose:

• Ensures correct segment pairing

• Prevents D-D or V-V joining

What is combinatorial diversity?

Random pairing of:

• Heavy chains + Light chains

Heavy and light chains are:

• On different chromosomes

• Assembled independently

This multiplies diversity.

What is junctional diversity?

Occurs at V(D)J joining sites.

Mechanism:

• DNA hairpin cleavage

• Random nucleotide addition

• Exonuclease trimming

Creates enormous variability.

What is terminal deoxynucleotidyl transferase (TdT)?

• Adds random nucleotides

• Major contributor to diversity

• Can cause out-of-frame joins

What is Artemis?

• Opens DNA hairpins

• Required for proper joining

What is allelic exclusion? ▫ How does it happen? (what is the general mechanism, i.e. how does it relate to productive rearrangements?)

Each B cell expresses:

• ONE heavy chain allele

• ONE light chain allele

Ensures:
➡ Single specificity

Mechanism:

• If productive (in-frame) rearrangement occurs → stop further rearrangement

• If nonproductive → try second allele

▫ Using this same mechanism, why-in the above bullet point-are most light chains κ as opposed to λ?

Order:

1. κ rearranges first

2. If successful → stop

3. Only if κ fails → λ rearranges

Therefore:
➡ Most antibodies are κ

How does the heavy chain variable region attach to the heavy chain constant region? ▫ (splicing vs. recombination)

• VDJ recombination creates variable region.

• Attachment to constant region:
  ➡ Via RNA splicing, NOT recombination

What is the significance of the order of the constant regions? What role might alternative splicing play?

• Constant regions arranged sequentially:
  IgM → IgD → IgG → IgA → IgE

• Closest constant regions are expressed first:
  ➡ Naïve B cells express IgM and IgD

• Alternative splicing determines:

• Membrane vs secreted form

• IgM vs IgD expression

What is an αβ T cell?

αβ T Cells

• Majority of T cells

• Recognize peptide + MHC

• Need successful β chain rearrangement first

What is a γδ T cell?

• Recognize unconventional antigens

• Barrier immunity

• Must successfully rearrange γ and δ

• Less common

What are the similarities/differences between B cell receptor and T cell rearrangement?▫ Remember that the two are largely the same (particularly the actual recombination part), but there are some differences that are reflected in the functions of B cells and T cells

Similarities:

• V(D)J recombination

• RAG dependent

• Junctional diversity

• Allelic exclusion

Differences:

BCR	TCR
Can be secreted	Always membrane-bound
Undergoes somatic hypermutation	Does not
Class switching	No class switching
Effector function via antibody	Effector via cytokines/killing

What are the components of the TCR complex/CD3 complex? What are the functions of the TCR/CD3 complex?

Components:

• TCR α chain

• TCR β chain

• CD3 γ

• CD3 δ

• CD3 ε

• ζ chain

Functions:

1. Transport TCR to surface

2. Signal transduction

3. Contains ITAMs

Receptor diversity comes from:

1. V(D)J recombination

2. Heavy/light pairing

3. Junctional nucleotide addition

Controlled by:

• RAG

• RSS

• 12/23 rule

• TdT

• Allelic exclusion

Purpose:
➡ Generate millions of unique receptors
Risk:
➡ Autoimmunity or SCID if defective

What are the tenets of the clonal selection theory?

1. Each lymphocyte expresses one unique antigen receptor before antigen exposure.

2. Antigen selects (does NOT create) specific lymphocytes.

3. Binding → clonal expansion.

4. Activated clones differentiate into:

   • effector cells

   • memory cells.

5. Self-reactive lymphocytes are eliminated or inactivated

Why are tenets relevant to this discussion?

B cell development generates huge receptor diversity first, then selects useful and safe clones through:

• checkpoints

• positive/negative selection

What are differences between stem cells and stromal cells?

Stem Cells	Stromal Cells
Hematopoietic origin	Bone marrow support cells
Become immune cells	Provide developmental environment
Differentiate	Support differentiation

In what way are stromal cells supportive? (Physical contact/soluble factors)

• Physical contact

• Soluble cytokines

What are the important examples of each? (SCF/Kit and IL-7)

• SCF (Stem Cell Factor) ↔ Kit receptor

• IL-7 → promotes B cell survival & proliferation

Pro B-Cell

• Heavy chain gene rearrangement begins:

  • D → J recombination

  • then V → DJ

• RAG enzymes active.

Pre-B Cell

• Heavy chain successfully produced.

• Forms Pre-B Cell Receptor (pre-BCR).

• Proliferation occurs.

• Light chain rearrangement begins.