LEC 7-9

The variable region is encoded by … that each encode only a part of the Ig domain

gene segments

What is somatic DNA recombination?

the gene segments are rearranged during the development of each lymphocyte to form a complete and unique variable region coding sequence (unique antigen receptors)

What does the V gene segment encode?

CDR1 and CDR2 loops, and a part of CDR3 loop

What is the CDR3 loop formed by?

end of V gene segment and the beginning of the J gene segment

What encodes all of the IgH chains, Ig L lamba and Ig L kappa chains?

three separate loci

What is found at the 5’ end of each Ig locus?

cluster of V gene segments

What is found at varying distance from the 3’ end of the V gene segments?

several J segments

What is between V and J segments in the H chain?

D segments

What is found at the 3’ end of J segments?

C (constant) region genes

What is the difference in the C region genes between kappa and lamba?

Lamba → 4 C lamba regions between J segments

Kappa → single C kappa region at the 3’ end of J

What is the sequence of events of the formation of V regions?

Somatic recombination - starts with germline DNA, D-J rearranged + DNA joined

Transcription - V-J or V-DJ joined rearranged DNA

Splicing - primary transcript RNA (introns are removed)

Translation - mRNA formed

Polypeptide chain - fully assembled Ig heavy and light chain

D segment must join … segment first before joining …

J segment

V segment

What is combinatorial diversity?

antibody diversity arising from different combinations of V, D and J segments

(only V, J in light and V,D,J in heavy chains)

How is the rearrangement of V, D and J gene segments is guided by what?

by flanking DNA sequences called recombination signal sequences (RSSs)

What is the structure of a RSS?

segment (V/D/J) - heptamer - 23 OR 12 base pair spacer - nonamer

What is the 12/23 rule?

Gene segments can join only when one has a 12-bp RSS spacer and the other has a 23-bp RSS spacer.

What recognizes the 12/23 RSS and brings the appropriate gene segments together during V(D)J recombination?

V(D)J recombinase

How are V and J segments joined?

the intervening DNA is deleted and the V and J segments are ligated together

What is synapsis in V(D)J recombination?

Bringing selected coding segments and their RSSs together by chromosomal looping event

What is the signal vs the coding joint?

• Coding joint: Contains the coding segments (V and J, or V, D, and J in theheavy chain). Thesesegments are joined together to form the functionalvariable-region sequence of the antigen receptor.

• Signal joint: Contains the recombination signal sequences (RSSs), which arejoined together and usuallyexcised as a circular piece of DNA.

What lymphocyte-specific enzyme creates double-stranded DNA breaks at the RSS-coding sequence junctions?

V(D)J recombinase

V(D)J recombinase is composed of which two molecules?

RAG 1 and RAG 2 (recombination activating gene)

RAG genes are expressed only in …

developing B and T cells

RAG protein is inactivated in …

proliferating cells

After the formation of ds breaks, … must be opened at the coding junctions where … may be added or removed from coding wend to create an even greater diversification. The broken coding ends and signal ends are brought together and ligated by ds break repair process that is found in all cells called …

hairpins

nucleotides

non homologous end joining (NHEJ)

Which proteins bind to the ds breaks?

Ku70 and Ku80

What is the role of Ku70 and Ku80?

recruits catlytic subunit DNA dependent protein kinase (DNA-PK)

What is the role of DNA-PK?

it phosphorylates and activates Artemis

What is the role of Artermis?

its an endonuclease that opens up the hairpins

After the hairpins are open which lymphoid specific enzyme adds nucleotides to broken DNA ends?

terminal deoxynucleotidyl transferase (TdT)

What ligates/joins the DNA ends?

DNA ligase IV and XRCC4

What is the end result of VDJ recombination?

imprecise coding joint and precise signal joint

What is the difference between combinatorial diveristy and junctional diversity?

combinatorial diversity uses different V, D, J segments

junctional diversity uses different N and P nucleotides used in each clone

What happens when DNA hairpins form during V(D)Jrecombination?

Heptamer signal sequences are ligated to form a signal joint. The coding ends remainas hairpins.

Which enzymes open the DNA hairpins, and what does it produced?

The Artemis–DNA-PK complex opens the hairpins at random positions, producing single-stranded DNA ends.

The single stranded ends may have complementary nucleotides forming short …

palindromic sequences (P - nucleotides)

What are N-nucleotides, and which enzyme adds them?

N-nucleotides are random, non-templated nucleotides added to the DNA ends by terminaldeoxynucleotidyl transferase (TdT).

What happens when the two coding ends cometogether?

The single-stranded ends pair by complementary base pairing where possible.

What happens to unpaired nucleotides at thejunction?

removed by exonuclease

How is the final coding joint formed?

DNA synthesis fills gaps, and DNA ligase seals thebackbone, producing a coding jointthat may retain P- and N-nucleotides.

How does junctional diversity increase antibodydiversity?

Random addition and removal of nucleotides at V, D, and J segment junctions create unique DNA

sequences, increasing antibody variable-region diversity.

Why can junctional sequences help identify individual B-cell clones?

Each B-cell clone can have a distinctive V(D)J junctional sequence, which can serve as a molecular marker for tracking that clone.

… and … are derived from the same pre-mRNA transcript a and are both expressed on the surface of mature B cells

IgM and IgD

How can a mature B cell express both IgM and IgD without undergoingclass-switch recombination?

The B cell produces one primary RNA transcript containing both Cμ and Cδ. Through alternative RNA splicing, VDJ is joined to either Cμ to produce IgM or Cδ to produce IgD. The DNA remains unchanged.

How does a B cell produce membrane-bound versus secreted IgM from the same heavy-chain gene?

Through alternative RNA processing. Membrane-bound IgM mRNA retains the M1 and M2 exons, which encode the membrane anchor. Secreted IgM mRNA uses an upstream cleavage/polyadenylation site, excluding M1 and M2 and producing a soluble antibody.

TCRalpha locus is roughly homologous to … - V and J segments only

Ig Light chain

TCRbeta locus is roughly homologous to … additonital D segments

Ig Heavy chain

The germline organization of the human TCR alpha and beta loci takes place in the …

thymus

What are T-cell receptor excision circles (TRECs), how are they formed, andwhat can they be used as amarker for?

small circular DNA molecules formed when DNA is excised during TCR gene rearrangement in developing T cells in the thymus.

They serve as markers of newly produced T cells that have left the thymus.

How are TRECs used in newborn screening for Severe Combined Immuno deficiency (SCID), and what does a very low TREC level suggest?

Newborn screening measures TREC levels in blood. Very low or absent TRECs suggest reduced or absent new T-cell production and may indicate SCID or another condition affecting T cells.

BCRs bind directly to …

epitopes on proteins, carbs or lipids via CDRs in variable region

Where can BCRs recognize antigens?

soluble antigens or antigens on cell surfaces

TCR recognize …

peptide fragments found on MHC

What is MHC restriction?

a T-cell receptor (TCR) recognizes an antigen only when the antigen fragment is presented on the correct MHC molecule.

CD4 → MHC II

CD8 → MHC I

Example: A CD8⁺ T cell will recognize a viral peptide when it is presented on MHC I, but it will not recognize that same peptide presented on MHC II.

BCRs and TCRs are generated by …

V(D)J recombination

• combinatorial diversity (random V, D, J assembly)

• junctional diversity (P- and N- nucleotides)

BCRs are formed by … and … chain pairing

heavy, light

TCRs are formed bu … and … chain pairing

alpha, beta

What is the outcome of making multiple BCRs and TCRs?

each with unique antigen binding site

What are the two mai nfunctions of activated effector T cells?

1. expand cohorts of effector T cells

2. activate their effector functions at sites of antigen encounter

How are antigens concentrated to initiate immune responses?

Captured at their site of entry and transported to secondary (peripheral) lymphoid organs, throug which naive T cells constantly circulate.

What do T cells recognize, and how are antigens presented to them?

T cells recognize short peptide antigens displayed on MHC molecules on cell surfaces (cell-associated antigens), not soluble, cell-free antigens.

How do MHC molecules present antigens to different T-cell classes?

Display antigens from different cellular compartments to different classes of T cells.

What are the two key properties of antigens recognized by T cells?

• Most T cells recognize only short peptides.

• CD4⁺ and CD8⁺ T-cell receptors recognize peptide antigens displayed by MHC molecules (MHC restriction).

What is antigen processing?

the generation of peptides from native proteins

What are the two caterogies of major intracelulular compartments, seperated by membranes?

1. cytosol

2. vesicular compartments (involved in endocytosis and secretion)

How are antigens from the cytosol and vesicular compartments processed and presented?

• Cytosol → MHC I → CD8⁺ T cells: Intracellular proteins are broken into peptides, transported into the ER, and loaded onto MHC I for presentation to CD8⁺ killer T cells.

• Vesicular compartments → MHC II → CD4⁺ T cells: Proteins taken up from outside the cell by endocytosis are broken into peptides in vesicles and loaded onto MHC II for presentation to CD4⁺ helper T cells.

Which cells express MHC I?

almost all somatic cells and immune cells, except RBCs

What does it mean by direct presentation via MHC I?

Direct presentation means a cell presents peptides from proteins inside itself on its own MHC I molecules.Example:

1. A virus infects a cell.

2. Viral proteins inside the cell are broken into peptides.

3. The peptides are loaded onto MHC I.

4. MHC I displays the peptides on the cell surface for recognition by CD8⁺ killer T cells.

Cytosolic pathogens are microbes that…

replicate in the cytosol or contiguous nuclear compartment (nucleus)

Cytosolic pathogens are degraded in the …

Peptides bind to …

Presented to …

Effect on presenting cell …

cytosol

MHC class I

Effector CD8 T cells

cell death

Intravesiscular pathogens are microbes that …

survive and replicate in membrane-bound vesicles (lysosomes or endosomes). NOT in cytosol or nucleus. remain inside host vacuoles after phagocytosis

Intravesicular pathogens are degraded in …

Peptides bind to …

Presented to …

Effect on presenting cell …

endocytic vesicles (low pH)

MHC II

effector CD4 T cells

activation of macrophage to kill intravesicular bacteria and parasites

Extracellular pathogens and toxins are microbes that …

proliferate outside of cells

Extracellular pathogens and toxins are degraded in …

Peptides bind to …

Presented to …

Effect on presenting cell …

Endocytic vesicles (low pH)

MHC II

Effector CD4 T cells

activation of B cells to secrete Ig to eliminate extracellular bacteria/virus/toxins

DCs take up … antigens, process them and load peptides onto … for presentation to …

exogenous

MHC I

CD8 T cells

What is cross presentation?

The dendritic cell takes up material from outside itself (an infected cell) but presents those peptides on MHC I → CD8 T

why is it called that: because normally MHC I (intracellular) and MHC II (extracellular)

What is the purpose of cross-presentation?

Because even though infected viral cells can present viral peptide on MHC, they can only present them to ACTIVATED CD8 T cells.

Cross-presentation allows dendritic cells to activate naïve CD8⁺ T cells against viruses and tumours that do not directly infect the dendritic cells, enabling the resulting effector T cells to kill the affected cells.

Why are infected somatic cells unable to activate naive CD8 T cells?

because they lack co-stimulatory molecules (ex: B7) that are needed to activate them

What is cross presentation especially important in?

anti-viral CD8 T cell responses (when virus DOES NOT infect dendritic cell) and anti-tumour immunity

Another exception is autophagy which moves … and … proteins into … which are then degraded in … and peptides get loaded onto …

cytosolic and nuclear proteins

vesicles

lysosomes

MHC II

What is the outcome of autophagy?

CD4 T cells can recognize antigens (self ags, viral ags) that woudn’t normally reach MHC II → boosts antimicrobial defense

What are the two exceptions in antigen presentation?

1. Autophagy

2. Cross-presentation

What happens in autophagy, what is the antigen source, what is it presented on, what does it activate?

Cytosolic/nuclear proteins are shuttled into vesicle → degraded by lysosomes

self proteins, intracellular pathogens

MHC II

CD4 T cells

What happens in cross-presentation, what is the antigen source, what is it presented on, what does it activate?

Exogenous antigens (from dead/infected cells) loaded onto MHC I by DCs

Virsues infecting non-APCs, tumours

CD8 T cells

primes naive CD8 T cells (cross-priming)

What is protein degradation in the cytosol carried out by?

proteasome

What is a proteasome?

a large, multicatalytic protease complex that generates most MHC I presented peptides

What is the proteasome made up of?

20S catalytic core (cylindrical complex)

two 19S regulatory caps one at each end

What is the function of the 20S core?

breaks proteins down into peptide fragments

What do the two 19S regulatory caps do?

one binds and delivers proteins; the other prevents premature peptide/protein exit

How are cytosolic proteins tagged for degradation?

by ubiquitin in the ubiquitin-proteasome system

What is the purpose of the ubiquitin-proteasome system?

degrades cytosolic proteins into short peptides for processes including MHC I presentation

What are the 5 steps of the ubiquitin-proteasome system?

1. ubiquitination

2. proteasome degradation

3. TAP transporter

4. Loading onto MHC I

5. Immune recognition

How are target proteins tagged for degradation?

several ubiquitin molecules attach to the target, forming a polyubiquitin chain

What does a K48-linked polyubiquitin chain signal?

targets the attached protein for proteasomal degradation

What is ubiquitin, and what does K48 mean?

Ubiquitin is a small protein, attaches to other ubiquitins by linkage at lysine (K) residues at position 48 → forms polyubiquitin chain on the target protein

What occurs in step 2 - proteasome degradation?

cuts proteins into small peptides (8-10 aa)

MHC I is 8-10 aa

During an infection what does IFN-y turn proteasome into?

immunoproteasome

What is the role of immunoproteasome?

makes peptides better suited for MHC I presentation

What is the role of TAP transporter in step 3?

Tap 1 and Tap 2 (transporters associated with antigen processing) transport peptides from the cytosole into the ER before binding to MHC I

In step 4 - mechanism of peptide processing and loading onto MHC I molecules, what are the four events that occur?

1. retention in the ER

2. peptide-loading complex forms

3. peptide delivery

4. stabilization and exit