Antibody Diversity and T Cell-APC Interactions

Question 1

B-CELL-RECEPTOR:
What are the 5 isotypes of the Constant region of an Ig chain?

Answer 1

µ, δ, γ, ε, α

Question 2

B-CELL-RECEPTOR:
How can constant and variable regions of heavy and light chains be combined?

Answer 2

-The same C(H) and C(L) region can be connected to millions of V(H) and V(L) regions

-The same V(H) can be connected to different C(H) regions

Question 3

B-CELL-RECEPTOR:
How are heavy and light chains encoded?

Answer 3

-The heavy chains are encoded together

-The light chains are encoded on different chromosomes and separately from heavy chains

Question 4

B-CELL-RECEPTOR:
What are the different region gene segments?

Answer 4

V segment - Variable
D segment - Diversity -> only in heavy chains
J segment - Joining
C segment - Constant

Question 5

B-CELL-RECEPTOR:
What is the composition of an Ig?

Answer 5

-Variable and constant region
-2 identical heavy chains [IgM (μ), IgD (δ), IgG (γ),
IgE (ε), IgA (α)] -> C regions: μ, δ, γ, ε, α

-2 identical light chains, kappa (κ) or lambda (λ)

Question 6

B-CELL-RECEPTOR:
How is the Kappa light chain locus structured?

Light chains include V, J, and C segments

Answer 6

1820 kb on Chromosome 2

-Multiple V(kappa) gene families separated by noncoding gaps
-Downstream of Vkappa genes are four J(kappa) segments
-The C(kappa) segments only have structural functions

Question 7

B-CELL-RECEPTOR:
How is the Lamda light chain locus structured?

Light chains include V, J, and C segments

Answer 7

1050 kb on Chromosome 22

-Multiple V(Lamda) gene families with noncoding gaps
-each J segment with a particular C segment

-loss of most of the Vλ segments in - only 5% of mouse Igs are lamda
-In humans 40% of light chains are Lamda

Question 8

B-CELL-RECEPTOR:
How is the heavy chain locus structured?

Answer 8

1250 kb on Chromosome 14

-Isotypes determine the type of Ig -> [IgM (μ), IgD (δ), IgG (γ),
IgE (ε), IgA (α)]

-V, D, J, and C segments -> C regions encode for the isotypes

Question 9

B-CELL-RECEPTOR:
How are gene segments recognized upon recombination?

Answer 9

-The gene segments are flanked by recombination signal sequences (RSS) -> RAG 1 and 2 slides through the DNA and recognize, cut, and join V, D, J, and C

RAG1 and RAG2 (recombination activating gene encode for RAG1 and 2 -> only in lymphocytes)

-other proteins are required too (Heptamer, Nonamer,..) -> they are not restricted to lymphocytes

Question 10

B-CELL-RECEPTOR:
What is the difference between the Kappa and Lambda light chains?

Answer 10

They are very similar, slightly different structure
-It provides variability
-Lambda serves as backup, in case Kappa doesn’t work (2 chances for Kappa bc of 2 chromosomes)

Question 11

TCR:
What are the chains that form T-Cell-Receptrors?

Answer 11

-αβ chains or gamma-delta (γδ) TCR
-αβ is preferred and attempted first, if it doesn’t work it will try gamma-delta

-most T-Cells have αβ chains; gamma-delta (γδ) TCR are less variable and often have other specific roles

Question 12

TCR:
For TCRs which one is the heavy and which is the light chain?

Answer 12

-αβ: β is the heavy chain with the D segment, α is light
-γδ: δ is the heavy chain with the D segment, γ is light

Question 13

What is the structural difference between BCR and TCR?
In terms of heavy, light chains, and the epitope binding site:

Answer 13

-BCR has 2 heavy chains and 2 light chains
-BCR has two binding sites that bind the epitope

-TCR has 1 heavy chain combined with 1 light chain
-TCR has 1 binding site for the epitope

-Recombination step (RAG1 and 2) is pretty much the same

Question 14

INCLASS QUIZ1:
Which TCR chains resemble the BCR lambda chain?

Answer 14

The lambda chain is light, so -> α and γ

Question 15

Explain the Loop mechanism of VDJ recombination:

Answer 15

RAG1/2 bring RSS sequences of gene segments together and forms a loop -> cleavage at the RSS sequence -> forms a hairpin

Artemis is messy and cuts the hairpin forming overhangs that are filled by repair enzymes generating palindromic nucleotides (sequence and reverse sequence)

Exonuclease don’t like overhangs -> they cut some nucleotides off (trimming) -> gap is filled by RAG1/2

Question 16

How does the VDJ recombination process contribute to variability?

Answer 16

Due to cleavage and hairpin formation of the gene segments, followed by random addition of nucleotides by RAG1/2

-In heavy chains (not in light chains) of BCRs and both TCR chains, Terminal deoxynucleotidyl transferase Tdt inserts non-template (N) nucleotides next to palindromic (P) nucleotides (or just between the segments)

Question 17

SUMMARY for possibilities of variability:

Answer 17

-gene segment arrengment
-for BCR: Each heavy chain can pair with a kappa or lambda?? (2 Chromosomes - 2 chances)
-Palindromic nucleotides -> by Artemis
-Exonuclease trimming
-for heavy chains of BCR and both TCR: Nontemplated nucleotides by Tdt

Question 18

In CLASS QUIZ 2:
Which enzyme is responsible for generating P nucleotides from hairpin cleavage?

Answer 18

-Artemis opens the hairpin and inserts for P nucleotides
(Terminal deoxynucleotidyl transferase Tdt for Nontemplated nucleotides -> just random nucleotides without a template

Question 19

In which process does the frameshift check occur?

Answer 19

2 chromosome copies the heavy chains and light chain

-Allelic exclusion: we do not want to express two receptors so 1 chromosome is turned OFF the other is turned ON
-> heavy chains first: for TCR β first, for BCR the heavy chain

-> if two attempts fail -> apoptosis

Question 20

What is the stepwise formation of B/T-Cell receptors?

Answer 20

-In B-cells, the heavy chain is made first and is paired with the surrogate light chain first (SLC) forming a pre-BCR -> if it works (TEST if there is no frameshift, stability test f.e.) the machinery starts again for light chain recombination

-In T-cells, the heavy β chain is formed first and paired with pre-Tα (helps stabilize it) -> if it passes the clustering process and forms a functional CD3 complex -> the α-chain recombination starts

Question 21

What is the meaning of double negative and double positive in the development of T cells mean?

Answer 21

In Corticomedullary junctions, the Cortex, and Subcapsular Cortex: Double negative
-Double negative (DN) means no CD4 and no CD8 yet
-> Recombination of TCR gene segments yields αβ or a γδ T cell
(during the process the T cells are tested to self-peptide binding - bc random gene arrangement can lead to self-peptide recognition)

-After recombination the cell double positive for CD4 and CD8

In medulla: double positive
-Positive/negative tolerance shifts the cell either to CD4 or CD8

Question 22

How is the formation of αβ or a γδ TCR ensured?

Answer 22

-With allelic exclusion -> shutting off one chromosome so that only one TCR is expressed - similar to BCR

First shot formation of β and see if it works, if not 2nd shot, if it does -> pre TCR formation with fake α, followed by alpha gen recombination and formation of alpha and check, if it works alpha will replace surrogate pre TCR-α, if not 2nd shot

Question 23

Which co-receptors (CD4, CD8) are formed during the development phase in the thymus?

Answer 23

Both are formed first, and the cell is tested for correct function at this stage -> Double Positive

-> So this is the positive selection, TEST of FUNCTION -> does it interact with any MHC? Does it interact with a peptide? (hopefully not self -> checked in NEGATIVE selection)

Question 24

Binding affinity in the selection process:

Answer 24

Positive selection: TCR binding MHC with low affinity to self-MHC are restricted
95% fail here -> don’t receive surviving signal -> apoptosis

Negative selection: TCRs binding with high affinity to self-MHC/peptide is restricted

Why do we want binding to self-MHC at all??? regardless of low or high affinity?? Why are low-affinity self-binding restricted, shouldnt that be good?

Question 25

How do Treg cells inhibit an immune response?

Answer 25

Tregs are a subset of CD4 cells:

-depletes the area of stimulating cytokines
-produce inhibiting cytokines -> inhibiting T cells
-directly kill T cells
-inhibit APCs

Question 26

How does a T-cell know if it is CD4 or CD8?

Answer 26

-Cortical thymic epithelial cells (cTECs) express high levels of MHC class I and II (exception -> epithelial cells usually express only MHC-I??)
The ones binding to loose or too tight to self-MHC are neglected

Is there a preference for each one to be tested first?? What if it can bind both?? -> both are tested at the same time, the one with lower affinity will be degraded

Question 27

What are the different paths to ensure self-tolerance, for negatively selected T-cells?

Answer 27

-Clonal arrest: no maturation, no proliferation signals (until it eventually dies)

-Clonal anergy: Inactivation

-Clonal editing: rearrangement of the T-α gene (V and J), to see if it can lower the binding affinity to self-MHC

-Clonal deletion: most common

Question 28

What happens to the next strongest self-MHC TCRs?

Answer 28

They are converted into anti-inflammatory Treg cells -> they circulate and bind self peptides to reduce autoimmune response

Question 29

What type of negative selection is taking place in the medulla?

Answer 29

After T-cells have chosen their path CD4 or CD8 and became single positive (SP) there is a second negative selection, tested against self-peptides

Self-peptide presentation from dendritic cells expressing MHC-I for
CD8 T cells and MHC-II for CD4 T cells

Self-peptide presentation from different tissues (TCR have only seen thymus peptides yet) by mTECs expressing AIRE

Question 30

IN class QUIZ 3: After maturation in the thymus, which organs do these naive T cells go to?

Answer 30

To the secondary lymph organs:
-> Lymph nodes, Spleen, MALT

Question 31

What are other Thymocytes?

Answer 31

-NK- Cells: No TCR
-NKT cells: express anti CD1 TCR (not as variable, doesn’t change that much) -> similar to TCR but binds to non-self lipids

• γδ T cells: also not very variable, important for fetal immunity, often reside within epithelial barrier as intraepithelial lymphocytes (IELs)

-Treg: CD4+ subset that helps to quench adaptive immunity