Diversity vs. Specificity - 9/3

Question 1

Immunoglobulins

Answer 1

(Ig) = antibodies

• all Abs are made by a single cell clone that has a unique (specific) antigen specificity
• 2 identical heavy chains, 2 identical light chains

Superfamily: Immunoglobulin IgM, Iga/b, T-cell receptor, HLA molecules

Question 2

Clonal Selection

Answer 2

• Gene rearrangement events occur in absence of antigen, this is called clonal selection.
• occurs in bone marrow or thymus. You make all of the B and T cells, they are released into peripherty, you are exposed to antigen and the antigen selects the clone specific for it. You hope you are exposed to a pathogen you have made a clone that the antigen can select.
• a clone is a lymphocyte of one specific specificity (pre-cursor) and its progeny. 10^7-10^11 different clones
• Clonal Selection Process:

1. Lymphocyte clones mature in generative lympoid organs (bone marrow and thymus), in absence of antigens
2. clones of mature lyphocytes specific for diverse antigens enter lympoid tissues
3. Antigen-specific clones are activated (“selected”) by antigens
4. Antigen- specific immune response occurs

Question 3

Major Mechanisms for Generation of Antibody Diversity

Answer 3

Primary Immunoglobluin Rearrangment:

1. Combinatorial Diversification: Multiple germ line genes: (have two copies, paternal and maternal): V-J or V-D-J recombinations
2. Junctional Diversity: Addition of nucleotides during process of D-J or V to DJ joining

Secondary Immunoglobulin Rearrangement:

   3. Somatic hypermutation: point mutations occuring in             fully assembled V-J and V-D-J regions during an                 immune response. Provides a significatnt source of            Ab diversity, occurs in periphery during immune                   response.

Question 4

Hematopoeisis

Answer 4

IL-3: Pluripotent Stem Cell –> Lympohoid Progenitor

• Made by T cells, multi-lineage cytokine

IL-7: Lymphoid Progenitor –> B lymphocyte or T lymphocyte

• necessary for survival of T cells
• Made by BM stromal cells, fibroblasts

Question 5

Heavy Gene organization

Answer 5

• located on chromosome 14 = VDJ
• Constant (C) genes: present for each class and subclass of immunoglobulin. Each C gene is composed of several exons, one for each domain and another for the hinge region
• Variable (V) region genes: composed of a leader and V exon. (about n=100)
• Joining (J) exons
• Diversity (D) exons (about n=23)
• this is not present on light chains
• all of the exons are separated by introns

Question 6

Kappa Chain Gene organization

Answer 6

• chromosome 2= VJ
• Constant region gene: Kappa light chain contains only ONE C region (this is because there is only one type of kappa light chain)
• V region genes have a leader exon and a V exon
• Several Joining (J) exons located between the V and C genes
• All exons are separated by introns

Question 7

Lambda Gene organization

Answer 7

• chromosome 22 = VJ
• C region genes: composed of 4 C region genes, one for each subtype of lambda chain
• upstream of each C gene there is an additional J (Joining) exon
• V region genes (n=30)
• eavh V region genes is composed of two exons, one (L) coding for leader and (V) coding for variable region
• The L, V, J and C exons are all separated by introns

Question 8

Allelic Exlusion

Answer 8

• both maternal and paternal chromosomes are present in the antibody genome
• however, they express the rearranged heavy-chain genes from only ONE chromsome, and the rearranged ligh-chain genes from only ONE chromosome
• This ensures that functional B cells never contain more than one V_HD_HJ_H and one V_LJ_L unit.

*** this is necessary for antigenic specificity- expression of both alleles would render the B cell multispecific.

Question 9

Making a Heavy Chain

Answer 9

1. one D region, joined with one J region, then one V gene is rearranged to the D/J region
2. Gene becomes transcriptionally activated because promoter is brought close to enhancer
3. pre-mRNA is spliced in the nucleus and reamining introns are removed- bringing the VDJ next to one of the C exons
4. mRNAs are translated in cytoplasm . The heavy chain is assembled with a light chain in the ER and the Ig is secreted via secretory proteins

Question 10

mechanism of DNA rearrangments on Heavy Chain

Answer 10

• Recombination Signal Sequences (RSS)- flank the V,J,D exons and function in recombination
• Rag1 and Rag2- are recombination enzymes that catalyze the recomination event. They pull together the RSS’s that flank the V,J,and D exons and cut them, resulting in the exons being removed.

Junctional Diversity:

• between the cleavage site of D and J there is a hairpin cleavage, resulting in a sticky end. Random Nucleotides are inserted by Terminal deoxyribonucleotidyl transferase (TdT) without need for a template. This creates Junctional Diversity.
• P NT’s: added to asymmetrically cleaved hairpins in templated manner
• N NT’s: added in a non-templated manner

–> This leads to further diversity in the thir hypervariable region (Idiotype)

Question 11

Pre-B Receptor

Answer 11

• A complex of Igu heavy chain and surrogate (imposters) light chains with Ig-alpha and Ig-beta
• Rearrangement must be successful from pro-B to pre0B in order for cell survival: heavy chain must work - bone marrow checks it for proper 3-D confimation.
• If successful the pre-B cell cell will proliferate (only 1 in 3 have successful rearrangment)
• if unsuccessful = apoptosis

Question 12

Making a Kappa light chain

Answer 12

• no D region
• KAPPA IS ALWAYS MADE BEFORE LAMBDA
  1. one of the V genes is joined with one fo the J genes and introns are removed in between.
  2. gene is transcriptionally active
  3. Pre-mRNA is spliced in nucleus and introns are removed, resulting in mRNA with contiguous VJ and C exons
  4. mRNA is translated in cytoplasm and transported to the ER
  5. light chain is assembled with a heavy chain in the ER and the Ig is secreted bia route of secretory proteins.

Question 13

Receptor Editing

Answer 13

• nonproductive kappa light chain gene rearrangements can be rescued by further gene rearrangement.
• if the first is non-productive:
• 5’ Vk gene segment can recombine with an unused 3’ Jk gene segement to replace it
• can theoretically happen up to five times for each joining region, however if it grabs the fifth joining region on the first try there are no others, so it moves onto Alpha chain
• if none of the kappa recombinations work, then it moves onto the lambda chain.

Question 14

Alternative Splicing of Heavy Chain mRNA: Cμ & Cδ

Answer 14

1. The pre-mRNA is processed (spliced) in the nucleus and the remaining introns, including those between the exons in the C genes, are removed. The pre-mRNA can be processed in two ways, one to bring the VDJ next to the Cμ gene and the other to bring the VDJ next to the Cδ gene. The resulting mRNAs have the L, V, D, J and Cμ or Cδ exons contiguous and will code for a µ and a δ chain, respectively.
2. mRNAs are translated in the cytoplasm and the heavy chain is assembled with a light chain in the endoplasmic reticulum and the immunoglobulin is secreted via the normal route of secretory proteins.

Question 15

Immature B cells: BCR

Answer 15

• BCR: SIgM & Iga & Igb
• Immature B cells are “checked” by stromal cells by being presented bits and pieces of self (HLA)
• “Negative selection” - if immature B cells react acidly with self-Ags, then they are targeted for deletion by apoptosis
• “Positive Selection” - if they bind with low avidity, clones are selected for release into the periphery

Question 16

Pseudogenes

Answer 16

• accumulated mutations: sometimes these mutations are obtained from junctional diversification, and prevent it from encoding functional proteins (i.e. added frameshifts, nonsense, stop codons)
• pseudogenes have many rearrangements, significant number will be nonfunctional.
• these rearrangements are important though because they provide combinatorial diversification, and 10^11 different antibodies are present
• only 12.5% of lymphocytes actually make it to the end

Question 17

Junctional Diversity

Answer 17

• Somatic recombination (VDJ joining), results in the addition of N and P nucleotides - the addition of random nucleotides is the largest contribution to diversity at the V/D, D/J, or V/J segments.
• this diversity occurs at the “hypervariable region”
• the recombination is not always perfect and can result in frameshifts.
• However, these inaccuracies can triple the diversity of what your own genetics are capable of producing

Question 18

Clonal Selection (Again!)

Answer 18

The process of gene rearrangement of the heavy and light chains and the combinatorial association of these chains occurs during B cell development in the bone marrow and is independent of antigen.

Clones of B cells expressing all of the possible antibody specificities are produced during development and antigen simply selects those clones which have the appropriate receptor. The selected clones are then activated, proliferate and differentiate into antibody secreting plasma cells.

Question 19

Secondary Rearrangment Diversity

Answer 19

Somatic Hypermutation:

• Introduction of point mutations in the V regions of the Ig genes resulting in an expansion of the antibody repertoire to generate high-affinity antigen-specific antibodies.
• 10^3 to 10^4 times higher than normal spontaneous mutation rates

CD40:CD40L required for affinity maturation: T-dependent Ag

And then MAGIC HAPPENS!

Question 20

Story Time

Answer 20

Kid playing dress up in closet…..

• can only choose from mom’s closet or from dad’s closet. (germline)
• makes decision to go to moms: goes in and shuts the door, once he has made the decision to go in, he can’t go back (allelic exclusion)
• Puts on a shirt at random (D gene), puts on bloomers (J gene), puts on shoes at random (V gene) = DJV
• Goes to look in mirror (IgM and surrogate light chain being checked by stromal cells for confirmation in space: 3-D check)
• Goes to Dad’s closet to make a real light chain (allelic exclusion)
• puts on Dad’s vest at random and goes to check himself in mirror. (immature B cell with heavy chain on surface which works, and a light chain that has been rearraranged, V and J, must be checked by stromal cells)
• Vest doesn’t cut it, he is sent back in to change. (Receptor Editing, occurs on light chain only)
• Comes out with a tie, and he looks good. (Does a last check for self, if responds to self it is deleted, if not it is released into periphery as a mature naive B cell )

Question 21

SCID

Answer 21

= SevereCombined Immunodeficiency- “Bubble Kids”

• can be due to mutations in the RAG genes, thus could not rearrange germline for both B and T cells (3-4% of SCID cases)
• 50% are due to X-linked mutations on cytokine receptors IL-7
• affected patients usually present with life-threatening opportunistic infections in early infancy, it is lethal if tehy do not receive a bone marrow transplant

Question 22

X-linked Agammaglobulinemia

Answer 22

• B cell development pathology
• “Bruton’s Agammaglobulinemia”
• Bruton Tyrosine Kinase (Btk) delivers signal from Pre-BCR for survival of cell
• recurrent infections: Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus, enteroviruses, Giardia, etc.