Lymphocyte Development

Question 1

What are the phases of B Cell Development?

Answer 1

Phase 1) antigen receptor expression (somatic recombination & allelic exclusion)

Phase 2) elimination of self-reactive cells

Phase 3) terminal antigen-stimulated differentiation

Question 2

Describe the BCR.

Answer 2

• Immunoglobulin
• Y-shaped: light chain and heavy chain
  
  • variable regions of light and heavy chains make up the two antigen binding sites
  • constant region of light and heavy chain
• Can be membrane-bound or soluble

Question 3

Where does B cell development occur?

Answer 3

Primarily in the bone marrow

Question 4

How is diversity introduced to the light chain locus of Ig?

Answer 4

• SOMATIC RECOMBINATION
• variable region (V and J regions) of light chain germline chromosome is in a segmented form that cannot be expressed as it stands
• one gene from each region are combined to form final variable region of light chain
• results in combinatorial diversity (320 different combinations)

Question 5

How is diversity introduced to the heavy chain locus of Ig?

Answer 5

• SOMATIC RECOMBINATION
• variable region (V, D, J regions) of heavy chain germline chromosome is in a segmented form that cannot be expressed as is
• one gene from each region are combined to form final variable region of heavy chain
• results in combinatorial diversity (10,000 different combinations)

Question 6

How many Ig types can be made through somatic recombination (combinatorial diversity) and junctional diversity?

Answer 6

combinatorial diversity: 3.3 million different antibody combinations

total # possible Ig: 1x10¹⁴

Question 7

Desribe V(D)J recombinase enzyme.

Answer 7

• machinery used in somatic recomination: binds to recombination signal sequences in DNA
• enzymes of DNA recombination and repair found in all cells
  
  • DNA ligase IV, TdT
• lymphocyte-specific components
  
  • RAG-1 and RAG-2 complex

Question 8

What happens in DNA cleavage and repair in the immunoglobulin loci?

Answer 8

• RAG complex creates hairpin and cleaves
  
  • P (palindromic) nucleotides
• Tdt randomly adds nucleotides
  
  • N (non-templated) nucleotides
• adds factor of 3 x 10⁷ to overall diversity
• creates Junctional Diversity

Question 9

What is allelic exlcusion?

Answer 9

• B Cell Receptor
  
  • inhibition of heavy chain recombination
  • results in one Ig specificity per B Cell
• T Cell Receptor
  
  • inhibition of beta chain recombination
  • ensures T cell expresses only one TCR beta chain

Question 10

What are the steps to Ig gene rearrangement & antigen receptor expression?

Answer 10

1. heavy chain VDJ rearrangement
2. expression of heavy chain w/ surrogate light chain to test functionality
3. if successful, heavy chain rearrangement stopped (allelic exclusion)
4. proliferation of pre-B cells
5. light chain VJ rearrangement
6. light chain expressed w/ heavy chain to form final BCR

Question 11

What happens at the pre-B cell stage of B cell development?

Answer 11

• pre-B receptor is expressed: tests rearranged heavy chain with surrogate light chain

Question 12

What determines the isotype of Ig?

Answer 12

• Constant (C) region
• each C region encoded by different gene
• IgM and IgD are first C regions on heavy chain to be expressed
  
  • co-expressed by differential RNA splicing
• can be followed by other Ig types

Question 13

How are self-reactive B cells eliminated?

Answer 13

• Clonal Deletion: if immature B cell (expressing IgM) in bone marrow reacts with multivalent self-antigen, it undergoes apoptosis
• Anergic B Cell: if immature B cell in bone marrow reacts with soluble self-antigen, it becomes an inactive anergic cell but still goes into the periphery

Question 14

How do B and T cells compare in longevity?

Answer 14

B Cells: relatively short-lived

T Cells: relatively long-lived

Question 15

What are the phases of T cell development?

Answer 15

• Phase 1: arrival in the thymus and antigen receptor expression
• Phase 2: positive selection
• Phase 3: negative selection
• Phase 4: terminal antigen-stimulated differentiation into effector and memory cells

Question 16

Describe the TCR.

Answer 16

• alpha &beta dimer
• variable region & constant region
• single antigen-binding site

Question 17

How is diversity introduced to the TCR alpha and beta loci?

Answer 17

• Somatic recombination
• Combinatorial Diversity
  
  • alpha chain: V, J gene segments recombined from germline gene
  • beta chain: V, D, J gene segments recombined from germline gene
• Junctional Diversity
  
  • new nucleotides generated at joins between gene segmetns (RAG complex)

Question 18

What are the steps in TCR locus recombination?

Answer 18

1. beta chain locus undergoes recombination
2. beta chain is expressed on cell surface with surrogate alpha chain
   
   • pre-T cell: CD4+ and CD8+, pTα
   • pre-Tα receptor binds to beta chian, halts beta chain rearrangement
3. signals proliferation and recombination of TCRα locus

Question 19

What is postitive selection?

Answer 19

• weeding out T cell receptors that cannot **interact with MHC molecules bound to peptide **
• reglates expression of CD4 and CD8 molecules
• only ~2% of T cells make it thru positive selection

Question 20

Describe the steps in positive selection.

Answer 20

• double positive T cells interact with thymic epithelial cells
• TCR binds MHC class I (w/self peptide) **-OR- **TCR binds MHC class II (w/ self peptide) on epithelial cell based on specificity
  
  • those that bind MHC I will only express **CD8 **
  • those that bind MHC II will only express CD4
• ​cells that do not successfully bind undergo apoptosis

Question 21

What is negative selection?

Answer 21

• elimiating of thymocytes that bind too strongly to self-peptide:MHC complex
• central tolerance: only T cells tolerant of self survive

Question 22

Describe the steps in negative selection.

Answer 22

• mediated by bone marrow- derived dendritic cells and macs in cortico-medullary junction and medulla

Question 23

What do cyclosporin A (CsA) and FK-506 (tacrolimus) do?

Answer 23

• Immunosupressive Drugs: inhibit the activity of calcineurin
  
  • ​direct: blocks activation of NFAT
  • consequence: blocks synthesis of IL-2

Question 24

What does Rapamycin (sirolimus) do?

Answer 24

• Immunosuppresive drug: inhibits signaling from IL-2 receptor
  
  • Direct: blocks p70^S6 kinase involved downstream of IL-2 signaling
  • Consequence: blocks T cell proliferation and acquisition of effector functions

Question 25

What is ZAP-70 deficiency? What is its molecular basis? Symptoms?

Answer 25

• autosomal recessive immunodeficiency disease, SCID
• lack of ZAP-70 results in complete lack of CD8+ T cells and CD4+ T cells are non functional
• Symptoms: frequent infections, failure to thrive

Question 26

What is a mitogen? Examples?

Answer 26

substance that stimulates proliferation of T and B cells

ex. bacterial superantigens, mitogenic lectins, pharmacological activators

Question 27

How do bacterial superantigens (sAG) work? What’s their clinical relevance?

Answer 27

• act as glue between TCR Vß region and non-polymorphic region of MHC protein
  
  • TCR: specific Vß but any antigen specificity
  • MHC: Class II, any peptide attached
• activate up to 1/5 of T cells in body, results in massive release of cytokines and moderate to severe illness

Question 28

What are some examples of sAg? Symptoms?

Answer 28

• Toxic Shock Syndrome
  
  • pathogen: Staphylococcus aureus superantigen (TSST-1)
  • symptoms: hypotension, organ failure, fever
• S. aureus food poisoning
  
  • pathogen: S. aureus enterotoxins (SEA, SEB, SEC, SED, SEE)
  • symptoms: food poisoning effects

Question 29

How do mitogenic lectins work? Examples? Usefulness?

Answer 29

• plant-derived carbohydrate-binding proteins that crosslink T cell surface receptors, mimicking antigen stimulation; does not require APCs
• examples:
  
  • T Cells:
    
    • concanavalin A (ConA)
    • phytohemagglutinin (PHA)
  • T & B Cells:
    
    • pokeweed mitogen (PWM)
• useful for in vitro T cell activation (funcitonal assays)

Question 30

How do pharmacological stimulators work? Example? Usefulness?

Answer 30

• bypass TCR & APCs for T cell activation; activates transcription factors for IL-2 production
• phorbol myristate acetate (PMA) with ionomycin
  
  • ​PMA: activates NF-kB and AP-1
  • ionomycin: activates NFAT
• useful for in vitro T cell activation (funtional assays)