Lymphocyte Development Flashcards
What are the phases of B Cell Development?
Phase 1) antigen receptor expression (somatic recombination & allelic exclusion)
Phase 2) elimination of self-reactive cells
Phase 3) terminal antigen-stimulated differentiation
Describe the BCR.
- 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
Where does B cell development occur?
Primarily in the bone marrow
How is diversity introduced to the light chain locus of Ig?
- 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)
How is diversity introduced to the heavy chain locus of Ig?
- 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)
How many Ig types can be made through somatic recombination (combinatorial diversity) and junctional diversity?
combinatorial diversity: 3.3 million different antibody combinations
total # possible Ig: 1x1014
Desribe V(D)J recombinase enzyme.
- 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
What happens in DNA cleavage and repair in the immunoglobulin loci?
-
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
What is allelic exlcusion?
- 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
What are the steps to Ig gene rearrangement & antigen receptor expression?
- heavy chain VDJ rearrangement
- expression of heavy chain w/ surrogate light chain to test functionality
- if successful, heavy chain rearrangement stopped (allelic exclusion)
- proliferation of pre-B cells
- light chain VJ rearrangement
- light chain expressed w/ heavy chain to form final BCR
What happens at the pre-B cell stage of B cell development?
- pre-B receptor is expressed: tests rearranged heavy chain with surrogate light chain
What determines the isotype of Ig?
- 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
How are self-reactive B cells eliminated?
- 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
How do B and T cells compare in longevity?
B Cells: relatively short-lived
T Cells: relatively long-lived
What are the phases of T cell development?
- 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
Describe the TCR.
- alpha &beta dimer
- variable region & constant region
- single antigen-binding site
How is diversity introduced to the TCR alpha and beta loci?
- 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)
What are the steps in TCR locus recombination?
- beta chain locus undergoes recombination
- 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
- signals proliferation and recombination of TCRα locus
What is postitive selection?
- 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
Describe the steps in positive selection.
- 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
What is negative selection?
- elimiating of thymocytes that bind too strongly to self-peptide:MHC complex
- central tolerance: only T cells tolerant of self survive
Describe the steps in negative selection.
- mediated by bone marrow- derived dendritic cells and macs in cortico-medullary junction and medulla
What do cyclosporin A (CsA) and FK-506 (tacrolimus) do?
- Immunosupressive Drugs: inhibit the activity of calcineurin
- direct: blocks activation of NFAT
- consequence: blocks synthesis of IL-2
What does Rapamycin (sirolimus) do?
- Immunosuppresive drug: inhibits signaling from IL-2 receptor
- Direct: blocks p70S6 kinase involved downstream of IL-2 signaling
- Consequence: blocks T cell proliferation and acquisition of effector functions
What is ZAP-70 deficiency? What is its molecular basis? Symptoms?
- 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
What is a mitogen? Examples?
substance that stimulates proliferation of T and B cells
ex. bacterial superantigens, mitogenic lectins, pharmacological activators
How do bacterial superantigens (sAG) work? What’s their clinical relevance?
- 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
What are some examples of sAg? Symptoms?
- 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
How do mitogenic lectins work? Examples? Usefulness?
- 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)
- T Cells:
- useful for in vitro T cell activation (funcitonal assays)
How do pharmacological stimulators work? Example? Usefulness?
- 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)
