Immunology Chapter 8

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Immunology Chapter 8
2012-03-27 12:41:03
Lymphocyte Development

Chapter 8
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  1. Distinguish between central and peripheral lymphoid tissue.
    • 1. Central - Bone marrow and Thymus gland
    • 2. Peripheral - lymph nodes, spleen, mucosal lymphoid tissue
  2. Where are B cells developed and what cell type negative selection occurs in this tissue
    • 1. Bone marrow - Be cell development
    • 2. Bone marrow - Immature B cells bound to self cell-surface Ag is removed
    • 3. Peripheral organs - Mature B cells bind to Ag activate
    • 4. Peripheral organs - Activated B cells give rise to plasma and memory cells
  3. What are the stages of Be cell development beginning from a stem cell in the bone marrow?
    Stem cell - early pro-B cell (D-J) - late pro-B cell (V-DJ) - large pre-B cell (VDJ) - small pre-B cell (V-J) - immature B cell (VJ) - mature B cell
  4. What is the surrogate light chain and what roles does it play in B cell maturation?
    • 1. H-chain VDJ rearrangement must be tested for functionality
    • 2. Surrogate L-chains help form the complete test receptor until L-chain rearrangement takes place
    • 3. Surrogate - λ5 and VpreB chains made from nonrearranging genes
    • 4. This test pre-B cell receptor mediates transition from pro-B cell to pre-B cell
  5. What is X-linked agammaglobulinemia and what key protein is defective in this B cell deficiency?
    • 1. Mutation in BTK gene results in Btk deficiency
    • 2. Btk - Bruton's tyrosine kinase required for pre-B cell receptor signaling during transition from pro-B cell to pre-B cell.
  6. What is allelic exclusion and how is it accomplished at the Ig gene level
    • 1. The state in which only 1 of the 2 alleles of a gene is expressed in a diploid cell
    • 2. Prevents B cell from producing 2 receptors of different specificities
    • 3. Pre-B cell receptor - reduces VDJ recombinase activity by inhibitin RAG-1 and RAG-2 genes
    • 4. Pre-B cell receptor - targets RAG-2 protein for degradation during S phase of pro-B cell replicaiton
    • 5. Pre-B cell receptor - reduces access of H-chain locus to recombinase activity.
  7. What is meant by isotypic exclusion and how is this accomplished for Ig L-chain genes?
    • 1. The expression of only o1 type of L-chain - κ or λ - by an individual B cell
    • 2. κ locus rearranges before the λ locus
  8. What is the order of protein expression logic in B cell development and how does this relate to Ig gene expression?
    • 1. RAG-1 and RAG-2 - Recombinase proteins essential for BCR/TCR V(D)J recombination
    • 2. TdT - Terminal deoxynucleotidyl transferase enzyme that inserts nontemplated N-nucleotides into the junctions between TCR/BCR gene segment V-regions during assembly
    • 3. λ5 and VpreB - subunits of the surrogate L-chain used in the pre-B cell receptor
    • 4. Igα/β CD45R, and Btk - important in signal transduction during B-cell maturation
  9. What are the steps in Ig gene rearrangement at which developing B cells can be lost?
    • 1. Late pro-B cell - if H-chain rearrangement (V-DJ) is nonproductive on both chromosomes
    • 2. Pre-B cell - if κ/λ rearrangement on both chromosomes is nonproductive
  10. How have transgenic mice been used to dissect the mechanisms of B cell tolerance?
    • 1. The use of mice to express genes for self-reactive B cell receptors to determine the fate of the immature B cell
    • 2. 4 possible outcomes - clonal deletion, receptor editing, anergy, immunological ignorance
    • 2. Clonal deletion/receptor editing - transgenic mice with anti-H-2Kb receptors that also express H-2Kb molecules never express the anti-H-2Kb Ig as sIgM or edit the receptor via RAG proteins
  11. What is receptor editing and how can this mechanism rescue B cells from apoptosis
    • 1. B cell receptor is self-reactive
    • 2. RAG protein sponsors V-J L-chain rearrangement
    • 3. Rearrangement to a non-self-reactive receptor
    • 4. Rescues B-cell cell from clonal deletion
  12. Where do T cells develop and where does positive and negative selection occur?
    • 1. T cell progenitors develop in the bone marrow and migrate to the thymus
    • 2. +/- selection occurs in the thymus
    • 3. Mature T cells migrate to the peripheral lymphoid tissue
    • 4. Activated in the peripheral lymphoid tissue
  13. Describe the basic anatomy of the thymus
    • 1. Situated in anterior thorax superior to heart
    • 2. Lobular each with outer cortex and inner medulla
    • 3. Cortex - immature thymocytes associated with cortical epithelial cells
    • 4. Medulla - mature thymocytes undergoing +/- selection associated with medullary epithelial cells
    • 5. Macrophages, dendrites an d Hassall's corpuscle in medulla remove -selected thymocytes
  14. Describe the role of the thymus vs. bone marrow stem cells in the production of mature T cells using adoptive transfer experiments.
    • 1. nude mice - T cells don't develope due to defective thymus epithelium
    • 2. scid mice - T cells don't develope due to defective Ag-receptor gene rearrangement
    • 3. Grafting scid thymus tissue into nude mice restores T cell development
    • 4. Grafting nuce bone marrow cells into scid mice restores T cell development
  15. What are the 2 distinct lineages produced in the thymus?
    • 1. Double-negaitve thymocytes - Early CD3:T cell receptor population in thymus lacking CD4 and CD8 receptors
    • 2. Minority lineage - γ:δ T cells lacking CD4 and CD8 receptors at maturity including iNKT cells
    • 3. Majority lineage - &alpha:β with both CD4 and CD8 receptors called double+ thymocytes
    • 4. Large active double+ thymocytes develope into small resting double+ thymocytes
    • 5. These differentiate into inactive thymocytes with CD4 and CD8 receptors
  16. Correlate the stages of α:β T cell development with the program of gene rearrangement and expression of cell surface proteins
    See Figure 8.20
  17. What are the locations in the thymus for the various development stages of thymocytes
    • 1. DN1 enters from blood stream at cortico-medullary junction
    • 2. DN2 adhere via CD44 to cortical epithelial cells in the cortex
    • 3. DN3 migrate to outer cortex
    • 4. DN4 loose CD44 surface protein and dissociate from cortical epithelia cells in cortex
    • 5. Immature double+ thymocytes migrate to cortico-medullary junction
    • 6. Mature CD4 and CD8 thymocytes move to medulla where they leave thymus
  18. What are the competing processes at work in determining whether a TCR becomes γ:δ vs α:β?
    • 1. DN T cells simultaneuously rearrange their γ, δ, and β TCR genes
    • 2. If a γ:δ receptor successfully develops before β chain rearrangement produces a pre-T-cell receptor then Erk suppresses β rearrangement and commits the cells to the γ:δ lineage
    • 3. If the opposite is true, the pre-T-cell receptor pairs with pTα whihc signals suppression of γ:δ rearrangement and commits the cell to the α:β lineage
    • 4. The cell passes from DN3 to DN4 to DP stage where the TCRα chain rearranges
    • 5. α chain rearrangement deletes the γ genes and produces a mature α:β T cell receptor
  19. Why does the β chain rearrange first in an α:β T cell and what consequence does this have upon success or failure?
    • 1. β chain must pair with pTα as test receptor for functionality
    • 2. If no successful β chain is produced then apoptosis unless a successful γ:δ rearrangement occurs or further rearrangement
  20. That is the pTα chain and what role does it play in T cell development?
    • 1. surrogate α chain
    • 2. Pairs with β chain to make test receptor analogous to pre-B cells receptor
    • 3. Once paired, Rag is repressed and β chain rearrangement stops
    • 4. DN3 goes to DN4 with proliferation
    • 5. α chain rearrangement commences
    • 6. Selection and differentiation of CD4 and CD8
  21. What is the temporal patter of expression of the following proteins important in T cell development: RAG-1, -2, TdT, pTα, CD3?
    • 1. RAG-1, -2 - DN2->DN3 during β D:J and V:DJ recombination and DN4->DP during α chain recombination
    • 2. TdT - from Stem cell through BP TCR with CD4:CD8 receptors ; N-nucleotide addition during rearrangement
    • 3. pTα - Surrogate α chain; mostly during β recombination
    • 4. CD3 - cell signaling throughout T cell development in thymus
  22. Why is the α chain gene able to undergo repeated rearrangements?
    • 1. Due to the multiplicity of the V and J (~60) gene segments at the αchain locus
    • 2. editing more likely to occur following unsuccessful rearrangement
    • 3. Rearrangement continues until selection by self-peptide:self MHC complex
    • 4. Allows for successive and simultaneous testing of receptors with same β chain
  23. Explain how bone marrow transfer experiments demonstrate positive selection and MHC restriction
    • 1. Bone marrow donor mice - MHCaxb F1 hybrid.
    • 2. Irradiated bone marrow chimera recipients - MHCa and MHCb
    • 3. Immune T cells responded to APCs respective to the MHC genotype
    • 4. Demonstrates T cell +selection in thymus
  24. How do transgenic TCR mice conclusively demonstrate that self-peptide:self-MHC complexes are necessary for T cell maturation to CD4 and CD8 naive T cells?
    • 1. TCR genes specific for a known MHC genotype are introduced into a mouse
    • 2. If the mouse has that MHC molecule in its background then the TCR will differentiate from DP to CD4 or CD8
    • 3. If the mouse does not have that MHC molecule in its background the it will not differentiate - apoptosis
  25. Describe how transgenic TCR mice demonstrate that the MHC molecules recognized by the TCR determine co-receptor specificity
    • 1. Mice TCR transgenic for MHC class I molecules differentiate into the CD8 co-receptor
    • 2. Mice TCR transgenic for MHC class II molecules differentiate into the CD4 co-receptor
  26. How does targeted gene disruption demonstrate that thymic cortical epithelial cells mediate positive selection?
    • 1. Normal mice produce CD4 and CD8 T cells
    • 2. When MHC class II expression is eliminated by TGD, only CD8 T cells mature
    • 3. In MHC class II negative mice with MHC class II gene expressed only on cortical epithelial cells in the thymus, CD4 T cells mature in normal numbers
    • 4. Mutant MHC class II with a defective CD4 binding site, no CD4 maturation occurs
  27. Describe an experiment using transgenic mice that demonstrates that negative selection occurs in the thymus
    • 1. Mice TCR-transgenic for artificial peptide ovalbumin:MHC class I
    • 2. Inject ovalbumin
    • 3. DP thymocytes in thymus die by apoptosis
    • 1. Female mice TCR-transgenic for a natural self peptide found only in male mice
    • 2. Thymocytes die in male mice at the DP stage
    • 3. Thymocytes mature normally in females due to absence of male-only peptide
  28. Explain the role of AIRE in generating central T cell tolerance
    • 1. AIRE drives transcription of protiens in the thymus that are otherwise only produced in other peripheral tissues.
    • 2. This creates a self-shadow in the thymus where resulting in negative selection of T cells that are self-reactive
  29. What is the role of APCs in driving negative selection of T cells?
    • 1. MHCaxb F1 bone marrow is grafted into parental strain MHCa
    • 2. Bone marrow derived DCs and Mfs express both MHCa and MHCb
    • 3. MHCa chimeric mice tolerate grafts of MHCb
    • 4. DCs and Mfs must have presented MHCb for negative selection to TCR
  30. What is the affinity hypothesis and how does it account for positive and negative selection of CD4+ T cells and CD4+CD25+ T cells
    • 1. Affinity hypothesis - the strength of self-peptide:MHC binding determines positive and negative selection
    • 2. Low affinity binding - +selection
    • 3. High affinity binding - -selection
  31. Differentiate between marginal zone B cells, B-1 cells and B-2 cells in terms of location and ability to respond to particular Ag
    • 1. Marginal Zone B cells - located in spleen; respond to carb and protein Ag and sometimes require TH cells
    • 2. B-1 Cells - located in peritoneal and pleural cavity fluid; respond to car Ag and may respond to protein Ag and do not require TH cells
    • 3. B-2 Cells - located in secondary lymph organs; may respond to carb Ag and do respond to protein Ag and require TH cells
  32. What are follicular DCs and how do they present Ag?
    • 1. Distinct from classical DCs
    • 2. Capture Ag as a complex of Ag:Ab:complement
    • 3. Complex remains on surface and is presented to B cells
  33. How do chemokines drive the organization of peripheral lymphoid tissues?
    • 1. Developing lymph node secretes CCL21 which recruits DCs
    • 2. DCs then secrete CCL18, 19 which recruits T cells and B cells
    • 3. B cells induce differentiation of and recruit FDCs
    • 4. FDCs secrete CXCL13 which drives B cells to organize into follicles and recruits more B cells
  34. What is meant by peripheral tolerance and how have transgenic mice been used to demonstrate how it works?
    • 1. The elimination or inactivation of self-reactive lymphocytes that encounter their autoAg de novo in the periphery
    • 2. B-cells in mice transgenic for H-2Kb MHC class I molecule and H-2Kb expression restricted to the liver underwent apoptosis in the periphery
  35. Why do immature B cells often die when they reach the periphery and what are the signals that will rescue a B cell from this fate?
    • 1. There are many more immature B cells than there are follicles to accomodate them
    • 2. Immature B cells that do not enter a follicle eventually die (>50% every 3 days)
    • 3. BAFF - signal from follicle necessary for B-cell survival
    • 4. Syk - involved in signaling from the BCR that helps B cells mature