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Distinguish between central and peripheral lymphoid tissue.
- 1. Central - Bone marrow and Thymus gland
- 2. Peripheral - lymph nodes, spleen, mucosal lymphoid tissue
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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
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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
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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
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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.
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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.
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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 MHCb3. Immune T cells responded to APCs respective to the MHC genotype
- 4. Demonstrates T cell +selection in thymus
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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