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2013-11-13 11:30:11

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  1. What are the two processes involved in the preparation of peptide antigens in a cleft on MHC molecules w/ brief description?
    • Antigen processing: generation of peptides from native proteins
    • Antigen presentation: display of the peptide by the MHC at the cell surface
  2. What are the three types of pathogens (by area found) w/ examples
    • Cytosolic: viruses, L. monocytogenes
    • Intravesicular: M. tuberculosis, Leishmania (replicate inside macrophages)
    • Extracellular: toxins and various pathogens (internalized before being digested)
  3. Type of pathogen (by area) / presented on / recognized by.
    • Cytosolic pathogen: MHC I, CD8 Tcyto
    • Intravesicular/Extracellular: MHC II, CD4 Thelper
    • cross presentation: peptides are presented on the the "unexpected" MHC molecule
  4. How do Dendritic cells differ from macrophages and B cells re: APC?
    • Dendritic cells: activate both CD8 and CD4
    • Macrophages: activate CD4 cells that act back on that macrophage
    • B cells: activate CD4 cells that will be helper cells for antigen's antibody production
  5. Overview of MHC I pathway w/ all important details
    • Proteasome breaks down virus in cytoplasm
    • Peptide fragments are transferred to lumen of ER via TAP complex
    • MHC class I are present in the ER until they bind peptides
    • Peptide editing: various peptides are "tried" until a peptide is stably bound
    • *note- some DRiPs will be stable, and will be bound
    • *note- most peptides will not bind, and will be transported back into the cytosol
    • Proper binding results in a stable conformation, and the transportation of MHC I + peptide complex to membrane
  6. Describe constitutive proteasome structure and function.
    • Composed of one 20S catalytic core and two 19S regulatory caps
    • Ubiquitin modification of protein is detected by the 19S cap
    • Protein is introduced to 20S catalytic core where degradation occurs
    • Functions in normal protein turnover
  7. Describe immunoproteasome structure and function
    • The immunoproteasome is present in cells stimulated by IFN γ which...
    • Produces peptide fragments which have preferred anchor residues for MHC I and for TAP transport
    • Causes 20S proteasomes to bind to immunoproteasome regulator caps
    • Upregulation of ERAPP allows further trimming of peptides
  8. What are immunoevasins? Describe the potential mechanisms
    • proteins released by viruses that prevent the appearance of peptide:MHC I complexes on the surface of infected cells in an attempt to evade CD8 Tcyto
    • Target TAP transporter
    • Inhibit peptide loading by MHC I in ER
    • Ubiquinate immature MHC I complex (tag for destruction)
  9. What are the types of antigens processed and presented by MHC II?
    • Extracellular pathogens/proteins: internalized to endocytic vesicles
    • Intracellular pathogens: replicate inside vesicles in macrophages
    • Antigens that bind to sIg on B cells: internalized by receptor-mediated endocytosis
  10. Overview of MHC II pathway with all important details
    • Antigens are contained within endosomes
    • Endosomes become increasingly acidic (via vacuolar ATPase) activating acid proteases that degrade the antigens into peptides
    • Invariant chains prevent peptides from binding to the MHC II in the ER and deliver the CLIP:MHC II complex to the low-pH endosome
    • CLIP: a peptide that "blocks" other peptides from binding MHC II
    • The two endosomes are fused
    • HLA-DM releases CLIP from MHC II, and allows peptide editing to occur
  11. Give the two examples of antigen cross-presentation.
    • Non-infected dendritic cells present Ag from external source on MHC I to activate CD8 Tcyto (otherwise how to Tcyto target these pathogens?)
    • Loading of cytosol-derived proteins onto MHC II molecules to activate CD4 Thelper (otherwise how could the B cells become activated?)
    • *most likely due to autophagy
  12. What are the two properties of MHC that make it difficult for pathogens to evade?
    • polygenic: contains several different genes in every individual, which creates a range of peptide specificities
    • polymorphic: each person has variant alleles within the population. HIGHLY polymorphic
    • *note- evading MHC presentation would be detrimental to the host and incredibly beneficial to the pathogen
  13. Describe the polygeny and polymorphism of MHC molecules in detail with proper terms.
    • polygeny
    • MHC genes are called HLA (Human Leukocyte Antigen) genes
    • There are 3 MHC I α genes (HLA-A/B/C)
    • There are 3 pairs of MHC II α:β genes (HLA-DP, DQ, DR)
    • *note- the HLA-DR cluster contains a few extra β chains, which increases variability
    • *note- most polymorphism is restricted to the peptide-binding cleft, altering anchor residues for a given MHC molecule
    • polymorphic
    • There are over 800 different alleles for HLA genes in the population, and most will be heterozygous for these genes
  14. Describe the interplay between MHC and T cells  and the medical issues that can be caused
    • MHC Restriction: another name for dual specificity
    • Before leaving the Thymus T cells must undergo positive selection for your MHC molecules
    • Alloreactive T cells: recognize and react to non-self MHC molecules (any nucleated cell has MHC I).
    • Alloreactivity common in transplantation.
  15. How do superantigens work?
    • Superantigens bind (intact) to MHC II molecules on the surface of a cell and the Vβ region of the TCR
    • In this way they stimulate large number of T cells...
    • releasing cytokines (IFN γ) and causing septic shock
    • Preventing T cells from specifically binding to antigens
  16. What are the three protein kinases in humans?
    • Tyrosine protein kinases (most common for BCR/TCR)
    • Serine protein kinases
    • Threonine protein kinases
    • (all have -OH group that is phosphorylated)
  17. What are the two "types" (re: association with receptor) of kinases, and how do they function?
    • Receptor kinases: (covalent) one protein has receptor and kinase
    • Upon ligand binding the receptor/kinase protein dimerizes.autophosphorylates
    • Non-receptor kinases: (noncovalent) two proteins
    • 1. The kinase and receptor are noncovalently associated at the membrane and dimerize/autophosphorylate after ligand binding
    • 2. The kinase is associated with a membrane protein unassociated with the receptor.  The two are only brought together after ligand binds to the receptor by co-receptors
    • *note- this is most important to lymphocyte receptors
  18. What enzyme does the opposite job of a kinase?
    protein phosphatases
  19. What are the important domains involved in the creation of multi-protein receptor complexes
    • SH2: binds a phosphotyrosine (most relevant to the Tyr kinase pathways we've been discussing)
    • eg pYXXZ (phosph.Tyr, any, any, hydrophobic)
    • Different SH2 domains prefer different AA combinations
    • SH3: recognizes proline
    • PH: recognize PIP3
  20. Scaffolds vs. Adaptors
    • Both are used by Tyr Kinases to assemble multi-protein signalling complexes
    • Scaffolds: bring multiple proteins to receptor
    • phosphorylated by the kinase in multiple places
    • Can recruit many different proteins on these sites
    • Adaptors: link two molecules
    • are bound to a different signalling proteins in cytoplasm
    • Bind to phosphorylated kinase, linking the signalling proteins to the receptor
  21. What are small G proteins? Example? (not HOW they work)
    • AKA small GTPases
    • Distinct from large heterotrimeric G proteins
    • Act as molecular switches in pathways leading from Tyr kinase-associated receptors (act downstream)
    • Ras, Rac, Rho, and Cdc42 are examples
  22. How do small G proteins work?  What enzymes regulate them?
    • GDP-bound form is inactive, GTP-bound form is active
    • Guanine-nucleotide exchange factors (GEFs): induce conformational change in small GTPase to catalyze exchange of GDP for GTP (activation)
    • GTPase-activating proteins (GAPs): accelerate the intrinsic hydrolysis of GTP->GDP (deactivation)
    • *note- each G protein has its own specific GEFs and GAPs to retain pathway specificity
  23. What are the two pathways for intracellular signalling proteins to be recruited to the plasma membrane? w/ description
    • Tyr protein kinase pathway: Tyr phosphorylation of the receptor or associated scaffold followed by recruitment of SH2-domain containing signalling proteins or adaptors
    • Subsequent recruitment of GEFs can activates associated small GTPases that can act on downstream targets
    • Phosphatidylinositol kinase pathway: local phophatidylinositol phosphorylation recruits signalling proteins (PIP2 -> DAG/IP3) (PIP2 -> PIP3)
    • PI3-kinase is most important, converting membrane PIP2 to membrane PIP3 which is recognized by proteins with a PH domain
  24. What are the two general mechanisms for signal termination?
    • Dephosphorylation by phosphatases
    • Degradation of signaling proteins by ubiquitin-proteasome system
  25. Describe the ubiquitination of proteins involved in signalling and the potential outcomes
    • Dephosphorylation by phosphatases
    • Degradation of signaling proteins by ubiquitin-proteasome system
    • *note- polyubiquitination at Lys 48 causes degredation, but at Lys 63 activates some signalling pathways
  26. What is the name for the molecules that amplify the signal within a cell? Give common examples generated in Tyr kinase pathways
    • Second messengers
    • Ca2+ ions and a variety of membrane lipids/soluble derivatives
  27. What does the functional ("true") TCR look like w/ general functions
    • [TCRα:β + CD3 (γ+δ+ε) + ζ] + [CD4/CD8]
    • α:β: bind the antigen
    • CD3 & ζ: have ITAMs for phosphorylation (signal function)
    • The entire complex is needed for stability and transport to the membrane (electrostatic interactions)
    • *note- each ITAM has two Tyr residues that become phosphorylated when receptor binds ligand, allowing SH2 domain recruitment
  28. What does the functional ("true") BCR look like w/ general functions
    • sIg + [Igα +Igβ]
    • sIg: bind the antigen
    • Igα and Igβ: have ITAMs for phosphorylaton (signal function)
    • The entire complex is needed for stability and transport to the membrane (hydrophillic interactions)
  29. How are co-receptors involved in the signaling pathway (TCR)?
    • CD4 and CD8 help in two ways
    • 1. LCK is noncovalently bound to the coreceptor
    • During ligand binding co-receptors are brought close, and LCK phosphorylates the ITAMs on the TCR
    • 2. Co-receptors stabilize the interaction between TCR and MHC:peptide complex, giving time for signal to be generated
  30. Give the TCR signalling overview with all important interactions and outcomes
    • Co-stimulatory molecules are brought to TCR during ligand binding
    • LCK on co-stimulatory molecules phosphorylats the ITAMs on TCR
    • ZAP70 recognizes P'd ITAMs via SH2 domain and P's other molecules (scaffolds)
    • PLC-γ is bound to membrane by PIP3 and scaffolds
    • *note-PIP3 kinase (req for PIP3) is activated by the costimulatory (CD28 to CD80/86) signal
    • PIP3 also recruits Itk to phosphorylate PLC-γ
    • PLC-γ acts as enzyme on PIP2 to create membrane-bound DAG and cytoplasmic IP3
    • DAG leads to Ras activation and PKC-θ
    • IP3 leads to Ca2+ entry
  31. Describe each of the potential signalling activation pathways of TCRs (what PLC-γ enacts) with some detail
    • Ca2+: Ca2+ leaves ER, and binds to calmodulin, which binds to calcineurin, which dephosphorylates NFATs
    • The now activated NFATs move to nucleus and bind promotor elements
    • Ras: Ras activated by GEF, tiggers 3-kinase relay that results in activation of transcription factor AP-1
    • PKC-θ: recruited to membrane and then results in NFκB activation by (P by PKC-θ causes removal of inhibitor from NFκB)
    • *note- NFAT, AP-1, and NFκB all bind to the promoter region of IL-2, and all are required to stimulate transcription
  32. compare/contrast BCR and TCR signalling pathways (specifically the homologous enzymes)
    • TCR signalling: LcK (brought by CD4/CD8) P's the ITAMs (CD3+ζ) which are recognized by ZAP-70 to initiate signalling
    • Coreceptor CD4/CD8 brings LcK
    • BCR signalling: Fyn, Blk, Lyn (associated with Igα and β) P's the ITAMs (Igα and β) upon ligand binding/receptor clustering which are recognized by Syk to initiate signalling
    • Coreceptor CD19, 21, 81 complex brings PI3Kinase, but PI3 kinase is not involved in activation (no "2 signal" requirement)
  33. What are the stages of B cell development w/ names and overview of what's happening (image recap)
    • Stem cell: receives stromal cell signalling
    • Early pro-B cell: H-Chain D-J rearrangement
    • Late pro-B cell: H-Chain V-DJ rearrangmement
    • Large pre-B cell: Test H-Chain w/ surrogate L-chain (VpreB & λ5) + Igα, Igβ ("pre-B receptor")
    • *Allelic exclusion
    • Small pre-B cell: L-Chain V-J rearrangement
    • *Allelic exclusion AND isotypic exclusion
    • *30-60 cells with same H-chain, different L-chain result
    • Immature B cell: negative selection, IgM expressed on surface
    • Mature B cell: IgD and IgM made from alternatively spliced H-chain transcripts
  34. Different between progenitor cells and precursor cells
    • Progenitor cells: initiate rearrangement of heavy chain. 
    • Cells become precursor cells
    • Precursor cells: initiate rearrangement of light chain.
    • Cells become immature cells
  35. What are stromal cells? What is their function?
    • specialized non-lymphoid CT in intimate contact w/ developing lymphocytes
    • 1. form specific adhesive contacts with lymphocytes (provides signalling within lymphocytes)
    • 2. provide cytokines and chemokines which control lymphocyte differentiation and proliferation
  36. How do pre-B cell receptors get tested for successful rearrangement?  What are the results?
    • 1. Incorporating the rearranged heavy chain into a receptor on the cell surface w/ surrogate light chain (VpreB and λ5)
    • 2. Assembly of invariant proteins Igα and β on the cell surface
    • Successfully formed Pre-B cell receptors dimerize and generate signals to halt further rearrangement (allelic exclusion) and initiates transition to large Pre-B cell
  37. What are the three ways that allelic exclusion is promoted by the pre-B receptor
    • 1.Reduces V(D)J recombinase activity (lower RAG-1 and RAG-2 expression)
    • 2. Reduces levels of RAG-2 by targeting remaining for degredation
    • 3. Reduces access access of the H-chain locus to the recombinase machinery
  38. Why does light chain rearrangement in pre-B cells have a higher chance of generating an intact light chain? (than H-chain)
    • 1. If VJ rearrangement of κ-chain genes on one chromosome fails, repeated rearrangements of unused V and J will occur until rearrangement is productive
    • 2. If this fails then VJ rearrangement of κ genes is tried on the second chromosome
    • 3. If this fails then λ rearrangement may succeed (also has second chromosome to try)
    • *note-same is true for α segments over β segments
  39. Allelic vs isotypic exclusion.  Descriptions and when they occur
    • Allelic exclusion: a successful rearrangement silences the unsuccessful chromosome
    • B cells- Large pre-B (after H-chain) and Small pre-B (afer L-chain)
    • T cells- β and delayed α
    • Isotypic exclusion: a successful rearrangment of a light-chain isotype (κ or λ) prevents the other from being expressed
    • B cells- Small pre-B (after L-chain)
    • T cells- none
  40. Describe the differences in autoreactivity testing between B cells and T cells
    • B cells: immature B cell is tested for autoreactivity in the bone marrow
    • Negative selection dictates that B cells will be tolerant of self antigents
    • T cells: immature T cell is tested for autoreactivity in the Thymus
    • Negative selection dictates that the T cells will be tolerant of self antigens
    • Positive selection dictates that the T cells will recognize self MHC molecules
  41. What are the 5 possible fates of immature B cells in response to self-antigens.
    • Normal maturation: no strong reactivity to self antigen
    • Clonal deletion: cell death by apoptosis (after attempt at editing)
    • Receptor editing: the successful production of a new receptor
    • Anergy: perminantly unresponsive, will later die
    • Immunological ignorance: no response to self-antigen test, but will react if presented with the self-antigen (eg crystallin only in lens)
  42. Similarities and Differences between B cell and T cell development
    • Similarities: orderly and stepwise rearrangment of antigen-receptor genes with testing
    • selection is dependent on interaction with other cells (stromal or thymal)
    • Differences: T cell has two selection processes
    • T cell has two distinct lineages (γ:δ and α:β) which express different ag-receptor genes
  43. What are the stages of α:β T cell development w/ names and overview of what's happening (image recap)
    • Stem cell: receives thymic stromal cell signalling
    • DN1: expresses no marker proteins
    • DN2: β chain VJD rearrangement (analogous to H-chain)
    • DN3: Pre-T receptor is tested using surrogate α chain
    • *allelic exclusion
    • DN4: cell proliferation
    • Large DP: α chain VJ rearrangement occurs
    • *expresses both CD4 and CD8
    • Small DP: 2 TCRs are present on the surface (invariant pTα and CD3 molecules make up "pre T cell receptor")
    • both TCRs undergo + and - selection
    • *allelic exclusion occurs
    • SP: a single TCR is now presented on the surface along with EITHER CD4 (helper) or CD8 (killer)
    • *only cell in medulla
    • Mature T cell:
  44. Describe the cellular architecture of the thymus and what stages of T cells appear where.  How does this factor into selection?
    • Cortex: composed of epithelia that express both MHC I and II (crucial role in positive selection)
    • contains only immature thymocytes and scattered macrophages
    • Medulla: contains only mature single-positive thymocytes with dendritic cells and macrophages
    • Site of negative selection
    • dendritic cells and macrophages express co-stimulatory molecules not present in cortex
  45. Describe the positive and negative selection of T cells
    • DP cells will die in 3-4 days unless they undergo selection
    • Positive selection: ~30% of thymocytes will be able to recognize self-peptide:self MHC
    • takes place in cortex
    • determines if cell will be CD4 or CD8
    • Negative selection: Those who recognize the complex too strongly undergo apoptosis (~2% survive at the end)

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