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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
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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)
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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
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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
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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
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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
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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
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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 TcytoTarget TAP transporter
- Inhibit peptide loading by MHC I in ER
- Ubiquinate immature MHC I complex (tag for destruction)
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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
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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
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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
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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
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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
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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.
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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
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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)
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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
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What enzyme does the opposite job of a kinase?
protein phosphatases
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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
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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
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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
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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
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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
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What are the two general mechanisms for signal termination?
- Dephosphorylation by phosphatases
- Degradation of signaling proteins by ubiquitin-proteasome system
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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
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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
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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
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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)
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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
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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
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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
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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)
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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
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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
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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
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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
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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
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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
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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
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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
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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)
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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
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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:
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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
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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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