VL 6 (Katja Hanack) Flashcards
Bone marrow cell –> effector cells
Name cell types of the innate and adaptive immunsystem.
Which cells belong to both?
Innate Immune system:
* Macrophage
* Dendritic cell
* Mast cell
* Natural killer celll
* Complement protein
* Neutrophile
* Eosinophile+basophile
* Granulocytes
Adaptive Immune System:
* B-cell (antibodys)
* T-cell (CD4+ and CD8+)
Innate Immune system & Adaptive Immune System:
* Natural killer T cell
* yd T cell
What is so special about the innate immunsystem?
- immediat after birth
- Specificities are inherited in the genome
- Expressed by all cells of a particular type (e.g. macrophages)
- Triggers immediate response
- Recognizes broad classes of pathogens
- Interacts with a range of molecular structures of a given type
- PAMP´s (Pathogen associated molecular paterns) e.g: Flagellin, LPS (Lipopolysaccheride)
–> molecules associated with groups of pathogens
–> small olecular motifs conserved with class of microbes
–> recognition by PRR (pattern recognition receptors) activate the innate immune response and protect the host from infection
How does it work?
1. Immediate after birth (0-4 h)
2. Infection
3. Recognition by performed, non-specific effectors (e.g RRR)
4. Removal of infectious agents (e.g: Phagocytosis, Lsysis)
Whta is special about the adaptive immune system?
- Able to discriminate between even closely related molecular
structures - Encoded in multiple gene segments
- Requires gene arrangement
- Clonal distribution
- Able to recognize highly specific pathogenic structures
- Immunological memory
- main function is to distinguish between foreign and self
—> requieres several selection steps in the development of T and B cells
How does it work:
1. Adaptive immunity (after 96 hours)
2. Infection
3. Transport of antigen to lymphoid organs
4. Recognition by native T and B cells
5. Clonals expansion and diffrentiation of effector cells
6. Removal of infectious agents
T cell and B cell development (adaptive immunsystem)
T cells
- development in thymus
- positive and negative selection step
T cells development:
1. T cell precursor migrate into Thymus (lack T-cell receptor (TCR))→double-negative (DN)
2. Pre-TCR expression as cells progress through DN2-4
3. Cell proliferation during DN4 – double positive (DP) transition
4. αβ-TCR+CD4+CD8+ thymocytes interact with cortical epithelial cells that express MHC I/II molecules associated with self- peptides
5. DP fate depends on TCR – self-peptide-MHC ligands interactions
→too little signaling
→delayed apoptosis (death by neglect) →too much signaling
→acute apoptosis (negative selection); medulla
→intermediate level of TCR signalling
→effective maturation (positive selection)
6. Fully mature T cells leave medulla (thymus)
B cells
- development in the bone marrow
- selection by receptor editing
B cell development:
1. bone marrow: common lymphoid progenitor (CLP)
→ pro-B- cell
→pre-B-cell (pre-BCR, comprised: Ig heavy chain + surrogate light chains (VpreB or V5))
→immature-B-cell (mature BCR = IgM, comprised: rearranged heavy-/light-chain genes; Ag-binding)
2. B cell selection process to prevent any further development of self-reactive cells (receptor editing + clonal deletion)
3. checkpoint completed
→ transitional B cells leave bone marrow
→maturation in secondary lymphoid organs
4. immune response
→ Ag-specific B cells develop into
→ plasma (antibody-secreting) cells or memory B cells
Peptid loading on MHCI and MHCII
MHC I:
proteasomal degradation of cytosolic, nuclear, mitochondrial, ER proteins
→peptide fragments
→TAP-binding (ABC-family transporter)
→peptide translocation in ER lumen
→MHC I folding, assembly in ER with help of calnexin
→dissociation from calnexin
→peptides loaded onto MHC I residing in TAP-associated peptide loading complex (PLC; contains: tapasin, ERp57, calreticulin)
→MHC I completes folding
→MHC I dissociates from TAP
→transport through secretory pathway to plasma membrane
MHC II:
MHC II αβ dimers associate with invariant chain (li) in ER
→Li-MHC II-complexes internalized by clathrin-mediated endocytosis
→transport to intraluminal vesicles (ILVs) of MVB
→in ILVs: li proteolysis→Li-fragment: class II-associated invariant chain peptide (CLIP) in MHC II peptide-binding groove →CLIP removement from CLIP-MHC II complexes by HLA-DM enzyme
→peptide binding to MHC II
→transport to plasma membrane in tubulovesicular endosomes
Endogeneous and exogenous processing of antigens
endogeneous
proteasome degradation of Ag
→smaller peptides
→transport to ER via transporter associated with Ag processing (TAP) →peptide attached to major histocompatibility complex (MHC) class I →cell surface
→presentation to CD8- T cell
exogenous
taken into cell by phagosome
→joined to MHC II
→cell surface
→presentation to: CD4+ T cell, some Ag: CD8- T cell via cross-presentation
MHC II related activation of T helper cells
- TCR - peptide/MHC-II-complex (APC); CD4 transfers signal of Ag-contact to T-cell
- costimulation: B7 (APC) – CD28 (T-cell) → living, proliferation
- differenziation: cytokines, IL
→t-effector- cells
role of signal 2:
* immunological tolerance to self Ag
* active transport of signaling proteins in T cell plasma membrane to contact site between T cell - APC
Th1
* produce: IL-2/10; IFN-γ; TNF-β
→increased cell-mediated response against bacteria & protozoa through macrophages, Tcyt triggered by IL-12
Th2
* produces: IL-4/5/6/9/10/13
→humoral immune response against extracellular parasites (e.g. helminths) through B-cell, mast cell, eosinophil triggered by IL-2/4
Branches of adaptive immune response
Molecules of the immune system: Cytokines
- molecules of cellular communication
- immunmodulating agents“ (interleukins, interferons etc.)
- each cytokine has a cell-matching receptor
- Cytokines can act autocrine, paracrine or endocrine
—> binds the same cells
—> binds to neighbouring cells
—> releases in the blood stream
Receptor signaling after cytokine engagement:
JAK-STAT signaling cascade
* cytokine binding to cytokine receptor type I/II (lack intrinsic tyrosine kinase activity)
* JAK1-3, TYK2 associate with intracellular part of cytokine receptors
→activated
→JAKs phosphorylate Tyr residue of receptors + STAT TFs (STAT1-4/5A/5B/6) →P-STAT dimerize + translocation to nucleus
→transcriptional activators for diverse target genes with STAT-binding motifs →enhancement: cell proliferation, survival, differentiation
Smad signaling cascade:
* Ligand binding to type 1/2 receptors
→type 1 receptor activated through phosphorylation by type 2 receptor →receptor-regulated Smads (R-Smads; Smad1-3/5/8) phosphorylation at C-terminus
→R-Smad conformational change
→complex formation with Smad4
→complex accumulation in nucleus
→cooperate with other transcriptional co-regulators
→modifiy target gene transcription
Molecules of the immune system: Chemokines
- metasitasis
- inflammation
- cell recruitment
- angiogenesis
- leukocyte trafficking
- wound healing
- lymphoid organ development o Th1/2 differentiation
Molecules of the immune system: antibody molecules
- globular glycoproteins in serum/other body fluids, synthesized & secreted by plasma cells (terminally differentiated B cells)
- Basic structure (in most cases): correspond to IgG of mammals
–> Y-shaped
–> MW: 150 kDa
–> 2 identical heavy & 2 identical light chains - 5 classes + subclasses in homo sapiens, mus musculus with different structure & function (IgM/A/G/E/D)
Screening of Antibodys by ELISA
- Cultivation of freshly fused hybridoma cells in 96 well plate
- Culture supernatants are tested weekly fort he right Ab secretion via ELISA
- Positive cell clones are recloned by limited dilution
- Stable clones are
→ Ab producing hybridoma cell (3-4 months)
Types:
* Direct ELISA
* Indirect ELISA
* Sandwich ELISA
* Competitive ELISA
Immuneprecipitation (IP)
Classic IP
Highest capacity
Some restrictions in Ab types used
Ab eluted with Ag
protein A, G, A/G = Ab-binding proteins
* bind constant Ab domain
→Ab orientation (Ag-binding domain orientated away from support)
* different Ab affinities
* different IgG subclasses
* 4 protein A, 2 protein G Ab-binding domains
* Ab isn ́t covalently bound to Ab-binding proteins→release of Ab-Ag comples during elution
Cross-link IP
Significant binding capacity loss
Some restrictions in Ab types used o Ab retained on resin
small, amine reactive homobifunctional protein cross-linkers (e.g. DSS, BS3)
steps:
–> Ab coupled to protein A, G, A/G agarose beads
–> Incubation with cross-linkers
–> cross-linkers covalently attach Ab to protein A, G, A/G
Direct IP
* Some binding capacity loss
* Suitable for all Ab
* Ab retained on resin
Binds Ab directly to insoluble, solid support
→no protein A, G, A/G
bound amine residues to agarose
→direct, covalent Ab-binding
(→no co-elution) in random orientation
https://info.gbiosciences.com/blog/bid/179306/protein-immunoprecipitations-which-method-to-use
Flow cytometer
http://labs.umassmed.edu/socolovskylab/research~flow_cytometry.html
