Immunity Flashcards
(12 cards)
Immune system overview: evolution, hierarchy, inflammation. Examples of differentiation pathways
Immune system costly- >10% mammalian genes involved. Relies on ability to recognise self vs not self.
Evolution: start w/ restriction enzymes; phagocytosis+ non-self-rejection around level of sponges, just before animal branch node, adaptive response in animals (innate response hyperbolic, adaptive much steeper+ faster- ‘memory’). Complement system of innate+ adaptive arms.
Hierarchy: hematopoietic cells-> myeloid (bone tissue) or lymphoid (spleen, thymus, lymph nodes) stem cells.
Inflammation= localized reddening, swelling, heat, pain (response to injury, infection) due to coordinated invasion of area by immune cells, incl mast cells, monocytes, lymphocytes, etc. Bleeding-> inflammation-> proliferation-> remodelling
Immune cell activation and polarisation; complement system
Immune cell activation+ polarization: polarization= guided differentiation/specialization driven by molecular cues, usually in situ. Cues can be DAMPs/PAMPs (danger/pathogen associated molecular patterns) and extracellular ligands (cytokines). Cascade/ molecular cues guide polarisation+ f(x)/ immune cells-> specific coordinated immune response.
Complement system (pathogen lysis, inflammation activation, phagocytosis) enhances immune system, consists of circulating peptides from hepatocytes, triggered by protease activation-> convert pre-complement peptides-> active forms. Durected by engagement of antigens on targets w/ antibodies or PAMPs. Deployment can be via classical pathway (Ag-Ab complexes)/lectin/alternative pathways-> inflammation, phagocytosis, cell lysis/inactivation. Diff triggers-> diff molecular cascades, activation reg by effector proteins, specific outcomes depend on initial trigger-> opsonization, inflammation, cell lysis.
Innate immune system
Innate immune system: macrophages+ granulocytes (polymorphonuclear leukocytes incl neutrophils, eosinophils, basophils). Monocytes polarise-> macrophages dependent on specific signals.
Neutrophils= most abundant leukocyte, phagocytes, short-lived, degranulate to release anti-microbial proteins, neutrophil extracellular traps (NETs)- pus.
Eosinophils, basophils: trigger allergic inflammation+ anti-parasitic f(x), secrete histamine+ cytokines+ chemicals (ie ROS) that drive inflammation, contain heparin (prevent blood clotting)
Dendritic cells link innate+ adaptive arms. Immature DCs polarise to mature DC in response to danger/infection signals, migrate to lymph node-> presentation of antigens coupled to MHC-II, exp molecules stimulating T+ B-cell activation
Natural killer cells- crossover of adaptive+ innate. Recognise specific stress signals on cells-> apoptosis. Cytotoxic granules in cell release perforin to e.g. cancer cell, release cytokines.
Adaptive immune system: MHC, Lymphocytes and VDJ recombinase
Major histocompatibility complex (MHC) tell adaptive immune cells they don’t harbour viral/bacterial infections. MHC class I found on all cells (self-cell passport- overridden by viral antigens so if both presented, cell killed). MHC class II on APCs.
Lymphocytes B (bone marrow)+T (thymus). Lymphoid cells go though dev stages ensuring differentiation to specific functional cell types, large repertoire of clones expressing immunoglobins/ TCRs reactive toward different antigens, production of cell clones not reactive to host antigens. (-ve selection against T cells that recognise self antigen)
V(D)J recombinase- collection of enzymes mediating genomic re-arrs. Key enzymes= recomb activating genes (RAGs). Targets= recombination signal seqs (RSSs) @ VDJ gene segments. Termination of process triggered during B/T cell dev- RAG1/2 exp. Vast repertoire/binding diversity for TCRs+ antibodies through re-arr of TCR+ immunoglobin loci. Each T cells exp single TCR seq; each B cell clone makes 1 antibody seq
T cells and Plasma+memory B cells (adaptive immune system)
Plasma+ memory B cells-activation by antigen binding-> co-stimulation-> cytokine help-> B cell clonal expansion+ differentiation. secrete antibodies-> neutralisation (block viral binding sites, coat bacteria), agglutination of microbes, precipitation of dissolved antigens (all 3 enhance phagocytosis), activation of complement system leading to cell lysis. Produce durable response
T cells: e.g. CD8 cytotoxic T-cells directly kill cells exp foreign proteins; CD4 helper T-cells refine immune response: naïve T cell becomes-> Th1 T-cells (activate macrophages, CTLs), Th2 (activate B cells), Th17 (recruit macrophages, neutrophils), Treg (suppress immune responses), Maturation by naïve T-cells from thymus-> lymph node-> ACP cross-presentation triggers effector cells-> lymph node releases specific cells to target (ie virally infected/ tumour cells). Activation can be by activating co-stimulatory receptors (many, inhibitory ones exist), endogenous TCR… T cell f(x) regulated by integration of activity of stimulatory receptors. T cell effector+ memory responses crucial for sustained tumour killing (perforin)
Antibody structure and vaccination
Structure: 12 domains. Each tetramer of 2 identical heavy+2 light chains, stabilise w/ S-S. Each chain has 1 variable Ig domain @ N-term. V region of H+L chain associate-> antigen binding site. L chain has 1 constant domain, H chain has 3 (CH1,2,3). CH1 associates CL, CH2+CH3= Fc domain, drive dimerization, interact receptors driving effector f(x)s.
Variable domains incl extra loops @ top- hypervariable AKA complementarity determining regions (CDRs)- rest of beta sheet=framework. 3 CDRs per chain. CDR3 on H chain most variable. Variability @ DNA level by recomb
Fc region: constant domains relinked to effectors w/ Fc receptors +/ complement system, -> phagocytosis/cell lysis via complement system. Neonatal Fc receptor (FcRn) protects IgG antibodies from degradation
Vaccination: flu haemagglutinin binds sialic acid residues on target cell, essential for invasion. Immunity from antibodies binding+ blocking sialic acid binding site. Mutations in haemagglutinin recognised by antibody yearly. Covid vaccinations by exposure to surface spike proteins
antibodies as research tools: developing antibodies, scFv fragments and ELISA
dev/antibodies: polyclonal antibodies- animal injected w/ antigen-> immune response-> serum contains pool of diff antibodies binding epitopes on antigen. Monoclonals (Nobel)- antibody producing B cells from immunized moused fused w/ myeloma (B cell cancer) cells, -ve selection so unfused cells die, ELISA to find individual hybridoma clones producing antigen reactive antibodies.
scFv fragments derived from library-> phage display, incubation, biopanning+ characterization (repeating cycles)- Nobel prize for directed enzyme evolution+ phage display.
Epitope tagging allows antibody-based techniques: Sandwich ELISA (capture antibody on surface w/ blocking buffer, add antigen, then antibody for antigen w/ enzyme, then add substrate producing coloured product measurable by colorimetry (wash between steps))- also direct indirect, competitive ELISA.
Antibodies as research tools: westerns, IP, IF, flow cytometry, FACS
Western blot: separation, transfer to membrane, stain (Ponceau, antibody), visualise (e.g. chemofluor)
Immunoprecipitation: add antibody to cell lysate, incubate, add protein A/G beads+ incubate-> antigen-antibody-bead complex, wash+ elute, purify. Then analyse w/ Western, Staining, mass spec etc
Immunofluor- direct (1 antibody) or indirect (2 antibody). Multiplex diff antibodies linked to diff fluor, amplify signal w/ secondary antibodies. Also immunohistochemistry
Flow cytometry: mix of cells bound by antibodies raised against protein X/Y, each antibody coupled w/ diff fluorophore. Visualise cell in flow sheath w/ laser-> spatial distribution of tagged proteins
Fluorescence activated call sorting (FACS)- mix/cells bound by antibodies to protein A/B bound to diff fluorophores passed through super thin tube 1 by 1. Laser signal-> detector triggering current targeted @ protein+ sorting using charged plates.
Antibody potential uses as research tools
scFv fragments, ELISAs, Western blots, Immunoprecipitation, Immunofluorescence, Flow cytometry, FACS
Therapeutic antibodies: humanised antibodies and Rheumatoid arthritis
Humanised antibodies: murine, chimaeric (-ximab), humanised (-zumab) or human (-umab). To make humanised: inactivate mouse heavy+ light chain Ig genes (embryonic stem cells)-> mouse train not producing IgG. Intro genes encoding H+L chain of human IgG into mouse ES cells-> transgenic mouse w/ both human+ mouse IgG. Cross 2 transgenic strains-> some progeny exp only human IgG.
Rheumatoid arthritis in part due to persistent TNF-alpha binding its receptor-> sustained inflammatory response+ autoimmunity. Humira/Adalimumab treatment (also approved to treat lupus, psoriasis, Crohn’s) derived from scFv library- dev antibody binding+ neutralising TNF-alpha from phage display.
Immunity: EGFR pathway, Herceptin, inferring immune checkpoints and Antibody drug conjugates
EGFR pathway blocking antibodies target cancer: deregulated EGF receptor f(x)-> several cancers (colorectal, breast, head&neck)- EGF-> ligand-induced dimerization-> activate pathway. ErbB2 binds activated EGF receptor-> signalling. Antibodies disrupting EGFR/ErbB2 heterodimerization target specific cancers, e.g. Cetuximab, Pertuzumab.
Herceptin targets cancer cells by: binding HER2 (receptor over-exp in 20% breast cancers), deactivates oncogenic signalling, promotes antibody-dependent cell cytotoxicity (ADCC).
Inferring immune checkpoints earned Nobel. Integration/ combined signals from stimulatory& inhibitory receptors dictates T cell responses. Theralizumab= antibody raised against CD28, a T-cell co-activator- stimulate T-cell killing leukemia; had good pre-clinical pharmacodynamics (mice, rates, macaques), but trialling in humans @ 1/500 dose-> cytokine overload-> fluid in lungs, toxin buildup in kidney, blood flow slowed
Antibody drug conjugates (ADCs) target cell surface tumour associated antigens. Internalisation+ trafficking to lysosome-> release of drug+ cell death- bridge/ linker to drug usually in constant region. HER2 directed ADC (Enhertu) efficacy found v high (median survival 8.4->12.5 months compared to non-targeted drug).
Immune therapies: Adoptive T cell therapy/T-cell transfer therapy, Chimeric antigen receptor, Bi-specific T cell engagers
Cell therapies= treatments relying on potentiating immune cell f(x) against cancer cells
* Adoptive T-cell therapy: isolate tumour infiltrating lymphocytes (TILs), activation, expansion, re-infusion
* Chimeric antigen receptor (CAR)-based therapies: remove blood from patient to get T cells, make CAR T cells in lab, grow, infuse into patient- bind cancer+ kill them. CAR ectodomain-scFv fragment directed towards TAA. CAR endodomain- functionality conferred through chimeric domains. Main issues= requirement for autologous T cell transplantation- solve w/ allogenic transplantation of NK-CAR cells or off-the-shelf therapeutics.
* Bi-specific T cell engagers, BiKEs, etc- off-the-shelf therapeutics w/ capacity for multi-modality, multi-valency, multi-functionality.
