Immuno 2 Flashcards
major vs minor histocompatibility complex
strong immune response, major determinant for rapid TRANSPLANT/GRAFT REJECTION, differentiate self from foreign cells –> respond to foreign ag vs less immunogenic and don’t elicit ab, involved in self/nonself recognition but lesser extent than major HC
MHC locus vs MHC I locus vs MHC II locus vs codominant expression
located in region of chrm 6 vs classical: HLA/B/C, nonclassical: HLE/F/G vs HLA-DP/DQ/DR vs all MHC genes from each chrm = expressed –> each person expresses 6 class I and 6 class II
classical MHC I vs nonclassical MHC II
on all nucleated cells and PLTs, presents ag to CD8+ T cells vs on more limited cellular expression; HLA-G = immunosuppressive
MHC structure
heterodimer of 2 proteins w/ at least 1 = transmembrane, has a cavity where self and nonself peptide binds to => peptide-binding groove (PEPTIDE MUST BE IN GROOVE for MHC proteins to fold) (mult peptides can bind to single MHC); can display 1 type of peptide at a time
2 lvls of MHC restriction: self restriction vs ag restriction
TCR only recognizes peptide presented on self HLA vs TCR recognizes specific ag on self HLA
MHC I vs MHC II
MHC-encoded alpha chain and beta2-microglobulin NOT encoded in MHC locus, on all nucleated cells and PLTs, presents ag to CD8+ T cells vs both MHC-encoded alpha and beta chain, on APCs, presents ag to CD4+ T cells
ag processing: endogenous vs exogenous
class I, processing intracellular proteins on MHC I for CD8+; proteins are in cyto –> proteasome digest proteins into peptide in cyto –> peptide diffuse to ER nd bind to TAP –> TAP loads peptide on HLA-A/B/C –> MHC-peptide complex transported to cell membrane vs class II, processing extracellular proteins on MHC II for CD4+; ag phagocytosed –> invariant chain transported to MHCII Compartment (MIIC) w/ CLiP in binding groove –> MIIC and endosome fuse –> peptide loaded onto HLA-DP/DQ/DR –> MHC-peptide complex transported to cell membrane
cross presentation
exogenous ag end up on MHC I: DC phagocytoses ag –> processed peptide or protein escape phagolysosome –> can bind to TAP –> loaded on MHC I –> presented to CD8+; CRITICAL in responding to cytosolic infxn (viruses and tumors)
CD1
MHCs can only present proteins –> CD1 = MHC-like molec that presents lipid ags –> presents to NKT cells
pres of carb ags
endocytosed glycoproteins go thru MHC II: peptide part = inserted in MHC II, carb part = presented to CD4+
immunodominant epitope
peptide ag that has the highest affinity to MHCs
MHC and B cells vs T cells
B cell phagocytoses and present ag to MHC II => exogenous pathway –> activate CD4+ T cells –> CD4+ T cells bind to peptide-MHC complex on B cells –> release cytokines to activate B cell fxns –> clonal expansion and differentiation vs anchor residue of peptide binds to pocket of MHC peptide groove –> side chain of aa out of groove and binds to TCR –> loops of TCR bind to invariant ridge of MHC peptide binding groove –> conformational change (lock and key, ag restriction)
superantigen
bind external surface of TCR beta chain on T cell and MHC II on APC (not peptide groove) –> activate CD4+ cells –> massive cytokine prod –> systemic inflam response; ex: bacterial exotoxins
characteristics of B/T ag receptors (4)
multimeric (BCR have light/heavy chains, TCR have alpha/beta or gamma/delta chains), have variable regions that bind ag w/ high specificity, have constant regions for signaling, have no intrinsic signaling abilities –> need additional receptors
each locus has a complete set of what genes?
variable genes, joining genes, constant genes (VJC)
somatic recombination
irreversible. BCR have heavy chains/TCR have beta chains w/ VJCD genes; BCR have light chains/TCR have alpha chains w/ VJC genes; VDJ recombinase/RAG1&2 recognize recombination signal sequence (RSS) –> randomly cleaves and bind DNA segments
allelic exclusion
when somatic recombo starts on 1 chrm, it inhibits recombo on other chrm –> ensures 1 BCR/TCR is expressed on each B/T cell
variable region vs constant region
for specificity –> binds to single specific ag; it’s specific via combo of 3 CDRs at ag binding site vs for functionality –> conserved regions bind to accessory molec
pos selection vs neg selection
Allow maturation of cells that respond to ag and self MHC correctly vs not allowing maturation of cells that don’t respond to ag and self correctly –> apoptosis
seq of events of T cell maturation (7)
- Expression of CD25
- T cell progenitors express CD3 but not CD4 or 8 => double neg
- TCR VDJ genes rearrange
- Expression of CD3,4, AND 8 => double pos
- if they react to self MHC-ag –> pos selection OR if they don’t react to self MHC-ag –> neg selection –> apop
- Expression of either CD4 OR CD8 chosen at corticomedullary jxn => single pos
- mature naive T cells go to circ
alternative/nonconventional T cells: gamma/delta T cells vs NK T cells
both still mature in thymus, have TCR, and respond more rapidly than conventional alpha/beta T cells. express CD3 and CD8, conc in skin and mucosa, recognize phosphor-ag and stress proteins (dmged cells, heat shock protein) vs express CD3, CD4, and killing receptors of NK cells, recognize glycolipid ag
seq of events of B cell maturation (4)
- BCR VDJ rearrangement until surface igM receptor expressed
- if fxnal heavy chain expressed –> go make light chain; if not –> apop
- if fxnal light chain expressed –> leave bone marrow; if not –> try again –> if still not –> apop
- transitional B cell expresses surface igD –> mature naive B cell –> goes to secondary lymph organ
steps of T cell immune response (8)
- ag recognition
- T cell activation
- T cell proliferation
- T cell differentiation - become right effector for ag
- T cell migration
- T cell effector fxn - kill/control pathogen
- T cell contraction - dec response
- T cell memory
how to fully activate T cell? (3 signals)
Binding of TCR (by MHC-peptide complex)
Binding of costimulatory molecules (CD80/CD86 on APC to CD28 on T cell) (CD40 on APC to CD40L on T cell)
Binding of cytokine receptor
impt adhesion molec for T cell activation?
ICAM-1 (CD54) on APC to LFA-1 (CD11a+CD18) on T cell
NFAT. cyclosporine?
nuclear factor of activated T cells. IP3 stimulates Ca2+ release from ER –> inc intracellular Ca2+ opens Ca2+ channel –> Ca2+ binds to calmodulin to activate calcineurin (a phosphatase) –> calcineurin dephosphorylates cytoplasmic pNFAT, activating it –> active NFAT translocates to nucleus and initiates transcription for T cell activation (IL-2)
CYCLOSPORINE INHIBITS CALCINEURIN
NF-kB
DAG activates PKC –> PKC phosphorylates IκB (an inhibitor of NF-κB) –> phosphorylated IκB releases NF-κB –> NF-κB translocates to nucleus and initiates transcription for T cell activation (IL-2)
PI3-kinase/Akt
Lck activates PI3 kinase (PI3K) associated with CD28 costimulatory molec –> PI3K phosphorylates membrane-bound PIP2 to generate PIP3 –> PIP3 activates Akt –> Akt translocates to nucleus and initiates transcription for anti-apoptotic proteins
CD4 Th1 vs Th2 vs Th17 vs Treg cells
induced by IL-12 (from APC) or IFN-γ (from NK cell); produces IFN-γ –> activator effector fxn; inc ab isotype switch and MHC II; inhibits Th2 vs induced by IL-4; produces IL-4 (activate mac, isotype switch), IL-5 (eos), IL-13 (activate mac, mucus prod), some IL-10, isotype switch to igE by binding FcER to mast cells or eos –> fight allergy or parasite and mast cell degranulation; IL4 and IL10 inhibit Th1 vs induced by IL-6, IL-23, TGF-β; produces IL8 (to recruit neu and promote their response), IL17, IL22; increased in pts w/ autoimmune dz vs induced by TGF-β and RA by resident macs; express CD25 (IL-2 receptor) and transcription factor FoxP3; produce IL10, TGF-β, IDO; natural Tregs differentiate in thymus based on avidity, induced Tregs differentiate in periphery based on FoxP3
NKT cells vs CD8 Tc cells
Express T markers (CD3, CD4, few CD8) and NK markers (CD56, CD16, NKG2D, FasL)
o CD16 – Fcγ receptor triggering antibody-dependent cellular cytotoxicity (ADCC)
o NKG2D – bind MHC I; contains ITAM motif
o FasL recognizes Fas (CD95) on stressed self cells
Bind lipid ag –> protect against mycobacteria and fungi
Effector functions
o Cytolysis through NK killing receptors and CD16
o Secretion of cytotoxic proteins (ex: perforin)
o Secretion of cytokines
vs
Activated by TCR binding of Ag-MHC I complex on APC
Recognize same self ag or same MHC I
Primary goal = cytolysis of altered self cells, cytotoxic proteins, cytokine prod
apop pathways: perforin/granzyme pathway vs Fas/FasL-induced pathway vs TNF-mediated death pathway
Perforin monomers = released w/ perforin polymerase to form perforin channel in target cell –> Enzymes granzyme and granulysin from CD8 CTL enter perforin channel –> Binding of granzyme, granulysin, or cytokines initiate apop of target cell vs CD8 CTL and NK cells upregulate Fas ligand to bind Fas receptor/CD95 –> induce cellular stress –> activates caspase cascade –> apop vs CD8 CTL and NK cells produce TNF-α to bind its receptor death domain –> activates caspase cascade –> apop
drug targets for T cell activation
o Cyclosporine – inhibits calcineurin
o IL-2 receptor antibodies or antagonists – prevent IL-2 signal transduction –> no T cell growth
o JAK inhibitors – block cytokine signaling
o TNF-α inhibitors
primary effector cells for killing?
ag specific Tc cells and non ag-specific NK cells
3 signal hypothesis
T cell must receive three distinct signals during activation:
Binding of TCR (by MHC-peptide complex)
Binding of costimulatory molecules
Binding of cytokine receptor
-if not all 3 signals delivered –> anergic
describe TCR binding vs binding of costimulatory molec vs binding of cytokine receptor (part of 3 signal hypothesis)
Lck kinase (associated with CD4 and CD8) phosphorylates CD3 zeta chain at Tyr residues in ITAM domains –> TCR-CD3-zeta complexes to cluster –> IL-2 receptor/CD25 go from low affinity to high affinity for IL-2 prod –> clonal expansion vs CD80/CD86 or CD40 on APC bind to CD28 or CD40L on T cell; CD80/86 can bind to CTLA-4 but it’s an inhibitory signal –> turn off immune response; ICAM-1/CD54 on APC bind to LFA-1/CD11a+CD18 on T cell for adhesion vs lead to replication and differentiation of T cells
ag properties. what makes a good ag?
a substance that specifically binds to ab or TCR; may or may not elicit immune response, typically a protein or glycoprotein > carb > lipid, hapten, aa, DNA. Highly folded, unevenly charged, greater than 1kD in size
immunogen vs epitope/determinant vs hapten
an ag eliciting B/T cell response vs small portion of ag bound to B/TCR (single ag can have mult epitopes, some epitopes elicit stronger response => immunodominant) vs small portion of ag bound to ab (unable to initiate response by itself –> must be part of larger molec aka carrier to be immunogenic)
Know the structure of ab
light chain, heavy chain, variable region, constant region, disulfide bond. Fab region contains ag binding site, Fc/constant region is diff for each isotype
junctional diversity
during VDJ recombo, gaps = left by RAG –> P nucleotides fill in sticky ends, N nucleotides fill in gaps
describe B cell ag pres itself. follicular APC vs subcapsular sinus APC
B cells captures ag in 2ndary lymph organ –> NOT endocytosed but presented on BCR surface –> ag transferred to BCR –> 2nd receptor = TLR or Fc receptor –> igM prod, B cell switch isotypes, become plasma and memory cells. bind smaller Ag that filter into follicles vs bind larger Ag and Ag-Ab (immune) complexes
describe B cell ag pres to T cell
B cell captures exogenous ag –> loaded onto MHC II –> naive T cell recognizes it and = activated and gives costim to B cell –> B cell = activated –> secretes ab, switch isotypes, becomes plasma cell
describe B/T cell interaction in LN
- activation: T cell activated in T cell zone of LN while B cell activated in B cell zone
- Migration: Both T and B begin producing chemokines and move toward each other
- Contact: Meet OUTSIDE follicle in parafollicle; B cell gets Th help; Abs made
- Outcomes: B and T cells stay outside follicle → B cell becomes plasma cell, but short-lived and produce low levels of Ab
- B cells return to follicle → proliferate and differentiate to plasma cell → germinal center forms –> high Ab level production
germinal center rxn
Naïve B cells in follicle (mantle zone) are activated by CD4 Th cells in parafollicle, then re-enter follicle –> Go to dark zone, and begin proliferating –> Non-dividing progeny of B cells go to light zone; interact with FDC and CD4 Tfh until selected
affinity maturation
B cells transition from dark zone to light zone –> Contact FDC presenting Ag and mutate Ab binding site –> If affinity increases, B cell selected to survive –> Can re-enter dark zone and proliferate, then cycle back to light zone, going through cycle repeatedly
isotype switching
occurs in light zone, irreversible process.
- Cytokine signal from CD4 Tfh (major source) and FDC (minor source)
- IgG – induced by IFN-γ
- IgE – induced by IL-4 and IL-13
- IgA – induced by TGF-β - Activation-induced cytidine deaminase (AID) produced
- AID recombines DNA → cuts VDJ from Cµ gene to a new constant gene
- Intervening DNA (i.e., constant regions) looped out and excised
- New isotype secreted
multimeric vs monomeric ab
bound by J chain, igM = pentamer, igA = dimer in mucosa vs igA, igG, igD, igE in blood/circ
valency vs affinity vs avidity
of binding sites vs binding strength b/w single ag epitope and single ab binding site vs total binding strength of all ab-epitope pairs in multivalent Ag-Ab interactions
describe ab degranulation
used for large ag that are too big to be endocytosed; mostly igE; igE binds Ag –> eos or mast cell binds IgE via FcεR, OR IgE binds FcεR on mast cell or eos –> then binds to allergen
Either way: cell releases granules by exocytosis –> ag degraded
describe ab-Dependent Cellular Cytotoxicity
- IgG binds Ag on target cell
- FcγRIII (CD16) on NK cells and CD8 CTL bind IgG Fc
- Crosslinking of FcγR activates NK cell or CD8 cell to degranulate and release contents granules containing cytotoxic proteins
- Target cell killed by cytotoxic processes, e.g., perforin-granzyme
describe primary vs secondary response
- B cell contacts Ag, generally in LN
- IgM produced 48-72 hrs after Ag exposure to B cell in LN
- B cell does somatic hypermutation (affinity maturation)
- isotype switching → more long-lived response
- As Ag is cleared, contraction occurs → reduced B cell #s
- Memory cells are made
vs - exposure to same Ag
- Memory cells activated, proliferate (clonal expansion), and secrete IgG
- Reactivated memory cells contact Ag on FDCs and continue somatic hypermutation to inc ab affinity
- Contraction and generation of memory cells
ab feedback
process to downregulate ab prod. at high ab levels, ab bind to polyvalent ag –> ag/ab complex –> polyvalent ag bind to BCR and Fc portion of ab bind to CD32 –> activates ITIM domain –> ITIM dephosphorylates and inactivates BCR signaling
major sites of mucosal immunity/MALT
GALT, NALT, BALT, GU & rectal mucosa, mammary glands
specialized structures of mucosal tissues vs challenges in gut mucosa
epithelial structures (villi, cilia) and lymphoid structure (in submucosal layer under epithelium; organized but not encapsulated like LN; contains B/T cell, ILC, DC, and mac) vs large SA to protect and discrimination b/w microbial communities –> used specialized mucosal immunity
describe role of specialized epithelial cells for gut mucosa
absorptive epith cells - absorb nutrients, secrete cytokines, produce beta defensins in colon; goblet cells - produce mucin –> sweeps contaminated mucus, prevent contact of microbes w/ epithelium; microfold/M cells - ag transport to DC in Peyer’s patch w/o processing => transcytosis; Paneth cells - secrete alpha defensins for cytotoxicity and activate neu, CD8, & NK cells
what’s the key interaction in activating DCs w/ gut epithelium?
TLR binding P/DAMP
homing
after APC captures ag, APC travels to mesenteric LN –> activate naive B/T cells –> T cells induced by APC in GALT return to intestines; processed by retinoic acid (from vit A absorbed by DC); integrin allows T cells to home
neonatal Fc receptor: FcRn
expressed on placenta and gut epithelium in neonates, expressed on endothelial cells and macs in adults; transfers igG across cells while protecting it from lysosomal degradation –> transfers maternal igG to baby and prolongs half-life of igG
describe igA prod in gut mucosa
B cells producing igA = in Peyer’s patch and mesenteric LN. M cell captures ag and present to naive B/T cell –> ag-specific B cells = activated –> DC secrete RA for B cell gut homing and secrete factors for igA isotype switch
poly-Ig receptor
ab produced by GALT B cells must be transported from lamina propria to gut lumen: intestinal epith cells produced poly-Ig receptor –> binds igA dimers and igM pentamers thru domain J chain –> transcytosis –> poly-Ig receptor cleaved –> part of poly-Ig receptor bound to ab protects igA and igM from degradation in gut lumen
alveolar macs
majority of free cells in alveolar spaces, poorly phagocytic; express lots of inhibitory receptors; nml phenotype = antiinflamm –> secrete IL10, TGF-β, NO –> inhibit activation of pulm DC and T cell
describe GU mucosa
multi-layered epithelium w/ strat sq cells, no lymphoid follicles, little igA –> more igG; have mucus-producing cells, produce antimicrobial peptides, have regional DC/B/T cells
clonal anergy vs clonal ignorance vs tolerance vs tolerogen
failure of B/T cells to react to specific ag vs self ag = ignored even when B/T cells recognize ag = functional vs lack of response by ADAPTIVE immune response vs ag recognized by ADAPTIVE immune cells but induces cells to be anergic
central tolerance vs peripheral tolerance
occurs w/ immature lymphocytes in primary lymph organ vs occurs w/ mature lymphocytes in circ and secondary lymph organ
T cell tolerance vs B cell tolerance
elim or activation of self reactive T cells vs B cells
T cell central vs peripheral tolerance
in thymus; 2 mechanisms: neg sel and generation of natural Treg; central selection factor = avidity of TCR binding to MHC self peptide complex; AIRE = autoimmune regulator protein - activates expression of ag for tissues outside of thymus vs ag dependent process; 3 mechanisms: anergy (lack of CD80/86 (costim) on APC –> can’t bind CD28 on T cell; binding inhibitory receptors like CD80/86 bind CTLA4 on T cell), suppression (natural and induced Treg cells expressing CTLA4 & PD1 or releasing perforin and granzymes => contact dependent, or secreting IL10, TGF-β, IDO => contact independent), deletion (apop intrinsic pathway: strong binding of self ag or growth factor deprivation –> Bak/x oligomerize –> activate caspase 9 –> activate caspase 3 –> apop; apop extrinsic pathway: ligand binds to death domain of receptors –> activate caspase 8 –> activate caspase 3 –> apop)
another way to induce T cell tolerance: tolerize DC
Tolerized DC doesn’t respond to activation signals –> can’t traffic from tissue to 2ndary lymph organ to activate T cells
B cell central tolerance vs peripheral tolerance
in bone marrow; 2 mechanisms: receptor editing (self ag = recognized w/ high affinity –> light chain removed –> RAG1/2 reactivated to make those light chains –> pass checkpoint or apop), deletion (apop pathways) vs 3 mechanisms: anergy (lack of costim), deletion (apop pathways), follicular exclusion
immunoprivileged tissues
tissues w/ no immune response b/c of potential dmg of inflamm and dysfunction from the immune response. ex: eye, placenta, brain, testes
why need tolerance? what happens if there was no tolerance?
restores homeostasis. self reactive B/T cells become activated
molecular mimicry
when ag has epitope very similar to self ag –> T cell reacts to both ag and self uninfected tissue
CBC vs w/ diff
count # RBCs and WBCs vs same w/ CBC but also count %age of WBCs
how to read Serum Electrophoretograms?
1st peak = albumin (hyper = dehydration, hypo = inc in other blood proteins like ab
2nd peak = acute phase proteins –> early inflamm
3rd peak = igM –> early humoral response, and some acute phase proteins
4th peak = igG –> established humoral response
abnl results: High third peak = increase in IgM –> new infxn, Wide 4th peak = broad, poly-clonal response, Narrow 4th peak = monoclonal response –> B cell lymphoma or leukemia
pos vs neg acute phase proteins
Made by liver in early inflammation (especially in systemic), Appear in α peak (and some in β), Includes: CRP (c-reactive protein), SAA, fibrinogen, ferritin, haptoglobulin vs Decrease in serum during acute phase response, Compensate for increase in positive acute phase proteins to keep total protein conc and blood vol at the same level, Includes: albumin, transferrin
titration based assays. neutralization activity
serum dilution generates value as patient’s titer of interest (directly or reciprocal); higher the [Ab] –> more serum must be diluted to reach the cut-off point; greater dilution = higher reciprocal value. how much of Ag-specific Ab can neutralize target; If Ab is neutralizing –> less activity noted in assay (due to lower virus titers/less toxic activity)
how to produce polyclonal ab vs monoclonal ab
Host animal = immunized and boosted with Ag –> when Ab titers = high, immune serum = removed –> serum is used or Ab is purified from it vs Host animal (mouse) = immunized –> Spleen = removed and lymphs = isolated –> Lymphs fused with myeloma (cancer cells) –> Surviving cells are screened for Ab production => hybridomal (mix of ca cells + spleen cells) –> Individual Ab-secreting cells isolated by culture
advantages vs disadvantages of making polyclonal ab. why do monoclonal ab?
Rapid and inexpensive, generate Abs with diverse specificities and functionality (broad levels of protection), Can re-bleed host as blood volume is reconstituted vs With repeated administration, patient develops Ab response against therapeutic Ab since it is from a non-human host, Only have anti-serum (not B cells) –> dependent on host for continued production. immunotherapy: prevent dz and tx dz
purification procedures w/ ab: affinity chromatography vs immunoprecipitation
bind Ab to column matrix to then purify vs Ab = mixed w/ Ag-containing solution or cell expressing Ag –> Ab = linked to particle or bead that can have moiety that would bind Ab –> Centrifuge beads –> elute bound material
agglutination assays
Ab is used to crosslink Ag present on particle surface –> forming Ag-Ab matrix and producing visible aggregates. used for Blood typing, Autoimmune disease (RF factor, SLE), Bacterial infection testing, Hemagglutination and hemagglutination inhibition. semiquantitave
Enzyme Immunoassay (EIA)/Immunofluorescent Assay (IFA)
qualitative. detect either Ag or Ag-specific Ab in a patient’s sample; ex: ELISA
lateral flow assays
qualitative. sample added and pulled thru test cartridge via lateral flow established by absorption pad –> Analyte in sample binds to conjugate (Ab specific for analyte labeled with reporter molecule) –> Sample flows across the test line of analyte-specific Ab –> Binds analyte-conjugate complex THROUGH ANALYTE –> Sample flows across the control line of anti-species Ig –> Binds analyte-conjugate complex THROUGH Ab –> Colorimetric reaction develops if anything is bound at test or control lines. ex: at-home pregnancy tests, rapid at-home COVID tests
western blot
qualitative. sample (infected cell/tissue, serum) is subjected to protein electrophoresis –> proteins = separated by size and charge –> Proteins = transferred to a membrane that is probed with Ab specific for Ag of interest. Has diagnostic uses (HIV, Lyme disease, HLA typing)
isolation leuk subsets
by size/density: Sample containing mix of cells is layered on a solution with defined densities –> Sample is centrifuged –> cells migrate to density that matches their own
by ag specificity: FACs (fluorescent activated cell sorting) or MACs (magnetic-activated cell sorting). Cell = labeled with Ab conjugated with fluorescent or magnetic particle that is used to “direct” its isolation
DCF-DA assay
measures [O] burst. Passively diffuses into cell –> Hydroxyl, peroxyl, or other ROS cleave DCF-DA to fluorescently active form –> Amount of fluorescence is proportional to amount of ROS generated
how to measure phagocytic activity?
Phagocytic cells exposed to small beads labeled with fluorescent molecule –> Cells = examined at various times post-incubation to see how many beads have been phagocytosed (by flow cytometry or fluorescent microscopy)
how to measure lymphocyte proliferation?
measure cell’s ability to proliferate when stimulated in cell culture. Isolate lymphocytes –> Place in culture with stimulus (ex: mitogen: substance that stimulates mitosis) –> Add reporter (radioactive thymidine or dye) –> Measure uptake of reporter as a measure of mitotic rate OR measure amount of cytokine produced by EIA
flow cytometry. what is it used for?
measure cytokine prod in each cell. Isolated cells = stimulated with peptide(s) of interest with chemical during incubation that prevent protein secretion –> Cells are fixed and permeabilized –> stained with labelled Ab –> Anti-cytokine Ab can enter permeabilized cell –> Fluorescent signals are then measured by flow cytometry. Used for immunophenotyping (tell what ca stage), fluorescent activated cell sorting (isolate cells of interest), epitope mapping of B/T cell response
immunohistochemistry vs immunocytochemistry
using Abs to label cells within TISSUE section vs using Abs to label parts of individual CELLS
risk factors of autoimmune dz
pmhx of autoimmune dz, fhx, genetics (mutations, failure of tolerance, genetic polymorphism), environ exposure, gender (hormone levels - women higher risk than men), dysregulated immune rxns
what 3 scenarios can lead to autoimmunity?
- APC reacts highly to ag –> activates self reactive T cell
- T cell thinks healthy tissue = ag d/t molecular mimicry (Guillain Barre syndrome - destruction of myelin sheath, Celiac dz - react to gliaden (gluten peptide)
- Indirect damage by CD4-produced cytokines or Direct damage by CD8 CTL d/t proinflamm response
what are the major effector molec of autoimmune dz?
ab –> forming immune complexes or binding to tissue activating complement and ADCC. and highly specific HLA polymorphic alleles –> MHC II affecting CD4 T cell activation
Systemic Lupus Erythematosus (SLE)
Ab-mediated with Ag-Ab complexes deposited throughout body. from UV exposure, drugs, HLA DQ8, HLA DR3, complement, or TNF pathway. sxs: constitutional (fatigue, fever w/o infection, headache and/or weight loss), cutaneous (rash, photosensitivity), musculoskeletal (arthritis, arthralgia), organ-specific (nervous system, renal, cardio-pulm, and GI). pathophysio: UV –> apop –> proinflamm response –> ANA and anti-dsDNA ab made –> high ag-ab immune complexes all over body –> high Th17 and IFN-alpha from DC, low Treg and hyperactive B cells. dx: pts meeting >/= 4 of 17 criteria of Systemic Lupus international Collaborating Clinics (SLICC), presence of ENAs (antiRo, antiLa, antiRNP). tx: anti malarial
Rheumatoid Arthritis
Slow and progressive systemic inflammatory dz involving 3+ joints –> musculoskeletal disability, can lead to ag/ab immune complex deposition in lung and kidney. from gender (more women than men), genetics (HLA and non HLA), environ (smoking, periodontitis). pathophysio: self reactive B/T cells destroy synovial joints –> ag/ab complexes deposited –> proinflamm cytokine prod –> synovial fibroblasts proliferate –> activate more osteoclasts; can become extra-articular disease with affected tissues including lung, kidney, eyes, skin, heart, nervous system, GI. dx: Rheumatoid factor (antibodies to IgG), Anti-CCP Ab. tx: non biological anti-rheumatic drugs, adjunct therapy, biological anti-rheumatic drugs (TNF inhibitors, JAK STAT inhibitors)
Autoimmune Diabetes
aka Type 1.5 DM or latent autoimmune diabetes of the adult (LADA), slow and progressive. from genetic (HLA and on HLA), stress d/o, enterovirus infxn, Celiac dz. pathophysio: pancreatic beta cells = destroyed –> can’t make insulin (often misdz as TIIDM), autoreactive ab made. dx: excessive thirst, frequent urination, blurry vision, weight loss; Onset of diabetes at age > 30 years, presence of circulating islet autoantibodies, Insulin independence for ≥ 6 months after diagnosis. tx: get insulin, statins, antihypertensives, exer
Hashimoto’s Thyroiditis
painless goiter, generally symmetrical; may also present with hypothyroidism with or without goiter. from fhx of autoimmune association, environ (high iodine intake, low selenium, pollutants, prior HCV) or genetic (HLA or non HLA); cigarette smoking = dec risk. pathophysio: environ dmg to thyroid –> ag in thyroid –> APCs activate B/T cells –> B/T cells infiltrate thyroid and dmg thyroid, promoting apop. dx: eval TSH, free thyroxine (T4), Ab to thyroid peroxidase (TPO), anti-thyroglobulin Ab. tx: levothyroxine
Myasthenia Gravis
Autoimmune disorder of neuromuscular junction. from use of penicillamine (anti-rheumatic drug), fhx (1st degree family member with SLE), genetics (HLA association (HLA-B*08) and non HLA). pathophysio: B cells produce autoab to Ach receptor –> block or destroy AChR at postsynaptic neuromuscular junction –> Ach can’t bind to AChR; Achesterase degrades Ach –> sxs of dz. dx: clinical pres (skel muscle weakness); testing Abs to AchR, muSD, or LRP4 in serum; edrophonium chloride testing (inhibits breakdown of Ach by interfering with Achesterase –> improves muscle weakness). tx: Achesterase inhibitor –> lets Ach bind to AchR, corticosteroids for immunosuppression
cell surface/membrane bound TLRs: TLR1 vs TLR2 vs TLR4 vs TLR5 vs TLR6
Mono/macs, DC, PMN, B cells; ligand = bacterial lipoproteins vs mono/macs, DC, PMN, mast cells; ligand = bacterial and fungal glycolipids and lipoproteins like peptidoglycan, lipoteichoic acid, lipoabarinomannan; forms heterodimer w/ TLR1 or TLR6; good for gram pos and mycobacteria infections vs mono/macs, DC, PMN, mast cells, epithelial cells; ligand = LPS (bacterial lipopolysaccharide) of gram neg bacteria; binds DAMPs vs mono/macs, DC, PMN, epithelial cells; ligand = flagellum vs mono/macs, PMN, mast cells, B cells; ligand = lipopeptides (eg. Mycobacterium, Mycoplasma)
cytosolic/endosomal TLRs: TLR3 vs TLR7 vs TLR8 vs TLR9
DC, NK cells, B cells; ligand = viral dsRNA —> good for viral infections, artificial ligand/agonist = polyl:C (polyinosinic-polycytidylic acid) vs mono/macs, DCs, PMN, B cells; ligands = ssRNA —> good for viral infection, synthetic ligand/agonist = imiquimod, R848; expression = inc and induced on stimulated APCs vs mono/macs, DC, PMN, mast cells; ligand = viral ssRNA —> good for viral infection, agonist = imiquimod, R848; expression = inc and induced on stimulated APCs; not basally expressed by cells but inducible vs mono/macs, DC, PMN, NK cells, B cells; ligand = unmethylated CpG oligonucleotides (ODN) from bacteria and viruses, agonist = CpG ODN or vaccine adjuvant; can also be activated by apoptotic DNA
how to fight extracellular bacteria vs intracellular bacteria vs mycobacteria?
humoral immunity: ab, complement, neu and mac; CD4 T cells (differentiation, isotype switch) and CD4 Th17 cells (recruit neu) vs cell-mediated immunity: mac, NK cells; CD8 T cells, CD4 Th1 cells (produce IFN-gamma) vs cell-mediated immunity, IFN-gamma, TNF-alpha, NK T cells
how to fight viruses?
Type I IFNs (IFN-α and IFN-β), NK cells (ADCC), mac; CD8 T cell (IFN-gamma, TNF-alpha), CD4 Th1 cell (cytokine prod to promote CD8 T cell); ab can bind virus and neutralize it but virus can escape endosome before getting degraded => ag dependent enhancement
how to fight extracellular vs intracellular parasites?
phagocytes, cytokines, complement overall. humoral immunity: igE –> FcER on eos, mac; CD4, NK (ADCC) vs cell mediated immunity: mac, NK; CD4 Th1, CD8, gamma/delta T cells
how to fight fungi?
phagocytes w/ dectin PRR, ILC making IL17, complement; CD4 helper cells, CD4 Th17, CD8, ab
how to fight tumors?
NK, macs, eosinophils, cytokines; CD4, CD8, B cells, antibody, cytokines
how can microbes evade immunity?
polysacch capsule, toxins, ag variation, intracellular survival, suppress immune response, extracellular enzymes degrading immune molecules
M1 vs M2 mac activation
bind PRR –> crosslink receptors (FcR or CR) –> IFN-gamma from other macs/CD4&8/NK stimulates M1 (classically activated) vs IL4 and IL13 from Th2 stimulates M2
