biol 485 exam 2

what do APCs recruit?

• helper T cells

• cytotoxic T cells

how do B cells protect the body?

• antibodies bind to virus → can’t bind to cell surface

• cells not affected by virus

what cell is cytotoxic T cell targeting?

• internal antigens inside cell

• infected cells, cancer

• induces apoptosis

what are some of the mechanisms of action by antibodies?

• bacterial toxins → neutralization

• bacteria in extracellular space → opsonization

• bacteria in plasma → complement activation

what is the structure of an antibody?

• quaternary → 2 heavy + 2 light

how is there antigen specificity in an antibody?

• interaction between light/heavy chain variable regions

• Fab (fragment of antigen binding)

how is antibody effector activity possible?

• interaction of the constant regions of the heavy chain

• Fc (fragment that crystalizes)

how can antibody diversity be encoded in the genome?

• original thought → one gene = one protein

  • estimated that different antibody types generated is > human genome

• recombination of the locus only in somatic cells (B cell)

how how can a finite amount of genetic info make different binding sites?

• variable region = changes

• VDJ recombination

where does the isotype switching happen?

• lymph node and spleen

• same variable region → changed constant region

what are the region segments in the light chain?

• Variable

• Joining

• Constant

what are the heavy chain locus region gene segments?

• Variable

• Diversity

• Joining

• Constant

how many loci are on the light chain?

• two

• V will join J through somatic recombination

• on either lambda or kappa locus

what is the mechanism of the VDJ recombination?

• recombination signal sequences (RSS)

  • has a nonamer and heptamer separated by 12 or 23 bp spacer (12/23 rule)

• 12 bp RSS pair with 23 bpRSS in opposite orientation for recombination to happen

• opposite orientation - heptamer → nonamer OR nonamer → heptamer

• heptamer is always closest to segment being recombined

how do the regions get clustered together?

• V region has a promoter upstream → if activated → transcription occurs → RNA splicing happens (C gets spliced to VJ)

• to get rid of unnecessary regions → recombination

RAG 1 + RAG 2

• RAG 1 = recombination activating gene 1

• RAG2 = enzymes → complex that recognizes RSS sequences

• RAG ½ = cleave DNA at junction between RSS and variable coding region

what is the process of RAG1 and RAG 2 acting on a light chain locus?

1. RAG ½ binds to RSS 12 bp spacer of V1

2. then it binds to RSS 23 bp spacer of J2

3. cleave between RSS and V1 + RSS and J2

4. join at signal joint (RSS together) then join at the coding joint

does RAG ½ give a clean cut?

• no nucleotides (palindromic or N) are added/lost to give more variability at VD, DJ, or VJ (light chain)

• junctional imprecision

• N is added random

• P is added due to asymmetry

what are some fo the mechanisms to generate antibody diversity in B cells?

• multiple gene segments

• heavy/light combinatorial diversity

• P nucleotide addition

• exonuclease trimming

• nontemplated N nucleotide addition

isotypes

• classes of antibody

• IgM (big/bulky hard to get into nooks)

• IgD

• IgG ( neutralization/blocking things)

• IgA (secrete on mucosal surfaces/mucus layers)

• IgE (mast cells/degranulation)

class switch recombination

• conserved (C) gene segments

• added after transcription during mRNA splicing step

• heavy chain locus has multiple c segments

• VDJ of heavy will splice with most downstream C segments C mew and Cdelta

• only AFTER class switch recomb can VDJ fuse with C

pre-BCR + BCR complexes

• recombine heavy chain locus → if successful expresses as dimer with surrogate light chains → light chain recombine → mature BCR complex

what is allelic exclusion and why is it important?

• ensures that each developing B cell synthesizes only one heavy and one light chain

• makes sure that there is only one receptor being presented (monoclonal)

T cell receptor

• one constant and one variable region

• variable regions constitute antigen binding site

• complementarity determining regions (CDRs)

  • highly variable region

  • interact with MHC I and II

cross presentation

• pAPCs (dendritic)

• only do it when its licensed to do so

• phagocytosis → make peptides → on MHCII and MHCI

MHCI

• every nucleated cell

• internal proteins

• CD8 + T cells

  • cytotoxic T cells

• short peptides

• alpha 1 +2' binding pocket (one protein)

• endogenous pathway

MHCII

• pAPCs

• external proteins

• CD4 + T cell

  • T helper cells

• longer peptide

• alpha-1 and beta-1 binding pocket (two different chains)

• exogenous pathway

what do cytotoxic T lymphocytes do?

• look for infected and transformed cells to kill

• CD8 + T

what do helper T cells do?

• activate other immune cells

• for B cells

  • proliferation

  • differentiation

  • class switch recombination

what are the regions that interact with peptide or MHC I or II?

• complementarity determining regions (CDRs)

  • on the highly variable regions

what makes the peptide binding groove in MHCI and II?

• MHCI → a1 and a2 domain

• MHCII → alpha 1 and beta 1 domains (from the a and b chains)

what is the order in which VDJ rearrangement occurs in T cells?

1. double negative (DN) T cells (neither CD4 or CD8)

2. recombines TCR on beta chain locus first → pre-TCR

3. pre-TCR → signal for proliferation →T cell express CD4 + CD8 (double positive(DP))

4. DP cells initiate alpha chain rearrangement → mature TCR complex

5. if TCR binds MHCI → CD4 if MHCII → CD8 = single positive T cells

what is MHC restriction?

• T cells → restricted in recognizing peptides only in context to MHC → restricted to one MHC allele

• establish if a T cell will become CD4+ or CD8+

how does allelic diversity work in context to MHCI?

• different individuals load different sets of viral or bacterial peptides

• also can display altered self peptides from cancer cells → CTL killing

can macrophages express MHCII by themselves?

• no need to be stimulated by cytokines in order to present MHCII

How do B cells become activated when presenting MHCII?

• TCR activate B cells

what are some of the features that help maximize the variety of peptides?

• codominant expression - maternal + paternal expressed at the same time

• polygeny - multiple genes → same function

• polymorphism - many different alleles circulating in population

how many different versions can one individual present of MHCI and II

• 6 different MHCI

• 12 different MHCII

how can MHC expression change with conditions?

• genetic regulatory components

  • promoters → increased MHC I + II

• viral interference

  • shut down MHCI expression →bind B2-microglobulin

• cytokine-mediated signaling

  • IFN-a and TNF-a increases MHC expression

  • corticosteroids and prostraglandins downregulate MHC expression

endogenous paathway

1. errors in translation → proteosome degradation of proteins

2. peptides from cytoplasm → ER by TAP complex

3. chaperones help fold and move it close to TAP

4. once it is loaded it’s released to secretory → expressed on surface

what are chaperones?

• proteins that help other proteins fold

• calnexin, calreticulin, ERp57

where does MHC start in each pathway?

• rough er

where does each pathway get its proteins from?

• cytoplasm (endogenous)

• exogenous

how does each pathway turn those proteins into peptides?

• proteosome (endogenous)

• phagocytosis + acidification (exogenous)

how do the peptides get into the same space as the MHC?

• TAP complex (endogenous)

• fusion of exosome with endosome (exogenous)

exogenous pathway

• antigen gets phagocytosed with antibody

• antigen gets degraded into peptides

• CLIP (class II associated invariant chain) binds MHCII to prohibit MHCI binding

• acidify to degrade and peptides can bind for expression

what must happen to thymocytes before and after leaving the thymus?

• before

  • V(D)J recombination to make a random TCR

  • positive selection (for a proper TCR)

  • negative selection ( not recognizing self)

• after

  • functionally mature T cells but naive

what is the path of T cell development in the thymus?

• HSC precursor cells enter → uncommitted w/ no TCR

• undergo V(D)J and positive selection

• commit to either CD4 or CD8

• migrate to medulla → negative selection (central tolerance)

• TCRs that are both tolerant and MHC-restricted

what is the receptor that commits T cells to their lineage?

• arrive in thymus → can still become NK cells, dendritic, b or T

• Notch → signaling activates GATA-3

• also can commit in vitro without the thymus present

what are the two main stages of T cell development and their brief steps?

• early phase development

  • commitment of HSC to T cell lineage

  • VDJ

  • expansion of T cells that have rearranged one TCR gene (beta selection

  • completion of VJ recombination

• late phase development

  • positive selection (proper signaling TCRs)

  • negative selection (recognize self MHC)

  • lineage commitment (either CD4+ or CD8+)

what are the double negative stages?

• thymic precursors (CD44+ and CD25-) ← DN1

• notch (expressed by thymic epithelium) begins to commit → turns on CD25 ← DN2

• undergoes VDJ recombination to express pre-TCR ← DN3 + turn off CD44 expression

• proper signaling through pre-TCR leads to maturation ← DN4 + loss of CD25 expression

what is the sequence if proper pre-TCR signaling is successful by DN3?

• maturation to DN4

• proliferation in the subcapsular cortex

• suppression of further TCR beta rearrangement

• express both CD4 + 8

• cessation of prolif.

• initiation of TCR alpha-chain rearrangement

pre-TCR

• recombine TCR beta chain and if successful → beta chain w/ pre-Talpha protein = pre-TCR

• if DN3 unsuccessfully rearranges both beta chain loci → apoptosis

mature TCR complex

• replaces pre-TCR complex at the surface

• TCR-alphabeta/CD3 complex

• turn into double positive T cells (DP) → undergo positive and negative selection

what do double positive T cells undergo?

1. positive selection (low-intermediate affinity) → MHC restriction

2. negative selection (high affinity)

3. lineage commitment (either CD4+ or CD8+)

what are some of the characteristics of positive selection?

• occurs in the cortex of thymus

• self peptide MHC I or II

• affinity model of selection

  • no binding → death by neglect

  • high affinity → apoptosis

  • low-intermediate affinity → positive selection + maturation

how does positive selection ensure MHC restriction?

• cortical thymic epithelial cells (cTECs) mediate positive selection

  • express self peptide on both MHCI + II

• if DP thymocyte binds to self MHC on cTEC → survival + differentiation

• if fails → die by neglect

what does affinity model support?

• the strength of the signal received is critical

  • fetal thymocytes (FTOC) are in vitro with peptides added exogenously

  • the MHCI on thymic epithelial cells (cTECs) have no, low or high affinity for their peptide

what are some of the characteristics of lineage commitment?

• the DP thymocytes are tested for low-intermediate binding to self-MHC in the cortex of the thymus

• expression of the CCR7 chemokine receptor to move to medulla

• result in silencing of either CD4 or 8

what are the models for lineage commitment?

• instructive

• stochastic

• kinetic signaling

instructive model?

• TCR/CD4 or TCR/CD8 instructs the T cell to commit to a lineage

stochastic model?

• randomly downregulate CD4 or cd8 → cells with the wrong MHC specificity will die

kinetic signaling model?

• downregulate CD8 by default

  • TCRs specific to MHC II will maintain continuous reaction through TCR/CD4/MHC II binding → commit to CD4

  • specific to MHCII dirupted interaction through TCR/CD8/MHC I due to low CD8

what are the other lineages that DP thymocyte could commit to?

• NKT cells

• intraepithelial lymphoytes (IELs)

• regulatory T cells (Treg)

what are some of the characteristics of negative selection?

• occurs in (central tolerance) → medulla of the thymus

• occurs through clonal deletion → apoptosis is induced that bind too strongly to self-MHC

• thymic macrophages in both cortex and medulla

• medullary thymic epithelial cells ( mTECs)

what is AIRE?

• autoimmune regulator → transcription

  • induces expression of many tissue specific proteins in mTECs

  • leads to expression of tissue-specific antigens (TSAs)

what are some other mechanisms that maintain self-tolerance?

• negative selection

• peripheral tolerance

• development of regulatory T cells

  • high affinity interactions during negative selection

how do regulatory T cells inhibit other T cells?

• depletion of local area → stimulation of cytokines

• production of inhibitory cytokines (IL-10 and TGF-B)

• inhibiting activity of pAPC

• killing other T cells

what are the signals needed for T cell activation?

• signal 1 → antigen-specific TCR engagement (TCR + CD4 binding)

• signal 2 → contact with costimulatory ligands on APC

  • costimulatory signals → needed for optimal T cell activation + proliferation

• signal 3 (needed for FULL activation) → cytokines directing T-cell differentiation

what is the complex in the center of the immunological synapse with CD4 and CD8?

central supramolecular activating complex, cSMAC

what is the complex that allows for high affinity interactions that allow for stability?

• peripheral supramolecular activating complex, pSMAC

what happens during signal 1?

• TCR binds to peptide

  • relies on CD3 for signal transduction

• CD4 binds to MHCII away from the peptide

what does T-cell activation begin with?

• tyrosine kinase Lck

• CD4 + CD8 cytoplasmic tails bring Lck to TCR complex

• interacts with CD3 → phosphorylates tyrosines on ITAM (immunoreceptor tyrosine-based activation) motifs → phosphorylated ITAM (docking sites for ZAP-70) → Lck phosphorylates ZAP-70 → phosphorylates LAT + SLP-76

• transcription factors - AP1, NF-kappa-B, NFAT together will turn on survival for T cell

what provides the second signal required for T cell activation?

• by the CD28 coreceptor binding to CD80 or CD86 on the pAPC

  • induce IL-2 → for T cell proliferation

  • Bcl-2 → survival

• activation of DCs or macrophages through PRRs will lead to increased expression of CD80/86

• if the T cell only gets 1 signal it will be difficult to activate (anergic)

for signal 2 (T cell activation) what is the significance of coinhibitory molecules?

• activate T cell → replicates 24 hours → turns on its own brakes

• CTLA-4 will outcompete CD28 for CD80/86 binding → blocking signal 2

• CTLA-4 = brakes (has a higher affinity)

what is the significance of the third signal (T cell activation)?

• keep the T cell proliferating/survival and differentiation

• polarizing cytokines → direct naive T cells to become specific effector T cells (specialized recruiter)

how does the third signal (T cell) polarize T cell, CD4+ to specific subpopulations of T cells?

• APCs sample then send out cytokines to polarize T cells into specific subsets

  • APC recognize → give specific job

what is the outcome of activation + proliferation of T cell activation?

• memory + effector cells

• effector - short lived and dead by the end of the response

• memory cells - long lived and quick to respond at a second exposure

what is the type 1 response of T helper cell subsets?

• viral infection + intracellular pathogens

• Th 1 + 17

• ex. TLR3 on DC → Il-12 production → promote Th1 differentiation of naive helper T → Th1 activate CTLs + APCs + B cell differentiation IgG

what is the type 2 response of T helper cell subsets?

• parasite + bacterial infections

• Th 2,22,9

• ex. worms stimulate PRRs (TLR2/4) → stimulate mast cells → IL-4 production → polarize Th2 → eosinophils + B cell differentiation to IgE

Th1 + Th17

• Th1

  • intracellular pathogens

  • macrophages

  • tissue inflammation

  • GATA3

  • stimulate B cells IFN-y (IgG1/IgE)

• Th17

  • extracellular pathogens (bacteria, fungi) barrier tissues

  • autoimmunity, tissue inflammation

Th2 + Th22 + Th9

• Th2

  • parasites

  • eosinophils

  • allergy

  • T-Bet

  • stimulate B cells

• Th22

  • extracellular

  • inflammatory skin disease

• Th9

  • extracellular + worms

  • autoimmunity