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FHS Paper 1 > Immune recognition > Flashcards

Immune recognition Flashcards

1
Q

Early observations in TCR signalling

A
  1. If inhibit try phoshatates, induce TCR phosphorylation and signalling without pMHC
  2. TCR is small compared with other surface molecules

=> kinetic segragation model (1990s/2000s)

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2
Q

Predictions of kinetic segregation model

A
  1. Tyr phosphatases segregate from TCR on ligand engagement
  2. Truncation of phosphatase enzymes inhibits their segregation and thus inhibits TCR triggering
  3. Elongation of pMHC decreases segregation of phosphatases
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3
Q

Varma et al 2006

A

Overlayed fluorescence of CD45 and TCR distribution at immunological synapse between T cell and artificial APC

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4
Q

Other NTR triggering mechanisms proposed

A
  1. Induced proximity/ aggregation
    - Low surface density of pMHC and T cells only need a couple of pMHC to trigger makes this unlikely- how can a single TCR-pMHC induce TCR aggregation?
    - Unlikely to be TCR/co-receptor heterodimerisation because not all TCR signalling requires co-receptor
  2. Conformational change -> reveals tyr residue
    - Allosteric? structural studies have not found conserved allosteric changes associated with binding
    - Mechanical mechanism? Evidence of CD3 conformational change with binding but unclear how mechanical force would transfer. Also, if mechanical mechanism, accessory receptor-ligand interactions (CD2, CD58) would reduce force on TCR, but these interactions enhance signalling.
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5
Q

Kohler et al 2010

A

Studies NKCs and target cells. Introduced ligands for activator and inhibitory receptors into target cells -> affects cell lysis.
Elongation of inhibitory ligand decreased efficacy of inhibition.
When have longer activator ligands, shorter inhibitory ligands are less effective.
If same size, ligands colocalise => proximal membrane signal integration (allows localisation of abnormal cells and response polarisation)

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6
Q

Yokosuka et al 2012

A

Optimal PD1 inhibition of TCR requires matched size and colocalisation.
Authors varied size of PD1 and measured IL2 produced.
Long forms of PD1 lost colocalisation and inhibition; shortening restores.

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7
Q

Signal integration TCR and CD28

A

Can integrate in nucleus so do not need colocalisation

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8
Q

Arase et al 2002

A

Paired activator and inhibitory NTRs result from gene duplication, possibly due to host-pathogen arms race.

Authors studied mice (have Ly49Rs on NKCs rather than KIRs)

  • inhibitory R Ly49I binds MHC-I
  • murine CMV expresses decoy receptor m157 which engages the inhibitory receptor (so MHC-I can be down-regulated in infected cells without consequence, escaping NKCs and T cells)
  • some mouse strains express a paired activator version of the inhibitory receptor (Ly49H) -> binds decoy receptor so activates NKC vs virally infected cells
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9
Q

Yang et al 2016

A

APCs only express a few copies of any given foreign pMHC.
Authors found 8-46 specific pMHCs in in vitro HIV infection

~10^12 T cells in periphery with ~10^8 unique TCRs

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10
Q

3 key studies in 90s

A

[Evavold 1991] [Hogquist 1994] [Lyons 1996]
T cells from mouse spleen -> mix with APCs loaded with different peptide concentrations (mixed lymphocyte reaction).
Good T cell response to wt Ag but abolished with single AA mutation
=> T cells very specific

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11
Q

Don Mason 1998

A

Theoretical calculations assuming ~10^8 unique TCRs can produce effective immunity.
Recognise peptide 8-14AA length, with 20 proteinogenic AAs.
Predicts ~10^5 to 10^6 pMHC per TCR

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12
Q

Ignatowicz 1996, 1997

A

Mice achieve comprehensive T cell repertoire with selection by a single peptide.
Their T cells respond to different, unrelated peptides.

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13
Q

Wooldridge et al 2012

A

Decamer CPLs. 9.36 x 10^12 peptides used to quantify degeneracy of patient derived CD8 TCR.
Subsampled likely peptide candidates from defamer CPLs, then incubate with T cells and C1R-A2 cells.
ELISA for MIP1beta.
Used mathematical algorithm weighting peptides based on circumstances of T cell activation.

~500 peptides within factor of 2 of optimal agonist
~60,000 within factor of 10
~1.3x10^6 within factor of 100

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14
Q

Functional consequences of TCR degeneracy

A
  • solution to providing comprehensive immunity whilst conserving resources
  • polyclonal responses
  • molecular mimicry -> autoreactivity
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15
Q

T cell discrimination models

A
  1. Occupancy model
    - assumes activation determined by number of bound TCRs, immediate signalling. pMHC potency depends on Kd. Most R-L interactions in body.
  2. KPR model
    - activation based on number of TCRs bound for sufficient duration
    - due to proofreading time between binding and signalling (biochemical steps e.g. ITAM phosphorylation, binding and phosphorylation of ZAP70)
    - unbinding reverses biochemical steps
    - amplifies differences between Koff of different ligands => higher discrimination
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16
Q

Yousefi et al 2019

A
  • Optogenetic system using phyB as a ligand, so TCR binding controlled by light
  • red -> phyB cycles between bound and unbound (rate depends on intensity)
  • mathematical modelling indicated that half life of ligand-TCR interactions determines signalling downstream of TCR
  • artificial system not using pMHC, however their system did induce Ca and Erk MAPK signalling and led to up regulation of CD69 (T cell activation marker)
17
Q

Pettmann et al 2021

A
  • model of T cell discrimination and promiscuity
  • T cells expressing 1G4 TCR (recognises peptides expressed in many cancers, Kd~8uM)
  • altered peptide with series of point mutations to decrease affinity then plotted dose-response curve => gradual reduction in response as decrease affinity
  • at 15% response cut-off, correlation between p15 (peptide conc) and Kd
  • gradient gives index of discriminatory power, alpha
  • for TCR, a = 2 whereas a = 1 for other receptors in body tested (e.g. IL13R, M3, c-Kit). a = 2 => 10x decrease affinity overcome by increase ligand conc by 100x. BCR a = 1.3 (intermediate) but more data needed.
    => evidence against occupancy model (which would have a = 1 due to linear relationship between affinity and concentration)
  • authors calculated 2.7 biochemical steps with proofreading time of 2.8s
  • authors also did meta-analysis of existing murine and human data- the 3 90s studies showed a = 9 whereas all more recent data gave a ~ 2
  • attributed to inaccurate measurements of binding using surface plasma resonance (which struggles to accurately measure very week TCR-pMHC interactions)
18
Q

Kohanim et al 2020

A
  • proposed an immune surveillance mechanism consistent with enhanced but imperfect discrimination
  • auto reactive T cells kill abnormal hyper secreting endocrine cells to maintain homeostasis
  • e.g. T cell may recognise unregulated proteins in the pathway connecting a mutation to hyper secretion of insulin in beta cells in T2DM, then kill those cells
  • autoimmunity upon over expression of self-antigen
19
Q

KPR to account for optimal affinity

A
  • enhance TCR affinity for cancer therapeutics
  • these receptors are actually less sensitive than WT
  • once bind Ag, TCR could signal for a period of time then something limits it (e.g. internalised with ligand) => so ligands with supra-physiological affinity would prevent TCR returning to membrane
20
Q

Huang et al 2013

A

T cell antigen discrimination is sensitive (able to respond to ~1-10 pMHCs on an APC)

  • authors showed that a single pMHC can trigger cytokine secretion from T cells
  • mixed CD4 T cells with APCs bearing qdot-labelled pMHC
  • cytokine captured on T cell surface to allow detection by fluorescent antibodies
  • T cells responded to even a single pMHC
  • as increase the number of pMHCs, more T cells are able to respond on average
21
Q

TCR sensitivity remarkable in light of two observations

A
  1. TCR/pMHC lifetime is short (seconds) and is subjected to molecular forces
    - [Huppa et al 2010] - solution lifetime of recombinant proteins 1.24s; membrane lifetime with T cells 0.109s
  2. Cytokine production requires TCR/pMHC interactions for several hours
    - [Huppa et al 2003] - simultaneously tracked TCR-CD3 complex and PI3K activity in single T cells using 3D video microscopy
    - despite rapid internalisation of most TCR-CD3, TCR-dependent signalling was still evident up to 10hrs after conjugate formation
    - blocking this interaction caused dissolution of the synapse and proportional reduction in IL2 production and cellular proliferation
22
Q

Molecular mechanisms of T cell Ag sensitivity

A
  • microvilli-like protrusions (actin-mediated)
  • adhesion receptors e.g. LFA1, CD2 (binds CD58 which is expressed on all cells in body)
  • coreceptors (if block, need more Ag for same response)
23
Q

Trade-off between T cell sensitivity and discrimination

A

^ KPR time increases discrimination (since increase fold-difference between pMHC) but decrease sensitivity (as probability of signalling for any pMHC is decreased)

24
Q

Farfan & Dushek

A

T cell Ag sensitivity mechanisms

25
Q

Newby et al 2016

A

mathematically calculated 10^9 seconds for CD45 to spontaneously segregate from a small 100nm membrane patch

26
Q

Carbone 2017

A

In vitro reconstruction of CD45 spontaneous segregation -> 15 mins

T cell signals in seconds

27
Q

Sage 2012

A

Microvilli-like protrusions push on nearby cells -> close membrane apposition and CD45 segregation

28
Q

Bachmann 1997

A

LFA-1 and CD2 increase T cell sensitivity in functional studies

29
Q

Wang et al 2018

A

HCMV can decrease CD58 expression (ligand for CD2) -> decreased recognition and killing of infected cells by CD8 T cells

30
Q

Patel 2017

A

KO of CD58 in cancer cell lines impaired recognition and cytolysis

31
Q

Jiang et al 2011

A

CD8 modulates TCR-pMHC. Micropipette adhesion assay

32
Q

Dushek et al 2008

A

Mathematical analysis showing that a single pMHC serially binds multiple clustered TCRs before diffusing away.
Serial binding increases sensitivity

33
Q

Liu et al 2014

A

TCR-pMHC subjected to tensile forces, which would increase kOFF ordinarily. Forces may be due to long surface molecules.

Hypothesis that TCR-pMHC interactions for catch bonds rather than slip bonds (so applied force rather increases lifetime). Authors observed this for OT1 TCR binding its ligand, OVA-pMHC.

FHS Paper 1 (4 decks)

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