![Why
does
it
all
ma]er?
Mechanis&c
Underpinning
for
T
cell
Sensi&vity
These
studies
help
us
to
understand
the
physical
parameters
that
define
whether
a
pMHC
will
be
s&mulatory
or
not.
They
also
help
us
to
understand
the
exquisite
sensi&vity
of
T
cells
for
low
densi&es
of
agonist
pMHC.
Sensi&vity
is
important
because
professional
APCs
like
DCs
might
not
express
large
densi&es
of
a
par&cular
cognate
pMHC
for
a
T
cell.
Also,
a
scanning
T
cell/APC
interac&on
in
the
lymph
node
is
rather
short
lived
(~minutes),
so
the
T
cell
needs
to
be
able
to
signal
and
stop
migra&ng
if
even
a
small
amount
of
cognate
pMHC
is
found.
Possible
Mechanism
of
Quan&ta&ve
T
cell
Help
for
B
cell
Matura&on.
Review
re:
role
of
IS
in
T
cell
help
for
B
cells-‐-‐Dus&n.
Mol
Cell.
2014
Apr
24;54(2):255-‐62.
B
T
Ag
Y
Y
AnAgen
gathering
Efficiency
α
BCR
affinity
AnAgen
presentaAon
#
pMHC
α
BCR
affinity
TCR
signals,
acAvaAon
#
CD40L
α
#
pMHC
seen
T
B
T
cell
help
#
CD40L
α
B
cell
prolif
A
2014
ar&cle
suggested
that
exocytosis
of
TCR
in
the
cSMAC
may
contribute
to
long-‐term
regula&on
of
B
cell
responses
to
T
cell
help
las&ng
beyond
the
&me
frame
of
direct
T-‐B
interac&ons.](https://image.slidesharecdn.com/biom514cellsignalingneumann2017fullpghandout-170818220924/85/Immunoreceptor-Signaling-Lecture-Slides-72-320.jpg)
![Signal Transduction Dynamics
Dolmetsch, et al. Nature 392, 933-936(30 April 1998)
• T cells also exhibit Ca2+ oscillations and spikes during activation
• Ca2+ clamp method can reproduce arbitrary oscillation amplitudes
and periods (left)
At low levels of Ca2+, oscillatory [Ca2+]i
increases the number of cells expressing
an NFAT reporter.](https://image.slidesharecdn.com/biom514cellsignalingneumann2017fullpghandout-170818220924/85/Immunoreceptor-Signaling-Lecture-Slides-91-320.jpg)
Immunoreceptor Signaling Lecture Slides
- 1. 1. An&gen Receptor Signaling with focus on TCR signaling & the Immunological Synapse BIOM 514, Cell Signaling Apr. 1 & 6, 2016 Aaron Neumann, Ph.D. (Pathology) CRF327, akneumann@salud.unm.edu Abbas, Lichtman & Pillai, 8th ed., chapters 7, 9 and parts of 4 & 12
- 2. OVERVIEW OF LECTURE MATERIAL • Lecture 1: An&gen Receptor Signaling and the T cell Immune Synapse • Lecture 2: Cytokine Receptor, Notch and Innate Immunoreceptor Signaling. Regula&on of signal dynamics • Problem Set: Spillane & Tolar
- 3. Basic Concepts in Signal Transduc&on
- 4. Common events that govern signaling
- 5. • Signal ini&a&on • Ligand binding to membrane receptors leads to receptor conforma&onal changes and/or aggrega&on, resul&ng in phosphoryla&on of receptors (or associated intracellular signaling proteins) • Signal propaga&on • Recruitment of adaptor proteins to phosphorylated receptors ini&ates signaling cascades that take the signal to the nucleus • Structural mo&fs allow for specificity of protein interac&ons • Signal termina&on • Phosphatases can block signaling • Proteins are degraded (ubiqui&na&on) General principles of membrane receptor signaling
- 6. Types of receptors that regulate immune cell signaling
- 7. Protein Phosphorylation KINASE PHOSPHATASE Serine Threonine Tyrosine Kinases: Transfer the terminal phosphate of ATP to the hydroxyl group of a tyrosine residue in the substrate protein Phosphatases: Remove phosphate moieties from tyrosine residues (dephosphorylation) Phosphoryla&on regulates protein ac&vity and provides a binding site for other proteins.
- 8. Motifs Bind to Other Motifs in Proteins 3 examples Src homology 3-domain (SH3) Src homology 2-domain (SH2) Regulatory domain Binds to: Phospho- tyrosine P X X P Proline-rich stretch Pleckstrin homology domain (PH) PIP3 Phospho- inositols P X X X These motifs become very important in building local assemblies of signaling proteins on adaptor proteins. How do mul)ple protein/protein interac)on domains improve cell signaling?
- 9. The importance of protein domains/motifs for signaling. Fig 6.3 Janeway © 2001 Garland 400 - 500 nm 1.0-1.5um Illustration of the Problem Confocal Observation Volume for Fluorescence Correlation Measurements of Cytosolic Signaling Protein Dynamics To scale, a typical membrane associated protein signaling domain would be about this size…pretty tiny. The concentration of cytosolic signaling intermediaries is typically such that there are only 1-3 molecules in this volume! Volume: ~1 fL The probability of receptor-intermediary interaction is quite low (takes ms to diffuse to receptor in this volume, but binding/unbinding is on µs timescale.) Adaptor proteins form signaling complexes that promote and stabilize protein-protein and protein lipid interactions.
- 10. Overview of ITAM signaling systems • Ini&a&on: Membrane proximal signaling events • Output: Transcrip&on factor ac&va&on • Finally, we will focus in detail on the process of T cell ac&va&on via the TCR to inves&gate how one important ITAM signaling system works.
- 12. ITAM Immunoreceptor Tyrosine-‐based AcAvaAng MoAf YXXL/I(X)6-8YXXL/I ITAM Sequence Tyrosine Leucine/ Isoleucine *ITAMs do not have intrinsic kinase activity *The phosphorylated ITAM motif recruits kinase
- 13. There are many more ITAM receptors… Note that many of these non-TCR/BCR ITAM signaling systems use FcRgamma chain or DAP12 as the signaling partner that actually provides the ITAM motif. A receptor/signaling chain complex must form.
- 14. What are the early, receptor proximal events in ITAM signaling generally? ITAM/receptor complex Src family kinase ITAM pTyr ITAM Recruits active Syk/ZAP-70 Phosphorylate downstream adaptors and signaling proteins
- 15. ITAMs are phosphorylated by Src Family kinases Including: Src, Yes, Lyn, Fyn, Blk, Lck pTyr in C-term interacts with SH2 domain, locking protein in an inactive conformation (CskàpY505 on lck) Dephosphorylation of this Tyr (CD45, SHP-1) leads to conformational change that allows for unfolding & kinase activity Myristoylation: promotes membrane localization Regulatory tyrosine
- 16. • Phosphorylation of the ITAM domains creates binding sites for other protein domains called SH2 (Src Homology 2 domain) • This enables recruitment of signaling proteins to activated receptors – Signaling proteins are brought near the kinases and can in turn be activated – Or binding to a phosphotyrosine may activate the proteins by altering protein conformation (allosteric activation) – Syk, ZAP-70 • Proteins can be phosphorylated on three classes of amino acids: – tyrosines (receptors), serine/ threonine (downstream), or histidine
- 17. These kinases are ESSENTIAL for immune responses. ZAP-70 defects lead to Severe Combined Immunodeficiency (autosomal recessive). Syk KO mice die after birth of severe haemorrhaging. B-lineage cells cannot form (lack of signaling from pre-BCR complex, no clonal expansion or maturation of pre-B cells). Syk & ZAP-70 are key kinases in ITAM cascades TWO Src homology 2 domains www.nimr.mrc.ac.uk/.../thirty2/kinases/ Mouse embryos that lack the cytoplasmic tyrosine kinase Syk develop lethal hemorrhages at midgestation www.mpi-muenster.mpg.de/nvz/kiefer.shtml
- 18. Mocsai, et al. Nature Reviews Immunology 10, 387-402 SHP-1 Cbl ITAM/Syk Signaling Engages Many Downstream Effectors
- 19. Mocsai, et al. Nature Reviews Immunology 10, 387-402 Syk’s many pTyr sites control its activity and interactions, both on and off of the ITAM-containing receptor
- 20. Where is this all going? Nuclear Translocation of Transcription Factors Figure 9-14 Abbas © 2005 Elsevier
- 21. Figure 9-14 Abbas © 2005 Elsevier NFkB and NFAT are activated in the cytosol and translocate into the nucleus to begin transcriptional activation. AP-1 is composed of fos and jun. Jun is activated in the cytosol and translocates. Fos must be transcribed by Elk, which is translocated.
- 22. Receptor ligation ITAM Syk/ZAP70 Cell Type Specific Program NFAT NFkB AP-1 activation We will examine TCR signaling as a model to understand what lies in the middle and how this sensitive immunoreceptor system gets triggered. input output
- 23. TCR signaling • The biochemical signals that are triggered in T cells by antigen recognition are transduced not by the TCR itself but by invariant proteins called CD3 and ζ which are non-covalently linked to the antigen receptor to form the TCR complex. – Highly variable antigen receptor + invariant signaling proteins • What advantages does this give?
- 24. Peptide binding pocket Variable region / Complementrarity-determining region (CDR) Disulfide bond Positively charged amino acids: Lysine in α chain Lysine + arginine in β chain • Interact with neg residues in CD3 and ζ TCR Structure
- 25. Antigen affinity (Kd): 10-5 to 10-7 M (i.e., OT-I TCR 5.9 uM; Immunity. 1999;10:227–37.) Low affinity = need for adhesion molecules TCR Structure
- 26. The TCR complex
- 27. • Facilitate signaling • Provide secondary signals • Adhesion molecules Accessory molecules See also Abbas Figure 7-9B
- 28. Coreceptors: bind MHC molecules and enhance TCR signaling • CD4+ respond to Class II MHC: cytokine-production helper cells, extracellular microbes • CD8+ respond to Class I MHC: CTLs, eradicate intracellularly infected cells • Signal transduction via Lck: Interaction of CD4/CD8 with MHC brings CD4/CD8-associated Lck close to TCR the complex, which then phosphorylates the ITAMs of CD3 and ζ
- 29. • Close proximity of CD4/CD8- associatedLck activated ITAMs • CD3 associated Fyn may activate ITAM : TCR binding to MHC may transduce conformational change to activate Fyn • Zap-70 is related to Syk in the BCR system More details…
- 30. Signaling pathways triggered by TCR activation
- 31. CosAmulatory receptors: recognize molecules on APC and iniAate second signals • Receptor tails have structural motifs that serve as docking sites for adaptor molecules (such as PI-3 kinase or Grb2) once tyrosines are phosphorylated • Functional consequences are different for each receptor **Can be activating or inhibitory
- 32. Example of Costimulatory receptor function: After binding ligands for TLRs, Dendritic cells will express B7
- 33. • TCR provides specificity • Coreceptors enhance signaling by bringing Lck in close proximity of ITAMs • Costimulatory receptors initiate activating or inhibitory signals and play a key role in the outcome of APC interaction Activation of T cells involves the integration of signals from multiple receptors…
- 34. OrganizaAon of signaling at the T-‐APC contact On the scales that are accessible within the limits of conven&onal fluorescence imaging, we realize that the T-‐APC contact forms an organized (but very dynamic!) structure termed the “immunological synapse”. Supramolecular Ac&va&on Complex (SMAC) Distal (dSMAC) Peripheral (pSMAC) Central (cSMAC) Many important intermolecular and intercellular events happen in the SMAC. We will start at the molecular level and build our way up, focusing on the literature on T cell immunological synapses from the past decade. Huppa, Davis. Nature Reviews Immunology 3, 973-‐983 (December 2003)
- 35. Molecular & Cellular Interac&ons Relevant to TCR Triggering • TCR-‐CD3 complex structure • Models of TCR ac&va&on by cognate pMHC • Biophysical considera&ons regarding forma&on of ac&va&ng T-‐APC interfaces • Why it all maeers
- 36. The TCR complex Immunity Volume 24, Issue 2 2006 133 -‐ 139 Michael S. Kuhns , Mark M. Davis , K. Christopher Garcia If you had to experimentally demonstrate that this is the correct TCR complex structure, how would you do it? Call, et al, Cell. 2002 Dec 27;111(7):967-79.
- 37. Kuhns & Davis, Cell. 2008 Nov 14; 135(4): 594–596; Xu, et al. Cell. 2008 Nov 14;135(4):702-‐13. Basic residues (+) in the zeta and epsilon tails promote membrane associa&on in inac&ve TCR-‐CD3 complexes by binding to acidic lipids (e.g., phospha&dylserine). Membrane associa&on buries the ITAM tyrosines in the membrane and denies kinase access, preven&ng ac&va&on in the absence of pMHC.
- 38. P. Anton van der Merwe & Omer Dushek Nature Reviews Immunology 11, 47-‐55 (January 2011) Ini&a&on of TCR signaling is likely to require mechanical forces or reorganiza&on of the lipid bilayer to relieve the associa&on of CD3 complex ITAM domains with the membrane. Mechanical ac&va&on Note that in this example, the force is applied perpendicular to the membrane (piston-‐like mo&on). More recently tangen&al force models have been considered (covered later). Lipid reorganiza&on Ca2+ influx may also be involved in amplifying ITAM signaling by locally compe&ng the electrosta&c interac&ons between CD3 tails and membrane.
- 39. Problem: How much cognate pMHC does it take to sAmulate a T cell, parAcularly if the pMHC is rare? The story starts in Switzerland in the mid 90s…
- 40. The Serial Triggering Model Observed by flow cytometry: • Number of pMHC on an APC • Number of internalized TCR (assumed internalized = ac&vated) àCalculated # TCR ac&vated per pMHC present Determined: Each pMHC must ac&vate and cause the internaliza&on of up to 200 TCR. àThis led to the Serial Triggering model where one pMHC could serially ligate and ac&vate many different TCRs. Implies: agonis&c pMHC needs short bound life&me to engage many TCRs during the course of a T-‐APC encounter. T cells scan over DCs for cognate pMHC. They must find it in <10min (50% of contacts last < ~2.5m) if they are going to stop and be ac&vated. Is serial triggering realis8c during scanning T-‐APC interac8ons? Must know: What is the life8me of an agonis8c TCR-‐MHC interac8on? Celli, et al. JEM vol. 202 no. 9 1271-‐1278 (Valituu et al. Nature. 1995 May 11;375(6527):148-‐51.) Dura&on of T-‐APC encounters With cognate pMHC Without cognate pMHC
- 41. Stone, et al. Immunology. 2009 Feb; 126(2): 165–176. TCR-‐pMHC Bound life&me t1/2 range from less than 1 s to ~30 s Solu&on binding data for various TCR/pMHC complexes The TCR-‐pMHC with fastest kine&cs could account for serial triggering leading the T cell to stop, but many agonis&c pMHC seem too slow. For instance, a pMHC with 20 s bound life&mes could visit <30 TCR during a T-‐APC encounter, not hundreds.
- 42. Xie, et al. Nature Immunology 13, 674–680 (2012) Is serial triggering really necessary? Photocrosslinkable pMHC were aeached covalently to TCR during a T-‐APC interac&on. àThey can’t dissociate, so if serial triggering is required, these “pMHC (XL)” should be less s&mulatory than their standard, non-‐crosslinked counterparts, “pMHC (Std)”. Result: pMHC (XL) is more s8mulatory than pMHC (Std). • More prolonged Ca2+ flux • Greater IL-‐2 secre&on While serial triggering may happen to some extent, it does not appear to be so essen&al as originally thought.
- 43. Are there any thermodynamic or kineAc parameters derived from soluAon measurements of TCR-‐pMHC binding that correlate with sAmulatory potency? • When it became generally feasible to measure solu&on binding kine&cs of TCR-‐ pMHC with commercially available SPR instruments, there was much interest correla&ng affinity, kon and koff of pMHC for TCR with biological ac&vity • The affinity (in solu&on binding measurements) of TCR for agonist pMHC is rela&vely weak, typically in the Kd=1-‐100uM range. Mod. From: Stone, et al. Immunology. 2009 Feb; 126(2): 165–176. Bound state life&me There were correla&ons between binding affinity and kine&cs in some limited systems but overall there was no universally strong correla&on between solu&on binding parameters and ac&vity. Agonist, weak agonist and antagonist pMHC can have very similar solu&on binding affinity and rate constants
- 44. More recently, single molecule imaging invesAgaAon has pushed the limit of resoluAon to allow tesAng triggering capability of ever smaller numbers pMHC Manz, et al. (groves lab) PNAS May 31, 2011 vol. 108 no. 22 9089-‐9094 Circles and triangles: T cells with two different TCRs Titrated bio&nylated MCC-‐pMHC and measured number pMHC at synapse (x axis) Recorded Ca2+ flux in T cells Irvine, et al (Davis Lab) Nature 419, 845-‐849(24 October 2002) Ca2+ fluxes can be triggered by <10 cognate pMHC in an immune synapse Using a microfabricated ar&ficial bilayer that constrained the number of pMHC that TCRs could see to only a few, Ca2+ flux in the T cell required very small numbers of pMHC
- 45. How does the TCR know to acAvate signaling in response to very low levels of pMHC? Pseudodimer model: dimers of congnate and non-‐congate pep&de pMHC promote signaling Molecular MechanotransducAon: pMHC applies a torque to TCRαβ, which is transmieed to CD3εγ and CD3εδ Dwell Time: What determines if a pMHC-‐TCR interac&on is s&mulatory is how long the pMHC “dwells” near a single TCR, repe&&vely binding it. We’ll look at three mechanis&c models of TCR s&mula&on arising from the recent literature. Note that these models are not mutually exclusive alterna&ves but may be looking at different aspects of the same process as different research groups look at TCR triggering from diverse backgrounds and perspec&ves.
- 46. How does the TCR know to ac8vate signaling in response to very low levels of pMHC? Pseudodimer model: dimers of cognate and non-‐cognate pep&de pMHC promote signaling P. Anton van der Merwe & Omer Dushek Nature Reviews Immunology 11, 47-‐55 (2011) CD4 contacts an&genic pMHC but also brings lck into proximity to TCR interac&ng with dimerized “self” pMHC, ac&va&ng both TCRs’ signaling Cohran et al. Immunity. 2000 Mar;12(3):241-‐50. Soluble single chain MHC have been engineered and loaded with defined pep&des that are chemically x-‐linked to the MHC monomeric >=dimeric Monomeric pMHC bind TCR but are not s&mulatory, but >=dimer pMHC is s&mulatory. à Suggests that single pMHC are not s&mulatory and you have to at least present a dimer of pMHC (but this is soluble pMHC, not in a T-‐APC contact)
- 47. How does the TCR know to ac8vate signaling in response to very low levels of pMHC? Pseudodimer model: dimers of cognate and non-‐cognate pep&de pMHC promote signaling PepAdes to be presented to 5C.C7 (MCC reacAve) T cells Krogsgaard et al. Nature 434, 238-‐243(10 March 2005) Standard agonis&c pep&de Single AA subs&tu&ons (altered K5) ER chaperone that binds this MHC prior to an&gen pep&de loading Self pep&des that bind this MHC (Ca2+ flux) dimeric pMHC • K5 monomer is not s&mulatory, but K5-‐K5 is. • Some of the altered K5 and self pMHC (those that are recruited to T-‐APC contacts) can support signaling from heterodimers with K5. • These self pMHC are not agonis&c when homodimerized. • Some altered K5 and self pMHC can’t support signaling with K5 (i.e., b2m).
- 48. How does the TCR know to ac8vate signaling in response to very low levels of pMHC? Molecular MechanotransducAon: pMHC applies a torque to TCRab, which is transmieed to CD3εγ and CD3εδ N-‐glycans top side FG loop of b chain TCRab rises above shorter, more rigid CD3 dimers FG loop of TCRβ C domain is important for TCR ac&va&on, is well structured and approaches CD3εγ Op&cal traps are used to apply force through bead-‐bound ligands to the TCR. Forces applied are in the 10s of pN range, typical of the forces applied at cell-‐cell and cell-‐ substrate interfaces Kim et al. Front. Immunol., 18 April 2012 | doi: 10.3389/fimmu.2012.00076 Wang et al. Immunol Rev. 2012 Nov; 250(1): 102–119.
- 49. How does the TCR know to ac&vate signaling in response to very low levels of pMHC? Molecular MechanotransducAon: pMHC applies a torque to TCRab, which is transmieed to CD3εγ and CD3εδ Tangen&al force applied through a trapped ligand-‐bead, measured Ca2+ flux 17A2=Non-‐agonis8c an8-‐CD3 With force Without force Ligand on bead: non-‐agonis&c mAb pMHC Kim et al. J Biol Chem. 2009 Nov 6;284(45):31028-‐37. For mAb and pMHC, you only see Ca2+ signal if you pull tangen&ally on the bead-‐ligand Model: • TCR-‐pMHC bonds form • T cell translates rela&ve to APC • Tangen&al force on TCR complex via pMHC (yellow) • TCR Cβ (blue) pivots on its TM domain • FG loop (magenta) pushes down on CD3 dimers • Presumably causes changes in CD3 tails that promote their ac&va&on Ca2+ flux
- 50. How does the TCR know to ac8vate signaling in response to very low levels of pMHC? Dwell Time: What determines if a pMHC-‐TCR interac&on is s&mulatory is how long the pMHC “dwells” near a single TCR, repe&&vely binding it. • This concept is different from serial triggering (one pMHC binds and ac&vates many different TCR in series). • Dwell &me considers the &me that a pMHC spends bound to a single TCR, dissociates, but then rebinds that TCR before diffusing away. • In this model, a strongly agonis&c pMHC would be able to dwell for a long &me, thus integra&ng a lot of signaling through the bound TCR. Dwell &me depends on: • TCR/pMHC binding kine&cs (determines half-‐life of bound complex) AND • Diffusion of TCR and MHC in the membrane (determines how likely it is that TCR and MHC will move too far apart to bind before rebinding can occur) Govern & Chakraborty. Immunity. 2010 Feb 26;32(2):141-‐2.
- 51. How does the TCR know to ac8vate signaling in response to very low levels of pMHC? Dwell Time: What determines if a pMHC-‐TCR interac&on is s&mulatory is how long the pMHC “dwells” near a single TCR, repe&&vely binding it. • In solu&on, you would never see TCR dwelling on pMHC because solu&on diffusion is much too fast. Typically approx tens to 100 um2/s for proteins. • In a membrane, transmembrane protein diffusion is much slower (and constrained to the 2D bilayer). Typical protein diffusion in membranes is ~0.1 um^2/s. Known: kon and diffusion coefficients Based on a model of binding+diffusion in a membrane, calculated a predicted average number of rebindings. Note that the predicted number of rebindings is rela&vely small Govern et al used two different TCRs and a variety of pMHC with known affini&es and binding rate constants for those TCRs as well as known ability to s&mulate T cell prolifera&on and cytokine produc&on. Govern et al. Proc Natl Acad Sci U S A. 2010 May 11;107(19):8724-‐9.
- 52. How does the TCR know to ac8vate signaling in response to very low levels of pMHC? Dwell Time: What determines if a pMHC-‐TCR interac&on is s&mulatory is how long the pMHC “dwells” near a single TCR, repe&&vely binding it. Govern et al. Proc Natl Acad Sci U S A. 2010 May 11;107(19):8724-‐9. They knew the kon and diffusion coefficients of the TCR and pMHC used. From this they could use a model of binding/diffusion in a 2D membrane to calculate the dwell &me of each pMHC/TCR ( “ta”). ta for TCR/pMHCs was the best predictor of T cell prolifera)ve response. EC50 (uM) For prolifera&on pMHC agonis&c strength: Strongest Moderate weakest
- 53. Dwell &me theory of TCR-‐pMHC interac&ons • Depends on – Forward kine&c rate constant for binding – Diffusion of TCR and pMHC
- 54. Dwell Time Modeling: Goals • How does the membrane diffusion environment influence TCR-‐pMHC interac&on dynamics? – Does changing D (diffusion coefficient) from solu&on values (100 um^2/s) to membrane values (0.1 um^2/ s) impact molecular dwell &mes • How does changing the forward rate constant for binding influence TCR-‐pMHC interac&on dynamics? – Does changing pON (probability of binding in one model step) from lowàmoderateàhigh values impact molecular dwell &mes
- 55. How the model sets up and runs Ini&alize and execute model Control how binding and diffusion works Quan&ta&ve outputs about binding Size and &me informa&on about model you’ve run Free TCR (red circle) Bound TCR (yellow circle) Free pMHC (green square) Agent based model of TCR and pMHC binding in apposed membranes
- 56. Run the Netlogo model of TCR-‐pMHC interac&on with • D=0.1, 100 um^2/s • Low, moderate and high probability of binding RESULTS
- 57. Triplicate runs pOFF=0.01 in all 250s dura&on, 10ms &me resolu&on 10nm binding radius 100 TCRs and pMHCs in ~15 um^2 membrane D Diffusion Coefficient (um^2/s) Membrane Solu&on
- 58. Now that we’ve considered the state of knowledge regarding molecular TCR-‐pMHC interacAons, let’s think about the cell-‐cell interface environment in which these interacAons occur… What are some important features of this environment that bear on signaling processes? Achieving close cellular apposiAon Micro/nanoscale organizaAon of signaling components Mechanics of the Immune Synapse
- 59. Achieving close cellular apposiAon. What are the challenges? Huppa, Davis. Nature Reviews Immunology 3, 973-‐983 (December 2003) Molecular InteracAons at the T-‐APC immunological synapse This figure helps to summarize cell biological informa&on, and to be fair, that was all it was intended to do in the review cited below. But from the standpoint of physical interac&on between two cells, it has some major omissions and inaccurate representa&ons. What aspects of the immune cell interface are not represented or not accurately represented here?
- 60. Achieving close cellular apposiAon. What are the challenges? Casasnovas et al. Proc Natl Acad Sci U S A. 1998;95(8):4134-‐9. Yin et al. Proc Natl Acad Sci U S A. 2012 Apr 3;109(14):5405-‐10. LFA-‐1 ICAM-‐1 dimer There is no glycocalyx illustrated on either cell, but this is an important repulsive barrier that resists apposing two cells closer than ~100nm. 40 nm T-‐APC adhesion via LFA-‐1/ICAM-‐1 is important for stabilizing the cell-‐cell interface, but this would space the cell membranes ~40nm apart The TCR-‐pMHC complex is only ~15nm, so engagement of TCR will require membranes to be pushed into ~15nm separa&on.
- 61. Achieving close cellular apposiAon. What is the solu8on? T cell invadopod like protrusions (ILP) Sage et al. J Immunol. 2012 Apr 15;188(8):3686-‐99. Top: T cells siung on an Ag-‐pulsed APC probe the APC with ILPs even before Ca2+ flux. Red arrows show sites where membrane targeted fluorescent protein in the T cell is pushed into the APC membrane. Le€: TEM image shows the interdigita&on of the synapse between APC and T cell, including sites of apparent T cell ILP ac&vity (red arrows). ILPs are ac&n dependent and are thought to push the T cell and APC membranes close enough to engage TCR-‐pMHC binding.
- 62. Achieving close cellular apposiAon. What is the solu8on? Kine&c segrega&on. Sequen8al engagement of different sized receptors accompanied by reorganiza8on of their membrane distribu8ons. ICAM-‐1 LFA-‐1 T APC TCR pMHC CD45 James, vale. Nature. 2012 Jul 5;487(7405):64-‐9. ICAM-‐1 LFA-‐1 T APC TCR pMHC CD45 During ini&al T-‐APC adhesion, large ICAM-‐1/LFA-‐1 interac&ons predominate. TCR can’t be engaged at this distance. Large nega&ve regulatory phosphatases are allowed in the contact. ILPs form small close contacts where TCR-‐pMHC can engage. Adhesive interac&ons between CD2 and CD58 can stabilize these close contacts (CD2/ CD58 is about the same intermembrane length as TCR/ MHC). As close contacts grow, the energy cost of bending membranes is balanced by mul&ple binding interac&ons to stabilize and enlarge areas of TCR engagement. Large ectodomain proteins are excluded (e.g., CD45)
- 63. Micro/nanoscale organizaAon of signaling. Recruitment of LAT to ac&vated TCR complexes is a key early event in TCR signaling. How do we prevent LAT from prematurely assembling at TCR complexes? Methods: fluorescence super resolution imaging and TEM with gold beads on membrane sheets and whole cells. Both methods are in good agreement, so I’m showing just the TEM immunogold. • TCR complex has been shown to form nanoclusters in resting T cells. • Lat forms similarly sized clusters. • The size of both clusters is in the 40-300nm range. • Estimated 5-20 TCR per cluster Small CD3ζ Large Lat Non-‐ac&vated T cell membrane ac&vated T cell membrane Lillemeier, et al. Nat Immunol. 2010 Jan;11(1):90-‐6.
- 64. Micro/nanoscale organizaAon of signaling. Top: Ripley’s func&ons and their deriva&ves are a way to show that objects (i.e., small and large immunogold beads) are clustered on certain length scales. • The peak of the Ripley’s L(r)-‐r curve is the length scale of greatest clustering. • Ac&va&on on cognate pMHC surfaces increases the amount and length scale of clustering for CD3 and Lat. Boeom: Bivariate Ripley’s sta&s&cs show the probability that CD3 and Lat are clustered together at different length scales. • The shaded areas represent the regions of the plot where the observed distribu&on of CD3 and Lat are not dis&nguishable from random with 99% confidence. • If the lines go outside of these regions, then the paeern colocaliza&on of CD3 and Lat shows evidence of clustering (above the shaded area) or segrega&on (below the shaded area). • On a non-‐ac&va&ng surface, Lat clusters are segregated from CD3. • On an ac&va&ng surface, they become co-‐aggregated
- 65. Micro/nanoscale organizaAon of signaling. Complimentary evidence of Lat and TCR complex co-‐ aggrega&on a€er ac&va&on came from fluorescence cross-‐correla&on spectroscopy (FCCS) measurements. Orange lines show the likelihood of observing co-‐ diffusion of Lat and CD3ζ in living cell membranes. • CD3/Lat co-‐diffusion is not seen in T cells on a non-‐ ac&va&ng ar&ficial membrane. • Placing agonis&c ligand on the bilayer markedly increases the co-‐diffusion of TCR complex and Lat. Therefore, several lines of evidence show that: 1. TCR and the key signaling adaptor Lat are preclustered in T cell membranes on tens to hundreds of nanometers scales . 2. Prior to s&mula&on, Lat and TCR complex are segregated from one another in separate nanostructures. 3. When a cognate pMHC is seen, Lat becomes co-‐aggregated with TCR nanoclusters.
- 66. Mechanics of the immune synapse. So, TCR organizes into clusters when ac&vated. What happens to TCR clusters in the immunological synapse? cSMAC pSMAC dSMAC TCR TCR The cSMAC is rich in TCR, while CD45 and other nega&ve regulatory phosphatases tend to be in the dSMAC. This ini&ally led to the idea that TCR signaling was occurring in the cSMAC. CD45 Subsequent imaging work at high spa&al and temporal resolu&on clarified that signaling from the TCR actually happens as ac&vated TCR forms into microclusters, largely in the pSMAC, during the ini&al spreading phase of synapse forma&on. In this phase TCR is sampling for an&gen and forming microclusters. These TCR microclusters then move laterally into the cSMAC. This happens when the synapse stops spreading and develops a strongly contrac&le pSMAC. In this phase, TCR is signaling, causing the cell to stop and integrate signal and eventually being downregulated. We will consider: How do TCR microclusters move? What actually happens in the pSMAC?
- 67. How do TCR microclusters move? An actomyosin flow develops in the pSMAC. This is similar to the “tractor” that forms at the leading edge of migra&ng cells and moves them forward. F-‐ac&n forms into bundles at the edge of the synapse and myosin IIA mediated contrac&on causes the F-‐ac&n to contract inward toward the cSMAC. Recruitment of GEFs to the synapse is important for upregula&ng Rac and Cdc42 GTPase dependent ac&n nuclea&on (e.g., via Arp2/3 complex). Rho GTPase regulates myosin IIA mediated contrac&on in the pSMAC. Babich et al. 2012 // JCB vol. 197 no. 6 775-‐787 Kumari et al. Biochim Biophys Acta 2014 Feb;1838(2):546-‐56. Mechanics of the immune synapse.
- 68. How do TCR microclusters move? Ar&ficial membranes have been made with underlying metal barriers. When transmembrane proteins (i.e., TCR complex) encounter the barrier, they must go around, not through. Examples of TCR microcluster trajectories as they move toward the cSMAC and encounter barriers show that the TCR gets hung up on the barriers and must slide along the barriers to con&nue centripetal mo&on. In contrast, actomyosin mo&on is beneath the membrane and thus not effected by the barriers. Ac&n flows past the barriers. This suggests “fric&onal coupling” of the actomyosin flow to the TCR complex as a mechanism of TCR cluster mobility. The exact mechanism of coupling is currently unknown. Mechanics of the immune synapse. Demond et al. Biophys J. 2008 Apr 15;94(8):3286-‐92.
- 69. Do moving TCR clusters transduce force as part of their signaling? Mechanics of the immune synapse. TCR-‐pMHCs being dragged from pSMAC to cSMAC by actomyosin flow will experience force in the piconewton range. T cells respond with greater TCR signaling when presented with an&-‐CD3 on s&ff surfaces rela&ve to so€er surfaces, which may relate to the amount of force experienced by the TCR. Huppa, etal. Nature. 2010 Feb 18;463(7283):963-‐7. Time (sec) of TCR-‐agonist pMHC interacAon Huppa et al used a FRET probe between a pMHC and an&-‐ TCR monovalent an&body. FRET signal was only observed when the TCR-‐pMHC complex was bound, allowing measurements of TCR-‐pMHC binding &mes. Found that synap&c TCR-‐pMHC binding &mes are 3-‐12 &mes shorter than the solu&on value. F-‐ac&n inhibitors (prevents actomyosin transport of TCR clusters) increased TCR-‐pMHC binding &mes. Suggested that the force applied to TCRs in the synapse as they are dragged by actomyosin flow can shorten their half &me of binding to pMHC.
- 70. What is the mechanical nature of the TCR-‐agonist pMHC bond? Mechanics of the immune synapse. Depoil and dus&n. Trends Immunol. 2014 Nov 17;35(12):597-‐603. Liu et al. Cell. 2014 Apr 10;157(2):357-‐68. OT1 Types of bonds • Slip bonds are linearly more likely to break (shorter life&me) with increasing force. • Catch-‐slip bonds become stronger (longer life&me) with increasing force, but only up to their rupture force (then they become slip bonds). Biomolecular Force Probe (BFP) • A rigid bead is coated with a low density of pMHC such that only a single pMHC will contact a T cell when the bead touches it. • The bead is aeached to an RBC. • The RBC/bead is repeatedly brought up to the T cell membrane, then retracted. • The force applied to the TCR can be calculated from the RBCs membrane deforma&on during retrac&on.
- 71. What is the mechanical nature of the TCR-‐agonist pMHC bond? Mechanics of the immune synapse. Liu et al. Cell. 2014 Apr 10;157(2):357-‐68. Antagonist pMHC Agonis&c pMHC Agonist pMHC strength Le€: Agonis&c pMHC consistently display catch-‐slip bonding with TCR. The rupture force is correlated with the agonis&c “strength” of the pep&de. Right: Antagonis&c pep&des form slip bonds with TCR. T cells integrate the total &me single TCRs are subjected to catch bonded pulling force (10 pN, OVA pep&de). Strong T cell Ca2+ fluxes require integra&on of 10 s of catch bonded &me per minute of total BFP s&mula&on.
- 72. Why does it all ma]er? Mechanis&c Underpinning for T cell Sensi&vity These studies help us to understand the physical parameters that define whether a pMHC will be s&mulatory or not. They also help us to understand the exquisite sensi&vity of T cells for low densi&es of agonist pMHC. Sensi&vity is important because professional APCs like DCs might not express large densi&es of a par&cular cognate pMHC for a T cell. Also, a scanning T cell/APC interac&on in the lymph node is rather short lived (~minutes), so the T cell needs to be able to signal and stop migra&ng if even a small amount of cognate pMHC is found. Possible Mechanism of Quan&ta&ve T cell Help for B cell Matura&on. Review re: role of IS in T cell help for B cells-‐-‐Dus&n. Mol Cell. 2014 Apr 24;54(2):255-‐62. B T Ag Y Y AnAgen gathering Efficiency α BCR affinity AnAgen presentaAon # pMHC α BCR affinity TCR signals, acAvaAon # CD40L α # pMHC seen T B T cell help # CD40L α B cell prolif A 2014 ar&cle suggested that exocytosis of TCR in the cSMAC may contribute to long-‐term regula&on of B cell responses to T cell help las&ng beyond the &me frame of direct T-‐B interac&ons.
- 73. Possible Mechanism of Quan&ta&ve T cell Help for B cell Matura&on. Choudhuri et al. Nature. 2014 Mar 6;507(7490):118-‐23. TCR F-‐acAn 3um TCR int. overlay TEM 500nm Vesicles T cell PM Fluorescence-‐TEM correla&ve study: TCR intensity is found where cSMAC microvesicles are seen Tg TCR T cell on ar&ficial membrane with cognate pMHC and ICAM-‐1. Microvesicles are present outside the T cell near the cSMAC loca&on. Microvesicles are produced by ESCRT-‐dependent exocytosis (similar to HIV budding). B cells expressing cognate pMHC internalize Tg TCR at immunological synapses and ac&vated PLCg is found in proximity B cell captured TCR. Purified microvesicles display total TCR in propor&on to the density of cognate pMHC used to ac&vate the T cell. Germinal Center Light Zone Dark Zone T B Hypothe)cal Model
- 74. 2. Cytokine Receptor, Notch and Innate Immunoreceptor Signaling. Regula&on of signal dynamics BIOM 514, Cell Signaling Apr. 3, 2015 Aaron Neumann, Ph.D. (Pathology) CRF327, akneumann@salud.unm.edu Abbas (7th ed.) Chapter 7,9, parts of 11
- 75. OVERVIEW • Cytokine Receptors—Jak/STAT signaling • Notch Signaling • NFkB—a central immune response transcrip&on factor • Innate Immunoreceptor Signaling: Fc Receptors, Toll like Receptors, C type lec&n receptors • ITIM antagonism of ITAM signaling • Dynamical considera&ons in signaling
- 77. JAK/STAT Pathway Type I and II cytokine receptors • Cytoplasmic tail contains tyrosine • Jak is attached to receptor tail • Clustering of receptors leads to Jak- mediated phosphorylation of the receptor tail • STAT can now bind and be activated • STATs dimerize (homo- or heterodimerization) and translocate to the nucleus where they stimulate transcription, changing gene expression • Cellular response is different depending on the combination of receptor, Jak and STAT activated
- 78. Radtke, Immunity Volume 32, Issue 1 2010 14 -‐ 27 Notch signaling impacts lymphocyte development and activation How Notch signaling works: 1. Export of Notch to the plasma membrane • Furin-‐like protease cleavage • Glycosyla&on by the fringe glycosyltransferases 2. Binding ligand on opposing cell membrane • Jagged • Delta like ligands (Dll) 3. Cleavage at the plasma membrane • ADAM (A Disintegrin And Metalloprotease) • γ-‐secretase 4. Release of Notch intracellular domain into the cytoplasm 5. Nuclear Transloca&on 6. Binds to CSL transcrip&on factor and recruits coac&vatoràgene transcrip&on 7. Proteosomal degrada&on 1 2 3 4 5 6 7
- 79. Radtke, Immunity Volume 32, Issue 1 2010 14 -‐ 27 Notch signaling impacts lymphocyte development and activation Notch1 signaling in T cell development • Maintains a stem cell popula&on (HSC) that seeds the thymus • Suppresses B lineage development in DN stage • Supports prolifera&on at the DN4àDP transi&on due to Notch signaling that supports prolifera&on and survival. Notch in peripheral T cells • Expression increases and Notch intracellular domain is seen a€er TCR ac&va&on • Occurs even without APC sources of Notch ligand • T cells don’t seem to express much Notch ligand How is Notch ac8va8on happening?? • Jagged-‐Notch may drive Th2 differen&a&on
- 80. Radtke, Immunity Volume 32, Issue 1 2010 14 -‐ 27 Notch signaling impacts lymphocyte development and activation Impact of Notch Signaling: Regulates lineage commitment during development through transcrip&on factor ac&va&on Drives signaling that Promotes • Prolifera&on • Metabolism • Survival Using pathways that influence • Apoptosis (p53, PI3K/Akt) • Cell Cycling (p27Kip1) • Metabolism (mTOR)
- 82. The history of Toll-like receptors — redefining innate immunity Luke A. J. O'Neill, Douglas Golenbock & Andrew G. Bowie Nature Reviews Immunology 13, 453–460 (2013) doi:10.1038/nri3446 TLRs & signaling (Abbas Ch4) MyD88, TRIF ê TRAFs ê Trxn factor (NFkB, IRFs)
- 83. Nature Reviews | Immunology Dectin 2 ITAM HDM allergens Malassezia sp. CARD9 BCL-10 MALT1 IKK IKK IKK I B p50p65 P Proteasome Nucleus Cytoplasm Fungal hyphae FcR P P SYK P P SYK Mincle Myeloid DC Cysteinyl leukotrienes ? ? _ _+ _ _ + p50p65 Tnf and Il6 FcRγ results in NF-κB activation10,58 , although a role for the SYK-CARD9–BCL-10–MALT1 complex has not yet been confirmed. Thus, dectin 2 triggering alone might induce an adaptive immune response, which is supported by data showing that dectin 2 recognition of fungal hyphae from C. albicans, Trichophyton rubrum and Microsporum audouinii leads to TLR-independent production of the pro-inflammatory cytokines TNF and IL-6 (REF. 10). Furthermore, dectin 2 recognition of house dust mite allergens activates SYK through FcRγ to gen- erate cysteinyl leukotrienes, an important mediator of allergic inflammation in the lungs58 . Notably, recogni- tion of Histoplasma capsulatum β-glucans by dectin 1 also leads to the production of leukotrienes59 , suggesting that a common SYK-dependent pathway is involved in leukotriene synthesis after CLR triggering. The related CLR mincle also pairs with FcRγ and induces gene transcription through the SYK–CARD9– BCL-10–MALT1 complex12 . In macrophages, recogni- tion of dead cells by mincle through the endogenous ligand SAP130 (SIN3A-associated protein, 130 kDa) mediates CXC-chemokine ligand 2 (CXCL2) and TNF production in a SYK- and CARD9-dependent manner, which induces neutrophils to migrate into damaged tis- sues12 . Mincle also interacts with α-mannosyl PAMPs expressed by the pathogenic fungus Malassezia spp. and induces gene transcription and TNF production without the involvement of TLRs, further suggesting that mincle, similarly to dectin 2, couples FcRγ-signalling to NF-κB activation18 . The similarities of mincle downstream sig- nalling with the dectin 1 pathway suggest that both CLRs couple SYK activation to NF-κB activation through the CARD9–BCL-10–MALT1 complex. In contrast to dectin 2 and mincle, BDCA2 does not induce TLR-independent cytokine production even though it also pairs with FcRγ. The unusual signal- ling pathway induced by BDCA2 might be because it is expressed only by pDCs, whereas dectin 2 and mincle are expressed by myeloid-derived antigen presenting cells (TABLE 1). A recent study has shown that lymphoid and myeloid cells have differential requirements for CARD proteins in BCL-10-mediated NF-κB activation32 , which might explain why dectin 2 and mincle couple FcRγ-signalling to NF-κB activation and BDCA2 does Figure 4 | Signalling by dectin 2 and mincle leads to cytokine expression. BothDC-associatedC-typelectin2 (dectin2)andmacrophage-inducibleC-typelectin(mincle) pairwiththesignallingadaptormoleculeFcreceptor γ-chain(FcRγ) throughthepresenceofapositivelycharged aminoacidresidueintheirtransmembraneregions.The phosphorylationoftheimmunoreceptortyrosine-based activationmotifs(ITAMs)ofFcRγ followingC-typelectin receptor(CLR)activationservestorecruitspleentyrosine kinase(SYK)andinducessignallingpathwaysthatmodulate cytokineexpression.Dectin2bindstopathogen-associated molecularpatterns(PAMPs)expressedbyfungalhyphae, andminclebindstoα-mannosylPAMPsonMalasseziaspp. fungi.BothsignallingpathwaysleadtoToll-likereceptor (TLR)-independentproductionofcytokinessuchastumour necrosisfactor(TNF)andinterleukin-6(IL-6);dectin2 triggeringisknowntoresultinnuclearfactor-κB(NF-κB) p50–p65activation,andmincletriggeringinducesaCARD9 (caspaserecruitmentdomainfamily,member9)-dependent signallingpathway.Similaritieswiththedectin1signalling REVIEWS P Nature Reviews | Immunology Dectin 1 P P SYKRas PP RAF1 CARD9 BCL-10 MALT1 IKK IKK IKK NIK IKK I B p50p65 RELB RELBp100 p52 RELB RELB p52 RELBp65 P RELBp65 P RELBp65 P Proteasome p50p65 P Ser276 p50p65 P p65 PCBP Ac Ac Nucleus Cytoplasm p50p65 P Ac Ac Il6 and Il10 p50 p50 p50REL p50REL REL p65 P Ac Ac Il12b Il1b Ccl17 and Ccl22 p50p65 P Ac Ac Il12a Il23p19 Fungia b ? Or Inactive NF- B p50REL Figure 3 | Dectin 1 signalling through SYK and RAF1 directs NF-κB-mediated cytokine expression. a | The binding of fungi to DC-associated C-type lectin 1 (dectin 1) induces phosphorylation of the YxxL (in which x denotes any amino acid) motif in its cytoplasmic domain. Spleen tyrosine kinase (SYK) is recruited to the two phosphorylated receptors, which leads to the formation of a complex involving CARD9 (caspase recruitment domain family, member 9), B cell lymphoma 10 (BCL-10) and mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1); this induces the activation of the IκB kinase (IKK) complex through an unknown pathway. IKKβ phosphorylates inhibitor of NF-κBα (IκBα), thereby targeting it for proteasomal degradation. This results in the release of nuclear factor-κB (NF-κB; consisting of either p65–p50 or REL–p50 dimers), which then translocates into the nucleus. SYK activation also leads to the activation of the non-canonical NF-κB pathway that is mediated by NF-κB inducing kinase (NIK) and IKKα, which target p100 for proteolytic processing to p52; this subsequently leads to nuclear translocation of RELB–p52 dimers. In a SYK-independent manner, dectin 1 activation leads to the phosphorylation and activation of the serine/threonine protein kinase RAF1 by Ras proteins, which leads to the phosphorylation of p65 at Ser276. Phosphorylated Ser276 serves as a binding site for the histone acetyltransferases CREB-binding protein (CBP) or p300 (not depicted) to acetylate (Ac) p65 at different lysine residues. Ser276-phosphorylated p65 also dimerizes with RELB to form inactive dimers that cannot bind DNA, and hence attenuates the transcriptional activity of RELB. b | Binding of acetylated p65 to the Il10 (interleukin-10) enhancer and Il6 REVIEWS PSYK TLR9DNA Endosome MYD88 MYD88 TLR9 DNA Endo BDCA2 DCIR P P P BLNK BTK PLC 2 ? Ca2+ mobilization TLR pathway SHP1 or SHP Plasmacytoid DCa b ITAM FcR P P ITIM _ _+ Ifna, Ifnb, Tnf and Il6 Ifna Tnf Cytoplasm Nucleus Similar to DC-SIGN, neither DCIR nor been shown to induce immune respons own, but instead modulate signalling Figure 2 | Signalling by BDCA2, DCIR and M protein (BDCA2) leads to the recruitment of sp tyrosine-based activation motif (ITAM) of the p theactivationofacomplexconsistingofBcell which induces Ca2+ mobilization. The signalling downregulation of Toll-like receptor 9 (TLR9)-in (TNF) and interleukin-6 (IL-6) by plasmacytoid d the recruitment of myeloid differentiation prim TLR-induced cytokines. b | Activation of DC im compartments, where TLR8 and TLR9 reside. T (ITIM) recruits the phosphatases SH2-domain-c the activation of an unidentified signalling pat TNF production or TLR9-induced IFNα and TNF c | Cross-linking of myeloid C-type lectin-like r ITIM and the recruitment of SHP1 or SHP2. MIC signal-regulated kinase (ERK). However, it is no TLR4-induced IL-12 production. LPS, lipopolys molecule; TRIF, TIR-domain-containing adapto ITAM Signaling Pathways in the Innate Immune System: C-type Lectins Nat Rev Immunol. 2009 Jul;9(7):465-‐79. hemITAM Classically involved in recognition of pathogen surface carbohydrates FcRgamma coupled ITAM signaling. In some cases, ITAM signaling antagonizes TLR signaling.
- 84. Fc Receptors
- 85. Important Functions of Fc receptors
- 86. Mast Cell Signaling: the cornerstone of allergic inflammation Gilfillan, 2006 Nat. Rev. Immunol
- 87. The ITAM signaling pathway is similar
- 88. ITIM-containing Receptors Recruit PHOSPHATASES • SHP1, SHP2 – SH2 containing tyrosine phosphatases • SHIP – SH2 containing inositol phosphatase
- 89. ITAM/ITIM crosstalk in B cells Secreted IgG can form complex with antigen Crosslinks BCR and FcγRIIB FcγRIIB recruits SHIP that hydrolyses a phosphate on PIP3 and terminates signaling Proposed control mechanism to stop antibody production
- 90. Signal Transduction Dynamics The linear pathways for signal transduc&on presented in textbooks can be misleading. • Cross-‐talk amongst signaling pathways influences signal outputs. • Dynamical nature of signal varia&on with &me is important. For instance, human immature Dendri&c Cells exhibit spontaneous oscilla&ons of intracellular Ca2+ in the res&ng state. These are lost as the cell matures. 400 800 1200 1600 0 200 400 600 Ca2+ iIntensity(AU) Time (sec) DIC Fluo-4 + ionophore
- 91. Signal Transduction Dynamics Dolmetsch, et al. Nature 392, 933-936(30 April 1998) • T cells also exhibit Ca2+ oscillations and spikes during activation • Ca2+ clamp method can reproduce arbitrary oscillation amplitudes and periods (left) At low levels of Ca2+, oscillatory [Ca2+]i increases the number of cells expressing an NFAT reporter.
- 92. Signal Transduction Dynamics Dolmetsch, et al. Nature 392, 933-936(30 April 1998) These three transcription factors respond similarly to continuous Ca2+ amplitude, but have different behavior depending upon oscillation frequency. Thus, different transcriptional programs could be controlled by Ca2+ signal dynamics. Encoding oscillations is usually a matter of some negative feedback in the signaling system operating with a delay. For instance, the IP3R releases store Ca2+, but is also inhibited by the rise in cytosolic Ca2+. How would the cell decode oscillatory signals?
- 93. Signal Transduction Dynamics TF-A: high Ca2+ affinity, short half-life TF-B: low Ca2+ affinity, long half-life time %maxsignal Calcium, TF-A, TF-B TF-A gets activated by very little Ca2+ and decays rapidly. Its signal will be oscillatory unless the period of Ca2+ oscillation is shorter than the half life of TF-A. TF-B only gets activated by high levels of Ca2+ (at the peak of the oscillation), but it degrades slowly. This causes its signal to be persistent. With this system, you could have regimes where only TF-A or both were activated and TF-A could be periodic or persistent. This would be controlled by the amplitude and period of the oscillatory stimulus.
- 94. Extra slides on BCR signaling (if there is Ame)
- 95. BCR signaling • Antigen binding domain is a surface-expressed immunoglobulin with the same antigen specificity that the B cell will secrete • Signaling occurs through associated Igα and Igβ ITAM containing proteins • Multi-subunit receptor with variable antigen receptor + invariant signaling molecules
- 97. Nature Reviews Immunology 13, 475–486 (2013) doi:10.1038/nri3469 B cell Ac&va&on at APC contacts involves microclusters and cytoskeletal dynamics • BCR ac&va&on and microcluster forma&on • B cell ac&n mediated spreading on APC, integrin ac&va&on • Centripetal mo&on of microclusters on MT • Lysosome recritment • Contact site contrac&on How is an&gen acquired? • Soluble an&gens may diffuse into secondary lymphoid organs and be acquired from fluid phase. • APCs like subcapsular macs and follicular DCs present large par&culate an&gens How do B cells obtain an8gens from surfaces?
- 98. Nature Reviews Immunology 13, 475–486 (2013) doi:10.1038/nri3469 B cells polarize lysosomes toward an APC contact MTOC Lysosome Laser ablation of a single lysosome does not impact overall lysosome polarization to the synapse Ablation of the MTOC causes the lysosomes to disperse. What is the purpose of this with respect to antigen acquisition?
- 99. Macrophages contacting LDL aggregates also target lysosomes to extracellular contact sites for the purpose of degradation and material extraction Abigail S. Haka et al. Mol. Biol. Cell 2009;20:4932-4940 All LDL aggr. Extracellular LDL aggr. Lyso contents released Acidification of extracellular contacts with agLDL due to lysosomal release
- 100. Mechanisms of intercellular exchange of proteins FEBS Letters Volume 583, Issue 11, pages 1792-1799, 14 MAR 2009 DOI: 10.1016/j.febslet.2009.03.014 http://onlinelibrary.wiley.com/doi/10.1016/j.febslet.2009.03.014/full#feb2s0014579309001872-fig2 B cells have also been proposed to gather Ag from APC by “trogocytosis”. The B cell exerts force on the APC membrane sufficient to rip out the antigen and some accompanying membrane. Other mechanisms of protein transfer are also known, though their contribution to B cell Ag gathering is less clear.