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Immunology > Week 10 > Flashcards

Week 10 Flashcards

1
Q

What does SH2 domains typically recognize?

A

A phosphorylated tyrosine and often the amino acid three positions away (pYXXZ)

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2
Q

What activates small GTPases?

A

When they are bound to GDP, they are inactive, but the exchange of GDP for GTP through GEFs, activates the small GTPases.

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3
Q

GEFs

A

Guanine-nucleotide exchange factors. Facilitate the exchange of GDP for GTP in GTPases, thereby activating them.

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4
Q

GAPs

A

GTPase-activating proteins that facilitate the conversion of GTP to GDP in small GTPases, thus inactivating them. Serve as a control for GTPase signaling.

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5
Q

Where are small GTPases typically located?

A

Bound to the cytosolic side of the plasma membrane. When activated, they recruit signaling proteins, thus localizing the signaling proteins to the plasma membrane.

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6
Q

Phosphatidylinositol kinases

A

Phosphorylate phosphotidylinostiol in the plasma membrane to propagate signaling. Often they are di- or tri-phosphorylated to create phosphatidylinositol 4,5-biphosphate (PIP2) or PIP3

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7
Q

phosphatidylinositol 3-kinase (PI 3-kinase)

A

Phosphorylates PIP2 into PIP3 and is often recruited by binding of its SH2 domain to phosphotyrosines in an activated receptor tail

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8
Q

What is a bacterial “infection”?

A

Presence of bacteria in the host PLUS disturbance of host structure or function by the bacteria or the host response to the bacteria

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9
Q

What is the strategy of an abcess?

A

Depriving bacteria of connective tissue for bacteria to hide. Compresses draining venules to prevent toxin circulation.

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10
Q

What are ante-antibodies?

A

Soluble effector components of the innate immune system that evolved before antibodies. Examples include C-type lectin, complement, C reactive protein, serum amyloid A.

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11
Q

What are the broad functions of the complement system?

A 
Opsonization
Chemotaxis and leukocyte activation
direct lysis of bacteria and pathogens
Augmentation of antibodies
clearance of apoptotic cells
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12
Q

Crry

A

membrane bound complement regulator. KO mice are embryonic lethal, as an abcess forms on the embryo.

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13
Q

What kills bacteria in gastric juice besides HCl?

A

Nitric oxide (also used in phagosomes). Bacteria in the sulci of tongue respire nitrite from ingested nitrite. Nitrite enters stomach and gets acidified into nitrous acid, which dismutates into nitric oxide.

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14
Q

Leukocyte Adhesion Deficiency

A

autosomal recessive disorder due to lack of CD18 (beta-2) integrin.CD18 binds to extracellular matrix proteins and leads to actin skeleton reorganization. Leukocytes cannot exit circulation to most body sites. Recurrent bacterial infections. Infected foci contain few neutrophils.

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15
Q

Azurophil granules

A

Bacterial permeability increasing protein. Lysozyme. Serpocidins (elastase, cathepsin G, protease 3, azurocidin), alpha defensins. All are pre-fromed.

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16
Q

myeloperoxidase

A

Uses circulating halides to make hypohalous acids from H2O2. The green color of pus.

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17
Q

Reactive Oxygen Intermediates

A

O2 (oxygen) -> O2- (superoxide anion) -> H2O2 (hydrogen peroxide) -> OH (hydroxyl radical) -> Water

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18
Q

Chronic Granulomatous Disease

A

Usually X-linked (gp91 deficiency). Failure of leukocytes to make ROS upon encounter of pathogens. Ineffective leukocytes accumulate in abscesses.

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19
Q

Functions of mast cells

A

Degranulation in response to IgE crosslinking or neuralpeptide signaling. Release histamine, serotonin, and leukotrienes to initiate vasodialtion. Release tryptases to degrade ECM.

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20
Q

PARs

A

Protease-activated receptors. In response to proteases, these will increae vascular endothelium permeability, promote chemotaxis in neutrophils,

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21
Q

MHC-I peptide length

A

8-10 amino acids long. cleft is closed off on both sides

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22
Q

Where is the majority of variability in MHC I?

A

The N-terminal of alpha-1 and alpha-2 domains forming the peptide binding cleft

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23
Q

Number of ‘pockets’ in MHC-I

A

Typically six pockets, named A-F. Each binds a particular flavor of amino acids, i,e. there are preferences.

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24
Q

Calnexin

A

Chaperone protein in ER that stabilizes MHC I before its association with B2 microglobulin

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25
Q

TAP protein

A

Heterodimer in ER membrane that shuttles peptides into the ER lumen for MHC I presentation. Contains an ATP-binding cassette for this activity.

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26
Q

Calreticulin

A

Soluble form of calnexin. Associates empty MHC-I with the peptide loading complex in the ER lumen.

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27
Q

Peptide loading complex

A

Loads peptides onto MHC-I. Consists of TAP, tapasin, etc.

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28
Q

ERAP1/ERAP2

A

will trim peptides in the ER to fit MHC-I better.

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29
Q

Methods of ‘immunoevasins’ produced by viruses that interfere with MHC I presentation

A

Blocking peptide entry to the ER
Retention of MHC I within the ER (tapasin interference)
Degradation of MHC I
Binding MHCI at cell surface

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30
Q

Phagocytosis size limit

A

1 micron or more in diameter

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31
Q

How are extracellular proteins delivered to the ER for cross-presentation in DCs?

A

Phagosomal:ER fusion near the cell membrane.

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32
Q

How is peptide diversity increased in cross-presentation?

A

Proteasomes are recruited to the endolysosome that degrade proteins into peptides, but there are also hydrolytic proteases in the endolysosome that can produce peptides that the proteasome alone may not make.

33
Q

What mediates the fusion of the ER and phagosomes for cross presentation to MHC I?

A

Sec22b (on ER) and Syntaxin 4 (on phagosome)

34
Q

LRO Pathway

A

Lysosomal Related Organelle pathway that is involved in cross presentation of peptides to MHC I through phagosomes. Delivers NAPDH oxidase to the phagosome to preserve an alkaline pH to prevent total degradation of contained proteins.

35
Q

ERGIC pathway

A

(ER-Golgi intermediate compartment) Fusion of phagosomes and ER for cross-presentation of peptides on MHC I. Uses SNARE proteins Syntaxin-4 and Sec22b Involves the ‘passing’ of the peptide loading complex into the endosome. Passes newly synthesized proteins from the ER to the Golgi and serves as “QC’

36
Q

ERC

A

Endosomal recycling compartment important for delivering MHC-I molecules to the endosome for cross-presentation

37
Q

Is MHC I or MHC II more stable?

A

MHC II is more stable. MHC I is more short-lived, so it can give a snapshot of the current conditions of the cell.

38
Q

How do superantigens work for MHc II?

A

They are potent T cell mitogens that bind to relatively conserved regions of the MHC II, and a particular Vb element (Vb8). They bind outside of the peptide groove. They activate all T cells expressing that particular Vb.

39
Q

What are some examples of superantigens?

A

Toxic shock syndrome (TSST-1), Viral SAG

40
Q

What does HLA-DM do?

A

Removes CLIP (cleaved invariant peptide) from MHC II to allow for peptide loading.

41
Q

What are some ways in which MHC-II expression/presentation is regulated?

A

Transcriptionally regulated through CIITA and boosted through IFNg signaling
TLR signaling also upregulates expression for about 24h
altered trafficking
reduced degradation

42
Q

What does HLA-DO do?

A

Expressed in B cells and mTECs. Binds to HLA-DM, prevents it from stabilizing newly synthesized MHCII, thereby preventing peptide loading at higher pH.

43
Q

MARCH-1

A

Turned off by TLR signaling. Ubiquinates MHCII, tagging it for degradation.

44
Q

What are some ways in which pathogens interfere with MHC II signaling?

A

Viral downregulation of CIITA
IL-10 induced by some pathogens upregulates MARCH-1
altered intravesicular trafficking

45
Q

LFA-1 ligand

A

ICAM-1

46
Q

What is cytoskeletal rigidity (or ligand rigidification) and why is it important?

A

The amount that a cell-surface receptor or ligand is associated with the cytoskeleton, allowing for or preventing free diffusion throughout the cell membrane. Has been shown to be important for T cell costimulation in the context of ICAM/LFA-1 interactions.

47
Q

How did Jenkins ‘accidentally’ discover costimulation?

A

Tried fixing splenocytes so that he wouldn’t have to sac a mouse every time he wanted to stimulate his T cells. He found that fixed splenocytes would not stimulate T cells in the presence of peptide. The restimulation of these T cells showed their anergy after the primary fixed stimulation.

He was later able to restore T cell proliferation to fixed splenocytes by adding an anti-CD28 antibody that would engage T cell CD28.

48
Q

What is the general costimulation concept?

A

If a naive T cell recognizes its cognate pMHC complex in the presence of costimulatory signaling, it will become activated. However, in the absence of costimulatory signals, a T cell recognizing its cognate pMHC complex will become anergic or tolerised.

Costimulatory molecules should not induce signals or responses on their own.

49
Q

What is the structure of CD28?

A

Disulfide-linked homodimer of Ig domains and trans/intracellular domain. Short cytoplasmic tail that contains unstructured signaling motifs (tyrosines and prolines).

50
Q

What is the ligand for CD28?

A

CD80 and CD86 (B7.1 and B7.2)

51
Q

Structural differences between CD80 and CD86.

A

CD80 is (noncovalently) dimeric, CD86 is monomeric. Both are upregulated on APCs in response to danger signals. CD86 comes up earlier than CD80. Other than that, they are highly homologous and bind to CTLA-4 with higher affinity than CD28.

52
Q

How is the CD28 ligand binding signal transduced through the cell?

A

cytoplasmic tails are phosphorylated and this modification is recognized by a number of signaling molecules, some of which overlap with TCR signaling (Lck, VAV3, PI3K, SLP76)

CD28 signaling seems not to be inducing unique signaling during TCR activation, but instead serves to augment pathways that are already activated through TCR signaling (Lck, ZAP70, etc)

53
Q

How does TIRF imaging work?

A

Total Internal Reflection Fluorescence (TIRF) uses a supported lipid bilayer containing activating molecules on a coverslip to visualize the location of cell surface molecules on cells interacting with the lipid bilayer. Only molecules interacting with the lipid bilayer can be visualized.

54
Q

Does CD28 form its own signaling domain?

A

Yes. In the presence of CD80, CD28 will cluster peripherally around the TCR upon activation. The CD28 molecules in this peripheral ring are in constant exchange, moving in and out.

55
Q

What was found in early CD28-/- in vivo experiments (1993/1996)?

A

Injection with a superantigen only lead to d3 expansion of CD4 and CD8 T cells in the presence of CD28 (and the presence of the correct VB component of the TCR).

56
Q

How were costimulatory molecules shown to have a role in autoimmunity/GvH?

A

Mice were treated with streptozotocin (to kill Beta cells) and transplanted with human pancreatic islets. Mice that were also treated with CTLA-4-Ig (to block engagement of CD28) for two weeks showed a severely delayed rejection of implanted cells, as measured by glucose levels (Lenschow, 1992).

57
Q

What was seen in CD80-/- in vivo experiments?

A

Severely reduced T cell proliferation, similar to CD28-/- phenotype. However, addition of CTLA-4-Ig had a heightened effect on the mice, due to the blocking of CD86 signaling.

dKO of both CD80 and CD86 also led to significantly less B cell CSR.

58
Q

What was unexpected about the effects of CD80/CD86 dKO on Tregs?

A

dKO mice on NOD background were MORE susceptible to diabetes. It turns out that these dKO mice lack Tregs, as they require stronger signaling in the thymus to develop.

59
Q

Structure of CTLA-4

A

Very similar to CD28 (homodimer, Ig domains, etc). Binds to same ligands (CD80/CD86) but is inhibitory.

60
Q

Which binds to CD80/CD86 with higher affinity: CD28 or CTLA-4?

A

CTLA-4. By a lot.

61
Q

What is abatacept?

A

CTLA-4-Ig, which was further developed into belatacept. Used to suppress transplant rejection through the blocking of CD80/86:CD28 signaling.

62
Q

Where in the cell is CTLA-4 in a resting T cell?

A

Most CTLA-4 is sequestered to an endosomal compartment. Upon activation of the T cell, CTLA-4 is trafficked to the synapse, where it competes with CD28 for costimulatory signaling.

63
Q

How does CTLA-4 inhibit costimulatory signaling?

A

No one is quite sure, but it may be through intracellular signaling. It could also be through molecular ‘crowding’, as one molecule of CTLA-4 can engage two CD80 homodimers at once (leading to a zipper-like formation that increases avidity and crowds out other molecules). This is supported by TIRF experiments that show low CD28 in high CTLA-4 areas of the synapse. (this doesn’t work with monomeric CD86)

Finally, CTLA-4 can strip CD80/CD86 from the surface of APCs and endocytose them. Tregs are good at this.

64
Q

Phenotype of CTLA-4-/- mice.

A

multi-organ autoimmunity - die within 3-4 weeks. Conditional KOs removing CTLA-4 on just Tregs also die relatively early-on.

65
Q

What happens to adult mice that have CTLA-4 in T cells knocked out?

A

They are relatively healthy since they have an expansion of both Tregs and Tconv cells. Also they are resistant to EAE.

66
Q

What are the three functions of CTLA-4 that can be observed in vivo by knocking out the gene in various contexts?

A

CTLA-4:

1) Attenuates Teff cells in a cell-intrinsic manner.
2) Mediates cell-extrinsic regulation of Teff by treg
3) Attenuates Tregs in a cell-intrinsic manner.

67
Q

Structure of PD-1

A

PD-1 is a member of the CD28 superfamily. Does not form homodimers. Upregulated in response to TCR stimulation (but transcribed, as opposed to CTLA-4). Works through intracellular signaling of cytoplasmic tail (ITIM and ITSM motifs)

68
Q

What is the concept of central versus peripheral costimulatory attenuation?

A

CTLA-4 appears to be more involved in central attenuation, which involves APCs interacting with T cells in the lymph nodes. Due to the PD-1 ligands expression, these are thought to act in peripheral attenuation, wherein T cells are inhibited at the point of non-APC cell recognition in tissues.

69
Q

What is the important signaling molecule for PD-1 signaling?

A

SHP2

70
Q

What are some markers for T cell exhaustion?

A

PD-1, LAG-3, CD244, CD160

71
Q

Phenotype of PD-1-/- mice in vivo.

A

PD-1-/- mice thrive, as opposed to CTLA-4-/- mice, but they have defects in controlling autoimmunity and develop lupus-like disorders later in life. OT1-PD-1-/- T cells transfered into RIP-OVA (expressing OVA on pancreatic cells) mice are more susceptible to diabetes. PD1-/- on NOD background are also more susceptible to diabetes.

72
Q

What is an example of T cell exhaustion being adaptive and compensatory?

A

PD-1 -/- T cells can sometimes be MORE exhausted, due to a compensatory upregulation of other exhaustion markers. This was shown in a model of P14 PD-1-/- T cells transferred into recipients that were infected with LCMV clones that typically promote T cell exhaustion.

73
Q

What are two general takeaways from the studies of genetic KOs for costimulatory molecules?

A

1) KO phenotypes must be interpreted with care. Timing of gene/protein deletion/depletion is key.
2) Signaling systems designed to maintain homeostasis are particularly tricky. Some are redundant, others (CTLA-4, Tregs) are not.

74
Q

How can you prove that cell membrane receptor:ligand pairs can bind in cis?

A

FRET imaging showing quenching of signal upon addition of second molecule into LUV. Also, TIRF imaging showing that addition of PD-1 to supported lipid bilayer can prevent PD-L1 on the same lipid bilayer from interacting with PD-1 on the T cell surface.

75
Q

What was the first immunotherapy for cancer?

A

Ipilimumab, which is an anti-CTLA-4 for treatment of melanoma

76
Q

What are the different mechanisms through which CTLA-4 and PD-1 checkpoint blockade therapies are thought to work?

A

anti-CTLA-4 will work in the lymph node, allowing for the activation of stronger responses to APCs that are carrying tumor antigen. anti-PD-1, however will prevent the ‘shutdown’ of Teffs at the tumor site itself through PD-1 ligands expressed by the tumor cell.

77
Q

Which checkpoint blockade therapies activate which subsets of T cells?

A

anti-CTLA-4 leads to CD4+ and CD8+ activation, while anti-PD-1 was mostly CD8+ T cell expansion. Both lead to expansion of exhausted T cells.

78
Q

Why are cancers with higher mutational rates more responsive to checkpoint blockade?

A

Higher mutational rates (from smoking, UV damage, etc), means larger pools of neoantigens, which widens the pool of potential cognate CD8+ T cells that can be rescued by checkpoint blockade.

Combination of radiation with checkpoint blockade may increase the number of neoantigens.

79
Q

What is ICOS?

A

Induced Costimulatory molecule - expressed on activated T cells and required for proper GC formation and Th cell differentiation. ICOL is the ligand.

Immunology (17 decks)

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