Lecture 9 Flashcards
Compare what antibodies Naive B cells express with what “class switched and Memory B cells” can express
Naive B cells express IgM and IgD
“class switched and memory B cells” may express IgG, IgA, or IgE
Name the 2 invariant signaling molecules that are associated with the BCR. What is the last subunit that is needed to form a complete BCR? (besides the 2 signaling molecules)
IgAlpha and IgBeta
The BCR also needs an Ag receptor
(these 3 things form a complete BCR)
Describe the method that IgAlpha and IgBeta are associated with the BCR. Also describe the structure that these 2 molecules contain so that they may participate in signaling functions.
IgAlpha and IgBeta are “non-covalently” associated with the BCR
IgAlpha and IgBeta contain “ITAMs” in their cytoplasmic tails (bc they are transmembrane proteins) and they use their ITAMs in order to participate in signaling functions
Describe the relationship between IgAlpha, IgBeta, and the Ag receptor in a BCR.
IgAlpha and IgBeta are REQUIRED for the “assembly and expression” of the Ag receptor
When a B cell encounters an Ag-complement or Ag-Ab complex, the BCR and Co-receptors can be “linked” by binding to the same antigen molecule. State the 3 Co-receptors that can become linked the BCR in this fashion.
CD19
CD21
CD32
True or False:
Co-receptors that bind to the same antigen as the BCR will enhance the signaling process of the B cell. explain.
False
Co-receptors that bind to the same antigen as the BCR can “enhance or inhibit” the signaling process of the B cell.
each Co-receptor has a different effect on the signaling of the B cell
Describe function of CD19, CD21, and CD32.
CD19: functions as the dominant signaling component of B cells
CD21: positively regulates B cell activation and serves the “lower the Ag threshold for B cell activation”
CD32: negatively regulates B cell signaling bc it has an ITIM subunit
What is another name for CD21?
CD21 = CR2 (complement receptor 2)
Describe the “2 signals” that have to occur to the B cell before B cell activation can happen.
Signal 1 is caused by the binding of the B cell to an Ag, via the BCR in the case of B cells.
(just this signal is not enough to activate the B cell)
Signal 2 is carried out by Costimulatory molecules on the surface of B cells
Beginning with the ITAMs of the BCR, describe what occurs within the B cell in order to activate PLCgamma2, Ras, and Rac.
The BCR engages the antigen, which results in a conformational change that makes the BCR-associated ITAMs accessible to Src kinases (Lyn, Fyn, and Blk)
The Src kinases then phosphorylate the ITAMs of IgAlpha and IgBeta, which allows them to act as a docking site
The SH2 domains on Syk tyrosine kinase allow it to dock to the ITAMs of IgAlpha and IgBeta, which activates Syk
Now that Syk is active, it phosphorylates “critical tyrosine residues” on BLNK (or SLP-64, same thing)
Phosphorylated BLNK recruits other enzymes to activate PLCgamma2, Ras, Rac and Btk (bruton tyrosine kinase)
True or False:
Once PLCgamma, Ras, and Rac are activated the rest of the signaling pathway in B cells is identical to the pathway in T cells. explain.
True
PLCgamma, Ras, and Rac all eventually stimulate NFAT, NF-KB, and AP-1. Just like what occurs in T cells.
What is another name for BLNK?
BLNK = SLP-65
In BCR signaling, signal initiation by Ag occurs by ___-____ of the BCR and is facilitated by the _____ for the BCR.
Cross-linking
Coreceptor
B cells express a receptor for C3d. What is the name of this receptor and explain the origin of the C3d.
C3d is formed via degradation of C3b, after C3b (came from the cleavage of C3 in the complement pathway) binds covalently to the microbe/Ag-Ab complex.
The receptor for C3d on B cells is CR2/CD21 (same thing)
When a C3d-Ag complex binds to a B cell, what 2 transmembrane proteins on a B cell recognize these respective subunits of the C3d-Ag complex?
The BCR transmembrane protein recognizes the Ag
The CR2 transmembrane protein recognizes the C3d (which is still bound to the microbe)
Out of CD19 and CD81, which of these has an ITAM? also state which of these CD’s is NECESSARY for the normal expression of the other CD.
CD19 has an ITAM
CD81 is necessary for the normal expression of CD19
In the case of a microbe being bound to the BCR complex and Co-receptors at the same time, the BCR complex consists of IgM, IgAlpha, and IgBeta. What 3 subunits make up the co-receptor complex that CR2 is involved in?
CR2 is expressed in a complex with CD 19 (which has an ITAM) and CD81
What is the main role that CD81 plays in the co-receptor complex?
CD81 is important for linking the CD21 (CR2), CD19, an CD81 complex to the cytoskeleton of the B cell
Compare the B cell response if a microbe binds to the B cell Ag receptor to when a microbe binds to the Ag receptor and the CR2 complex simultaneously.
The B cell response is greatly enhanced when the microbe is bound to both receptors simultaneously, as opposed to binding to just the Ag receptor alone.
State the 2 processes conducted by the CR2 complex that mediate the enhancement of the B cell reaction
- phosphorylation of potential substrates in the BCR complex via LYN (which is bound to CD19)
- Ca2+ mobilization by a PI3 Kinase dependent mechanism
Explain why, in the B cell coreceptor complex, CD19 does such a good job of amplifying BCR signaling. (2 reasons)
- CD19’s cytoplasmic tail becoming phosphorylated is what recruits a Lyn Kinase
The Lyn Kinase then greatly enhances the phosphorylation of the ITAM tyrosines in IgAlpha and IgBeta
- Phosphorylated CD19 activates PI3-Kinase, which activated Btk and PLCgamma2 on the inner leaflet of the PM of the B cell
(these 2 processes greatly enhance B cell activation)
The inhibitory signaling that controls B cell activation is essential to prevent collateral damage to the body caused by what 2 specific processes?
uncontrolled lymphoproliferation
inflammation
Name the receptor type and enzyme that work to mediate the activation of B cells via inhibitory signals. How
Inhibitory receptors (ITIMs)
E3 ubiquitin ligase
Beginning when a ligand binds to the extracellular ligand-binding domain of an inhibitory receptor, name the follow steps that occur in order to attenuate an inhibitory signal.
When the ligand binds to the inhibitory receptor, the cytosolic ITIM domain of the receptor is phosphorylated by a Src family kinase
Next, the p-ITIM uses it’s new phosphate to recruit a tyrosine phosphatase
After the phosphatase binds it’s SH2 domain to the p-ITIM, it dephosphorylates substrates and inhibits the activation of the B cell
Name the 2 “key inhibitory receptors” in B cells. which of these belongs to the SIGLEC (sialic acid binding Ig like lectin) family of lectins?
FcgammaRIIB (CD32)
CD22, which belongs to the SIGLEC family
Compare the cells that FCgammaRIIB and CD22 are found in
FcgammaRIIB is involved in signaling in activated B cells, DC’s, and Resident tissue macrophages
CD22 is an inhibitor that is expressed on B cells only
What are SHP and SHIP? also name a function they conduct that affects the kinase activity in lymphocytes and some innate immune cells. (be specific about the protein they affect)
SHP (SH2 domain-containing phosphatase) and SHIP (SH2 domain-containing inositol phosphatase) are tyrosine phosphatases
They remove phosphates from PIP3, which inhibits PI3-kinase activity
When a protein is being ubiquitinated, where do the ubiquitin subunits attach and what molecule recognizes the area of the substrate that the ubiquitin will attach?
the ubiquitin subunits attach to the lysine residues on the substrate
specific E3 ubiquitin ligases recognize these lysine residues
State the 2 different “ubiquitination shapes” that can be constructed on a substrate and describe the fate of each of these ubiquitination locations.
Lysine-48 type ubiquitination chains will target the protein/substrate for proteasome degradation
Lysine-63 type ubiquitination chains will NOT target the protein for degradation
What do most ACTIVE B cell responses require in order to occur? If it were a naive B cell, what would it need in order to become activated?
Active B cells require CD4+ T cell’s (helper T cells) to help them activate
A naive B cell would require a DC to help it become a mature B cell/activate
State the 6 steps that occur when a CD4+ T cell helps activate a mature B cell
- the “first signal” to the T cell occurs when the pMHC class II molecule on the B cell complexes with the TCR:CD3:CD4 complex on the T cell
- Then the CD4+ T cell is costimulated by the interaction of the CD80/86 on the B cell and the CD28 on the T cell
- Following costimulation via the CD28:CD80/86 complex, the activated T cell secretes “IL-4”
- Then CD40 on the B cell interacts with CD154 on the T cell
- The CD40:CD154 complex causes the expression of IL-4R on the surface of the B cell
- “SUPERPOSITION” of the BCR:TCR, CD40:CD154, and IL-4R:IL-4 complexes lead to the activation of the B cell
Cytokines have direct effects on Ig isotype switching. For the following cytokine signals, state what antibodies the B cell will produce:
No T helper Cell assistance:
T helper cell assistance & IFNgamma:
T helper cell assistance & IL-4:
T helper cell assistance & TGFbeta/BAFF:
No T helper Cell assistance: IgM
T helper cell assistance & IFNgamma: IgG subclasses (IgG1, IgG3)
T helper cell assistance & IL-4: IgE
T helper cell assistance & TGFbeta/BAFF: IgA
The signaling pathways that cytokines use are activated by ligand-induced _____ _____.
Receptor Clustering
Match the 5 main classes of cytokine receptor families to the following ligand groups.
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL-13, IL-15, IL-21:
TNF-alpha, TNF-beta, LT, CD40, FasL:
IL-1, IL-18:
IFN-alpha/beta, IFN-gamma, IL-10:
Chemokines:
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL-13, IL-15, IL-21:
Type I cytokine receptors (hemopoietin)
TNF-alpha, TNF-beta, LT, CD40, FasL:
TNF receptor family
IL-1, IL-18:
IL-1 receptor family
IFN-alpha/beta, IFN-gamma, IL-10:
Type II cytokine receptors
Chemokines:
7-transmembrane GPCR’s
Name the 2 criteria that different cytokine receptors are classified into families on the basis of.
- conserved extracellular domain structures
2. Signaling mechanisms
For the following Polarizing cytokine combination, state the transcription factor that will occur and the cell type that this combination will cause.
IL-2
TGFbeta
FOXP3
Induced T regulatory cell
For the following Polarizing cytokine combination, state the transcription factor that will occur and the cell type that this combination will cause.
IL-6
TGFbeta
RORgammat
T helper 17 cell
For the following Polarizing cytokine combination, state the transcription factor that will occur and the cell type that this combination will cause.
IL-4
GATA3
Th2 cell
For the following Polarizing cytokine combination, state the transcription factor that will occur and the cell type that this combination will cause.
IL-6
IL-21
BCL-6
Follicle T cell
For the following Polarizing cytokine combination, state the transcription factor that will occur and the cell type that this combination will cause.
IL-12
IFNgamma
T-bet
Th1 cell
Which 2 cytokine receptor families are the most similar? Briefly explain the similarity/difference between the signaling pathway(s) these cytokine receptor families use.
Type I and Type II cytokine receptor families
Type I cytokine receptors ALL use JAK-STAT signaling pathways
Type II cytokine receptors use either “JAK-STAT” or “JAK-STAT and Tyk-STAT” signaling pathways
Describe what differentiates the subgroups within the type I cytokine receptor family.
All type I receptors feature 1 gamma subunit that forms half of the extracellular receptor.
The other half is what determines the subgroups within the type I receptor family.
While the “variable half” of each subgroup is different, they are all similar enough to form a full extracellular receptor domain with the SAME gamma subunit.
Explain the JAK-STAT method of signaling (4 steps)
ligation of a cytokine to the receptor outside of the PM causes the oligomerization AND activation of the intracellular JAK domains
JAKs then phosphorylate a tyrosine residue on the receptor chain with which they are associated
STATs then bind, to the phosphate via an SH2 domain, to the recently phosphorylated receptor chain and are subsequently phosphorylated by the JAK proteins
STATs being phosphorylated causes 2 STATs to dimerize and move into the nucleus to alter gene expression of cytokine-regulated genes
What is another name for the Type II cytokine receptors? describe the transmembrane chains of this family (how many ligand and how many signal-transducing chains)
Interferon receptor family = Type II cytokine receptor family
they have 1 ligand-binding polypeptide chain and 1 signal-transducing chain
Tyk2 is involved with which cytokine receptor family? describe it’s function.
Type II cytokine receptor family
Tyk2 is very similar to JAK and activates STAT transcription factors in a similar manner
Which family of cytokine receptors features preformed trimers? explain what the ligands for these receptors look like?
The TNF receptor family has preformed trimers
The ligands for the TNF receptor family are also trimers and can be membrane bound (TNFRII) OR soluble (TNFRI)
Describe the extracellular domains of TNF family receptors.
they have cysteine-rich extracellular domains
State the 4 most important TNF receptors that are part of the TNF receptor family? what does the stimulation of these receptors usually lead to?
TNFRI (soluble)
TNFRII (membrane bound)
CD40 protein
Fas (First Apoptosis Signal)
Stimulation of this family of receptors may lead to apoptosis of the cell
Describe the 2 pathways and eventual outcomes after a TNF receptor is stimulated by it’s trimer ligand (2 options in this process)
TNF-RI ligand binds to the receptor which causes the “death domain” of the receptor to recruit TRADD (TNFRSF1-associated via Death Domain)
TRADD then recruits TRAF (TNF receptor associated adaptor proteins) that go on to do 1 of 2 things
- TRAF can activate Caspase-8 which initiates apoptosis
- TRAF can act as E3 ubiquitin ligase in order to activate the NF-KB and JNK MAP kinase pathways (which produce inflammatory mediators/survival proteins)
For TNF receptor signaling, which ligands usually cause apoptosis and which usually cause inflammatory mediators/survival proteins?
TNF-RI is the ligand that usually causes the production of inflammatory mediators/survival proteins
The other ligands for this family (TNFRII, LT, CD40, Fas) usually cause apoptosis
What is the common criteria that all members of the IL-1 receptor family (TLRs) all share?
All members share a Toll-like/IL-1 receptor domain (TIR domain), which a conserved cytosolic sequence
Explain what happens when a ligand binds to a TLR (IL-1 family) receptor.
Ligand engagement causes dimerization and the recruitment of MyD88 (which contains TIR domain)
MyD88’s TIR domain then binds to IRAK, which in turn links to TRAF6
TRAF6 is an E3 ubiquitin ligase which stimulates NF-KB
NF-KB influences gene expression of immune and inflammatory genes
True or False:
IL-1 receptor family receptors not only stimulate the NF-KB pathway, but they also stimulate the MAP kinase pathway. explain.
True
IL-1 family signals cause the phosphorylation of IRF3 and IRF7, which activate the MAP kinase pathway
What is the main criteria for receptors that belong to the chemokine receptor family?
They use GPCR’s in order to activate associated G proteins that carry out intracellular signaling
A lot of the end results of chemokine receptors have to do with adhesion, migration, and other cytoskeletal responses of the cell
Compare the active and inactive states of chemokine receptors
inactive state: Galpha is bound to GDP and forms a trimer with the Gbetagamma subunits that is attached to the inside of the PM
active state: Galpha is now bound to GTP and dissociates from the PM and the Gbetagamma subunits
Both Galpha and Gbetagamma subunits go to activate downstream pathways when the GPCR is in the active state.
What does the active Galpha subunit do in the chemokine receptor signaling mechanism?
the active Galpha subunit inhibits adenylyl cyclase in order to decrease intracellular levels of cAMP (this subsequently decreases the cAMP-dependent protein kinase activity)
What does the active Gbetagamma subunit do in the chemokine receptor signaling mechanism? (really has 2 main mechanisms)
active Gbetagamma activates Ras and PLC (Phospholipase C)
- Ras activates PI3Kgamma, which then goes to activate Akt (a protein kinase B)
- PLC hydrolyzes IP2 in order to generate IP3, which then causes the release of Ca2+ into the cell.
Then DAG and Ca2+ stimulate PKC (protein kinase C)
