Download MHC MOLECULES 1) Describe three important differences between

MHC MOLECULES
1) Describe three important differences between class I and class II MHC proteins?
Class I is a single chain bound to an invariant partner, 2-microglobulin, whereas class
II is a heterodimer
Class II is expressed only on APCs, whereas class II is ubiquitously expressed
Class I present 8-10 amino acid peptides, whereas class II presents 14-18 amino acid
Peptides
2) The following set of peptides are eluted from a purified MHC molecule. Which
class
of MHC molecule was it and what are its "anchor residues?"
VFTSKAINK
SFTIRTALK
QFTYFIVAK
RFTQKAIAK
RFTQFIVNK
EFTGKYFKK
F in position 2, T in position 3, and K in position 9.
3). Why would it make sense that Interferons would activate the expression of
LMP2 and
LMP7??
LMP2 and LMP 7 are involved in Class I antigen processing which is important in
activating CD8+ T cells which are most effective against viruses. Interferons are
produced in response to viral infections
4) Class II MHC molecules will traffic through the same ER that Class I molecules
are in yet they do not normally bind endogenous peptides.
a) How are Class II Molecules prevented from binding endogenous peptides in
the ER?
Through Invariant chain binding to peptide cleft. Ii is cleaved to leave the CLIP in the
Class II MHC groove.
b) How is this "blocking protein" removed from the Class II MHC groove to
allow an exogenous peptide to bind?
Class II MHC is loaded in the endosomes and lysosomes of APCs. The low pH in these
endosomes along with HLA-DM enables foreign peptide exchange with CLIP.
5) What is retrograde translocation and why do cells need this ability for Class I
antigen processing and presentation?
Retrograde translocation is the ability of proteins to transfer into cytoplasm from the ER.
This is necessary in the antigen processing of membrane proteins in the cytoplasm so that
they may become Class I antigens.
6) Tapasin deficient mice have Class I MHC that exit the ER but they do not have
normal peptide loading. How do you think this deficiency causes this result?
Tapasin plays a critical role in retaining Class I MHC molecules in the ER and allows
for proper loading of Class I MHC with peptide.
7) Why would CD1 (Class I like molecules) be found in the same endosomes as
Class II MHC molecules?
CD1 primarily present bacterial glycolipids which would be found in endosomes (with
exogenous antigens
T-CELL RECEPTOR
8) Compare the T cell receptor and the B cell receptor for antigen with regard to:
a) protein structure
usual picture of IgM vs. TCR
b) genetic of diversity
generated by V(D)J recombination. T cells do not undergo somatic mutation or class
switching.
c) associated polypeptides
TCR associates with CD3 complex and BCR associates with Ig- a and Ig- b
d) co-receptors
TCR uses CD4 and CD8, BCR uses CD19 recruited by CD21 (CR2, the complement
receptor)
e) recognition of antigen
BCR recognizes native antigen, usually a 3-dimensional surface
TCR recognizes a peptide-MHC complex where the peptide is a stretch of
contiguous amino acids.
9) What roles do CD4 and CD8 play in T cell antigen recognition?
They play essential roles by binding non-polymorphic regions of either MHC
class II or MHC class I respectively.
10) What is cDNA? What is the principle underlying subtractive cDNA cloning?
cDNA is a DNA copy of an RNA transcript. It can be either single or double stranded.
Subtractive cDNA cloning uses DNA hybridization to remove cDNA sequences in
common between two populations of cells allowing one to clone gene copies which are
uniquely expressed in a given cell type and not its "neighbor
11) How many different peptides can a class I MHC molecule bind if it requires the
structure XXXX(F or Y)XX(I, L, or M) ?
20 x 20 x 20 x 20 x 2 x 20 x 20 x 3 = 3.84 x 108
T-CELL SIGNALLING
3) Describe the general structure of a Src-family tyrosine kinase, indicating its
various functional domains.
Know about SH1, SH2 and SH3 domains and what they do. Also, regulatory
phosphorylation sites and myristylation site
a) Which Src family kinases are involved in coupling TCR engagement to
changes in gene activity?
Lck, fyn
b) Which Src family kinases are involved in coupling BCR engagement to
changes in gene activity?
Blk, lyn, fyn
c)What PTK in B cells plays a similar role to that of Zap-70 in T cells?
syk
4) How is NF-AT transcription factor activated?
Phospholipase Cg (PLC)is activated by phosphorylation. Active PLC hydrolyzes a
phospholipids component of the plasma membrane to generate the second messenger,
DAG and IP3. Ip3 mediates the release of Ca2+ from the endoplasmic reticulum. Ca2+
binds the protein calmodulin, which then associates with and activates the
Ca2+/calmodulin dependent phosphatase calcineurin. Active calcineurin removes a
phosphate group from NFAT, which allow this transcription factor to tranbslocate into
the nucleus.
5) What is an "adapter" molecule and how are adapters involved in signal
transduction?
Adapters lack PTK activity, but contain both SH2 domains and phosphorylatable
tyrosines. They serve as scaffolds for the building of signaling complexes.
6) What T cell signaling phenotype would you predict for a tyrosine to
phenylalanine mutation in an ITAM of the CD3γ chain?
The ITAMs on the CD3 chains get phosphorylated upon T cell activation by Lck and
serve as docking sites for the PTK named Zap-70. Zap-70 is the critical PTK for T cell
activation since in its absence, there is little phosphorylation and activation of
PLCgamma and the ras pathway doesn't turn on. The question is how will mutating the
CD3 gamma chain to an unphosphorylatable form effect these steps. I don't really know
the answer since I have never seen the experiment done, but I expect it will decrease
quantitatively TCR signaling, but won't abolish it since several of the other CD3 chains
have ITAMs which could still be phosphorylated
7) What is an SH2 domain and how does it work?
Src-homology 2 domain. Binds to specific phosphotyrosine-containing motifs in signaling
molecues (such as ITAMs) and helps PTKs find targets and scaffolds assemble signaling
complexes.
8) Why is cyclosporin used in the setting of organ transplantation? Would you
expect that a transplant patient would have to take cyclosporin forever, or just
around the time of the transplant?
Blocks TCR signal transduction by preventing NFAT activation (know the pathway).
Thus, host T cells would not activate against allograft material.. You must take the drug
forever to prevent alloreactivity.
9)What roles do SHP-1 and CD45 play in lymphocyte signaling? How does SHP-1
get
These proteins are protein tyrosine phosphatases. CD45 plays an activating role by
dephosphorylating Lck at its negative regulatory tyrosine residue. SHP-1 gets recruited
to the TCR complex via scaffold association and to the BCR complex via its interaction
with the cyto-domain of CD22. In both instances, it dephosphorylates PTKs, inactivating
them.
10) What are lipid rafts?
Lipid rafts are detergent insoluble patches of the cell membrane rich in cholesterol, GPIlinked proteins, glycosphingolipids, and signaling molecules. TCR and BCR get recruited
into lipid rafts upon initial B and T cell activation. Probably modulates signaling activity.