You have become interested in antigen presentation and processing, and decide to
ID: 208591 • Letter: Y
Question
You have become interested in antigen presentation and processing, and decide to pursue this interest in graduate school. You join a lab that has just crossed two well-characterized mouse strains, a female CBA mouse and a male C57BL/6 mouse to produce hybrid F1 pups. In order to begin your work, you must first look up the haplotypes of these strains so that you can determine what MHC alleles they express.
a). What are all of the class I MHC alleles that will be expressed in the mother, the father, and their F1 offspring?
b). Your PI shows you the work of a previous post doc who identified two viral epitopes (peptides) from the influenza virus. The post-doc purified class I MHC:peptide complexes from cells infected with influenza and then then separated the peptides from the MHC proteins. Sequencing of the eluted peptides found that both epitopes are derived from the nucleoprotein (NP) of the virus:
Peptide
Sequence
MHC class I from
NP366-374
ASNENMETM
H-2Db
NP147-155
TYQRYRALV
H-2Kk
You now want to determine if T cell activation by these peptides is in fact restricted to only the MHC class I proteins from which they were eluted (removed from).
To do this, you infect 3 mice (one from the mother’s strain (CBA), one from the father’s strain (C57BL/6), and an F1 offspring) with the influenza virus and isolate CD8 T cells from each of their spleens. You then set up a series of 51Cr release killing assays using target cells available in your lab. You find three target cell lines that you can use:
- EL4 cells (H-2b haplotype)
- P815 cells (H-2k haplotype)
- P815t cells (H-2k haplotype transfected with the H-2Db gene).
For the experiment, you will mix CD8 T cells isolated from your immunized mice with radiolabeled target cells as outlined in the table below.
If recognition by T cells of peptides really is restricted to the MHC protein from which they were eluted, what do you expect the results of this experiment to be? Enter your answer in the table below. (each answer should be “Yes” for killing of target cells by CD8+ T cells, or “No” for no killing).
Killing of target cells by CTLs from:
Target Cells
Mother
Father
F1
1
EL4 + influenza virus
2
EL4 + NP366-374
3
EL4 + NP147-155
4
P815 cells
5
P815 + NP147-155
6
P815t + NP147-155
7
P815t + influenza virus
c). (2 points) You start to notice a skin rash on some of your mice from strain CBA. You think T cells may be involved in the pathogenesis and are curious as to what antigens may be stimulating them. You decide to look at what peptides are being presented on MHC molecules for clues and you begin with class II MHC proteins. After specifically isolating IE:peptide complexes from cells by immunoprecipitation, you use acid to elute (remove) the bound peptides. You then separate the proteins using HPLC and sequence them using mass spectrometry. From this experiment you find two peptides that bind to the IE (ab) protein. Based on what you know about peptides that bind MHC class II vs. MHC class I molecules, describe two (2) reasons why you think these peptides are indeed binding to class II MHC. In your explanations, compare your reasons to what you know about class I peptides.
Note:. *Bold residues are anchor residues based on binding patterns known to exist for other peptides presented by IE
Peptide
Sequence
MHC class I from
NP366-374
ASNENMETM
H-2Db
NP147-155
TYQRYRALV
H-2Kk
Amino Acid Position in MHC groove: 123456 78910 HLQLAIRNDEELNKLLGKVT Peptide 1. Peptide 2. PGVTVKDVNQQEFVRALAAFLK Note:. "Bold residues are anchor residues based on binding patterns known to exist for other peptides presented by IEExplanation / Answer
Part a- there are three haplotypes H-2K, H-2D, and H-2L.
Partb-
Mother
Father
F1
In EL4 + influenza virus
No
No
No
EL4 + NP366-374
Yes
Yes
Yes
EL4 + NP147-155
Yes
Yes
Yes
P815 cells
No
No
No
P815 + NP147-155
Yes
Yes
Yes
P815t + NP147-155
Yes
Yes
Yes
P815t + influenza virus
Yes
Yes
Yes
In first case there is only H-2b haplotypes, that is why the CD8T cells cannot kill the influenza virus. Therefore, in other cases too, if the particular haplotype is present, then it can perform killings.
Part c-
The main difference in between MHC classes I and II is presence of anchor residues. MHC class I contains charged at 4th and 9th. The peptide 2 has V and L, which makes it with MHC class I.
The second reason is no one residue has negative charge in the peptide 2 at the region between 1-10.
Mother
Father
F1
In EL4 + influenza virus
No
No
No
EL4 + NP366-374
Yes
Yes
Yes
EL4 + NP147-155
Yes
Yes
Yes
P815 cells
No
No
No
P815 + NP147-155
Yes
Yes
Yes
P815t + NP147-155
Yes
Yes
Yes
P815t + influenza virus
Yes
Yes
Yes
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