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MHC restricted cytotoxic T cell responses to t o viral infection Introduction Major Histocompatibility Complex (MHC) restriction refers to the concept of T Cell Receptors (TCRs) recognising only MHC molecules presented on ‘self’ cells. It is particularly important in the body’s role of preventing autoimmunity and defence against invading pathogens. Within the thymus, T cells begin to differentiate – those that show too high or too low affinity for selfantigens undergo negative selection where they then die via apoptosis. Peter Doherty and Rolf Zinkernagel discovered MHC restriction in 1973 in Canberra, Australia. Later, they went on to receive the Noble Prize for Physiology or Medicine in 1996. Doherty and Zinkernagel found that TCRs would only interact with cells that had the same haplotypes as the MHC molecules presented on cells within one’s own body. This suggested the idea that the MHC-TCR interaction was antigen-specific due to experiments performed with mice with difference MHC polymorphisms. This assay aims to simulate those done in the 1970s by Doherty and Zinkernagel whereby a specific mouse’s spleen cells are mixed with splenocytes from different mice to assess the specificity of the interaction. Those cells that are mixed together that contain the same MHC haplotype, as well viral antigen specific CD8 T cells will result in that cell being killed. Further research can be undertaken in this area to expand knowledge on induction of specific non-responsiveness to foreign material entering a cell and in the area of autoimmune disease. Materials and Methods
Figure 1. Schematic Diagram of Experiment Set Up Results Part A. Results for level of fluorescence and cytotoxicity within mouse splenocytes
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Mean CPM value Effector : Target Ratio Uninfected Balb/c BALB/c x B10 mouse infected with MSV B10 mouse infected with MSV Unknown mouse infected with MSV (C3H x B10) mouse with MSV DBA/2 mouse with MSV Media Only (min release) 1% NP-40 (max release)
Mean % cytotoxicity
50 : 1
25 : 1
12.5 : 1
6.25 : 1
50 : 1
25 : 1
12.5 : 1
6.25 : 1
A
4017.33
4078.67
4078.67
4201.33
6.10
6.74
6.35
6.76
B
25576.00
22202.67
14658.67
7390.67
88.51
75.38
46.89
18.49
C
20485.33
13892.00
9476.00
5121.33
69.05
43.90
27.03
10.15
D
4048.00
3956.00
3833.33
4109.33
6.21
6.27
5.41
6.43
E
14137.33
9077.33
5213.33
3526.67
44.78
25.67
10.69
4.28
F
20853.33
12849.33
9108.00
5244.00
70.46
39.95
25.62
10.60
G
2422.67
2300.00
2422.67
2361.33
0.00
0.00
0.00
0.00
H
28581.33
28704.00
28520.00
29562.67
100
100
100
100
Table 1. Mean values for CPM and % Cytotoxicity at each dilution level for each mouse haplotype Part B. Relationship between mean % cytotoxicity and different mice 100.00 90.00 80.00
y t 70.00 i c i x 60.00 o t o t y 50.00 C % n 40.00 a e M 30.00
50:1 25:1 12.5:1
20.00
6.25:1
10.00 0.00 Uninfected BALB/c x B10 mouse Unknown (C3H x DBA/2 Balb/c B10 mouse infected mouse B10) mouse infected with MSV infected mouse with MSV with MSV with MSV with MSV Mouse Haplotype
Fig ure 2. Relationship between mean % cytotoxicity and each mouse haplotype Part C. Determining the MHC haplotype of the unknown mouse strain Mouse Effector Strain
H-2 Class 1 alleles
Uninfected Balb/c BALB/c x B10 mouse infected with
dd bd
Target mouse (BALB/c x B10) strain - MHC class 1 alleles bd bd
Cytotoxic Killing Weak Strong 2
MSV B10 mouse infected with MSV Unknown mouse infected with MSV
(C3H x B10) mouse with MSV DBA/2 mouse with MSV
bb
bd
Strong
kk
bd
Weak
kb dd
bd bd
Medium Strong
Table 2. Mouse effector strain respective alleles and their killing effects Discussion In order for MHC restriction to be successful both T cell receptor and MHC molecule must have the same haplotype. The cells taken from the BALB/c x B10 F1 mouse spleen require splenocytes with the same haplotype in order to be recognised other they will be undetected in a cell. The uninfected mouse (table 1) has low mean % cytotoxicity at all dilutions as the splenocytes recognise the target cells as ‘self’ and they are uninfected so there is no potential threat. The BALB/c x B10 mouse infected with MSV exhibits the H-2 haplotype so CD8 T cells will kill MSV since they share H-2 class 1 alleles. CD8 T cells are therefore restricted to MHC class type, (specifically class 1 and this case alleles b and d) and also to the type of virus. The B10 mouse infected with MSV also exhibits one haplotype of the two alleles and therefore high mean killing occurs. The unknown mouse infected with MSV (figure 2) results show weak cytotoxicity meaning recognition has been unsuccessful, as although it is infected with the virus, the cells are not being killed. Therefore, it is identified as having a differed haplotype – kk (table 2). The C3H x B10 mouse is heterozygous for the k and b haplotypes and so some killing does occur. DBA/2 mouse exhibits strong killing, as it is homozygous for the d allele. Without MHC restriction the body would not only not be able to differentiate between ‘self’ and ‘non-self’ cells within the body, but would also not be able to recognise potentially harmful pathogens too. The specificity of this concept is paramount due to the highly varied nature of viruses and the genes that code for the MHC molecules.
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References Bjorkman, P. J., M. A. Saper, B. Samraoui, W. S. Bennett, J. L. Strominger, D. C. Wiley. 1987. Structure of the human class I histocompatibility antigen, HLA-A2. Nature 329: 506
Zinkernagel, R. M., P. C. Doherty. 1997. The discovery of MHC restriction. Immunol. Today 18: 14
Parham, P. 2005. Putting a Face to MHC Restriction. The Journal of Immunology 174(1) 3-5