1

MHC restricted cytotoxic T cell responses to 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
bodys 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 self-
antigens 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 ones 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
mouses 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

2
Mean CPM value Mean % cytotoxicity
Effector : Target
Ratio
50 : 1 25 : 1 12.5 : 1 6.25 : 1 50 : 1 25 : 1 12.5 : 1 6.25 : 1
Uninfected Balb/c A 4017.33 4078.67 4078.67 4201.33 6.10 6.74 6.35 6.76
BALB/c x B10
mouse infected
with MSV
B 25576.00 22202.67 14658.67 7390.67 88.51 75.38 46.89 18.49
B10 mouse infected
with MSV
C 20485.33 13892.00 9476.00 5121.33 69.05 43.90 27.03 10.15
Unknown mouse
infected with MSV
D 4048.00 3956.00 3833.33 4109.33 6.21 6.27 5.41 6.43
(C3H x B10) mouse
with MSV
E 14137.33 9077.33 5213.33 3526.67 44.78 25.67 10.69 4.28
DBA/2 mouse with
MSV
F 20853.33 12849.33 9108.00 5244.00 70.46 39.95 25.62 10.60
Media Only (min
release)
G 2422.67 2300.00 2422.67 2361.33 0.00 0.00 0.00 0.00
1% NP-40 (max
release)
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























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
Target mouse (BALB/c x
B10) strain - MHC class 1
alleles
Cytotoxic
Killing
Uninfected Balb/c d d b d Weak
BALB/c x B10 mouse infected with b d b d Strong
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
100.00
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
M
e
a
n

%

C
y
t
o
t
o
x
i
c
i
t
y

Mouse Haplotype
50:1
25:1
12.5:1
6.25:1
3
MSV
B10 mouse infected with MSV b b b d Strong
Unknown mouse infected with
MSV
k k b d Weak
(C3H x B10) mouse with MSV k b b d Medium
DBA/2 mouse with MSV d d b d 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.
4
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