Identifying Haplotypes of Different Mouse Strains Using Mhc Restriction and Cytotoxic T Cells

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We were able to observe this reaction as the cells had been stained with BATDA. BATDA works by entering the cells and then undergoing esterification and becoming hydrophilic. This stops it from exiting the intact cell membrane. Therefore, when the cell has been lysed by the cytotoxic T cell, the dye spills out of the cell and it can be detected in the machine that reads ASSAYS. Each concentration of the effector to target cell ratio was repeated thrice to get a more accurate result. The readable assay, shown in Table 1 was converted to a line graph as shown in Figure 2. The line graph makes it easier for us to see the trends of the cytotoxicity rising as the concentration of effector to target cell ratio decreases. We can see that as there are fewer effector cells to the target cells, the immune response decreases. More importantly, however, it allows us to easily determine which strains of mice have stronger cytotoxic killing when compared to others. The line graph further allows us to establish a control that can be easily interpreted. The control in this experiment was the uninfected mouse strain as it lacked the independent factor, the infection.

This allowed me to fill in Table 2. By simply observing the values of the graphs in figure 2, it was easy for me to sort the immune responses as being strong, medium, weak or none.

As a snowball effect, I was then able to identify the haplotypes of the mice. The reasoning I used for this is explained more in the discussion section. As we can see, the BALB/c x B10 mouse strain produced the strongest cytotoxicity. We can assume that this must be the strongest reaction possible, apart from the 1% NP-40 detergent that lysed every single cell, as the haplotypes were the same. Next were the partial matches as seen in the orange, green and blue graphs. All these had medium to weak cytotoxicity, meaning that only one haplotype matched with the target cell, and not the other.

The control in the experiment was the uninfected BALB/c cell, as it did not contain the peptides from the MSV virus antigen. The unknown mouse that had the infection produced relatively the same trend, relatively, indicating that the haplotypes did not match with either alleles of the target mouse strain. Therefore, this left the C3H mouse strain, which had the alleles kk, and did not match with db.

The other forms of control was the 1% NP-40 and the media. The media contained no effector cells so it should have had no target cells being lysed, while the 1% NP-40 had a detergent, which should have lysed every single cell. This measure was taken to make sure that the target cells were normal, and that they didn’t all lyse spontaneously, nor were they invincible against the detergent.

Discussion

There are 2 conditions for the t cell to begin cytotoxic killing. Firstly, the MHC molecules must bind, meaning that the haplotypes should either match completely or partially. A complete match would be the Balb/c x B10 mouse with cells from the same strain. A partial match is when one of the haplotypes match but the other doesn’t, as shown in the B10 and target strain of BALB/c x B10. (4)

The other condition is that the peptide must also be recognized by the T cell receptor, so the splenocytes must actually be infected with the MSV. When both these conditions are met, the cytotoxic T cell is able to be activated and can kill the infected cell.

MHC restriction was demonstrated in the control. The uninfected Balb/c mouse would have had a partial binding, but because it was free of the infection, and hence the peptide, it did not trigger an immune response.

On the other hand, the unknown mouse infected with the MSV produced the same cytotoxicity as the uninfected BALB/c cells. This suggests that there was no immune response towards the unknown mouse, but because we know for sure that it has the peptide, we must assume that the T cell receptor did not recognize the MHC molecules. MHC restriction tells us that the haplotype cannot have either d or b alleles, leaving the mouse strain to be identified as homologous for C3H with kk alleles. The t cell receptors were unable to recognize the kk alleles which is why there was no immune response. (4)

It is important that activity of cytotoxic t cells is highly regulated in order to prevent the wrong cells from being killed (2). Having the peptide in the grove on the MHC is key, as this indicates that the cell has been infected. And because of the strong, medium, weak and no killing in addition to the presence of the infection, MHC restriction is supported, as there are varied levels of cytotoxic killing in all these strains.

References:

(1) PC Z. The discovery of MHC restriction. - PubMed - NCBI [Internet]. Ncbi.nlm.nih.gov. 2016 [cited 2 May 2016]. Available from: http://www.ncbi.nlm.nih.gov/pubmed/9018968?log$=activity

(2) G Allen R. Major histocompatibility complex class I-restricted cytotoxic T-lymphocyte responses in horses infected with equine herpesvirus 1. Journal of Virology [Internet]. 1995 [cited 2 May 2016];69(1):606. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC188619/

(3) Doherty PZinkernagel R. MHC Restriction and Cytotoxic T Lymphocytes. Concepts in Viral Pathogenesis. 1984;:53-57.

(4) Brenan M e. Analysis of haplotype preference in the cytotoxic T-cell response to H-Y. - PubMed - NCBI [Internet]. Ncbi.nlm.nih.gov. 2016 [cited 2 May 2016]. Available from: http://www.ncbi.nlm.nih.gov/pubmed/6971801?log$=activity