ID No: 24191175Friday 2PMAnthony NguyenExercise #3: MHC restricted cytotoxic T cell responses to viral infection

Student name: Anthony NguyenID No: 24191175Date: 26/04/2013Group No: 7Demonstrator: Chris Rodrigues

IntroductionMHC restriction was first described by Australian scientist Peter Doherty and colleague Rolf Zingeragel of Switzerland as the specific interaction of TCR (on a T cell) with the MHC and associated peptide. This interaction is important in T cell development in the Thymus and T cell activation in the periphery. MHC restriction can be demonstrated by exposing a variety of T cells (with different MHC backgrounds) from a mouse infected with the Moloney Sarcomas Virus (MSV) to MSV infected cells. T cells from a particular MHC background will be unable to recognise and kill infected cells from a different MHC background. However, those that are able to kill MSV infected cells are termed MHC restricted. To test the validity of specific interaction of TCR and MHC peptides, the experiment mentioned above will be examined in an in vitro assay. In this study, the target cells were prepared via infecting mice with 4 x 105 pfu (plaque forming units) of MSV and are labelled by adding a molecule, BADTA (bis-(acetoxymethyl)-2,2:6,2-terpyrid-ine-6,6-dicarboxylate), which is an acetoxymethyl ester of a fluorescence enhancing ligand. BADTA is hydrophobic ligand which enables it to penetrate the cell membrane, however, it is subsequently is converted into a hydrophilic ligand TDA upon entering the cell, trapping the ligand inside the cell. Upon cell lysis, the TDA is released and can be detected through the addition of Europium solution, which causes the formation of a fluorescent chelate. The amount of fluorescence serves as an indication of cell lysis (i.e. the amount of fluorescence is proportional to cell lysis) and can be measured by a fluorometer.

MethodPreparing the target cells: In a clean Eppendorf tube, add 1.0mL target cell solution with a concentration of 1 x 106 cells/ml and 20L of fluorescence enhancing substrate, BADTA. Leave the tube to incubate for 30 minutes at room temperature. While the target cells are incubating, prepare the effector cells and assay plate. After the 30 minutes of incubation, centrifuge the target cells at 500g for 5 minutes. Remove the supernatant and resuspend the cells (pellet) to a concentration of ~5 x 104 cells/ml.Preparing the effector cells:Adjust the effector cell concentration to 2.5 x 106 cells/ml in buffer solution. Note: All other cell suspensions for other effector cells from other infected mouse strains have already been diluted to 2.5 x 106 cells/ml by technical staffs.Preparing the assay plate: Add 100L of buffer solution to all wells in columns 4-12 in the plate.Aliquot 200L of the uninfected BALB/c splenocyte suspension (2.5 x 106 cells/ml) to each of the wells A1, A2, A3. Perform a series of dilution by first taking 100L from well A1 and mix with A4. Next, take 100L from A4 and mix with A7. After that, take 100L from A7 and mix with A10. Finally, suck up 100L from A10 and discard it. Repeat this procedure for diluting cells from A2 and A3.Repeat the series of dilution for each of the other rows with effector cells from the other 5 MSV infected mice in the order outlined in Figure 1, until there is 100L of each sample in each well.Adding the target cells:Add 100L of the prepared target cells (5 x 104 cells/ml) to each of the 96 wells.

Once the target cells are added to the plate, it will start the process of cellular interaction between effector and target cells. Once the reaction is finished, 200L of Europium solution is added to cause the formation of the fluorescent chelate of TDA released from cells on lysis. The fluorescence reading obtained will give an indication of the degree of cytotoxicity that has occurred.

Figure 1: The cytotoxicity assay of 96 (12 x 8) wells was prepared in such a way that tested subject under observation was repeated three times. In other words, columns 1, 4, 7, and 10 were treated as one experiment and columns 2, 5, 8, and 11 were treated as another. Rows A, G, and H acted as the control group. Rows B, C, D, E, and F act as the experimental group with various MSV infected mice (each with different effector cells).

Results

Figure 2: Data result of Group 7 represents the quantitative amount of fluorescence which corresponds to the level of cytotoxic activity of the cell.

Figure 3: Uninfected BALB/c mouse exposed to target cells

Figure 4: (BALB/c x B10) mouse with MSV exposed to target cells

Figure 5: B10 mouse infected with MSV exposed to target cells

Figure 6: Unknown mouse infected with MSV exposed to target cells

Figure 7: (C3H x B10) mouse with MSV exposed to target cells

Figure 8: DBA/2 mouse infected with MSV exposed to target cells

Figure 9: Media only (min release control) exposed to target cells

Figure 10: 1% NP-40 (max release control) exposed to target cells

Cell count:Target cellsCount 1Count 2Count 3

a441

b5135

c 1024

d872

e1134

Total:382916

Cells/ml:1.9 x 1071.45 x 1078.0 x 106

Average Cells/ml = 1.38 x 107 Cells/ml

Effector CellsCount 1

119

226

324

432

516

Total:117

Cells/ml:2.34 x 106

Count 2Count 3

a52

b85

c 82

d15

e73

Total:2917

Cells/ml:1.45 x 1078.5 x 106

Average Cells/ml = 8.45 x 106

DiscussionThe results of the unknown mouse showed no signs of cytotoxicityThe unknown mouse was the uninfected BALB/c mouse Reiterate the main points of the study Why was the experiment performed? What are you testing? How was this done? What will it address or demonstrate? Provide interpretation of the data Opportunity to discuss the results Broad statements in relation to the question or hypothesis tested Compare and contrast the results Written argument as to whether it answered the question or not Particular hypothesis not supported Incorrectly performed? Data didnt support the hypothesis? Page 1Page 2Page | 6