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IMM2011: Practical 3 MHC restricted cytotoxic T cell responses to viral infection Name: Emilie Page Student ID: 297094667 Group number: 3

There are two parts to this report you need to submit Part 1 and Part 2 ● A copy of this assessment sheet can be found in Moodle in the Practical classes section. ● You can use this template for your assessment submission. ● Use Part 1 of this template for your submission; complete a written report for Part 2, but you can remove the Part 2 template pages from your submission. ● Convert and submit a pdf of this assessment sheet (Part 1), and your written report (Part 2) via the dropbox provided in the practical classes section in Moodle. Please combine these into a single file. ● Tip: Do not leave this report to the last minute to start. ● You need to complete the online lesson (Practical 3 lesson: MHC restricted cytotoxic T cell responses to viral infection) before completing this assessment.

Part 1: Processing the raw data A. Complete the table below (2 marks) You will need to know which allele(s) each mouse strain or mouse cross is expressing. ● A ‘mouse cross’ is when two mice of different genetic backgrounds are bred. ● Remember – mice, just like us, inherit one HLA haplotype from each parent. You may wish to review ‘The MHC and antigen processing II’ l ecture for revision on the genetics of MHC inheritance.

BALB/c

H-2 haplotype d

H-2 class I alleles d d

B10

b

b b

C3H

k

k k

DBA/2

d

d d

BALB/c x B10

db

db

C3H x B10

bk

bk

Mouse strain

Copyright © Monash University 2020. All rights reserved. Except as provided in the Copyright Act 1968, this work may not be reproduced in any form without the written permission of the host Faculty and School/Department.

Page 1 of 4

B. Processing the raw data (2 marks) ● The raw data for the fluorescence is available to you on Moodle, within the M  oodle lesson. ● Complete Table 1 below that lists the processed raw data generated from your assay. o First determine the mean fluorescence count obtained for each set of triplicates (i.e. the raw data set) of your assay; o Use these values to determine the associated mean % cytotoxicity (use the formula in the unit guide), and complete the table. Table 1: Template for mean fluorescence and cytotoxicity analysis Mean fluorescent value Mice: effector cells

50:1

25:1

Uninfected BALB/c

3166.67

4433.33

4433.33

4566.67

1.731

6.736

6.345

2.080

B/c x B10) mouse infected with MSV

25466.67 25300.00

13066.67

6433.33

74.126

79.443

36.780

4.022

B10 mouse infected with MSV

15516.67 22566.67

11800.00

5566.67

41.824

69.919

32.315

3.120

4300.00

4166.67

4466.67

5.735

6.272

5.405

1.976

3H x B10) mouse infected with MSV

18300.00 14566.67

5666.67

3833.33

50.860

42.044

10.693

1.317

DBA/2 mouse infected with MSV

22666.67 22466.67

11333.33

5700.00

65.036

69.570

30.670

3.259

2500.00

2633.33

2566.67

31066.67 31200.00

31000.00

96400.00

Unknown mouse infected with MSV

Media only (min release) 1% NP-40 (max release)

4400.00

2633.33

12.5:1

6.25:1

Mean % cytotoxicity 50:1

25:1

12.5:1

6.25:1

C. Plotting data (2 marks) ● Data, such as that obtained in this experiment can be graphed in a number of ways. Some will be easier to interpret than others, and not all graph types are correct. ● It is important you choose the type of graph that is suitable for your data. ● The Microsoft program Excel will allow you to plot graphs, but it will not select the correct one or most appropriate for you. You should always consider what is the best way to present your data. Displaying data in the easiest way to interpret is very important. ● Also ensure that the colours and symbols you choose are easy to distinguish. ● Review the information on plotting graphs from your unit guide (pages 68-70). ● If you are not familiar with plotting graphs in Excel, there are some excellent tutorials available online. Graphing the mean % cytotoxicity ● Plot a graph that will best represent the data visually. Only one type of graph should be presented. ● Think carefully about the style. What style will allow you to most easily compare the cytotoxicity of the different mice over the different effector to target ratios? ● If not sure, try different styles and see which is easiest to interpret. ● All the mouse groups should be represented in the one  graph ● Do not generate a separate graph for each mouse. Copyright © Monash University 2020. All rights reserved. Except as provided in the Copyright Act 1968, this work may not be reproduced in any form without the written permission of the host Faculty and School/Department.

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● Do not include the data for the media alone or 1% NP-40 ● Label the axes appropriately, with Y-axis being % cytotoxicity and X-axis the effector:target ratio ● Make sure you include an appropriate and descriptive figure legend to support the figure.

D. Assessing the degree of cytotoxicity and determining the MHC haplotype of the unknown mouse strain (1 mark) ● Generate a table, similar to Table 2 and fill in the missing information using information available to you in the unit guide or from the online lesson, your understanding of MHC restriction, and analysis of the data you have analysed. ● The MHC class I haplotypes for each of the mouse strains are in your Table 1 at the start of this worksheet (also in the u  nit guide, and online lesson). ● List the degree of cytotoxicity (strong, medium, weak/none) that was observed with the cells from each of the mouse strains. Use the 50:1 ratio as the guiding reference on cytotoxicity achieved. ● List the MHC haplotype for each of the indicated mouse strains that were used as effector cells or target cells ● From your data and the haplotypes of the mouse strains listed in the unit guide, (1) determine the MHC haplotype of the unknown mouse strain that has been infected with MSV and gave you the result you achieved for that mouse, and (2) your reasoning. ● Think about what the experiment is measuring, and how it is being determined. Mouse strain effector cells

Effector cells

Target cells (BALB/c x B10)

Cytotoxic killing Enter one of the following Strong (≥ 60%) Medium (20-60%) Weak/none (≤20%)

MHC class I haplotype dd

MHC class I haplotype db

(BALB/c x B10) mouse infected with MSV

db

db

Strong

B10 mouse infected with MSV

bb

db

Medium

Unknown mouse infected with MSV (C3H x B10) mouse infected with MSV

kk

db

Weak

kb

db

Medium

DBA/2 mouse infected with MSV

dd

db

Strong

Uninfected Balb/c

Weak

Copyright © Monash University 2020. All rights reserved. Except as provided in the Copyright Act 1968, this work may not be reproduced in any form without the written permission of the host Faculty and School/Department.

Page 3 of 4

This is the end of Part 1. Some of the data you have generated here can be directly copied and used in Part 2 (although you must still show it in Part 1). When you have completed Part 2 (The Written Report), submit both Parts 1 and 2, as a single document, to the Practical 3 dropbox on Moodle.

Copyright © Monash University 2020. All rights reserved. Except as provided in the Copyright Act 1968, this work may not be reproduced in any form without the written permission of the host Faculty and School/Department.

Page 4 of 4

IMM2011 Practical 3: MHC restricted cytotoxic T cell responses to viral infection Emilie Page 29709466 Part 2: Discovering the unknown haplotype of a mouse infected with virus using cytotoxic (CD8+) T cell assay Introduction The purpose of the practical is to discover whether virus infected cells that are killed are Major Histocompatibility Complex (MHC) restricted, and to thus use this to determine the unknown haplotype (MHC) of an mouse which has been injected. In this practical, five mice of different strains have been infected with Moloney Sarcoma Virus (MSV). T cells are then taken, with one having an unknown MHC haplotype. A cytotoxic (CD8+) assay is used to observe the amount of cells which die, which can then be used to find the unknown haplotype. The experiment focuses specifically on the relationship of the MHC and the peptide that it is binded with. Restriction of the MHC molecules is a result of specificity between the T cell receptor (TCR) and its matching MHC-peptide (1). MHC restriction protects from the ‘suicide’ of its own cells by only allowing CD8+ T cells to recognise peptides from MHC Class I molecules (2).

Results The purpose of this practical is to determine the unknown MHC haplotype of a mouse. This is done through discovering whether the killing of infected cells is MHC restricted. Fluorescence is produced through the establishment of a cytotoxic (CD8+) assay, and this data is then processed. Higher fluorescence values indicate higher numbers of cells being killed, therefore fluorescence production is used to measure the cell death. Raw and Processed Data This table presents the raw mean fluorescent readings and the calculated cytotoxicity %. This was done using relevant formulas and excel. The mean fluorescence data was taken and used to calculate the cytotoxicity.

Table 1. Raw data of mean fluorescent value and the processed mean % cytotoxicity Mean fluorescent value

Mean % cytotoxicity

Mice: Effector Cells

50:1

25:1

12.5:1

6.25:1

50:1

25:1

12.5:1

6.25:1

Uninfected BALB/c

3166.67

4433.33

4433.33

4566.67

1.731

6.736

6.345

2.080

B/c x B10 mouse infected with MSV

25466.67

25300.00

13066.67

6433.33

74.126

79.443

36.780

4.022

B10 mouse infected with MSV

15516.67

22566.67

11800.00

5566.67

41.824

69.919

32.315

3.120

Unknown mouse infected with MSV

4400.00

4300.00

4166.67

4466.67

5.735

6.272

5.405

1.976

CH3 x B10 mouse infected with MSV

18300.00

14566.67

5666.67

3833.33

50.860

42.044

10.693

1.317

DBA/2 mouse infected with MSV

22666.67

22466.67

11333.33

5700.00

65.036

69.570

30.670

3.259

Media only (min release)

263.33

2500.00

2633.33

2566.67

-

-

-

-

1% NP-40 (max release)

31066.67

-

-

-

-

31200.00 31000.00 96400.00

1

IMM2011 Practical 3: MHC restricted cytotoxic T cell responses to viral infection Emilie Page 29709466

Graph 1. A comparative line graph of the calculated mean % cytotoxicity plotted over the effector:target ratio. The legend correlated to the matching coloured line on the graph. The x axis shows the effector:target ratio as labelled.

Table 2. The MHC class I haplotype of both the effector cells and the target cells, as well as the qualitative evaluation of the strength of cytotoxic killing observed. Strong is defined as ≥60%, medius is defined as 20-60%, and weak/none is defined as ≤20%. Mouse Strain: Effector Cells

Effector Cells MHC class I haplotype

Target Cells (BALB/c x B10) MHC class I haplotype

Cytotoxic Killing

Uninfected BALB/c

dd

db

weak/none

B/c x B10 mouse infected with MSV

db

db

strong

B10 mouse infected with MSV

bb

db

strong

Unknown mouse infected with MSV

kk

db

weak/none

CH3 x B10 mouse infected with MSV

kb

db

medium

DBA/2 mouse infected with MSV

dd

db

strong

2

IMM2011 Practical 3: MHC restricted cytotoxic T cell responses to viral infection Emilie Page 29709466 Discussion MHC restriction is a result of mature T cells binding to the MHC presented peptides (3). MHC molecules have different haplotypes, and thus are varied. Variation of MHC occurs where the peptide binds, and where the T cell receptor engages with the resulting MHC-peptide, which in turn results in MHC restriction (4). The purpose of this practical was to determine the unknown MHC haplotype of a mouse, and therefore make a conclusion on MHC restriction. It can be concluded that killing virally infected cells is MHC restricted. T cells from a particular MHC profile will NOT kill virally infected cells with a different MHC profile. This was found through discovering whether the killing of infected cells is MHC restricted. The cytotoxicity (CD8+) assay was performed, and the results shown in Table 1 and Graph 1, were processed and interpreted through the use of qualitative indicators, as shown in column 4 of table 2. As shown in table 2, the mouse with unknown haplotype was categorised as having weak/none, which means that the haplotype must be kk, as MHC Class I is codominantly expressed. To substantiate this, we can look at the other mice. The unknown haplotype has a very different MHC profile to the other observed mice, therefore d and b can be eliminated.

References 1. 2. 3. 4.

Zinkernagel, R., and P. Doherty. 1997. The discovery of MHC restriction. Immunology Today 1 8: 14-17. Liu, Z., and S. Tugulea.1998. Specific Suppression of T Helper alloreactivity by all-MHC class I - restricted CD8+ - T cells. International Immunology 10: 775-783. Buus, S., and A. Sette, SM. Colon. 1987. The relation between MHC restriction and capacity of Ia to bind immunogenic peptides. Science  235: 1353-1358. Corr, M., and AE. Slanetz. 1994. T cell receptor-MHC class I peptide interactions. Science 265:946-949.

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