MHC Restricted Cytotoxic T Cell Responses to Viral Infection

ID No: 24191175

Friday 2PM

Anthony Nguyen

Exercise !: M"# restricted cytotoxic $ ce%% res&onses to 'ira% in(ection )tudent na*e: Anthony Nguyen Date: 2+,-4,2-1! .rou& No: 7 De*onstrator: #hris /odrigues

ID No: 24191175

Introduction M"# restriction 0as (irst descried y Austra%ian scientist Peter Doherty and co%%eague /o%(  ingerage% o( )0it3er%and as the s&eci(ic interaction o( $#/ on a $ ce%% 0ith the M"# and associated &e&tide6 $his interaction is i*&ortant in $ ce%% de'e%o&*ent in the $hy*us and $ ce%% acti'ation in the &eri&hery6 &eri&hery6 M"# restriction restriction can e de*onstrated de*onstrated y ex&osing ex&osing a 'ariety o( $ ce%%s 0ith di((erent M"# acgrounds (ro* a *ouse in(ected 0ith the Mo%oney )arco*as 8irus M)8 to M)8 in(ected ce%%s6 $ ce%%s (ro* a &articu%ar M"# acground 0i%% 0i%% e una%e una%e to recogn recognise ise and i%% in(ecte in(ected d ce%%s ce%%s (ro* a di((er di((erent ent M"# acgrou acground6 nd6 "o0e'er those that are a%e to i%% M)8 in(ected ce%%s are ter*ed M"# restricted6 $o test the 'a%idity 'a%idity o( s&eci(ic s&eci(ic interaction interaction o( $#/ and M"# &e&tides &e&tides the ex&eri*ent ex&eri*ent *entioned *entioned ao'e ao'e 0i%% e exa*ined in an in vitro assay6 vitro assay6 In this study the target ce%%s 0ere &re&ared 'ia in(ecting *ic e 0ith 4 x 1- 5 &(u &%aue (or*ing units o( M)8 and are %ae%%ed y adding a *o%ecu%e ;AD$A is<acetoxy*ethy%< 22=:+=2>
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ID No: 24191175

Friday 2PM

Anthony Nguyen

 Adding the target cells: Add 1--@ o( the &re&ared target ce%%s 5 x 1- 4 ce%%s,*% to each o( the 9+ 0e%%s6 nce the target ce%%s are added to the &%ate it 0i%% start the &rocess o( ce%%u%ar interaction  et0een e((ector and target ce%%s6 nce the reaction is (inished 2--@ o( Euro&iu* so%ution is added to cause the (or*ation o( the (%uorescent che%ate o( $DA re%eased (ro* ce%%s on %ysis6 $he (%uorescence reading otained 0i%% gi'e an indication o( the degree o( cytotoxicity that has occurred6

Figure 1: $he cytotoxicity assay o( 9+ 12 x  0e%%s 0as &re&ared in such a 0ay that tested suect under oser'ation 0as re&eated three ti*es6 In other 0ords co%u*ns 1 4 7 and 10ere treated as one ex&eri*ent and co%u*ns 2 5  and 11 0ere treated as another6 /o0s A . and " acted as the contro% grou&6 /o0s ; # D E and F act as the ex&eri*enta% grou& 0ith 'arious M)8 in(ected *ice each 0ith di((erent e((ector ce%%s6 Results

Figure 2: Data resu%t o( .rou& 7 re&resents the uantitati'e a*ount o( (%uorescence 0hich corres&onds to the %e'e% o( cytotoxic acti'ity o( the ce%%6

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ID No: 24191175

Friday 2PM

Anthony Nguyen

Uninfected BALB/c mouse 5200 5000 4800

Experiment 1

4600

Experiment 2

4400

Experiment 3

4200 4000 3800 3600

50:1

25:1

12.5:1

6.75:1

Figure 3: ?nin(ected ;A@;,c *ouse ex&osed to target ce%%s

(BALB/c x B10) mouse with MSV 35000 30000 Experiment 1

25000

Experiment 2

20000

Experiment 3

15000 10000 5000 0

50:1

25:1

12.5:1

6.75:1

Figure 4: ;A@;,c x ;1- *ouse 0ith M)8 ex&osed to target ce%%s

B10 mouse infected with MSV 25000 20000

Experiment 1 Experiment 2

15000

Experiment 3 10000 5000 0

50:1

25:1

12.5:1

6.75:1

Figure 5: ;1- *ouse in(ected 0ith M)8 ex&osed to target ce%%s

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ID No: 24191175

Friday 2PM

Anthony Nguyen

Unknown mouse infected with MSV 5200 5000 4800 4600 4400 4200 4000 3800 3600

Experiment 1 Experiment 2 Experiment 3

50:1

25:1

12.5:1

6.75:1

Figure 6: ?nno0n *ouse in(ected 0ith M)8 ex&osed to target ce%%s

(C3 x B10) mouse with MSV 18000 16000 14000 12000 10000 8000 6000 4000 2000 0

Experiment 1 Experiment 2 Experiment 3

50:1

25:1

12.5:1

6.75:1

Figure 7: #!" x ;1- *ouse 0ith M)8 ex&osed to target ce%%s

!BA/2 mouse infected with MSV 25000 20000

Experiment 1 Experiment 2

15000

Experiment 3 10000 5000 0

50:1

25:1

12.5:1

6.75:1

Figure 8: D;A,2 *ouse in(ected 0ith M)8 ex&osed to target ce%%s

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ID No: 24191175

Friday 2PM

Anthony Nguyen

Medi# on$% & (min 'e$e#se cont'o$) 3500 3000

Experiment 1

2500

Experiment 2

2000

Experiment 3

1500 1000 500 0

50:1

25:1

12.5:1

6.75:1

Figure 9: Media on%y G *in re%ease contro% ex&osed to target ce%%s

1 *&"0 & (m#x 'e$e#se cont'o$) 34000 33000 Experiment 1

32000

Experiment 2

31000

Experiment 3

30000 29000 28000

50:1

25:1

12.5:1

6.75:1

Figure 10: 1H NP<4- G *ax re%ease contro% ex&osed to target ce%%s

#e%% count: Target cells

a b c  e  !ota": Ce""#$m":

Count 1 4 5 10 8 11 38 1.9 x 107

Count 2 4 13 2 7 3 29 1.45 x 107

Count 3 1 5 4 2 4 16 8.0 x 106

A'erage #e%%s,*%  16! x 1-7 #e%%s,*%

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ID No: 24191175

Friday 2PM

Anthony Nguyen

 Effector Cells

1 2 3 4 5  !ota": Ce""#$m":

a b c  e  !ota": Ce""#$m":

Count 1 19 26 24 32 16 117 2.34 x 106 Count 2 5 8 8 1 7 29 1.45 x 107

Count 3 2 5 2 5 3 17 8.5 x 106

A'erage #e%%s,*%  645 x 1-+ Discussion $he resu%ts o( the unno0n *ouse sho0ed no signs o( cytotoxicity$he unno0n *ouse 0as the unin(ected ;A@;,c *ouse /eiterate the *ain &oints o( the study • Bhy 0as the ex&eri*ent &er(or*edJ o o Bhat are you testingJ o "o0 0as this doneJ o Bhat 0i%% it address or de*onstrateJ Pro'ide inter&retation o( the data • &&ortunity to discuss the resu%ts o o ;road state*ents in re%ation to the uestion or hy&othesis tested o #o*&are and contrast the resu%ts Britten argu*ent as to 0hether it ans0ered the uestion or not • Particu%ar hy&othesis not su&&orted • Incorrect%y &er(or*edJ o Data didnKt su&&ort the hy&othesisJ o •

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