Materials

2.1.1 DMA

D M A - and D M B -cD N A s (accession num bers: x62744 and z23139) cloned in pCDM8 (Stratagene) were obtained from Adrian Kelly.

D Q A 1 * 0 3 0 1 SLud D Q B 1 * 0 3 0 2 -cD N A s w ere a gift from Dr. P eter Gregersen.

2.1.2 Peptides

The peptides KGLRKSNAAERRGPL, RAGHSSYTPLPG SNYSEGW HIS and GLRSVGASRHQGPL corresponding to the C -term ini o f the D R a (2 2 4 -2 3 8 ), DM P ( 2 4 2 - 2 6 3 ) an d D Q a ( 2 2 0 - 2 3 3 ) c h a in s , re s p e c tiv e ly , w ere sy n th e size d by the IC R F p e p tid e sy n th e sis service using Fm oc/tB u strategy and th eir pu rity was checked by HPLC analysis.

The pep tid es used for the peptide loading and p ep tid e exchange assays were a gift by Dr. Georg Malcherek.

2.1.3 Vectors

pA cU W 51-” K ozono” (K ozono et al., 1994) w as a g ift by D rs. P h ilip p a M arrack and John K appler. pA cSG 2 was purchased from

P h arm in g en . pC D N A 3.1-N eo, pC D N A 3.1-H ygro and pC R 2.1 w ere p u rc h a se d from In v itro g en . A m o d ified form o f pC E P4, c alle d p C E P 4 A and lacking EBNA-1 and OriP was obtained - from Dr. Isabel Correa, as well as pCEP4A carrying the gene for lacZ.

2.1.4 Cell lines

A ll B cell lin es, T2 and H T 1080 w ere m a in tain ed in R P M I su p p lem en ted w ith 10% PCS, 2 m M L -g lu tam in e and 10 U /m l p e n ic illin /s tr e p to m y c in . M ed iu m fo r th e c e ll lin e T 2 .D R 3 ad d itio n ally contained 0.5 m g/m l G418. 293 cells w ere grow n in D M E M c o n ta in in g G lu ta m a x , 10% P C S , an d 10 U /m l penicillin/streptomycin.

Sf9 cells w ere m aintained either as m onolayer cultures in 75 cm^ and 175 cm^ tissue cu ltu re flasks or in su sp en sio n in T ech n e sp in n e r flask s. T hey w ere grow n at 27®C in G race's m ed iu m su p p le m e n te d w ith g lu tam in e (2 mM fin al co n ce n tra tio n ), 10% PCS, 1% Amphotericin and 0.05 mg/ml Gentamicin.

T he cell lines used and their characteristics are listed in the table b e lo w .

Table 2.1

Cell line C haracteristic S o u rc e

Raji w ild typ e B u r k itt's lym phom a ICRF cell culture service

T2 Fusion cell line

(B L C L .174xC E M .T 2) that has a l a r g e h o m o z y g o u s d e l e t i o n within the M H C encompassing all o f the fu n c tio n a l M H C class II g e n e s

Dr. P eter Cresswell, Yale (Salter and Cresswell, 1986)

T2.DR3 T2 sta b ly tra n sfe c te d w ith the genes f o r DR3

D r. P e t e r C r e s s w e l l , Y a le (Riberdy an d Cresswell, 1992)

B L C L -P riess DR4

D Q A I W 3 0 I / D Q B I *0302

ICRF cell culture service

B L C L -S w e ig O O l DRAI m O I /D R B I *1011 D Q A I * 0 5 0 I/D Q B I *0301

as f o r Priess

BLCL-HID D RAI *OIOI/DRBI *0901

D Q A I * 0 3 0 I/D Q B I *0303

as f o r Priess

B L C L -J e s th o m DRAI m O l / D R B l ^0101 D Q A I W l O l / D Q B l *0501 as f o r priess BLCL-DEU DRAI m O l / D R B l *0401 D Q A I *0301/D Q B1 *0301 as f o r Priess

2 9 3 human epithelial kidney cell line as f o r Priess

H T 1 0 8 0 fib ro b la st cell line as f o r Priess

o varian cell line d e rive d f r o m S p o d o p t e r a f r u g i p e r d a (a rm y f a l l w o r m ) as f o r Priess 7 .9 .6 DRAI *0101/DRB1 *0301 DQAI *0501/DQB1 *0201 D M B - n e g a tiv e Dr. Elizabeth Mellins Stanford University (Morris et al., 1994) 74

2.1.5 Antibodies

The antibodies used in this thesis, their characteristics and references are listed below.

T able 2.2

A n t i b o d y R e a c t i v i t y C h a r a c t e r i s t i c S o u r c e

a n t i - D M 5 C I lumenal domain o f D M a mouse mAh, IgGI, dilute hybridoma