3 2 INTRODUCTION.
3.3. MATERIALS AND METHODS.
3.3.1. Synthetic Ras peptides an d recom binant proteins.
Ras peptides w ere synthesised using conventional solid phase Fmoc chem istry by the peptide laboratory of ICRF. The m u tan t an d norm al Ras p eptides have the amino acid sequences:
MTEYKLW VGAVGVGKSALTIQL, MTEYKLWVGAGGVGKSALTIQL,
respectively. As the peptides w ere highly hydrophobic, they w ere only p artially dissolved in 100% DMSO (Aldrich). For in vivo inununisation, p eptides dissolved in PBS w ere used. For in vitro restim ulation, peptides dissolved in 100% DMSO w ere used.
N orm al and m utant hum an K irsten Ras proteins w ere pro v id ed by th e W elcom e R esearch L aboratories. Both p ro tein s w ere p ro d u ce d as recom binant proteins in E.coli. M utant Ras protein h ad valine at codon 12 instead of glycine of norm al Ras protein.
3.3.2. Im m u n isatio n protocols.
Ras p e p tid e s w ere dissolved in PBS a n d m ixed w ith com plete freim d ’s adjuvant (Difco) in 1:1 (v /v ) ratio. The m ixture w as em ulsified by sonication (Branson Sonifier). Mice w ere im m unised by injection in tw o h in d footpads an d tail base w ith 200 p g Ras p e p tid es em ulsified w ith adjuvant. C ontrol mice w ere im m unised w ith ad ju v an t an d PBS. Two w eeks later, the mice were either sacrificed or boosted by injection in hind fo o tp ad s an d tail base w ith sam e am o u n t of Ras p e p tid es as the first im m u n isatio n . O ne w eek after boosting, p o p liteal an d in g u in al lym ph n odes w ere rem oved from 2-5 im m unised mice an d pooled.
3.3.3. G eneration of helper T cell lines and clones against Ras p eptides.
Single cell su sp en sio n s w ere p re p a re d from ly m p h n o d e s of im m unised mice in com plete T cell grow th m edium an d cu ltu red in 24 well plates at 10^ cells per well in the presence of 2 p g /m l Ras peptide. O ne w eek later, T cells w ere h arv ested an d cloned b y lim iting d ilu tio n as described in general m aterials and m ethods. The T cell lines an d clones w ere restim u lated w eekly in 2ml cultures in 24 w ell p lates containing
5x10^ T cells, 3x10^ irrad iated syngeneic feeder cells, 10% ra t Con-A supernatant and 2 ^ig/nU Ras peptide.
3.3.4. Proliferation assays.
The lym ph node cells from im m unised mice w ere resu sp en d ed in com plete T cell grow th m edium replacing the 10% PCS w ith 1% m ouse serum , and cultured in 96-U-bottomed plates a t 4x10^ cells per w ell in a total volum e of 200 |il in the presence or absence of Ras p ep tid e or protein. For m easuring proliferative responses of h elp er T cell clones, cloned T cells w ere cultured at 10^ per well w ith 3x10^ irrad iated syngeneic feeder cells in the presence or absence of Ras peptide. A fter 3 days incubation at
3 7 0c , 5% C O2, the plates w ere p u lse d w ith 1 p.Ci [ ^ H ] t h y m i d i n e
(Am ersham ) p er well. On day 4, the cells w ere harvested onto glassfibre p a p e r a n d th e th y m id in e in co rp o ra tio n w as m e a su re d by liq u id sc in tilla tio n counting. R esults are g iv en as th e m ean of trip lic a te d eterm in atio n s.
3.4. RESULTS.
3.4.1. Ras m u tatio n w as recognised specifically b y h elp er T cells w h en the Ras p ep tid e w as associated w ith H-2^ b u t not H-2^ m olecules.
C 3 H /H e (H -2^) m ice w ere im m u n ise d su b c u ta n e o u sly w ith synthetic Ras peptides corresponding to am ino a d d s 1-23 of norm al p21 Ras p ro tein or m u ta n t Ras changing glycine to valine a t position 12. The p rim ed T cells from d rain in g ly m p h n o d es w ere restim u lated in vitro w ith different concentrations of Ras p ep tid es an d proliferative responses w ere m easu red . Im m u n isatio n w ith m u ta n t Ras b u t n o t n o rm al Ras p ep tid es stim u lated Th responses. T cells of C 3 H /H e m ice im m unised w ith m u ta n t Ras p e p tid e specifically recognised m u ta n t Ras b u t n o t norm al Ras peptides (Fig. 3.1 A).
As a com parison, the Th responses to Ras peptides w ere analysed in C57BL/10 (H-2t>) mice as well. These mice show ed a different response p attern to the Ras peptides. Im m unisation w ith either m u ta n t o r norm al Ras p ep tid es in d u ced Th responses. R esponses in duced by m u ta n t Ras p eptide w ere consistently stronger than responses induced by norm al Ras p ep tid e. The Th responses in d u ced by m u ta n t Ras p e p tid e w ere n o t exclusively specific for th e im m u n isin g p e p tid e , b u t sh o w ed cross recognition of norm al Ras peptide (Fig. 3.IB). /
The different response p attern s of C 3 H /H e a n d C57BL/10 mice m ight be caused by the differences in eith er MHC genes o r non-M H C genes. To address this question BIO.BR mice, w hich have th e sam e H -2^ haplotype as C 3 H /H e and the sam e non-M H C genes as C57BL/10, w ere used for im m unisation. It was found th at BIO.BR mice show ed the sam e response p attern as C 3 H /H e mice (Fig. 3.1C). Therefore, the specificity of Th responses against Ras peptides w as determ ined by H-2 genes and not by non-H-2 background genes.
& 14000 12000 10000 8000 6000 4000 i m m u n i z a t i o n - R e s t i m u i a t i o n M u t a n t - M u t a n t M u t a n t - N o r m a l N o r m a h N o r m a l N o r m a l - M u t a n t P B S - M u t a n t P B S - N o r m a l 200 400 600 800 1000 1200 Ras peptide (ng/ml) B Ô 4 0 0 0 0 80000 60000 20000 I m m u n i z a t i o n - R e s t i m u i a t i o n M u t a n t - M u t a n t M u t a n t - N o r m a l N o r m a l - N o r m a l N o r m a l - M u t a n t P B S M u t a n t P B S - N o r m a l Ras peptide (ng/ml) 100000 80000 60000 40000 20000 I m m u n i z a t i o n - R e s t i m u i a t i o n M u t a n t - M u t a n t N o r m a l - N o r m a l M u t a n t - N o r m a l P B S M u t a n t N o r m a l M u t a n t P B S - N o r m a l 0.0 0.2 0.4 0.6 0.8 1.0 1.2
Ras peptide (ug/ml)
Fig. 3.1. T helper responses of C3H/He, C57BL/10, BIO.BR mice against mutant and normal Ras p e p tid es. C 3 H /H e (Fig.A) or C57BL/10 (Fig.B) or BIO.BR (Fig.C) m ice were immunised subcutaneously with 200 pg mutant or normal Ras peptides in complete Freund's adjuvant. Control mice were immunised with adjuvant and PBS. After 2 weeks, the draining lymph nodes cells were restimulated in vitro with the indicated concentrations of Ras peptid es. The proliferative responses w ere m easured by the incorporation of [^H] thymidine. The data are the mean of triplicates.
3.4.2. Ras peptide-specific Th cells were CD4+ and M H C class II restricted.
The p henotype of the Ras peptide-specific T cells w as analysed by conventional an tib o d y blocking assays. It w as fo u n d th a t m onoclonal antibodies directed against CD4 or M HC class II m olecules blocked the p e p tid e specific proliferative responses in either H -2^ mice or H-2^ mice (Fig. 3.2A and B). Furtherm ore, Ras peptide-specific T cells only recognised Ras peptide presented by syngeneic, b u t not class E -disparate APC. Fig. 3.3 show ed that a representative Ras-spedfic CD4+ CDS" clone from C57BL/10 mice responded to Ras peptide presented by C57BL/10 APC, b u t not D B A /2 (H-2<^) APC or C 3 H /H e APC. Together these d ata d em onstrated th at Ras peptide-specific Th responses w ere m ediated by CD4+ T cells an d restricted by MHC class U molecules.
3.4.3. M utant Ras peptide-specific Th cells recognised th e co rresponding intact Ras protein.
A lthough m u tan t Ras p ep tid e can be recognised specifically by T cells from C 3 H /H e mice, there is no guarantee th at the corresponding Ras pro tein h arbouring the m utation will be recognised. To be recognised by th e peptide-specific T cells, intact Ras protein needs to be processed in a w ay th at epitopes sim ilar to the synthetic Ras peptides are generated and p resen ted by MHC molecules. It w as im p o rtan t to know w h eth er such T cell epitopes w o u ld be generated d u rin g n atu ral an tig en processing of in ta c t Ras p ro tein . Thus, m u ta n t Ras p e p tid e p rim e d T cells w ere stim u lated by APC p ulsed w ith norm al or m u ta n t Ras proteins. Fig. 3.4 sh o w s th a t T cells from C 3 H /H e m ice im m u n ised w ith m u ta n t Ras p e p tid e recognised m u tan t Ras protein containing the sam e m u tatio n at position 12 as the im m unising peptide, b u t n o t norm al Ras protein.
I
120000 100000 80000 6 0 0 0 0 4 0 0 0 0 20000 .001 .01 Anti-CDS Anti-CD4 • n i l ' I ' I I 10 100 MAb (u g /m l) Bb
6 0 000 50000 4 0 0 0 0 3 0 000 20000 10000 ■ Antl-I-A B Anti-I-A □ No AbFig. 3.2. Phenotype of Ras peptide-spedfic Th cells. BIO.BR (Fig.A) or C57BL/10 (Fig.B) mice were immunised subcutaneously with 200 pg mutant Ras peptide. The draining lymph node cells were restimulated with 1 p g /m l mutant Ras peptide in vitro in the absence or in the presence of the indicated monoclonal antibodies (mAb). The concentrations of anti-CD4 (GK1.5) or anti-CDS (YTS169V) mAb varied as indicated (Fig.A). Anti-I-A^ mAb (TIB120) or anti-I-A^m Ab (H40-481.3) were used at 1 p g /m l. thymidine incorporation was measured on day 4.
I
50000 40000 - 30000 - 20000 - 10000 0 No peptide Normal Ras C57BL/10 C3H/He DBA/2Fig.3.3. The presentation of Ras peptide to a CD4+ clone by different MHC class II m olecules. A CD4+ CDS" T cell clone was established from C57BL/10 mice immunised with normal Ras peptide. Cloned T cells were cultured with medium alone or 1 |Xg/ml normal Ras peptide in the presence of irradiated C57BL/10 or C 3H /H e or D B A /2 spleen cells. p H ] Thymidine incorporation was measured on day 4.
12000 1 10000 • 8000 ■ I <-> 6000 • 4000 2000 15 20 0 5 10 immunization-Restimuiation Mutant-Mutant Mutant-Normal Normal-Normal Normal-Mutant PBS-Mutant PBS-Normal
Ras protein (ug/m l)
Fig.3.4. The recognition of intact p21Ras protein by Ras peptide-specific Th cells. C 3H /H e mice were inrununized subcutaneously with 200 pg mutant or normal Ras peptide or PBS. The draining lymph node cells were restimulated with either normal or mutant intact p21 Ras protein at the indicated concentrations. thymidine incorporation was measured on day 4.