Mercado de la industria de confección de prendas de vestir

Several groups have rep o r t e d that fibroblasts transformed with the v - Kl-rae

oncogene can be recognised and killed by natural killer (VK) cells and

these cells may play a n early role in host antitumour surveillance (Johnson

et al., 1985; Triable, 1986). These results are consistent w ith the

'missing s e l f hypothesis, that IK cells can recognise and eliminate cells

that fall to express s elf NHC class I molecules, suggested by Khrre et al.

(1986), as it is k n o w n that ras expression dawn regulates constitutive

expression and Induction of NHC antigens (discussed in section 1.6). The In

vivo studies reviewed by LJunggren and K&rre (1990) that support this theory also can be reconciled with the earlier observations of NSV/NLV

tumour regression. It has been found that only small not large tumour

lnocula can be rejected if the tumour cells are H-2 deficient as in the

case of KSV Infected or v - r a s transformed cells and the depletion of VK

cells with monoclonal a n t i bodies abrogates this response (Versteegh et si. ,

1989). Thus it is p roposed in the MSV/XLV tumour regression situation

described earlier, the large inoculum of KSV transformed cells in

preimmunized mice overwhelmed the limited nonadaptive VK cell response

resulting in tumour growth, whereas the large inoculum of MSV/KLV

transformed cells in preimmunized mice would be eliminated by the Tc

adaptive response specific for the MLV antigens as previously discussed. It

may also be possible that in the presence of the dominant immune response

to MLV antigens, the r e s ponses to res gene products are suppressed and thus

mice immunized with KSV/KLV Infected cells are unable to reject a tumour of

KSV transformed cells.

More recently Cheever et si. (1990) and Peace et si. <1991) have shown that

a T cell immune response to the mutated res protein can be e l icited by

immunization with synthetic peptides constructed to be identical to the

mutated portion of the rss protein. Peptides consisting of 12 or 13 amino

acid residues which corresponded to the amino a cid sequence from residue 5-

16 or 5-17 of p21 were constructed to contain the normal glycine at the

position corresponding t o residue 12 (termed Gly-12) or alternatively the

abberant substitution o f arginine (termed Arg-12> or serine (Ser-12).

Lymphocytes from C57BL/6 mice immunised with a single dose of the Arg-12

peptide were tested for a proliferative response to the above peptides in

vitro. A proliferative response against the Arg-12 peptide but not the Gly- 12 and Ser-12 peptides w a s found.

In conclusion although Tc responses against the MLV proteins are the main

cause of tumour regression in the KSV/MLV system, a n immune response can be

in the regression of tuaours has jet to be detersi ned and is under

investigation by fellow mem b e r s of the CSC research group at Warwick

University. One of the alas o f this study is to further investigate the

laaune response elicited by t h e XLV proteins.

1.8 Betroviral rectors and selection systei

As discussed in detail before the s a i n a i a of this project was to generate

tumour cell lines expressing well-defined antigens which could be used to

study the importance of IFI modulation of T cell mediated Immune responses

to these antigens. The antigens u s e d in this study were the gag and env

polypeptides expressed by Ki and X o MLV and the relative importance of

these two viral antigens in the Immune response to the MSV/KLV tumour is

also examined. The approach taken b y other groups to examine this response

has been to construct cell lines that express the Individual proteins in

the absence of other potential XLV antigens. As mentioned in section 1.7,

where the results from these gro u p s are examined, one problem with the

approach taken by these groups is that they use neomycin resistance to

select for positively transfected cells. It is proposed in this study that

the gene product of the neomycin resistance gene can itself elicit an

immune r espond ind evidence to support this theory is presented in section

4.7. 0 r -su objectives of this project is to establish a procedure for

selecting cells which have been successfully transfected with a gene of

interest without introducing o t h e r neoantigens, such as antibiotic

resistance selectable markers. Initially attempts were made to directly

select the cells expressing the protein of interest using fluorescent

labelled antibody and the fluorescence activated cell sorter (FACS). As

discussed in section 3.4 this was found not to be possible, therefore an

alternative method for selection w a s proposed. The procedure appears to be

successful and it is hoped may be u s e d in other studies.

The BBC class I D* selection system

The selection system established in this study is described in the

following text. The procedure was made possible by the relatively recent

availability of the cloned MHC class I gene D*- (Sher et al. , 1985) and the

monoclonal antibody HB19 (supplied by D. J. Naudsley) which can be used to

detect the H-2D* antigen. The cloned H-2D** gene Is transfected Into the H-

2DtJ negative eabryo fibroblast cell line C3H10TM (H-2k > with the DMA of

interest, in this case the gene encoding the MLV protein(s). Cells

expressing the H-2D** are sorted on the FACS after treataent with y-IFI. To

achieve a uniformly positive population of cells It is necessary to repeat

the sorting procedure, the actual nuab e r of sorts necessary being dependent

on the transfection and sorting efficiency. By ligating the DBA of interest

to the H-2D** gene it is passible to ensure that all cells that take up the

MHC gene also recieve the unselectable gene. The resulting cell line should

be positive for both the MHC c l a s s I antigen D* and the protein of

interest. The parental nouse strain <C3H/He> of the transfected cell line

C3H10TH would recognise the MHC D* antigen as foreign, therefore the

experiments to deteralne the laaunological importance of the antigen of

interest are performed in hybrid alee, which do not recognise the D* class

1 antigen as foreign. These nice are the FI progeny of a cross between

C3H/H* <H-2*> and C57BL6 (H-2»> a lee and thus they recognise both the

endogenous and acquired NHC antigens of the transfected cell line as self.

The proposal that this system can be used to examine the lmaune response to

proteins without actually interfering with the response they elicit is

supported by the data in section 3.4.

The V- selection system and the importance to tuaouriganlclty of point