Plan de mejora N°7 · Área: Planta de Sanitarios

A lm ost all of the data reported on somatic m utation is based on antibody responses to haptens conjugated to proteins. Affinity m aturation of these

responses is dependent on a(3 T cells. How ever, it is not clear at how m any different stages T cell help is required nor w hether somatic m utation can occur in the absence of aP T cells. Although germinal centres can form w ithout aP T cells (Chapter 4) and their formation is necessary for affinity m aturation, it may not be sufficient. It seems likely that a p T cells play an im portant p a rt in selecting a n d /o r expanding those B cells w ith im proved affinity for antigen. They m ay additionally have a direct effect on activation of the m u tatio n m echanism . W hich elem ents w ithin the germ inal centre induce som atic hyperm utation rem ain unknow n but the mechanism itself m ay not be entirely B cell specific. A recent report identified m utations in TCRa genes in germ inal centre T cells (Zheng et al, 1995).

A few studies have attem pted to characterise the antibody response to non­ p ro tein antigens. No evidence of som atic m utation w as found follow ing im m unisation w ith NP-ficoll (Maizels and Bothwell, 1985). In addition, a m uch w ider repertoire of V genes was used than that seen in the réponse to NP coupled to protein. Ficoll is classified as a TI-2 antigen and although there is evidence that one other TI-2 antigen, a(l-6) dextran can induce germ inal centre form ation (Wang, et al, 1994), ficoll does not (Goodlad and M acartney, 1995). The response to dextran also includes a range of V genes. H ow ever, some studies have reported an accumulation of point mutations in one set of V genes (Akolkar, et al, 1987, W ang, et al, 1990). All of these studies have u sed w ild type mice. The T cell p o pulation w hich participates in germ inal centre form ation in the response to dextran has not been characterised.

Bacterial lipopolysaccharide (LPS), a TI-1 antigen, elicits germ inal centre form ation in norm al mice (Goodlad and M acartney, 1995). It has n o t been established w hether this leads to affinity m aturation of the antibody response, although there is evidence that m em ory B cells are generated (Zhang et al.

1988). T cell help increases the secondary IgG titre (Liu, 1989 and C hapter 3). T C R a"/" mice form num erous germ inal centres b u t are unable to respond to protein antigens. Because of this, the hapten phO x coupled to LPS as a carrier was used to investigate the possibility that somatic m utation m ay occur in the absence of aP T cells. As it is not know n if h y perm utation occurs in the antibody response to LPS in w ild type mice, the response to phOx-LPS w as also examined in TCRa"*"/" animals.

Two approaches were used to determine if somatic m utations were present. In the first, following immunisation w ith phOx-LPS, hybridom as w ere m ade from spleen cells and were screened for the presence of phO x specific antibodies. cDNA from positive clones was then analysed to determ ine if the characteristic Vk-Ox1 and V y -O x l genes were used and, if so, if they contained m utations.

A lthough m any oxazolone specific clones w ere obtained, none u sed the characteristic 0x1 V genes and only a few clones expressed closely related sequences. These results provide evidence that B cell selection is radically different in the response to LPS as com pared to th at of p ro tein antigens. How ever, it is not clear from these data w hether somatic m utation occurs in this response. It m ay be that m utations occur b u t the population m ay not be expanded.

In order to address this issue, a second strategy w as em ployed. Following im m u n isa tio n , P N A ^ i B cells w ere sorted by FACS. cDNA from this population was amplified by PGR using prim ers specific for V^-O xl and Vh-

0x1. Thus, it m ight be possible to isolate germ inal centre B cells in w hich somatic m utations m ay have occured b u t w ould not be positively selected. A num ber of V genes w ithin this population w ere m em bers of the sam e V gene family as V ^-O xl or V y -O x l. V^-Oxl w as not found, b u t several sequences w ere identical to the germ line sequence of V y -O x l. A n u m ber of other

germ line genes w ere identified. In some cases it proved difficult to determ ine w hether a particular sequence represented a know n gene w hich contained m utations or w hether it was a previously u n rep o rted germ line gene in the sam e family. How ever, only a few clones contained substitutions consistent w ith the profile of intrinsic m utational 'hotspots'.

5.2 Results