DISCUSIÓN Y RESULTADOS.
4.2.3.6 PRINCIPIO DE AUSENCIA DE COACCIÓN O CONDICIONAMIENTO
and lowest rCD2 expressing cells. Cells of each population - (0 )to ta l/
unsorted, ( A )low rCD2, and (®)high rCD2 expressing - were plated into
96 well dishes at a density of 5x10^ cells/m l, and proliferation assessed
24hrs. after sorting in the presence of serial dilutions of IL2.
gene, in this case EFempty, since an excess w as used in co-transfection. The low rCD2 cells are probably a mix of untransfected and variably transfected cells, and thus m ight be expected to display different characteristics to the high rCD2 expressing cells. W hen proliferation of these populations is exam ined, there is no discernable difference in 3H Tdr incorporation in response to IL2 in the to tal an d hi rCD2 p o p u latio n s. H ow ever, in cells selected for lo rCD2 expression, a dram atic increase in proliferation is apparent. In all cases, the sam e num ber of cells were counted and plated, and unlike the m agnetic bead populations, dying cells were excluded by the FACS during sorting. The only obvious difference in these populations is the level of rCD2 expression, and this appears to inversely correlate w ith proliferative ability.
At this p o in t w e had m ajor reservations about continuing this stu d y since transfected CTLL did not proliferate well. A few experim ents w ere perform ed using m u ta n t Ras genes, b u t w ere un in terp retab le. Thus, this stu d y w as abandoned.
Chapter 6: Discussion
This series of experim ents w ith CTLL cells aim ed to establish a system that w ould allow expression of m u tan t Ras p roteins and exam ination of their effeects u pon IL2 proliferation. Clearly, CTLL can readily be transfected, as illu strated by the expression of the cell surface m arker rCD2. In addition, tran sien t expression of v-H a-ras w as able to prom ote AP-1 transactivation. H ow ever, a major problem was encountered w hen proliferation of transfected CTLL was examined, i.e. transfected CTLL did not proliferate.
This type of co-transfection approach can be used sucessfuly, as illustrated by a recent stu d y w hich identified a gene im p o rtan t in apoptosis(314); the key param eter seems to be the cell system used. CTLL are clearly too sensitive to m anipulation to be of use. A lthough Jurkat cells have been used successfully in this lab to study TCR signal transduction events(229), they unfortunately do not express IL2R. However, other IL2-responsive system s do exist. Thus, one possibility are BAF cells into which an IL2R P chain has been introduced. One group has used these cells to study IL2 regulation of Erk2 in response to IL2(135). These cells have also been u sed in stu d ies to exam ine the contribution of various dom ains of the IL2R p chain to prom ote IL2 responses, including proliferation. Thus, Satoh et alH73) described tw o m u tan t IL2R P chains: one bore a d eletio n of a m em b ran e -p ro x im a l serin e region. In tro d u ctio n of this receptor into BAF cells resu lted in abrogation of IL2- dependent proliferation. Another m utant contained a deletion of a m ore distal proline-rich dom ain, which could still induce IL2-dependent proliferation, but no longer induced IL2-dependent PTK activation, p21ras activation, or Jun/F os
in d u ctio n . A lth o u g h this d ata seem s to in d icate th a t p21ras activity is dispensible for IL2 proliferation, the serine-dom ain deleted receptor also did n o t induce PTK or p21ras activity. Furtherm ore, these cells do respond to serum , and thus m ay not be an ideal system for investigating ras involvem ent in IL2 p ro liferatio n . Thus, the q u estio n of p21ras in v o lv em en t in IL2- dependent proliferation is still unansw ered.
O ther IL2-responsive cells exist: e. g.(240) Kit225, which are IL2-dependent for grow th. In addition, they can be deprived of IL2 for several days w ithout significant loss of viability. Thus, it m ay be possible to exam ine the role of p21ras proteins in IL2 proliferation in Kit225 cells.
Chapter 7: Results - The regulation of p70 S6 kinase in T
lymphocytes
The im m unosupressants Cyclosporin A(CsA), FK506 and rapam ycin elicit their effects at different stages of the im m une response. CsA and FK506 interfere w ith TCR signal transduction leading to IL2 production(102). In contrast, rapam ycin does not affect IL2 p ro d u ctio n , b u t blocks T cell proliferation by interefering w ith events dow nstream of IL2R ligation(48, 101). The study of the effects of CsA and FK506 have been facilitated by the fact that specific transcription factors required for IL2 gene activation are sensitive to the actions of these compounds. Thus, reporter genes u n d er the control of the NFAT binding site from the IL2 enhancer are inhibited by the actions of CsA and FK506(49). This affect has been ascribed, in the case of NFAT, to the inhibition of the serin e/ threonine phosphatase calcineurin, which is required for NFAT, and thus also IL2 gene activity(53)(see fig u re 7.1).
In contrast to events induced by the TCR during T cell activation, relatively little is know n about signals initiated by the IL2R w hich drive cells to mitosis. The IL2R does induce the activation of genes, some of w hich are know n to play a role in mitogenesis. Thus, the oncogene c-myc and pim - 1(306, 315, 316) are induced upon treatm ent of T cell w ith IL2. In addition, various cyclins(171) and cyclin-dependent kinases(306) are also expressed. H ow ever, the kind of reductionist approach taken in the study of the IL2 gene in T cell activation may well be in appropriate here, as m any genes
C sA /F K 506