GENE EXPRESSION IN AN EX VIVO MODEL OF TOLERANCE

Abstract

P959 Aims: An understanding of the molecular basis of immune regulation will allow development of therapies both for exploitation of the immune response and for diseases caused by immune dysregulation. To identify critical regulatory factors in immunity we have used a mouse model wherein infectious regulatory tolerance is inducible by CD4/CD8 blockade in recipients of vascularised heart grafts. Once established, this transplantation tolerance is robust and isolated “tolerant” spleen cells show powerful immune regulatory properties, being able to impose donor-specific allo-tolerance upon fully immune competent naïve recipients. Methods: Using BALB/c-tolerant CBA [H-2k] mice, we analysed ex vivo spleen cell responses to irradiated donor (BALB/c [H-2d]) spleen cells at 48h and at 123h, the latter being 3h after boosting the cultures with donor antigen. For comparison, an identical ex vivo series of BALB/c-rejected CBA spleen cells were analysed. A compound comparison of four gene arrays (tolerance versus rejection, at 48h, and at 123h) used Affymetrix U74 chips and dChip analysis set to +/− 3 for identification of those genes showing enriched expression associated with tolerance, or with rejection. Results: Combined 48h and 123h arrays of the matched tolerant and rejected sample pairs gave 129 genes showing differential expression. To identify those genes that showed a biased expression in either tolerance, or in rejection, the results were ranked in three ways: those genes showing a positive shift from 48h to 123h; those genes with high expression at 123h; and those genes (tolerant) that showed a positive shift, whilst the rejection counterpart showed a negative shift, from 48h to 123h. Of those genes that did not fall into any of the three analyses above, most showed low expression levels at 123h, or did not change markedly between 48h and 123h. In rejection, there was a strong progressive amplification of IFNγ and granzyme B mRNAs. Of the eight genes that were strongly associated with tolerance, 3 are involved in chromosomal structure, in gene expression, and in cell cycling (H2A-X, SAP155, and cyclin B 1 respectively). Four others are involved with regulation of development of the immune response (BLNK, CCR6 and ELKL motif kinase) or of stem cells (axotrophin). Mice lacking axotrophin show agenesis of the corpus callosum due to abnormal axonal migration during development. Conclusions: In summary, we have identified a small number of genes associated with immune tolerance using an ex vivo model, some of which are known to be required for immune regulation. Others may also be involved in regulatory tolerance, for example in chromatin remodelling and epigenetic stabilisation of the tolerant phenotype. Taken together with our analyses of signal transduction in the same ex vivo model, where we find tolerance to be associated with STAT3, LIF and c-kit, we propose that regulatory tolerance involves acquisition of a relatively undifferentiated, non-aggressive phenotype that is epigenetically stabilised and may involve axotrophin.