increased expression of IkBa and, to a lesser extent, IkBb. TCR- induced nuclear translocation of c-Rel was seen for WT T cell blasts, with less marked further nuclear accumulation of c-Rel in IkBe2/2 T cells, suggesting that nuclear c-Rel was already closer to its maximum in the latter. Nuclear c-Rel was also present at higher levels in na¨ıve splenic IkBe2/2 B cells, relative to WT, and its early BCR-induced translocation was much more marked in IkBe2/2 B cells than WT (Figure 7). Again, accumulation of nuclear c-Rel occurred in IkBe2/2 B cells in the presence of higher IkBa expression relative to WT cells. c-Fos and NFAT2 were not detected in nuclei of resting or stimulated B cells or T cell blasts. When we studied functional outcomes of enhanced c-Rel nuclear translocation, we were surprised that IkBe2/2 T cells (na¨ıve or blast cells) did not make more IL-2 than WT cells when stimulated via TCR ex vivo, and showed only a modest increase in potential for IL-2 synthesis when stimulated with PMA and ionomycin (Figure 5E). However, these findings were consistent with a study which showed that inhibition of IL-2 expression in c- Rel2/2 relative to that in wild-type T cells was not detectable before 4 hours of TCR stimulation, that is, until the time when de- novo synthesised c-Rel would accumulate in the nucleus [26]. The authors also showed that basal and inducible transcription of IL-2 in T cells over-expressing c-Rel was between 2 and 3-fold greater than in control cells but that this difference was lost at four hours’ stimulation when the gene was very strongly upregulated. By way of comparison, basal and TCR-induced CD40 expression in c- Rel2/2 T cells were inhibited at all time-points, relative to wild- type, and c-Rel over-expression led to basal and inducible transcription of CD40 that was between 15 and 40-fold greater than that of controls throughout. We would argue that basal nuclear expression of endogenous c-Rel in T lymphocytes is actually unlikely to lead to enhanced IL-2 expression as a result of the kinetics of regulation of this gene: TCR-induced activation of IL-2 requires de novo synthesis of c-Rel, AP-1 and NFAT proteins and their coordinate binding at pIL-2. Peak expression of these factors coincides with maximum degradation of IkBe translocation in wild type cells. Thus at the time (from about 2 hours post- stimulation) when TCR-induced transcription of IL-2 would be expected to begin in earnest, there would be effectively no difference in IkBe between IkBe2/2 and WT cells. However, under circumstances in which its expression is increased above basal levels – in response to TNF, for instance, should this occur in vivo – a role for IkBe in negative regulation of IL-2 gene induction might become apparent. Further observations led us to explore c-Rel responsive genes besides IL-2 in IkBe2/2 lymphocytes. Firstly, na¨ıve splenocytes and LNCs of IkBe2/2 mice showed enhanced sensitivity to low concentrations of anti-CD3, by thymidine incorporation assay, and we consistently saw increased basal thymidine incorporation in IkBe2/2 splenocyte, LNC and T cell blast cultures (Figures 5F and 6A). 3H-thymidine uptake is usually taken as a correlate of IL- 2-dependent proliferation in T cell cultures, but it depends on the number of live cells in culture at the time of its addition – typically 48 hours into culture – and so it also depends on cell survival ex vivo [54]. Basal 3H-thymidine incorporation showed a reciprocal relationship with IkBe gene dose which was not due to IL-2. This might be explained either by increased basal proliferation or enhanced survival ex vivo of IkBe2/2 cells. That is, if the viability of IkBe2/2 cells exceeded that of WT cells, there would be more of the former able to take up thymidine 48 hours into culture. Secondly, LNs from IkBe2/2 mice, which often looked slightly larger than those from WT mice, contained significantly increased LNC numbers, with a trend to a higher proportion of B cells Regulation of c-Rel by IkBe in Murine Lymphocytes (Figure 6, B–D). Thirdly, B cells from IkBe2/2 but not WT mice survived for up to 8 days in conditions favouring T cell expansion and survival. From the data, we felt we were looking at a IkBe2/2 lymphoid cell phenotype characterised chiefly by enhanced survival mechanisms most clearly manifest in B cells. This interpretation accords with previous adoptive transfer experiments which showed a significant increase in splenic B cells of IkBe2/2 chimaeras as compared to wild-type or IkBa mutant ones [55]. Having established that na¨ıve IkBe2/2 splenic B cells showed increased basal nuclear expression and BCR-induced nuclear translocation of c-Rel compared to WT, we found that survival of these cells was significantly enhanced for up to 48 hours ex vivo in the presence or absence of BCR ligation (Figure 8A and 8D). We demonstrated significantly greater basal expression of CD40 in na¨ıve IkBe2/2 splenic B cells relative to WT which persisted for 24 or 48 hours in cells cultured in the presence or absence of anti-IgM and IL-4 respectively (Figure 8B and 8E). Finally, we observed a statistically significant persistence of BAFF-R expression in unstimulated IkBe2/2 B cells relative to WT for up to 48 hours culture ex vivo (Figure 8C), and enhanced BCR-induced upregulation of BAFF-R in IkBe2/2 compared to WT B cells, which was most marked at 24 hours, persisted for a further 48 hours (Figure 8D) and was also statistically very significant. The higher basal and inducible expression levels of these c-Rel-dependent pro-survival molecules is a likely mechanism for the enhanced survival of IkBe2/2 B cells ex vivo. CD40- and BAFF-R-dependent survival of B cells ex vivo could in turn account for the increased basal thymidine uptake ex vivo of IkBe2/2 splenocytes, LNCs and unstimulated T cell cultures, relative to WT, both through B cell survival and the ability of those B cells to deliver co-stimulation to T cells. Enhanced B cell co-stimulation might also explain the greater sensitivity of these cultures to low concentrations of anti-CD3. Doerre and colleagues proposed an important role for IkBe in differential regulation of genes responsive to p65 homodimers in stimulated B cells [9]. A recent study describes IkBe as having a specific role in regulating random variation of NF-kB activity from cell to cell such as to permit cellular heterogeneity and differentiation [56]. We propose that IkBe is a non-redundant regulator of c-Rel in both T and B cells and that IkBe deficiency or down-regulation leads to enhanced expression of c-Rel- dependent genes that permit survival of, at least, B lymphocytes. Given such effects on lymphoid cell survival, and the fact that c- Rel is a proto-oncogene, we wondered whether IkBe2/2 mice were predisposed to lymphatic tumours. However, no overt tumour was detected in lymphoid tissues of male or female mice aged up to 14 months. With hindsight, this is not surprising, since there are no reports of transformation mediated solely by over- expression of c-Rel in mice [23]. However, studies have implicated gene variants in the IkBe/c-Rel pathway in tumorigenesis: mutations in CD40, c-Rel and IkBe are associated with Hodgkin’s Disease, a malignant transformation of germinal centre B cells. In terms of other disease risks, rheumatoid arthritis-associated single nucleotide polymorphisms have been described in PKCh, which lies upstream of TCR-induced IkB degradation, in c-Rel, and in c- Rel target genes CD40 and TNFAIP3 [57,58,59]; and IkBe itself is upregulated at the mRNA level by TNF, whose over-expression is a feature of rheumatoid arthritis and other inflammatory diseases [52]. Our data suggest strongly that dysregulated expression of IkBe will alter c-Rel-dependent survival and function of lymphocytes, its under-expression or impaired function leading to enhanced lymphocyte survival, and its over-expression or resistance to degradation leading to impaired lymphocyte survival or function. PLoS ONE | www.plosone.org 15 September 2011 | Volume 6 | Issue 9 | e24504