its increased expression at d1 and d2 ex vivo, were also significantly higher in IkBe2/2 B cells (Figure 8B). Basal expression of BAFF- R was similar for WT and IkBe2/2 B cells. However, BAFF-R expression decreased with time in culture faster for WT than for IkBe2/2 B cells, so that there were significant differences in expression at d1 and d2 ex vivo (Figure 8C). In the case of B cells receiving a pro-survival signal via anti-IgM IL-4, there was a similar loss of viability in both WT and IkBe2/2 cells after one day in culture d1 of culture (Figure 8D). Viability appeared to recover between d1 and d2 and, at d2, was significantly greater for IkBe2/2 than for WT B cells. Basal expression of CD40 and its expression at d1 of culture in anti-IgM and IL-4 was significantly higher in IkBe2/2 B cells than WT B cells (Figure 8E). CD40 expression was increased at d2 and d3 to a similar level in both populations. Basal expression of BAFF-R was similar for WT and IkBe2/2 B cells and BAFF-R was strongly induced in both populations after 24 hours in culture, with significantly higher expression on IkBe2/2 compared to WT cells B (Figure 8F). The difference persisted and although less marked, it remained statistically significant at d2 and d3 in culture (Figure 8C, D). TNF had no effect on IkBe expression or survival ex vivo of WT B cells, to 72 hours (not shown). These data suggest a non-redundant role for IkBe in the regulation of c-Rel-dependent B lymphocyte survival mechanisms. Discussion While studying mechanisms whereby pre-culture in TNF of mouse T cell hybridoma 11A2 blocked subsequent IL-2 induction, we observed that inhibition of IL-2 was associated with suppressed nuclear translocation of c-Rel as well as increased expression and attenuated inducible degradation of IkBe, but not IkBa or -b. Nuclear expression of c-Fos, Fos B and NFAT2, and transcrip- tional activation of not only NF-kB, but AP-1 and NFAT/AP-1 reporter promoters, were also attenuated in TNF-treated cells. Deficits in AP-1 and NFAT expression and function are likely to contribute to reduced inducibility of IL-2 in TNF-treated cells both directly and indirectly, since induction of both IL-2 and c-Rel require AP-1 and NFAT2 [26,30,50,51]. Transient reconstitution of c-Rel in TNF-treated cells restored transcriptional activation of a c-Rel/AP-1-dependent reporter to untreated control cell levels but not to those of control cells also over-expressing c-Rel. Therefore c-Rel alone did not fully reconstitute the inhibitory effect of TNF at this promoter. It may be that effects of TNF on AP-1 and NFAT expression and transcriptional activity accounted for this deficit. TNF very likely had pleiotropic effects on 11A2 cells. The consistent observations that c-Rel translocation was strongly inhibited and that expression of IkBe, but not IkBa or -b, was strongly upregulated in TNF-treated cells led us to investigate the functional relationship of IkBe and c-Rel. Extending our studies to primary lymphoid cells, we found that IkBe appears to have a non-redundant role in regulation of c-Rel subcellular localisation in B lymphocytes and T cell blasts, and a negative regulatory role in B cell survival. We propose that IkBe regulates the latter, at least in part, by inhibiting c-Rel-dependent expression of anti-apoptotic molecules BAFF-R and CD40 in B cells. Reciprocity of IkBe levels and nuclear translocation of c-Rel held for both TCR- and TNF-R-activated pathways in 11A2 cells at all time-points of stimulation. Basal IkBe expression was increased and remained higher at all time-points of stimulation with PMA and ionomycin, or acute-on-chronic TNF, in TNF pre- treated cells relative to controls. When we examined IkB/NF-kB Regulation of c-Rel by IkBe in Murine Lymphocytes complexes by co-immunoprecipitation we observed that, whereas c-Rel associated constitutively almost exclusively with IkBb, newly synthesised c-Rel induced by PMA and ionomycin also associated with IkBa and IkBe. This stimulation-dependent shift in IkB/c- Rel complexes appears analogous to that (from IkBb to IkBa) documented for cytokine-treated T cells [49]. The amount of newly-synthesised c-Rel associating with IkBa reflected cytosolic levels of c-Rel (Figure 4A, panel 1 and Figure S4 A(ii)). By contrast, its association with IkBe reflected expression levels of IkBe in control and TNF-treated cells (Figure 4A, panel 7 and Figure S4 A(ii)). In addition, more IkBe co-immunoprecipitated with p65/c- Rel in TNF-treated than in control cell lysates of resting and PMA and ionomycin-stimulated cells (Figure 4B, panel 5 and Figure S4B). These were the only TNF-dependent differences in IkB/NF- kB complexes, and seem likely to be a consequence simply of the increased, stable expression of IkBe in TNF-treated cells. Although its association with IkBe is most marked for newly- synthesised c-Rel, IkBe over-expression also appeared to inhibit nuclear translocation of constitutively-expressed c-Rel. Most c-Rel was associated with IkBb in resting cells (Figure 4B, panel 4). Yet following TNF-R ligation, despite equivalent degradation of IkBb (and IkBa) in control and TNF pre-treated cells, very little pre- existing c-Rel reached the nucleus in the latter (Figure 2B panel 2; cf. Figure 2D panels 1–3). One explanation for this may be that IkBe, being so strongly expressed in TNF pre-treated cells, binds and inhibits c-Rel released in the wake of IkBb degradation. We found IkBe expression to be upregulated in TNF-treated cells, in all our experiments, at about the same time as another study identified IkBe as a gene directly inducible by TNF [52]). We also noted that IkBe appeared resistant to inducible degradation in TNF-treated cells in many but not all experiments. That there may be mechanisms of its regulation unique to IkBe is suggested not only by its structural difference from the other IkBs [8] but by IkBe-specific interactions with epsilon-regulatory proteins such as PP6 regulatory unit 1(PP6R1) and ankyrin repeat subunit (ARS)-A of PP6 which have recently been described. Knock-down of either the PP6R1scaffolding unit or of ARS-A (thought to mediate phosphatase substrate specificity) led to enhanced TNF-induced degradation of IkBe [18,19,20]. IkBe is expressed in T and B lymphocytes [9,47] and it has been shown that IkBe, like IkBb, prefers to bind c-Rel and p65 homo- or heterodimers. This is in contrast to IkBa which preferentially binds p65/p50 [8,10]. IkBe is expressed at high levels in splenic B cells, in which it has been suggested to have a role in regulating a subset of genes activated by p65 homodimers, since p65 homodimer-driven reporter gene expression increased as IkBe was down-regulated during B cell maturation [9]. The same authors had previously described constitutive nuclear c-Rel expression in IgG+ but not IgM+ murine B cell lines, with 10- fold lower expression of IkBe in the former.[53]. We found that culture of WT murine T cell blasts (8 days) or splenic B cells (3 days) in TNF did not upregulate IkBe above its already-high expression levels in the absence of TNF. It has further been reported that c-Rel is constitutively nuclear in mature but not na¨ıve B cells [23]. We extended our investigation of specific regulation of c-Rel by IkBe, and its functional outcomes, to studies in lymphoid cells of WT and IkBe2/2 mice. We argued that if IkBe had a unique role in regulation of c-Rel nuclear translocation, as our in vitro experiments suggested, c-Rel would accumulate in the nucleus of lymphocytes lacking IkBe, in the presence of normal levels of expression of IkBa and -b. We looked first in T cell blasts and found that c-Rel was present in the nucleus of resting IkBe2/2 and IkBe+/2 T cells in the absence of TCR signal (Figure 5A, B,C) and that this occurred despite PLoS ONE | www.plosone.org 14 September 2011 | Volume 6 | Issue 9 | e24504