Regulation of c-Rel by IkBe in Murine Lymphocytes expressed relative to values for resting IkBe+/+ T cells as indicated. (E) T cells blasts were stimulated, or not, for 6 hours with PMA and ionomycin in the presence of brefeldin A, labelled with anti-CD4-PECy7, fixed, permeabilised, labelled with anti-IL-2-FITC and analysed by flow cytometry. Shown are% IL-2+CD4+ in the live cell gate (mean +/2 SD, n = 3 experiments; one mouse per genotype per experiment). (F) LNC were cultured in triplicate with soluble anti-CD3 at the concentrations indicated. At 48 hours, 1 mCi 3H-thymidine was added to each well and culture continued for 18 hours prior to analysis of 3H-thymidine uptake. Mean +/2 SEM, n = 5 experiments (one mouse per genotype per experiment); p values refer to IkBe2/2 vs IkBe+/+, other P values were not significant. doi:10.1371/journal.pone.0024504.g005 for IkBe2/2 LNC (Figure 6A and 5F). However, this increase was not due to c-Rel-dependent basal expression of IL-2 in unstimulated cells, since it was not inhibited with blocking antibodies to IL-2R (data not shown). We also noted that LN were often larger in IkBe2/2 compared to WT mice (Figure 6B). Furthermore, total numbers of LN cells, but not splenocytes, were significantly higher for IkBe2/2 mice compared to heterozygote or WT (Figure 6C). Phenotypic analysis revealed a trend towards increased numbers of B220high (mature B) cells in the LN of IkBe2/2 mice (Figure 6D). We wondered whether increased cellularity of lymph nodes, increased B cell numbers and enhanced basal and TCR-stimulated thymidine uptake by LNC might be explained by c-Rel dependent effects on lymphocyte survival in IkBe2/2 mice. We therefore determined expression of the pro- survival co-stimulatory molecule CD40 in WT and IkBe2/2 T cell blasts by flow cytometry. Very few CD40+ T cells were detected. However, we found that a population of CD4 2 CD8 2 CD40+ cells, which persisted in day 8 cultures of IkBe2/2, but not WT, T cell blasts, were B220+ B cells (not shown). That is, IkBe-/2 B cells were surviving longer than WT B Figure 6. Increased basal thymidine uptake in splenocytes and LNC, increased LN cellularity and B cell numbers in IkBe2/2 mice. (A) Basal thymidine uptake by splenocytes and LNCs in IkBe+/+, +/2 and 2/2 mice (median, n = 10 experiments). (B) Enlarged axillary lymph node from an IkBe2/2 mouse compared to those from IkBe+/+ and IkBe+/2. (C) Total lymph node cell numbers, one mouse per IkBe genotype per experiment (mean +/2 SEM, n = 10). (D) LNC were labelled with anti-CD4-PECy7, anti-CD8-PE, anti-B220-FITC and anti-CD11b-APC before flow cytometric analysis. The percentage of each cell subset was multiplied by the total LNC number to give numbers for each cell subset (mean, n = 6 experiments). doi:10.1371/journal.pone.0024504.g006 PLoS ONE | www.plosone.org 11 September 2011 | Volume 6 | Issue 9 | e24504