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Constitutive CD40 signaling in B cells via LMP1/CD40 expression leads to prolonged survival and spontaneous cell division ex vivo (Homig-Holzel et al., 2008). In Igβ-deficient mice, constitutive CD40 signaling restores the peripheral B cell compartment to comparable B cell numbers as found in wild type mice (Hojer et al., in preparation). Consequently, we aimed to characterize the survival properties of LMP1/CD40//IgβΔGFP/Δ _{B cells in vitro and in vivo.}

3.3.4.1 LMP1/CD40//IgβΔGFP/Δ _{B cells show a prolonged survival in vitro}

To test whether constitutive CD40 signaling prolongs the survival of Igβ-deficient B cells in vitro, splenic B cells from all four genotypes were isolated and cultured for five days without additional stimulation. At day 1, 2, 3, and 5 of the culture, cells were stained with the vital dye TOPRO-3 and were subjected to flow cytometric analysis. From beginning of the culture, the survival of LMP1/CD40//IgβΔGFP/Δ _{B cells declined much slower than of the rapidly dropping Igβ}ΔGFP/Δ _B

cells, and thus similarly to IgβΔGFP/wt _{control B cells (Fig. 33A). While monitoring the percentage} of GFP+ _{cells over five days in culture, the percentages of GFP}+ _{cells in the Igβ}ΔGFP/Δ _culture

dropped rapidly, indicating a negative selection of Igβ-deficient B cells (Fig. 33B). LMP1/CD40 expression was observed to counteract that negative selection, as the percentages of GFP+ _{cells in} the LMP1/CD40//IgβΔGFP/Δ _{culture remained rather constant, similarly as in the culture of}

IgβΔGFP/wt _{control B cells. All together these results show that LMP1/CD40 expression prolongs} the survival of Igβ-deficient B cells in vitro, and indicate that constitutive CD40 signaling is able to rescue Igβ-deficient B cells from cell death to a certain extent.

3.3.4.2 Constitutive CD40 signaling enlarges the life-span of Igβ-deficient B cells in vivo To confirm a prolonged life-span of LMP1/CD40//IgβΔGFP/Δ _{B cells also in vivo, we performed}

bromodeoxyuridine (BrdU) pulse chase experiments. BrdU is a thymidine analog that is incorporated into replicating cells during the S-phase of the cell cycle by substituting for thymidine in all newly synthesized DNA strands. By applying BrdU to cell cultures or experimental animals (pulse), and subsequently detecting BrdU in the DNA with a specific antibody, cells that have entered the S-phase can be identified. After the pulse, the life-span of labeled cells can be monitored over a period of time without further labeling (chase). Since peripheral B cells are resting and barely proliferate, BrdU+ _{B cells in the periphery are derived} from transitional B cells emerging from the bone marrow, where they underwent several cycles of cell division during early B cell development (Forster and Rajewsky, 1990). For the in vivo assay, mice were fed with BrdU in the drinking water for 14 days (pulse) and were further observed

over a period of 56 days until day 70 (chase). Mice were analyzed on day 14 at the end of the pulse, and on day 42 and 70 during the chase. The percentages of BrdU+ _{splenic B cells were} determined by flow cytometry. The respective values are displayed with the graphs in figure 33C.

Figure 33 Constitutive CD40 signaling prolongs the life-span of Igβ-deficient B cells. (A, B) Splenic B cells of the indicated genotypes were enriched by depletion of CD43+ _{cells. Cells were cultured}

up to five days without stimulation and were analyzed for viability with TOPRO-3 incubation on day 0, 1, 2, 3, and 5 by flow cytometry. Percentages of viable (TOPRO-3-_{) cells were determined and mutant cells}

were identified by re-gating on GFP+_{. The graphs show (A) mean percentages of viable GFP-expressing}

cells with SD of three independent experiments in relation to the input at day 0, which were set as 100 % or (B) mean percentages of viable GFP+ _{cells of the respective genotypes over the time of the culture. (C)}

Mice were fed with 0.8 mg/ml BrdU in their drinking water for 14 days during the pulse period and BrdU+ _{B cells were tracked until day 70 during the chase period. Splenic samples were taken at day 14, 42,}

and 70. Percentages of BrdU+_{, B220}+ _{B cells were determined by flow cytometry. The graphs show the}

mean results (symbols) with SD of three different BrdU pulse chase experiments. The four different genotypes are indicated besides.

At day 14 of the pulse period, IgβΔGFP/Δ _{mice showed high percentages of BrdU}+ _{B cells (57 %).} In contrast, percentages of BrdU+ _{B cells in LMP1/CD40//Igβ}ΔGFP/Δ _{mice were much lower (37}

%) and similar to IgβΔGFP/wt _{control mice (32 %). During the chase period, Igβ-deficient B cells}

showed not only the lowest percentage of BrdU+ _{B cells after the pulse (23 %), but also the} slowest decline of labeled cells during the chase (12 % at day 70), evidencing a prolonged survival of peripheral LMP1/CD40-expressing B cells in vivo.

The increased percentage of BrdU+ _{B cells in Igβ-deficient mice at day 14 as well as the fast} decline during the chase period is most likely caused by a high influx of immature B cells from the bone marrow to the periphery, due to the diminished life-span of peripheral B cells deficient for Igβ (N. Uyttersprot, personal communication). By constitutive CD40 signaling, the survival of Igβ-deficient B cells was approximated to the survival of wild type B cells in vivo. Therefore, these results strengthen again the observations we made in vitro, namely that constitutive CD40 signaling is able to rescue Igβ-deficient B cells from cell death to a certain degree. However, this data also show that the ablation of Igβ results in the drop of survival of LMP1/CD40-expressing B cells to wild type level, evidencing that though constitutive CD40 signaling, pre-malignant B cells are still susceptible to the impairment of tonic BCR signals.