Wild-Type and LMP1 Transgenic Lymphoma Cells Do Not Require IL4 and Stat6 Signaling
To investigate whether IL4 independence was due to endogenous IL4 expression, IL4 transcription was assessed by an Rnase protection assay (RPA). IL4 transcription was detectable with control RNA and faintly in the mouse lymphoma cell line K46μ (Figure 4A). However, IL4 transcription was not detectable in CD19+ MACS-purified B cells from wild-type lymphocytes (unpublished data), LMP1 transgenic lymphocytes, or lymphoma cells, although the GAPDH and L32 controls were effectively protected (Figure 4A). Activated Stat6 (pStat6), a target of the IL4 receptor pathway, was detected in the wild-type and LMP1 transgenic lymphocytes (Figure 4B). In contrast, pStat6 was barely detected in either the wild-type or LMP1 transgenic lymphoma cells. However, the pathway was not disabled, as treatment of the lymphoma cells with IL4 induced Stat6 phosphorylation (Figure 4C).
Figure 4  Wild-Type and LMP1 Transgenic Lymphoma Cells Survive Independently of IL4/Stat6 Signaling in Culture
(A) Rnase protection assay for IL4 mRNA from purified B cells (CD19+) from WT and LMP1 transgenic splenocytes. The L32 and GAPDH housekeeping genes were used as a loading control. Arrow indicates the position of the protected probe.
(B and C) Immunoblot analysis of WT and LMP1 transgenic mice for activated pStat6 in (B) purified B cells (CD19+) at the time of harvest or in (C) whole splenocytes cultured with or without IL4. (B) Actin was used as a loading control, and the white line indicates that intervening lanes have been spliced out.
(D and E) MTS assay of (D) WT lymphocytes and (E) LMP1 transgenic lymphoma cells cultured with IL4, a neutralizing antibody to IL4, or a rat IgG isotype control at the indicated concentrations. Shown are the results from LMP1 transgenic lymphoma 3. The results are the mean ± SEM of triplicate samples from a single representative experiment that was repeated twice with similar results.   Although wild-type lymphocytes cannot be maintained in culture with IL4 supplementation alone (Figure 3A), slight enhancement in MTS activity could be detected if the cells were analyzed at an earlier time point, at 1 d (Figure 4D) versus 3 d (Figure 3A) post-harvest. The enhancement of MTS activity induced by IL4 in wild-type lymphocytes could be neutralized by the addition of IL4 antibody (Figure 4D). However, neutralizing antibodies to IL4 did not affect the MTS activity of LMP1 transgenic lymphoma cells (Figure 4E). In summary, the wild-type and LMP1 transgenic lymphoma cells grew independently of IL4 treatment and did not require Stat6 signaling.