LMP1 Promotes B-1a Lymphomas That Can Escape Allelic Exclusion
To determine if LMP1 signaling affects B cell differentiation and to immunophenotype the lymphomas that arise from LMP1 expression, surface Ig expression of heavy chains (IgM, IgG, IgD) and light chains (κ, λ) were analyzed by flow cytometry. Similar numbers of naïve (IgM+IgD+IgG−) splenic B cells with a strong bias towards κ light chain were detected from wild-type or LMP1 transgenic mice, indicating that LMP1 signaling does not affect B cell maturation (unpublished data). Flow cytometry analysis of the SCID-passaged wild-type and LMP1 transgenic lymphomas revealed an IgMhighIgDlow phenotype (Figure 2A), indicative of marginal zone, B-1, or memory B cells. B-1 cells are further separated into CD5+ (B-1a) and CD5− (B-1b) subsets. To differentiate between these cell types, lymphoma cells were further analyzed for the B-1a marker CD5. All (5/5) of the tested LMP1 transgenic lymphomas displayed an IgMhighIgDlowCD5+ phenotype (Figure 2A), an expression pattern that distinguishes B-1a cells. Interestingly, a spontaneous wild-type lymphoma also developed in B-1a cells (Figure 2A). These cells are an interesting population that is self replenishing with an increased likelihood to become malignant in aged mice [30]. Analysis of LMP1 transgenic mice before the development of lymphoma showed similar numbers of splenic B-1a (CD19+CD5+) and B-1b or B-2 populations (CD19+CD5−), indicating that LMP1 does not affect B cell differentiation (Figure 2B).
Figure 2  LMP1 Promotes B-1a Lymphomas That Can Escape Allelic Exclusion
(A) Flow cytometry analysis of splenocytes from a WT or LMP1 transgenic lymphoma for the pan–B cell (CD19), B-1a cell (CD5), and Ig heavy chain (IgM and IgD) and light chain (κ and λ) markers. Shown are the results from WT lymphoma 1 and LMP1 transgenic lymphoma 4. This analysis was repeated on four other LMP1 transgenic lymphomas (1, 2, 3, and 6) showing a similar B-1a phenotype, of which lymphomas 2 and 4 were also doubly positive for κ and λ light chains.
(B) Flow cytometry analysis of WT or LMP1 transgenic splenocytes for B-1a (CD19+CD5+) and B-1b or B2 subsets (CD19+CD5−). Percentages of B-1a and B-1b or B2 subsets are shown in each quadrant. This analysis was repeated on three other WT and two other LMP1 transgenic mice with similar results.
(C) Immunoblot analysis for κ and λ light chains of B cells (CD19+) purified from WT and LMP1 transgenic mice. Actin was used as a loading control.   Due to allelic exclusion, mature B cells that have been exposed to antigen will typically express only one heavy chain isotype (IgG, IgE, or IgA) and either a κ or λ light chain. Interestingly, 2/5 LMP1 transgenic lymphomas analyzed (lymphomas 2 and 4) were doubly positive for low levels of both κ and λ light chains (Figure 2A). Previous characterization of the LMP1 lymphomas had revealed that the lymphomas were clonal as determined by Ig heavy chain rearrangement [26], and analysis of κ chain rearrangement (Figure S1) of the samples analyzed in this study confirmed clonality. To further assess light chain expression, the passaged samples were tested by immunoblotting for κ and λ light chains (Figure 2C). Interestingly, very low levels of expression of both light chains were detected by flow cytometry. The low levels of expression may reflect a limitation of the total number of light chains that can be expressed on the surface of a B cell. In agreement with the flow cytometry analysis, LMP1 transgenic lymphomas 2 and 4 were also positive for κ and λ light chains by immunoblot analysis (Figure 2C), confirming that these lymphomas express both light chains. A previous study of mice that developed leukemia due to an expansion of self-reactive B-1a cells determined that the B-1a leukemias were also doubly positive for κ and λ light chains [31]. These findings indicate that expression of LMP1 in B-1a cells promotes the development of malignancy and can result in the aberrant escape from allelic exclusion.