Targeted Disruption of the S1P₂ Sphingosine 1-Phosphate Receptor Gene Leads to Diffuse Large B-Cell Lymphoma Formation

DLBCL immunophenotype in S1P₂^−/− mice. A, surface immunophenotype by FCM of a representative DLBCL from a S1P₂^−/− mouse and a histologically normal spleen from a littermate control S1P₂^+/+ mouse. Note the low/absent surface Ig, the loss of CD23, and the absence of CD138 in the DLBCL sample. B, AID expression in representative DLBCL from S1P₂^−/− mice, compared with purified normal GC B cells from two S1P₂^−/− and one S1P₂^+/+ mice. Mouse lung tissue was used as a negative control for the AID RT-PCR.

Clonal rearrangements of the IgH locus and evidence of SHM in DLBCL from S1P₂^−/− mice. A, Southern blot analysis of EcoRI-digested DNA from representative S1P₂^−/− DLBCLs, and control tail DNA, hybridized with a murine JH region probe. The germline immunoglobulin heavy chain EcoRI fragment is ∼6.5 Kb (arrow). *, clonal rearrangements of the IgH gene in the tumors analyzed. The predominant germline band observed in some tumors is most likely due to the presence of infiltrating normal cells (see text). B, mutation analysis of clonally rearranged IgV genes in lymphomas from representative S1P₂^−/− animals. Note that although Southern blot analysis in tumors #246 and #297 suggests the presence of two distinct clones, only one rearranged allele could be successfully amplified, possibly due to the usage of a less common V gene family member not recognized by the primers used, or to the presence of mutations in the primer binding site. C, nucleotide sequence alignment of the rearranged IgV gene from S1P₂^−/− DLBCL #245 (as obtained by direct sequencing) with the germline VH7183 and JH region. Dashes, sequence identities; the indicated nucleotides correspond to mutations. Note that since direct sequencing only detects clonally represented events, the observed mutations can be unequivocally attributed to the malignant clone.

Morphometric analysis of splenic GC formation in S1P₂^−/− mice. A, unimmunized mice; total area of BCL6+ aggregates in each size range [measured in pixels (1,000 pixels = 1 Kpx)] normalized for spleen section area. Inset, percentage of splenic B220+ PNA+ GC cells in old, unimmunized control and knockout mice measured by flow cytometry. The difference is significant (P < 0.0015). B, unimmunized mice; number of BCL6+ aggregates per spleen section area, calculated as described in A. The numbers presented are per average spleen section. *, ANOVA analyses indicated that both the size and number of >2 Kpx aggregates are significantly (P < 0.05) increased in S1P₂^−/− spleens. C, number and area of the GC aggregates in SRBC-immunized mice. Note the expected increase in larger aggregates for immunized control and knockout mice compared with values for unimmunized mice in A and B. n = 7 young control, 3 young S1P₂^−/−, 4 old control, 3 old S1P₂^−/−. Columns, mean; bars, SEM.