Figure S1. Flow cytometry measurement of peripheral blood B and T cells in indicated CLT RDBC chimeras and control mice. Figure S2. Comparative measurement of splenic and peripheral blood B-cell populations in the indicated CLT RDBC chimeras and control mice. Figure S3. CLT; AID-/- mice generated from germline breeding. Figure S4. Spleen size distributions of indicated CLT RDBC chimera cohorts. Figure S5. Southern blotting analyses of B-cell expansions from CLT (A) and CLT as well as CLT; AID-/- RDBC (B) chimeras. Table S1. CLT chimeras shown in Figure 2. Table S2. CLT and CLT; AID-/- chimeras shown in Figure 4.
ARTICLE ABSTRACT
The Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) contributes to oncogenic human B-cell transformation. Mouse B cells conditionally expressing LMP1 are not predisposed to B-cell malignancies, as LMP1-expressing B cells are eliminated by T cells. However, mice with conditional B-cell LMP1 expression and genetic elimination of α/β and γ/δ T cells (“CLT” mice) die early in association with B-cell lymphoproliferation and lymphomagenesis. Generation of CLT mice involves in-breeding multiple independently segregating alleles. Thus, although introduction of additional activating or knockout mutations into the CLT model is desirable for further B-cell expansion and immunosurveillance studies, doing such experiments by germline breeding is time-consuming, expensive, and sometimes unfeasible. To generate a more tractable model, we generated clonal CLT embryonic stem (ES) cells from CLT embryos and injected them into RAG2-deficient blastocysts to generate chimeric mice, which, like germline CLT mice, harbor splenic CLT B cells and lack T cells. CLT chimeric mice generated by this RAG2-deficient blastocyst complementation (“RDBC”) approach die rapidly in association with B-cell lymphoproliferation and lymphoma. Because CLT lymphomas routinely express the activation-induced cytidine deaminase (AID) antibody diversifier, we tested potential AID roles by eliminating the AID gene in CLT ES cells and testing them via RDBC. We found that CLT and AID-deficient CLT ES chimeras had indistinguishable phenotypes, showing that AID is not essential for LMP1-induced lymphomagenesis. Beyond expanding accessibility and utility of CLT mice as a cancer immunotherapy model, our studies provide a new approach for facilitating generation of genetically complex mouse cancer models. Cancer Immunol Res; 3(6); 641–9. ©2015 AACR.
