A Rapid Embryonic Stem Cell-Based Mouse Model for B-cell Lymphomas Driven by Epstein-Barr Virus Protein LMP1

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.

Figure 1. Schematic flow chart for generating mouse models of LMP1-driven lymphomas via RDBC

Mice with the indicated genotypes were bred and CLT embryonic stem (ES) cell lines were derived from the embryos. CLT ES cells were genotyped and injected into RAG2-deficient blastocysts to yield chimeric mice in which all peripheral lymphocytes derive from the injected ES cells. See text for details.

Figure 2. CLT RDBC chimeras develop LMP1-driven polyclonal expansions and clonal B-cell lymphomas

A, Kaplan-Meier curves showing survival of mice within a cohort of 21 CLT RDBC chimeras are plotted. B, Representative images of spleens from control (wild-type C57BL/6 mice) and CLT RDBC chimeras. Scale bar, 5 mm. C, Representative FACS analysis of splenic cells from control and CLT RDBC chimeras. The surface markers examined are indicated on the right. D, Southern blotting analysis of EcoRI-digested genomic DNA of samples separated from spleens (S) and livers (L) of the control and CLT RDBC chimeras for rearrangements of the IgH J_H region and downstream Cμ and Cδ constant region exons. Top: diagram represents IgH locus with positions of EcoRI sites, the locations of the J_H segments, and the position of the J_H probe used. Lower: Southern blot panel of DNAs from indicated control and tumor samples from spleens (S), livers (L) and purified splenic B cells (B). Red arrowheads indicate clonal J_H rearrangements. Molecular weight (kb) markers and probes are indicated on the left, and the running position of the germline (gl) 6.2 kb J_H locus-containing band is indicated on the right. The 7.8 kb EcoRI-digested fragment of exosc3 locus was probed in parallel and used as a loading control.

Figure 3. Generation of AID^−/− CLT ES cells by a CRISPR/Cas9 designer nuclease approach

A, Schematic diagrams of mouse AID genomic locus and AID knock-out allele with positions of indicated exons, BglII sites, Cas9/gRNA-targeting sites, and the probe used for Southern blotting. B, Southern blotting analysis of BglII-digested genomic DNA from parental CLT ES cells, one clone of CLT; AID^+/− ES cells, and two independent clones of CLT; AID^−/− ES cells. Molecular weight (kb) markers are indicated on the left. C and D, Real-time qRT-PCR (C) and Western blotting (D) analyses of AID expression in splenic B-cell populations from indicated CLT and CLT; AID^−/− RDBC chimeras. The mouse B-cell lymphoma cell line CH12F3 and splenic B cells purified from control mice (wild-type C57BL/6 mice) treated without (Naïve) or with indicated cytokines (CSR-activated) were used as the control for AID expression. In C, the AID expression level was normalized to the HPRT level. In D, tubulin was detected in parallel and used as a loading control. The asterisk (*) indicates a non-specific cross-reactive band. CIT: α-CD40⁺IL4⁺TNFβ

Figure 4. Comparative analysis of LMP1-driven polyclonal expansions and clonal B-cell lymphomas derived from CLT and CLT; AID^−/− RDBC chimeras

A, Kaplan-Meier curves showing survival of mice within cohorts of CLT and CLT; AID^−/− RDBC chimeras. Cohorts of 8 CLT RDBC chimeras and 16 CLT; AID^−/− RDBC chimeras were plotted. n.s. means statistically not significant by Gehan-Breslow-Wilcoxon test, p=0.1306. B, Representative images of spleens from CLT and CLT; AID^−/− RDBC chimeras. Scale bars, 5 mm. C, Representative FACS analysis of splenic cells from control, CLT and CLT; AID^−/− RDBC chimeras. Surface markers assayed are indicated on the right. D, Southern blotting analysis of EcoRI-digested genomic DNA of samples from spleens (S), livers (L) and purified splenic B cells (B) samples from the control, CLT and CLT; AID^−/− RDBC chimeras. Other details of the analysis are the same as described for Figure 2.

Figure 5. Transplantation of LMP1-driven B-cell population expansions derived from CLT and CLT; AID^−/− RDBC chimeras

A, Representative images showing the spleens and livers of wild-type (WT, C57BL/6xBALB/c, F1) and RAG2^−/−; γc^−/− mice transplanted with the indicated donor clonal B-cell expansions. Mice were analyzed 3 to 9 weeks after transplantation. Scale bar, 5 mm. B, Southern blotting analysis on EcoRI-digested genomic DNA of samples from spleens (S) and livers (L) of the indicated mice. 1^st and 2^nd labels indicate the primary and transplanted tumors, respectively. Other details are as described in the legend to Figure 1.