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. 2020 Jun 23;117(25):14421-14432.
doi: 10.1073/pnas.1921139117. Epub 2020 Jun 10.

Functional interplay of Epstein-Barr virus oncoproteins in a mouse model of B cell lymphomagenesis

Thomas Sommermann  1 , Tomoharu Yasuda  2 , Jonathan Ronen  3 , Tristan Wirtz  2 , Timm Weber  2 , Ulrike Sack  2 , Rebecca Caeser  4 , Jingwei Zhang  2 , Xun Li  2 , Van Trung Chu  2 , Anna Jauch  5 , Kristian Unger  6   7 , Daniel J Hodson  4 , Altuna Akalin  3 , Klaus Rajewsky  1
Affiliations

Functional interplay of Epstein-Barr virus oncoproteins in a mouse model of B cell lymphomagenesis

Thomas Sommermann et al. Proc Natl Acad Sci U S A. .

Abstract

Epstein-Barr virus (EBV) is a B cell transforming virus that causes B cell malignancies under conditions of immune suppression. EBV orchestrates B cell transformation through its latent membrane proteins (LMPs) and Epstein-Barr nuclear antigens (EBNAs). We here identify secondary mutations in mouse B cell lymphomas induced by LMP1, to predict and identify key functions of other EBV genes during transformation. We find aberrant activation of early B cell factor 1 (EBF1) to promote transformation of LMP1-expressing B cells by inhibiting their differentiation to plasma cells. EBV EBNA3A phenocopies EBF1 activities in LMP1-expressing B cells, promoting transformation while inhibiting differentiation. In cells expressing LMP1 together with LMP2A, EBNA3A only promotes lymphomagenesis when the EBNA2 target Myc is also overexpressed. Collectively, our data support a model where proproliferative activities of LMP1, LMP2A, and EBNA2 in combination with EBNA3A-mediated inhibition of terminal plasma cell differentiation critically control EBV-mediated B cell lymphomagenesis.

Keywords: B cell lymphomagenesis; EBNA; Epstein-Barr virus; LMP1; plasma cell differentiation.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Ebf1 and Rel are aberrantly activated in LMP1 lymphomas. (A) Experimental overview LMP1-L cohort. (B) Survival curve. (C) Representative M-FISH of t(11;12) translocations in LMP1-CLs 37 and 43. (D) Translocations in sorted (huCD2+) 2ryLMP1-Ls as determined by exome and Sanger sequencing (LMP1-L 31 and 43) or as suggested by CNVs in array CGH (LMP1-L 37). Arrows indicate ORF orientation (E and F) RNA sequencing of sorted splenic B cells (CD19+CD38+), tpLMP1 B cells (days 18 to 20 posttransplantation [p.t], huCD2+) and 2ryLMP1-Ls (huCD2+). (E) Heatmap showing fragments per kilobase of transcript per million mapped reads (FPKM)-normalized expression. (F) Principal component analysis on the 500 most-variable genes.
Fig. 2.
Fig. 2.
Ebf1 or Rel overexpression supports transformation of LMP1 B cells. (A) Experimental overview of A–G. (B) Growth curve of transduced (GFP+) cells. (C) Quantification of outgrowing single-sorted transduced (GFP+) cells on mouse embryonic fibroblast (MEF) feeder. Bars represent median outgrowth frequency. Number of biological replicates showing any outgrowth is indicated. Images show representative wells on day 21 post TAT-cre. (D) Survival curve of transplanted Rag2KOcγKO mice. (E–G) Analysis of Rag2KOcγKO mice transplanted with 3 × 105 GFP+ cells. Analysis was performed at symptom onset or between days 11 and 22 (control groups). (E) Representative organ images day 11 (RV-GFP/RV-Ebf1) and day 21 (RV-Rel). (F) Fluorescence-activated cell sorting (FACS) analysis of splenocytes from E. (G) Cell count of GFP+/huCD2+ cells in indicated organs. n = 2 (αCD40/IL-4 treated groups); n = 4 (iLMP1 RV-GFP); n = 5 (iLMP1 RV-Ebf1 or iLMP1 RV-Rel). (H) Human tonsillar GCBs were cultured on CD40-L/IL-21 feeder cells and transduced with RVs encoding GFP, LMP1-ires-GFP, Ebf1-ires-mCherry, or Rel-ires-mCherry. Starting day 5, cells were cultured on feeders without CD40-L/IL-21. Fold change of reporter+ cell number on day 12 over day 5 is presented; n = 12. All data are presented as mean ± SD. Significance was calculated by Welch’s t test (G) or one-sample t test (H). (*P < 0.05; **P < 0.005; ***P < 0.0005).
Fig. 3.
Fig. 3.
Ebf1 inhibits PC differentiation of LMP1 B cells. (A–D) Further analysis of 2ryLMP1-Ls from Fig. 1. (A) Heatmap showing FPKM-normalized expression of B cell and PC transcription factors (TFs) in RNA sequencing. (B) FACS analysis of splenocytes from tpLMP1 mice before lymphomagenesis (day 30 posttransplant). (C) FACS analysis of a representative splenic Ebf1high 2ryLMP1-L. (D) Quantification of CD138 expression on huCD2+ cells in B and C. Bars indicate median. n = 3 (tpLMP1 and Ebf1high LMP1-L), n = 9 (Relhigh LMP1-L). (E and F) RNA sequencing of iLMP1 B cells transduced on day 1 with RVs encoding GFP, Ebf1-ires-GFP, or Rel-ires-GFP. RNA was isolated from FACS-sorted (GFP+/huCD2+) cells on day 6 post TAT-Cre. Naive splenic B cells (CD19+/CD38+) from wild-type mice are presented as a control. (E) Heatmap showing FPKM-normalized expression. (F) Gene set enrichment analysis of a gene set differentially expressed in splenic PCs over GCBs (29) run in the space of genes differentially expressed between GFP- and Ebf1-transduced iLMP1 B cells on day 6.
Fig. 4.
Fig. 4.
EBNA3A inhibits PC differentiation. (A) iLMP1 B cells were transduced with RVs encoding mCherry or indicated EBNAs (reported by ires-mCherry). FACS-based quantification of mCherry+ cells day 7 over day 4 is presented (n = 5). (B–F) Analysis of Cd19-cre;R26EBNA3Astopf and Cd19-cre;R26BFPstopf mice. (B) Representative FACS analysis of spleens. (C) Survival curve. (D) Quantification of BFP expression in bone marrow pro (CD93+B220+IgM−IgD−CD19+c-kit+/low), pre (CD93+B220+IgM−IgD−CD19+c-kit−), immature (CD93+B220+IgM+IgD−), and splenic mature (CD93−B220+CD19+IgM+IgD+) B cells. n = 9 (Cd19-Cre;BFPstopf) n = 10 (Cd19-Cre;EBNA3Astopf). (E) Representative FACS analysis and quantification of BFP expression in bone marrow (CD138+CD267+) PCs; n = 12 (Cd19-Cre;BFPstopf) n = 14 (Cd19-Cre;EBNA3Astopf). (F) Elispot for total ASCs in sorted BFP+ bone marrow cells; n = 6. Images show representative wells with 4 × 104 cells. All data are presented as mean ± SD. Significance was calculated using a one-way ANOVA with P value adjusted via Dunnett (A), two-way ANOVA with Sidak’s multiple comparisons test (D), Mann–Whitney U test (E), and Welch’s t test (F). (*P < 0.05; **P < 0.005; ***P < 0.0005; n.s., nonsignificant).
Fig. 5.
Fig. 5.
EBNA3A inhibits PC differentiation and supports expansion of LMP1 B cells. (A–D) iBFP, iEBNA3A, iLMP1, and iLMP1/EBNA3A B cells. (A) Growth curve of bulk cultured cells. n = 4, n = 5 (iLMP1). (B) FACS-based quantification of BrdU uptake on day 6 post TAT-Cre. n = 6. (C) FACS-based quantification of active-caspase 3 on day 6 post TAT-Cre. n = 10. (D) Heatmap showing FPKM-normalized gene expression in RNA sequencing performed on sorted hucd2+ (iLMP1) or huCD2+BFP+ (iLMP1/EBNA3A) cells day 6 post TAT-Cre. Splenic naive (CD19+CD38+) B cells (from Fig. 3E) serve as a control. (E–H) Rag2KOcγKO mice were reconstituted with fHSPCs from Cd3εKO;Cd19-Cre;R26LMP1stopf (tpLMP1) or Cd3εKO;Cd19-Cre;R26LMP1stopf/EBNA3Astopf (tpLMP1/EBNA3A) mice. (E) Survival curve. (F) Representative image of spleens and quantification of splenic weight at symptom onset (tpLMP1/EBNA3A) or days 32 to 41 p.t. (tpLMP1); n = 3 (Rag2KOcγKO), n = 6 (tpLMP1), n = 10 (tpLMP1/EBNA3A). (G) Immunohistology on splenic sections day 36 p.t. (representative of three independent mice per group). Splenic section of a Rag2KOcγKO mouse is presented as negative control. (H) Elispot for total ASCs in sorted reporter+ splenic cells at symptom onset or days 32 to 41 p.t. (tpLMP1). Quantification was normalized to mean ASC frequency in tpLMP1 conditions. Images show representative wells with 1.5 × 105 cells. n = 6 (tpLMP1), n = 4 (tpLMP1/EBNA3A). All data are presented as mean ± SD. Significance was calculated using a Student’s t test (A–C and F) or Welch’s t test (H). (**P < 0.005; ***P < 0.0005).
Fig. 6.
Fig. 6.
EBNA3A blocks PC differentiation of LMP1 and LMP2A B cells. (A) Experimental overview of B–E. (B) Representative FACS analysis of spleens from donor animals and recipient animals at days 7 to 28 p.t. (quantification in SI Appendix, Fig. S6A). (C) Survival curve. (D) Representative images of LMP1/2A+ lymphomas arising in C. (E) Representative FACS analysis and quantification of CD19/CD138 expression on huCD2+GFP+ cells in lymphomas arising in C; n = 3. (F and G) iLMP1, iLMP1_2A, and iLMP1_2A/EBNA3A B cells analyzed on day 9 post TAT-Cre. (F) FACS-based quantification of CD138+/B220low PCs; n = 3 (iLMP1), n = 11 (iLMP1_2A), n = 9 (iLMP1_2A/EBNA3A). (G) Elispot for total ASCs among sorted reporter+ cells. Images show representative wells with 120 cells; n = 9. (H and I) Rag2KOcγKO mice were reconstituted with fHSPCs from Cd3εKO;Cd19-Cre R26LMP1_LMP2Astopf (tpLMP1_2A) or Cd3εKO;Cd19-Cre R26LMP1_LMP2Astopf;EBNA3Astopf (tpLMP1_2A/EBNA3A) mice. (H) Survival curve. (I) Representative FACS analysis of splenocytes from tpLMP1_2A/EBNA3A mice at symptom onset. Quantifications shows expression of LMP1_2A (huCD2+) and EBNA3A (BFP+) in whole splenocytes or CD19/CD138 expression on indicated populations. n = 7. All data are presented as mean ± SD. Significance was calculated using a Mann–Whitney U test (G and I), or one-way ANOVA with P value adjusted via Dunnett (F). (**P < 0.005; ***P < 0.0005).
Fig. 7.
Fig. 7.
Transformation of LMP1_2A B cells requires overexpression of EBNA3A and Myc. (A–H) Analysis of iEBNA3A, iLMP1, iLMP1_2A, and iLMP1_2A/EBNA3A B cells. (A) Representative FACS analysis and quantification of BrdU incorporation day 6 post TAT-Cre; n = 7 (iLMP1_2A), n = 6 (iLMP1_2A/EBNA3A). (B) RT-qPCR-based quantification of Myc expression day 6 post TAT-Cre; n = 17 (iLMP1), n = 14 (iLMP1/EBNA3A), n = 11 (iLMP1_2A and iLMP1_2A/EBNA3A). (C) Western blot of total cell lysates day 6 post TAT-Cre. Mychigh LMP1− LMP2A− mouse cell line 19pp serves as expression control (80). (D) Experimental overview of E–H. iEBNA3A cells were stimulated with αCD40 (2 μg/mL) and IL-4 (20 ng/mL) to allow transduction. (E) Growth curve of transduced (GFP+ or mCherry+) cells in bulk. (F) Representative FACS analysis and quantification of BrdU uptake on day 6 post TAT-Cre; n = 6. (G and H) Analysis of Rag2KOcγKO mice transplanted with 5 x 106 transduced cells; three independent donors per group. (G) Survival curve. (H) Representative organ images and FACS analysis of mice transplanted with RV-Myc transduced iLMP1_2A/EBNA3A B cells at disease onset. Quantifications show BFP/mCherry expression in huCD2+ cells and CD19/CD138 expression on huCD2+/BFP+/mCherry+ cells. n = 3. All data are presented as mean ± SD. Significance was calculated using a Student’s t test (A), Mann–Whitney U test (B), or paired Student’s t test (E and F). (*P < 0.05; **P < 0.005; ***P < 0.0005; n.s., nonsignificant).
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References

    1. Young L. S., Yap L. F., Murray P. G., Epstein-Barr virus: More than 50 years old and still providing surprises. Nat. Rev. Cancer 16, 789–802 (2016). - PubMed
    1. Taylor G. S., Long H. M., Brooks J. M., Rickinson A. B., Hislop A. D., The immunology of Epstein-Barr virus-induced disease. Annu. Rev. Immunol. 33, 787–821 (2015). - PubMed
    1. Swerdlow S. H., Webber S. A., Chadburn A., Ferry J. A., “Post-transplant lymphoproliferative disorders” in WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, (International Agency for Research on Cancer, ed. 4, 2008), pp. 343–349.
    1. Morscio J., Tousseyn T., Recent insights in the pathogenesis of post-transplantation lymphoproliferative disorders. World J. Transplant. 6, 505–516 (2016). - PMC - PubMed
    1. Kieser A., Sterz K. R., “The latent membrane protein 1 (LMP1)” in Curr. Top. Microbiol. Immunol., (2015), Vol. 391, pp. 119–149. - PubMed

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