CD4 + T cells are found within endemic Burkitt lymphoma and modulate Burkitt lymphoma precursor cell viability and expression of pathogenically relevant Epstein–Barr virus genes

Endemic Burkitt lymphoma (eBL) is an aggressive B cell cancer characterized by an IgH/c-myc translocation and the harboring of Epstein–Barr virus (EBV). Evidence accumulates that CD4 + T cells might contribute to eBL pathogenesis. Here, we investigate the presence of CD4 + T cells in primary eBL tissue and their potential dichotomous impact on an EBV-infected pre-eBL cell model using ex vivo material and in vitro co-cultures. In addition, we establish a novel method to study the effect of IgH/c-myc translocation in primary B cells by employing a CRISPR/Cas9 knock-in approach to introduce and tag de novo translocation. We unprecedently document that CD4 + T cells are present in primary eBL tumor tissue. Furthermore, we demonstrate that CD4 + T cells on the one hand suppress eBL development by killing pre-eBL cells lacking IgH/c-myc translocation in vitro and on the other hand indirectly promote eBL development by inducing crucial EBV Latency III to Latency I switching in pre-eBL cells. Finally, we show that while the mere presence of an IgH/c-myc translocation does not suffice to escape CD4 + T-cell-mediated killing in vitro, the CD4 + T-cell-mediated suppression of EBV’s Latency III program in vivo may allow cells harboring an IgH/c-myc translocation and additional mutations to evade immune control and proliferate by means of deregulated c-myc activity, resulting in neoplasia. Thus, our study highlights the dichotomous effects of CD4 + T cells and the mechanisms involved in eBL pathogenesis, suggests mechanisms of their impact on eBL progression, and provides a novel in vitro model for further investigation of IgH/c-myc translocation. Supplementary Information The online version contains supplementary material available at 10.1007/s00262-021-03057-5.

Overview of target locations for gRNAs targeting IgH and c-myc regions. b) LCL line was electroporated with IgH targeting or c-myc targeting RNPs. Cells were cultured for 3 days post electroporation, then gDNA was isolated and assessed for presence of mutations using Surveyor Assay kit.

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Supplementary Figure 4. Introduction of IgH/c-myc translocation using IgH and c-myc targeting CAS9 RNPs. a) Overview of gDNA PCR design used to detect IgH/c-myc translocations. b) LCL line was electroporated with RNPs targeting both IgH and c-myc regions. Cells were cultured for 3 days post electroporation, then gDNA was isolated and assessed for presence of translocation using gDNA PCR. c) Individual bands were cut out from the agarose gel, cloned into vector and sequenced.  Figure 5. Generation of ssDNA insert for introduction of GFP tag into IgH/c-myc translocation. a) Overview of plasmid used for generation of ssDNA insert. ssDNA was generated from PCR product, size and sequence were confirmed by gel electrophoresis (b) and Sanger sequencing (c  Figure 6. Confirmation of presence of IgH/c-myc translocation in CRISPR/CAS9 edited LCL lines a) Overview of gDNA PCR design used to detect IgH/c-myc translocations. b-d) LCLs from 4 donors either with or without IgH/c-myc translocation were cultured for 3 days. b) Percentage of GFP+ cells was determined by flow cytometry prior to harvesting. c) gDNA was isolated and the presence of the translocation was assessed by gDNA PCR. d) Dual probe FISH for IgH/c-myc translocation was performed. Representative images for each donor for both "wild type" and IgH/c-myc+ LCLs are shown.

Supplementary Figure 7. Effect of IgH/c-myc translocation on LCL gene expression. Western blot images
Western blot images for four donors used for band quantification used for graphs in Figure 6.

Supplementary Figure 8. Effect of IgH/c-myc translocation on LCL phenotype
LCLs from 4 donors either with or without IgH/c-myc translocation were cultured at same density for 3 days and compared to assess the effect of translocation on LCL phenotype. Different symbols represent different conditions, while different colors represent different TMC donors. P-values were calculated using paired t-test. p>0.05 not significant (n.s.). For flow cytometry experiments, isotype staining was used to determine positive cells.

cells on proliferation and viability of IgH/c-myc+ LCLs
LCLs either with or without IgH/c-myc translocation were cultured for 9 days either alone or in co-culture with various ratios of expanded autologous CD4+ T cells activated using anti-CD3/CD28 beads. At given timepoints cells were harvested, stained and analysed using flow cytometer. LCLs were pre-gated based on CD19 expression. Shown are mean  SD of mean percentage of positive cells from 3 TMC donors. P-values were calculated using two-way Anova with Tukey's test for multiple comparisons. p>0.05 not significant (n.s.), p<0.1*, p<0.01**, p<0.001***, p<0.0001****. a) Mean percentage of LCLs in co-culture was determined by CD19 and CD4 staining. b) Mean percentage of EdU+ LCLs was measured using Click-iT Flow Cytometry kit. c) Mean percentage of LCLs in different cell cycle stages was measured using EdU Click-iT Flow cytometry kit and FxCycle dye.

cells on c-myc and bcl6 expression in LCLs
LCLs were cultured for 7 days either alone or in co-culture with various ratios of expanded autologous CD4+ T cells activated using anti-CD3/CD28 beads. At given timepoints cells were harvested and LCLs were isolated using CD19+ beads and AutoMACS. Different symbols represent different conditions, while different colors represent different TMC donors. P-values were calculated using two-way Anova with Sidak's test for multiple comparisons. p>0.05 not significant (n.s.), p<0.1*, p<0.01**, p<0.001***. WB images used for quantification can be found in Supplementary  Figure 1. a) Total c-myc protein expression was assessed using Western blotting. Western blot image was quantified and normalized to -actin as loading control. Shown are mean  SD of normalized volume of c-myc band. b) c-myc and bcl6 expression was determined using qRT-PCR. Shown are mean  SD of dCt values normalized to geometric means of TBP and YWHAZ.