Fig. 1

Immune suppression induces transformation of LMP1/CD40 indolent B-cell lymphomas: To induce antibody-mediated T-cell depletion, LMP1/CD40-expressing mice were injected intraperitoneally every 4 days for three weeks with a mix of anti-CD4 (YTS 191.1.2), anti-CD8 (YTS 169.4.2.1), and anti-Thy-1 (YTS 154.7.7.10) antibodies in In VivoPure Dilution Buffer at a dose of 200 µg each (Bio X Cell; USA). For CsA treatment, wild type and LMP1/CD40-expressing mice were injected intraperitoneally daily for three months with placebo or 10 mg/kg of CsA (Sandimmun – Novartis; USA) diluted in 5% glucose. a Means and standard deviations of the spleen weights from the wild type (CD19_Cre) or LMP1/CD40-expressing mice immunosuppressed (αT and CsA) or not (Ctrl). b Absolute numbers of spleen cells per spleen. c Absolute numbers of B lymphocytes per spleen. d Frequency of cases with a clonal abundance greater than 10%. The clonal abundance was estimated after high-throughput sequencing of the VDJ regions. Transcripts were amplified by 5’RACE PCR using a reverse primer that hybridized within the µ CH1 exon as previously described.⁶ The amplicons were sequenced on an Illumina MiSeq sequencing system using the MiSeq kit Reagent V2 500 cycles. Repertoire analysis was performed using the IMGT/HighV-QUEST tool and the R software. Briefly, the VH, JH, and CDR3 segments were identified using HighV-QUEST. Based on these annotations, the reads were grouped into clonotypes that shared the same VH and JH genes and high CDR3 homology. Then, the relative abundance of each clonotype was calculated. e Hematoxylin and eosin staining of spleen sections from control LMP1/CD40-expressing mice (upper panel) or mice immunosuppressed with cyclosporine A (CsA) (lower panel). f Flow cytometry estimation of the spleen B-cell sizes from the Ctrl and CsA-LMP1/CD40-expressing mice. Upper panel, means and standard deviations of the forward scatter (FSC) for all studied mice. Lower panel, representative overlay of the FSC monoparametric histograms gated on B220⁺ B cells. g Means and standard deviations of the flow cytometry percentages of B220-positive spleen B cells expressing the CD80 and/or CD86 activation markers. h Right panel: Means and standard deviations of flow cytometry percentages of BrdU-positive B cells after in vivo BrdU incorporation. Left panel: Representative biparametric histogram showing the intensity of propidium iodide (PI, x axis) and BrdU (y axis) staining for the control and CsA-treated LMP1/CD40-expressing mice. The percentage of proliferating cells is indicated in the graph. For the in vivo proliferation assay, the mice were injected intraperitoneally with 2 mg of BrdU 18 h prior to cell isolation. The splenocytes were stained for B220, and the cell cycle phases were analyzed using the FITC-BrdU Flow Kit (BD Pharmingen). i Numbers of circulating white blood cells in the LMP1/CD40-expressing mice immunosuppressed (CsA) or not (Ctrl). j Flow cytometry percentages of circulating granulocytes (Gr-1⁺), B cells (B220⁺) and T cells (CD3⁺). k Upper panels: Flow cytometric estimation of circulating B-cell sizes from the Ctrl and CsA- LMP1/CD40-expressing mice. Overlay of FSC monoparametric histograms gated on B220 B cells (left panel). Means and standard deviations of the FSC for all studied mice (right panel). Lower panel: Representative lymphocyte morphology after May-Grünwald Giemsa staining of blood smears from the Ctrl and IS mice (magnification × 1000). For all experiments, at least four mice were studied for each condition. Significant differences are indicated by * (p < 0.05), ** (p < 0.01) and *** (p < 0.001)