Fig 2: Lineage switch phenotype is not detectable in parental leukaemia and is stable when CAR pressure is removed.(a) Primary E2a:PBX ALL was single-cell cloned by limiting dilution, expanded and analysed by flow cytometry. Heatmap of cell surface marker expression evaluated by flow cytometry. (b) CD45.2+ B220+ leukaemic blasts from sample 59–61 obtained at day 58 following CAR demonstrating intermediate B/myeloid phenotype expressing for CD22, CD11b and Gr1, but negative for CD19. (c–e) In vivo passage of post-CAR CD19- relapsed leukaemia, with E2a:PBX control, in absence of additional CAR treatment. (c) B/Myeloid biphenotypic sample 25–2 passaged twice off pressure, with re-emergence of a CD19+ population in 16/21 mice in 3rd passage (E2a:PBX control n=16, 2nd n=10, 3rd n=12). (d) Lineage switch samples 24–6 (n=5 per group) and (e) A001 (E2a:PBX control n=3, 2nd n=3, 3rd n=4) passaged twice. Respective survival curves for 1 of 2 experiments shown on the right.

Fig 3: Impaired survival of premalignant splenic B cells upon A1 knockdown. Total spleen cells from mice of the indicated genotypes were incubated in the presence or absence of the indicated mitogens for 24 h. The percentages of viable (Annexin V-) CD19+B220+ B cells in the Venus- or Venus+ fractions derived from DT-A1 mice or the total cell fraction of MYC or DT-FF mice (>90% Venus+ cells) was assessed by flow cytometry. Representative contour plots of mitogen-treated splenocyte cultures stained with anti-CD19 and anti-B220-specific antibodies and percentages of cells falling within each gate are shown. (b) Quantification of data shown in (a). To assess relative survival, the percentage of viable B cells was compared with the percentage of B cells identified straight after sacrifice. As these percentages differ, the bar graphs show the ratio of the percentage of B220+CD19+ B cells in culture after 24 h, divided by the percentage of the B220+ B cells present in the spleen on the day of sacrifice. All experiments were performed in technical duplicates. Bars represent means±s.e.m. (MYC n=8, DT-FF n=5, DT-A1 n=13, biological replicates, six independent experiments). ANOVA followed by Bonferroni post hoc test. **P?0.01, ***P?0.001. UN=untreated, CYT=cytokines (IL-2, IL-4 and IL-5), M+CD=anti-IgM and anti-CD40.

Fig 4: Ileal Btnl2-KO ?d IELs display an altered antibacterial response module compared to ileal WT ?d IELs.?d IELs from duodenum, jejunum, and ileum of cohoused 11-week-old Btnl2-KO and WT littermates (n = 3–4/genotype, each a pool of 2 mice) were sort-purified as CD45 + TCRß-TCR?d + cells. RNA sequencing was performed, and gene set enrichment analysis using NextBio. Gene ontology (GO) was employed to identify GO biological processes differentially enriched in Btnl2-KO and WT ?d IELs. a Volcano plot displaying genes differentially regulated between ileal Btnl2-KO and WT ?d IELs. The Horizontal dashed line indicates FDR = 0.05 and a vertical dashed line indicates |Fold Change | = 1.5. b Hierarchical clustering of top 50 differentially expressed genes between ileal Btnl2-KO and WT ?d IELs; duodenal and jejunal Btnl2-KO and WT ?d IELs were also included as a comparison. c Gene ontology enrichment analysis of most significantly impaired biological processes in ileal Btnl2-KO ?d IELs compared to ileal WT ?d IELs. d Hierarchical clustering of top dysregulated antibacterial response genes in ileal Btnl2-KO and WT ?d IELs within the combined top 3 GO processes; duodenal and jejunal Btnl2-KO and WT ?d IELs were also included as a comparison.

Fig 5: ER Stress Responses Are Similar across Plasma Cell Subsets(A)qRT-PCR analysis of ER stress response genesin plasma cell subsets. Data are cumulative from two individual experiments, each with three biological replicates of each plasma cell subset. Data are normalized to expression of HPRT. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005 by twoway ANOVA with post hoc Sidak’s multiple comparisons test.(B)Caspase-12 activation in plasma cell populations. Plasma cell populations were sorted and labeled for 1 hr with the fluorescent caspase-12 inhibitor ATAD-FMK. Representative plots (left) and quantification (right) are shown, with each data point representing cells from an individual mouse. Mean values ± SEM are shown. No significant differences were observed using one-way ANOVA.(C)Flow cytometric representative plots (left) andquantification (right) of p-eIF2a mean fluorescence intensities (MFIs) in plasma cell subsets. *p < 0.05 by one-way ANOVA with post hoc Tukey’s multiple comparisons test.(D) Human bone marrow plasma cells were treatedwith inhibitors to PERK (4 nM GSK2606414), GCN2 (500 µM SP600125 or indirubin-3’-monoxime), HRI (50 µM hemin), or PKR (100 µM imidazole-oxindole C16) for 1 hr and analyzed for intracellular p-eIF2a. Plots of p-eIF2a representative of two independent experiments are shown.(E) Schematic representation of mixed bone marrow chimera experiment to assess plasma cell population dependence on PERK (left). CD45.2+ chimerism values are shown (right) and are cumulative from two experiments. Each symbol represents a distinct mouse. ****p < 0.00005 by two-way ANOVA with post hoc Sidak’s multiple comparisons test.