Figure 5. MyD88-L265P- or... | American Society of Hematology

Figure 5.

$MyD88-L265P- or CARD11-L244P-driven immune evasion phenotypes prevent senescence-preferential immune clearance of lymphoma cells. (A) Lymphoma onset in recipient mice of stably MyD88-L265P;sh-scrambled (n = 6) or MyD88-L265P;sh-PDL (targeting both PD-L1 and PD-L2; n = 7) cotransduced Eµ-myc transgenic HSCs (P = .001). Of note, all lymphomas generated were GFP+ (data not shown). For knockdown efficiency, see supplemental Figure 4A. (B) In situ expression (with representative cases shown) of the indicated senescence-related markers of lymphomas as in panel A. SA–β-gal staining of cytospin preparations and lymphoma sections stained with hematoxylin and eosin and antibodies against H3K9me3 or Ki67. Scale bars, 100 µm. (C) Fraction of CD3+ cells within the overall population of viable cells in freshly isolated single-cell suspensions from n ≥ 3 individual enlarged lymph node samples per indicated genotype (with error bars denoting the standard deviation) analyzed by flow cytometry. (D) GSEA in RNA-seq–based GEP of GFP-sorted B-cell lymphoma cells as in panel A for the indicated gene signatures. (E) Cytotoxic potential of anti-PD1–derepressed splenic cytotoxic CD8a+ T cells coincubated with C12-RG+ vs C12-RG− fractions of the same, but ex vivo anti–PD-L1 antibody-treated MyD88-L265P B-cell lymphoma cells that developed in the same mouse the T cells were isolated from. Left, percentage of senescent viable GFP+ lymphoma cells after T-cell coculture and indicated antibody treatments. Right, viability of nonsenescent (proliferating) GFP+ lymphoma cells after T-cell coculture and indicated antibody treatments, n = 3 different tumor mice (with error bars denoting the standard deviation). (F) Tumor-free/OS, plotted in Kaplan-Meier format, of mice transplanted with manifest MyD88-L265P– or CARD11–L244P-driven lymphomas either engineered to stably coexpress sh-PDL (vs empty vector) at the time of transplantation (left), or exposed to an anti-PD1 antibody (vs mock) at the time of well-palpable lymphoma manifestation (right); n ≥ 3 samples per genotype and treatment arm. Asterisks indicate significance (P < .05).$

MyD88-L265P- or CARD11-L244P-driven immune evasion phenotypes prevent senescence-preferential immune clearance of lymphoma cells. (A) Lymphoma onset in recipient mice of stably MyD88-L265P;sh-scrambled (n = 6) or MyD88-L265P;sh-PDL (targeting both PD-L1 and PD-L2; n = 7) cotransduced Eµ-myc transgenic HSCs (P = .001). Of note, all lymphomas generated were GFP⁺ (data not shown). For knockdown efficiency, see supplemental Figure 4A. (B) In situ expression (with representative cases shown) of the indicated senescence-related markers of lymphomas as in panel A. SA–β-gal staining of cytospin preparations and lymphoma sections stained with hematoxylin and eosin and antibodies against H3K9me3 or Ki67. Scale bars, 100 µm. (C) Fraction of CD3⁺ cells within the overall population of viable cells in freshly isolated single-cell suspensions from n ≥ 3 individual enlarged lymph node samples per indicated genotype (with error bars denoting the standard deviation) analyzed by flow cytometry. (D) GSEA in RNA-seq–based GEP of GFP-sorted B-cell lymphoma cells as in panel A for the indicated gene signatures. (E) Cytotoxic potential of anti-PD1–derepressed splenic cytotoxic CD8a⁺ T cells coincubated with C₁₂-RG⁺ vs C₁₂-RG⁻ fractions of the same, but ex vivo anti–PD-L1 antibody-treated MyD88-L265P B-cell lymphoma cells that developed in the same mouse the T cells were isolated from. Left, percentage of senescent viable GFP⁺ lymphoma cells after T-cell coculture and indicated antibody treatments. Right, viability of nonsenescent (proliferating) GFP⁺ lymphoma cells after T-cell coculture and indicated antibody treatments, n = 3 different tumor mice (with error bars denoting the standard deviation). (F) Tumor-free/OS, plotted in Kaplan-Meier format, of mice transplanted with manifest MyD88-L265P– or CARD11–L244P-driven lymphomas either engineered to stably coexpress sh-PDL (vs empty vector) at the time of transplantation (left), or exposed to an anti-PD1 antibody (vs mock) at the time of well-palpable lymphoma manifestation (right); n ≥ 3 samples per genotype and treatment arm. Asterisks indicate significance (P < .05).

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