Myeloid loss of Beclin 1 promotes PD-L1hi precursor B cell lymphoma development

Abstract

Beclin 1 (Becn1) is a key molecule in the autophagy pathway and has been implicated in cancer development. Due to the embryonic lethality of homozygous Becn1-deficient mice, the precise mechanisms and cell type-specific roles of Becn1 in regulating inflammation and cancer immunity remain elusive. Here, we report that myeloid-deficient Becn1 (Becn1ΔM) mice developed neutrophilia, were hypersusceptible to LPS-induced septic shock, and had a high risk of developing spontaneous precursor B cell (pre-B cell) lymphoma with elevated expression of immunosuppressive molecules programmed death ligand 1 (PD-L1) and IL-10. Becn1 deficiency resulted in the stabilization of MEKK3 and aberrant p38 activation in neutrophils, and mediated neutrophil-B cell interaction through Cxcl9/Cxcr3 chemotaxis. Neutrophil-B cell interplay further led to the activation of IL-21/STAT3/IRF1 and CD40L/ERK signaling and PD-L1 expression; therefore, it suppressed CD8+ T cell function. Ablation of p38 in Becn1ΔM mice prevented neutrophil inflammation and B cell tumorigenesis. Importantly, the low expression of Becn1 in human neutrophils was significantly correlated with the PD-L1 levels in pre-B acute lymphoblastic lymphoma (ALL) patients. Our findings have identified myeloid Becn1 as a key regulator of cancer immunity and therapeutic target for pre-B cell lymphomas.

Keywords: B cells; Cellular immune response; Immunology; Neutrophils; Oncology.

Figure 1. Characterization and phenotypic analysis of Becn1^ΔM mice.

(A) SP size comparison between WT and Becn1^ΔM mice and SP/ body weight ratio (n = 4). H&E staining of SP sections from WT and Becn1^ΔM mice. Scale bars: 500 μm. (B) Lymphadenopathy in Becn1^ΔM mice compared with WT control. Inguinal (i), axillary (ii), and mesenteric (iii) LNs were examined. Data are representative of 3 independent experiments with 6- to 8-week-old mice (n = 2) in each group. (C) Total number of splenic CD45⁺CD11c⁺ DCs, CD4⁺ T cells, CD8⁺ T cells, B220⁺ B cells, CD11b⁺F4/80⁺ macrophages, and CD11b⁺Ly6G⁺ neutrophils from WT and Becn1^ΔM mice (n = 4). (D and E) Representative flow cytometry plots and statistical analysis of Gr-1⁺CD11b⁺ myeloid cells, Ly6G⁺CD11b⁺ neutrophils, and F4/80⁺CD11b⁺ macrophages in BM (D) and SP (E) of WT and Becn1^ΔM mice (n = 5). (F and G) Representative flow cytometry plots and statistical analysis of monocytic (Ly6C^hiLy6G^–) and granulocytic (Ly6C^intLy6G⁺) cells in BM (F) and SP (G) of 6-to 8-week-old WT and Becn1^ΔM mice (n = 5). Data represented in A and C–G were from 6- to 8-week-old mice and are presented as box plots, with lines representing median and error bars showing mean ± SEM. Statistical differences between groups were calculated using Student’s unpaired t test. *P < 0.05; **P < 0.01.

Figure 2. Cell type–specific regulation of Becn1 in proinflammatory signaling pathways and immune responses.

(A and B) Neu (A) or pMAC (B) from the periphery of WT and Becn1^ΔM mice were treated with LPS for the indicated time points, followed by IB with indicated antibodies. (C) ELISA measurement of TNF-α, IL-6, and IL-1β production by Neu and pMAC of WT and Becn1^ΔM mice treated with 100 ng/ml LPS for indicated time points (n = 4). (D) IB of pro–IL-1β expression in Neu or pMAC from WT and Becn1^ΔM mice. (E) Survival of WT and Becn1^ΔM mice (n = 10; female) treated with high-dose LPS (30 mg/kg, i.p.). (F) Plasma concentrations of TNF-α, IL-6, and IL-1β in WT or Becn1^ΔM mice (n = 5) at indicated time points after LPS treatment. (G) Survival of WT and Becn1^ΔM mice (n = 4; female) treated with PBS- or clodronate-containing liposomes to deplete macrophages or with anti-Ly6G antibody (1A8) to deplete neutrophils, followed by high-dose LPS treatment. (H) Heatmap representation of differential expressed genes in neutrophils isolated from Becn1^ΔM mice compared with WT controls: neutrophil-mediated immunity and IL-17–related cytokines (purple), chemokine receptor and neutrophil-chemotaxis (green), cell metabolism (blue). Data shown in A, B, and D are representative of 3 independent experiments with 6- to 8-week-old mice (n = 3; female) in each group. Statistical differences between groups were calculated using Student’s unpaired t test (mean ± SEM) (C and F) and Mantel-Cox log-rank test (E and G). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3. Becn1 ablation suppresses proteasomal degradation of neutrophil MEKK3 upstream of p38.

(A and B) Screening of Becn1 binding proteins in NF-κB and MAPK pathways in 293T cells. WCL, whole-cell lysate. (C) 293T cells transfected with 100 ng HA-MEKK3 along with increased amounts (0, 100, 250, and 500 ng) of FLAG-Becn1, followed by IB with indicated antibodies. (D) IB of MEKK3 protein expression in Becn1-deficient Neu and pMAC compared with WT controls. (E and F) WT or Becn1-deficient neutrophils were treated with LPS for the indicated time points, followed by IB with antibodies against MEKK3, TAK1 or p-p38 (E), and MKK3/6 signaling (F). (G) IB of 293T cells transfected with HA-MEKK3 along with empty vector or FLAG-Becn1 left untreated or treated with proteasome inhibitor MG132 (1 μM) or autophagy inhibitors 3-MA (5 mM) or CQ (10 μM). (H) IB of 293T cells transfected with WT or HA-MEKK3 (K69R, K79R, K174R, K273R, K299R, K397R, K435R) along with empty vector or FLAG-Becn1. (I) 293T cells were transfected with FLAG-MEKK3 WT or FLAG-MEKK3 K299R along with GFP-Becn1 and HA-ubiquitin K48 expression vectors, followed by immunoprecipitation with FLAG beads and IB with indicated antibodies. Data are representative of 3 independent experiments in 293T cells (A–C and G–I) and with 6- to 8-week old mice (n = 3; female) (D–F).

Figure 4. p38 or MEKK3 ablation restores Becn1^ΔM mouse phenotype so that it is similar to WT.

(A) SP size (left) and SP/body weight ratio (right) isolated from WT, Becn1^ΔM, and Becn1^ΔM:Mapk14^ΔM mice (n = 4). (B) Representative flow cytometry plots of Gr-1⁺CD11b⁺ neutrophils in WT, Becn1^ΔM, and Becn1^ΔM:Mapk14^ΔM mice. (C) ELISA measurement of TNF-α, IL-6, IL-1β, and IL-17A production in serum from WT, Becn1^ΔM, and Becn1^ΔM:Mapk14^ΔM mice treated with high-dose LPS. (D) Survival of WT, Becn1^ΔM, Becn1^ΔM:Mapk14^ΔM, and Becn1^ΔM:Il6^–/– mice treated with high-dose LPS. (E) Neutrophils isolated from WT, Becn1^ΔM, and Becn1^ΔM:Map3k3^Cas9 mice treated with LPS for the indicated time points, followed by IB with indicated antibody. (F) Representative flow cytometry plots of CD11b⁺Ly6G⁺ neutrophils in WT, Becn1^ΔM, and Becn1^ΔM:Map3k3^Cas9 mice. (G) ELISA analysis of TNF-α, IL-1β, and IL-17A production in serum from WT, Becn1^ΔM, and Becn1^ΔM:Map3k3^Cas9 mice treated with high-dose LPS. (H) Survival of WT, Becn1^ΔM, and Becn1^ΔM:Map3k3^Cas9 mice treated with high-dose LPS. Data are representative of 3 independent experiments with 6- to 8-week-old mice (n = 3; female) (B–D) and 10- to 12-week-old mice (n = 3; female) (E–G). Statistical differences between groups were calculated using 1-way ANOVA with Dunnett’s multiple comparison test (A, C, and G) and Mantel-Cox log-rank test (D and H). *P < 0.05; **P < 0.01.

Figure 5. Identification of MHC-I^hiB220^lo TdT⁺ pre-B cell lymphoma in Becn1^ΔM mice.

(A) Tumor formation in cervical LN (red arrows) in Becn1^ΔM mice. T, tumor; SLG, sublingual gland; SMG, submandibular gland. (B) Kaplan-Meier curves for tumor-free animals were calculated based on the tumor latency of the controls (n = 21; 11 females, 10 males; blue) and Becn1^ΔM mice (n = 40; 31 females, 9 males; red). (C) IHC staining of tumor sections from Becn1^ΔM tumor-bearing mice with indicated antibodies. Scale bars: 500 μm. (D) IHC staining of lung sections stained with B cell markers. T1, low antibody concentration; T2, high antibody concentration. Scale bars: 100 μm. (E) IHC staining of lung sections stained with anti–Bcl-6 (LN follicular B cells as a positive control). Scale bars: 100 μm. (F) FACS analysis of CFSE-labeled (APC-CFSE) B cells treated with LPS and IL-4 at indicated time points. (G) FACS analysis of B cell surface markers. Significant right-shift markers are indicated in red. (H) FACS analysis of cell populations in tumor with indicated antibody. (I) IHC staining of tumor sections with TdT and Ki67 antibodies. Scale bars: 100 μm. (J) FACS and statistical analysis of BM pre–pro B cell (B220⁺CD43⁺), pro–B cell (B220⁺CD43^lo), pre-B cell (B220⁺CD43^–), immature B cell (B220⁺CD43^–IgM⁺IgD^–), and mature B cell (B220⁺CD43^–IgM⁺IgD⁺) populations. (K) FACS analysis of B220⁺ B cell population in BM, SP, and LN of WT and Becn1^ΔM tumor-bearing mice. (L) Quantitative reverse-transcription PCR (qRT-PCR) of S1p1r and Cxcr4 mRNA in B220⁺ cells from WT (n = 5) and Becn1^ΔM tumor-bearing mice (n = 9). Data are presented as box plots, with error bars showing mean ± SEM (J and L) and are representative of 3 independent experiments (n = 3; female) (A, C–I, and K). Statistical differences between groups were calculated using Mantel-Cox log-rank test (B) and Student’s unpaired t test (J and L). *P < 0.05; **P < 0.01.

Figure 6. Becn1 deficiency induces B cell helper protumorigenic function of neutrophils.

(A and B) IHC staining or IF of tumor in cLN and lung stained with indicated antibody showing the interaction of B cells with neutrophils (arrows). DAPI, DNA-intercalating dye (nucleus, blue). Scale bars: 25 μm. (C) IHC staining of intestinal Peyer’s patch sections with indicated antibodies (green circle indicates B cell zone; arrowheads indicate neutrophils). Scale bars: 500 μm. Drawing illustrates a strip of neutrophils surrounding the edge of the B cell zone likely to support B cell proliferation and migration. (D) qRT-PCR of B cell helper neutrophil signature genes. (E) ELISA analysis of BAFF and IL-21 production by neutrophils after in vitro culture for 12 hours. IF shows accumulation of Gr-1⁺CD11b⁺ neutrophils in the tumor expressing IL-21. Original magnification, ×20. (F) IB of p38 activation in neutrophils from lymphoma (T) and lungs in Becn1^ΔM tumor-bearing mice compared with WT controls. N, normal. (G) Fluorescence and statistical analysis of NET formation in neutrophils using SYTOX dye (green). White arrows indicate NETs. Scale bars: 100 μm. (H) IF of tumor section with Ly6G (red) and CXCR3 (green) antibodies compared with WT LN. Scale bars: 25 μm. (I) IHC staining of tumor sections with anti-CXCL9 antibody compared with WT LN. Scale bars: 200 μm. Data are presented as box plots with error bars showing mean ± SEM (D, E, and G) and are representative of 3 independent experiments (n = 3; female) (A–C, F, H, and I). Statistical differences between groups were calculated using 1-way ANOVA with Dunnett’s multiple comparison test (D and E) and Student’s unpaired t test (G). *P < 0.05; **P < 0.01.

Figure 7. Neutrophil Becn1 levels correlate with PD-L1 in pre-B lymphoma.

(A and B) Heatmap of upregulated genes in B cells with selected genes confirmed by qRT-PCR (n = 4). (C) IB of B220⁺ cells with indicated antibodies after stimulation with CD40 agonist. (D) IB of B cells with indicated antibodies comparing B220^hi and B220^lo cells in tumor. (E) IF of tumor sections with anti–PD-L1 (red) and anti-B220 (green). Scale bars: 100 μm. (F) FACS analysis of PD-L1 expression in B cells from WT, Becn1^ΔM (T), and Becn1^ΔM: Il21^Cas9 DKO mice. (G) Immunoblot analysis of B cells from WT, Becn1^ΔM, and Becn1^ΔM: Il21^Cas9 DKO mice with indicated antibodies. (H) IF and statistical analyses of Becn1 expression in MPO⁺ cells with or without NET formation in pre-B-cell ALL compared with normal controls. Scale bars: 25 μm (normal, n = 6; pre-B-cell ALL, n = 10). Arrows indicate MPO⁺ neutrophils. (I) IHC of PD-L1 expression in pre-B cell ALL samples (n = 10) compared with normal controls (n = 6). Scale bars: 100 μm. LN, reactive lymphoid hyperplasia. (J) Correlation of IL-21, PD-L1, and neutrophil marker (LY6G6D) in B-cell ALL data set (TARGET). Data are presented as box plots with error bars showing mean ± SEM (B and H) and are representative of 3 independent experiments (n = 3; female) (C–G). Statistical analysis using 1-way ANOVA with Dunnett’s multiple comparison test (B) and Student’s unpaired t test (H). *P < 0.05; **P < 0.01; ***P < 0.001.