Progression from the Common Lymphoid Progenitor to B/Myeloid PreproB and ProB Precursors during B Lymphopoiesis Requires C/EBPα.

The C/EBPα transcription factor is required for myelopoiesis, with prior observations suggesting additional contributions to B lymphopoiesis. Cebpa expression is evident in common lymphoid progenitor (CLP) and preproB cells but is absent in proB and preB cells. We previously observed that marrow lacking the Cebpa +37 kb enhancer is impaired in producing B cells upon competitive transplantation. Additionally, a Cebpa enhancer/promoter-hCD4 transgene is expressed in B/myeloid CFU. Extending these findings, pan-hematopoietic murine Cebpa enhancer deletion using Mx1-Cre leads to expanded CLP, fewer preproB cells, markedly reduced proB and preB cells, and reduced mature B cells, without affecting T cell numbers. In contrast, enhancer deletion at the proB stage using Mb1-Cre does not impair B cell maturation. Further evaluation of CLP reveals that the Cebpa transgene is expressed almost exclusively in Flt3+ multipotent CLP versus B cell-restricted Flt3- CLP. In vitro, hCD4+ preproB cells produce both B and myeloid cells, whereas hCD4- preproB cells only produce B cells. Additionally, a subset of hCD4- preproB cells express high levels of RAG1-GFP, as seen also in proB cells. Global gene expression analysis indicates that hCD4+ preproB cells express proliferative pathways, whereas B cell development and signal transduction pathways predominate in hCD4- preproB cells. Consistent with these changes, Cebpa enhancer-deleted preproB cells downmodulate cell cycle pathways while upregulating B cell signaling pathways. Collectively, these findings indicate that C/EBPα is required for Flt3+ CLP maturation into preproB cells and then for proliferative Cebpaint B/myeloid preproB cells to progress to Cebpalo B cell-restricted preproB cells and finally to Cebpaneg proB cells.

Expression of LAG-3 and efficacy of combination treatment with anti-LAG-3 and anti-PD-1 monoclonal antibodies in glioblastoma.

Like in many tumor types, immunotherapy is currently under investigation to assess its potential efficacy in glioblastoma patients. Trials are under way to assess the efficacy of new immune checkpoint inhibitors including anti-PD-1 or CTLA4. We here investigate the expression and efficacy of a novel immune-checkpoint inhibitor, called LAG-3. We show that LAG-3 is expressed in human glioblastoma samples and in a mouse glioblastoma model we show that knock out or LAG-3 inhibition with a blocking antibody is efficacious against glioblastoma and can be used in combination with other immune checkpoint inhibitors toward complete eradication of the model glioblastoma tumors. From a mechanistic standpoint we show that LAG-3 expression is an early marker of T cell exhaustion and therefore early treatment with LAG-3 blocking antibody is more efficacious than later treatment. These data provide insight and support the design of trials that incorporate LAG-3 in the treatment of glioblastoma.

Absence of host NF-κB p50 induces murine glioblastoma tumor regression, increases survival, and decreases T-cell induction of tumor-associated macrophage M2 polarization.

High-grade gliomas harbor abundant myeloid cells that suppress anti-tumor immunity and support tumor growth. Targeting transcription factors, such as NF-κB p50, that mediate suppressive myeloid M2 polarization may prove therapeutic. GL261-Luc glioblastoma cells were inoculated into wild-type and p50-/- mice, followed by analysis of tumor growth, survival, tumor myeloid cells, and T cells. The absence of host p50 slows tumor growth and enables regression in 30% of recipients, leading to prolonged survival. Tumors developing in p50-/- mice possess a greater concentration of tumor-infiltrating myeloid cells (TIMs) than those in wild-type mice. TIMs are predominantly F4/80hi macrophages which, along with tumor-associated microglia, express increased pro-inflammatory M1 and reduced immune-suppressive M2 markers. In p50-/- mice, total tumor CD4 T cells are threefold more abundant, whereas CD8 T-cell numbers are unchanged, and both produce increased IFNγ and Granzyme B. Naïve splenic p50-/- CD8 T cells manifest increased activation, whereas naïve p50-/- and WT CD4 T cells show similar Th1, Th2, and Th17 polarization. Antibody targeting CD4, but not CD8, fully obviates the p50-/- survival advantage. Combined CD4 and CD8 T-cell depletion reverses myeloid M2 polarization in wild-type hosts, without affecting myeloid M1 polarization in p50-/- hosts. Finally, gliomas grow similarly in p50(f/f) and p50(f/f);Lysozyme-Cre mice, the latter having reduced p50 specifically in myeloid cells and tumor microglia. Thus, high-grade glioma T cells play a key role in directing M2 polarization of tumor myeloid cells, and reducing NF-κB p50 in both tumor myeloid cells and T cells may contribute to glioma therapy.

Adoptive transfer of immature myeloid cells lacking NF-κB p50 (p50-IMC) impedes the growth of MHC-matched high-risk neuroblastoma.

High-risk neuroblastomas harbor abundant myeloid cells that suppress antitumor immunity and support tumor growth. Macrophages lacking the inhibitory NF-κB p50 subunit adopt a pro-inflammatory phenotype. We now report that murine 9464D neuroblastoma cells, which express high levels of exogenous MYCN, grow slower in syngeneic p50(f/f);Lys-Cre mice that lack p50 in macrophages and neutrophils, compared with p50(f/f) littermates. Tumors in p50(f/f);Lys-Cre mice possess increased numbers of total and activated CD4+ and CD8+ T cells, and depletion of both of these T-cell populations accelerates tumor growth. Anti-PD-1 T-cell checkpoint blockade, or DNA methyltransferase and histone deacetylase inhibition, further slows tumor growth. In addition, adoptive transfer of immature myeloid cells lacking NF-κB p50 (p50-IMC), generated either from the bone marrow of p50-/- mice or via nucleofection of a p50 sgRNA:Cas9 complex into wild-type hematopoietic progenitors, also slowed growth of MHC-matched 9464D tumors but not of MHC-mismatched Neuro2A tumors. These findings further validate the utility of targeting myeloid NF-κB p50 as a strategy for cancer therapy and demonstrate activity of p50-IMC generated by gene editing of syngeneic marrow cells, a cell product relevant to clinical translation.

C/EBPα induces Ebf1 gene expression in common lymphoid progenitors.

C/EBPα is required for formation of granulocyte-monocyte progenitors (GMP) and also participates in B lymphopoiesis. The common lymphoid progenitor (CLP) and preproB populations but not proB cells express Cebpa, and pan-hematopoietic deletion of the +37 kb Cebpa enhancer using Mx1-Cre leads not only to reduced GMP but also to 2-fold reduced marrow preproB and >15-fold reduced proB and preB cells. We now show that IL7Rα-Cre-mediated deletion of the +37 kb Cebpa enhancer, which occurs in 89% of Ly6D+ and 65% of upstream Ly6D- CLP, leads to a 2-fold reduction of both preproB and proB cells, and a 3-fold reduction in preB cells, with no impact on GMP numbers. These data support a direct role for C/EBPα during B lineage development, with reduced enhancer deletion in Ly6D- CLP mediated by IL7Rα-Cre diminishing the effect on B lymphopoiesis compared to that seen with Mx1-Cre. Amongst mRNAs encoding key transcriptional regulators that initiate B lymphoid specification (PU.1, E2A, IKAROS, EBF1, FOXO1, and BACH2), only Ebf1 levels are altered in CLP upon Mx1-Cre-mediated Cebpa enhancer deletion, with Ebf1 reduced ~40-fold in Flt3+Sca-1intc-kitintIL7Rα+ CLP. In addition, Cebpa and Ebf1 RNAs were 4- and 14-fold higher in hCD4+ versus hCD4- CLP from Cebpa-hCD4 transgenic mice. Histone modification ChIP-Seq data for CLP indicate the presence of active, intronic Ebf1 enhancers located 270 and 280 kb upstream of the transcription start sites. We identified a cis element in this region that strongly binds C/EBPα using the electrophoretic mobility shift assay. Mutation of this C/EBPα-binding site in an Ebf1 enhancer-TK-luciferase reporter leads to a 4-fold reduction in C/EBPα-mediated trans-activation. These findings support a model of B lymphopoiesis in which induction of Ebf1 by C/EBPα in a subset of CLP contributes to initiation of B lymphopoiesis.

NF-κB p50-deficient immature myeloid cell (p50-IMC) adoptive transfer slows the growth of murine prostate and pancreatic ductal carcinoma.

BACKGROUND: Macrophages and dendritic cells lacking the transcription factor nuclear factor kappa B p50 are skewed toward a proinflammatory phenotype, with increased cytokine expression and enhanced T cell activation; additionally, murine melanoma, fibrosarcoma, colon carcinoma, and glioblastoma grow slower in p50-/- mice. We therefore evaluated the efficacy of p50-negative immature myeloid cells (p50-IMCs) adoptively transferred into tumor-bearing hosts. Immature cells were used to maximize tumor localization, and pretreatment with 5-fluorouracil (5FU) was examined due to its potential to impair marrow production of myeloid cells, to target tumor myeloid cells and to release tumor neoantigens. METHODS: Wild-type (WT)-IMC or p50-IMC were generated by culturing lineage-negative marrow cells from WT or p50-/- mice in media containing thrombopoietin, stem cell factor and Flt3 ligand for 6 days followed by monocyte colony-stimulating factor for 1 day on ultralow attachment plates. Mice inoculated with Hi-Myc prostate cancer (PCa) cells or K-RasG12D pancreatic ductal carcinoma (PDC)-luciferase cells received 5FU followed 5 days later by three doses of 107 immature myeloid cells (IMC) every 3-4 days. RESULTS: PCa cells grew slower in p50-/- mice, and absence of host p50 prolonged the survival of mice inoculated orthotopically with PDC cells. IMC from Cytomegalovirus (CMV)-luciferase mice localized to tumor, nodes, spleen, marrow, and lung. 5FU followed by p50-IMC slowed PCa and PDC tumor growth, ~3-fold on average, in contrast to 5FU followed by WT-IMC, 5FU alone or p50-IMC alone. Slowed tumor growth was evident for 93% of PCa but only 53% of PDC tumors; we therefore focused on PCa for additional IMC analyses. In PCa, p50-IMC matured into F4/80+ macrophages, as well as CD11b+F4/80-CD11c+ conventional dendritic cells (cDCs). In both tumor and draining lymph nodes, p50-IMC generated more macrophages and cDCs than WT-IMC. Activated tumor CD8+ T cells were increased fivefold by p50-IMC compared with WT-IMC, and antibody-mediated CD8+ T cell depletion obviated slower tumor growth induced by 5FU followed by p50-IMC. CONCLUSIONS: 5FU followed by p50-IMC slows the growth of murine prostate and pancreatic carcinoma and depends on CD8+ T cell activation. Deletion of p50 in patient-derived marrow CD34+ cells and subsequent production of IMC for adoptive transfer may contribute to the therapy of these and additional cancers.

Absence of myeloid Klf4 reduces prostate cancer growth with pro-atherosclerotic activation of tumor myeloid cells and infiltration of CD8 T cells.

The microenvironment of prostate cancer often includes abundant tumor-associated macrophages (TAMs), with their acquisition of an M2 phenotype correlating with local aggressiveness and metastasis. Tumor-derived M-CSF contributes to TAM M2 polarization, and M-CSF receptor inhibition slows prostate cancer growth in model systems. As additional cytokines can direct TAM M2 polarization, targeting downstream transcription factors could avoid resistance. Klf4 and C/EBPß each contribute to monocyte development, and reduced expression of macrophage Klf4 or C/EBPß favors their adoption of a pro-inflammatory M1 state. We find that a Hi-Myc C57BL/6 prostate cancer line grows more slowly in syngeneic Klf4(f/f);Lys-Cre compared with Klf4(f/f) mice when inoculated subcutaneously, but grows equally rapidly in C/EBPß(f/f);Lys-Cre and C/EBPß(f/f) hosts. In the absence of myeloid Klf4, TAMs have reduced expression of surface mannose receptor and Fizz1 mRNA, both M2 markers. Global gene expression analysis further revealed activation of pro-inflammatory, pro-atherosclerotic pathways. Analysis of tumor-infiltrating lymphocytes (TILs) demonstrated markedly increased activated CD8 T cell numbers, and CD8 T cell depletion obviated the inhibitory effect of myeloid Klf4 deletion on prostate cancer growth. These findings suggest that reducing expression or activity of the Klf4 transcription factor in tumor myeloid cells may contribute to prostate cancer therapy.

C/EBPß regulates sensitivity to bortezomib in prostate cancer cells by inducing REDD1 and autophagosome-lysosome fusion.

The purpose of this study was to ascertain the mechanisms by which advanced prostate cancer cells resist bortezomib therapy. Several independent studies have shown that cells are protected from proteasome inhibition by increased autophagic activity. We investigated whether C/EBPß, a transcription factor involved in the control of autophagic gene expression, regulates resistance to proteasome inhibition. In PC3 cells over-expressing C/EBPß, turnover of autophagic substrates and expression of core autophagy genes were increased. Conversely, C/EBPß knockdown suppressed autophagosome-lysosome fusion. We also found that C/EBPß knockdown suppressed REDD1 expression to delay early autophagy, an effect rescued by exogenous REDD1. Cells with suppressed C/EBPß levels showed delayed autophagy activation upon bortezomib treatment. Knockdown of C/EBPß sensitized PC3 cells to bortezomib, and blockade of autophagy by chloroquine did not further increase cell death in cells expressing shRNA targeting C/EBPß. Lastly, we observed a decreased growth of PC3 cells and xenografts with C/EBPß knockdown and such xenografts were sensitized to bortezomib treatment. Our results demonstrate that C/EBPß is a critical effector of autophagy via regulation of autolysosome formation and promotes resistance to proteasome inhibitor treatment by increasing autophagy.

The prolyl isomerase Pin1 modulates development of CD8+ cDC in mice.

BACKGROUND: Pin1 has previously been described to regulate cells that participate in both innate and adaptive immunity. Thus far, however, no role for Pin1 has been described in modulating conventional dendritic cells, innate antigen presenting cells that potently activate naïve T cells, thereby bridging innate and adaptive immune responses. METHODOLOGY/PRINCIPAL FINDINGS: When challenged with LPS, Pin1-null mice failed to accumulate spleen conventional dendritic cells (cDC). Analysis of steady-state spleen DC populations revealed that Pin1-null mice had fewer CD8+ cDC. This defect was recapitulated by culturing Pin1-null bone marrow with the DC-instructive cytokine Flt3 Ligand. Additionally, injection of Flt3 Ligand for 9 days failed to induce robust expansion of CD8+ cDC in Pin1-null mice. Upon infection with Listeria monocytogenes, Pin1-null mice were defective in stimulating proliferation of adoptively transferred WT CD8+ T cells, suggesting that decreases in Pin1 null CD8+ cDC may affect T cell responses to infection in vivo. Finally, upon analyzing expression of proteins involved in DC development, elevated expression of PU.1 was detected in Pin1-null cells, which resulted from an increase in PU.1 protein half-life. CONCLUSIONS/SIGNIFICANCE: We have identified a novel role for Pin1 as a modulator of CD8+ cDC development. Consistent with reduced numbers of CD8+ cDC in Pin1-null mice, we find that the absence of Pin1 impairs CD8+ T cell proliferation in response to infection with Listeria monocytogenes. These data suggest that, via regulation of CD8+ cDC production, Pin1 may serve as an important modulator of adaptive immunity.

Traf1 induction and protection from tumor necrosis factor by nuclear factor-kappaB p65 is independent of serine 536 phosphorylation.

Abnormal nuclear factor-kappaB (NF-kappaB) signaling has been attributed to the initiation and progression of cancer. Posttranslational modification of p65 facilitates optimal NF-kappaB signaling after activation. Here, we show that the phosphorylation of serine 536 was required for p65-mediated transcription and I kappa B alpha expression in fibroblasts. Furthermore, tumor necrosis factor (TNF) treatment slightly induced p65 phosphorylation, and both unphosphorylated and phosphorylated p65 translocated into the nucleus. The phosphorylation of serine 536 was not required for p65-mediated protection from TNF cytotoxicity and Traf1 induction in fibroblasts. Also, the corecruitment of p65 and RNA polymerase II to the Traf1 enhancer region did not require p65 phosphorylation. However, the corecruitment of p65 and RNA polymerase II to the Csf2 promoter required the phosphorylation of serine 536. These findings suggested that the requirement of serine phosphorylation at residue 536 and the distance between the NF-kappaB response element and the start of transcription may influence which genes will be transcribed.

Phosphorylation of RelA/p65 on serine 536 defines an I{kappa}B{alpha}-independent NF-{kappa}B pathway.

The association of the NF-kappaB p65/p50 dimer with IkappaBalpha plays a pivotal role in regulating its nuclear translocation and gene transcription. In addition, serine phosphorylation at various sites of the p65 subunit has been shown to be important in initiating transcription. Here we demonstrate that the regulation of nuclear translocation of p65 phosphorylated at serine 536 is not dependent on IkappaBalpha. Stimulation of either Jurkat or normal human T cells resulted in the nuclear translocation of phospho-p65 (Ser(536)). In addition, the phospho-p65 (Ser(536)) was not associated with either IkappaBalpha or p50, and the nuclear translocation of phospho-p65 (Ser(536)), but not total p65, was unaffected by the proteosome inhibitor MG-132, which blocks IkappaB protein degradation and prevents p65/p50 dimer nuclear translocation. Accordingly, the co-expression of a dominant negative mutant of IkappaBalpha blocked the transcriptional activity mediated by wild type but not the dominant positive p65 mutant (S536D). Furthermore, the transfection of the S536D form of p65 led to the induction of interleukin-8 transcription following stimulation, whereas the S536A form, which cannot be phosphorylated at this site, did not. Together, the findings suggest that p65 phosphorylated on serine 536 is not associated with or regulated by IkappaBalpha, that it has a distinct set of target genes, and that it may represent a noncanonical NF-kappaB pathway that is independent of IkappaBalpha regulation.