Up-regulation Of Blimp-1 Research Articles

A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation

Plasma cells (PC) are antibody-secreting cells and terminal effectors in humoral responses. PCs differentiate directly from activated B cells in response to T cell-independent (TI) antigens or from germinal center B (GCB) cells in T cell-dependent (TD) antigen-induced humoral responses, both of which pathways are essentially regulated by the transcription factor BLIMP1. The p38 mitogen-activated protein kinase isoforms have already been implicated in B cell development, but the precise role of p38α in B cell differentiation is still largely unknown. Here we show that PC differentiation and antibody responses are severely impaired in mice with B cell-specific deletion of p38α, while B cell development and the GCB cell response are spared. By utilizing a Blimp1 reporter mouse model, we show that p38α-deficiency results in decreased BLIMP1 expression. p38α-driven BLIMP1 up-regulation is required for both TI and TD PCs differentiation. By combining CRISPR/Cas9 screening and other approaches, we identify TCF3, TCF4 and IRF4 as downstream effectors of p38α to control PC differentiation via Blimp1 transcription. This study thus identifies an important signalling pathway underpinning PC differentiation upstream of BLIMP1, and points to a highly specialized and non-redundant role for p38α among p38 isoforms.

Blimp-1 Upregulation by Multiple Ligands via EGFR Transactivation Inhibits Cell Migration in Keratinocytes and Squamous Cell Carcinoma.

B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcriptional repressor and plays a crucial role in the regulation of development and functions of various immune cells. Currently, there is limited understanding about the regulation of Blimp-1 expression and cellular functions in keratinocytes and cancer cells. Previously we demonstrated that EGF can upregulate Blimp-1 gene expression in keratinocytes, playing a negative role in regulation of cell migration and inflammation. Because it remains unclear if Blimp-1 can be regulated by other stimuli beyond EGF, here we further investigated multiple stimuli for their regulation of Blimp-1 expression in keratinocytes and squamous cell carcinoma (SCC). We found that PMA, TNF-α, LPS, polyIC, H2O2 and UVB can upregulate the protein and/or mRNA levels of Blimp-1 in HaCaT and SCC cells. Concomitant EGFR activation was observed by these stimuli, and EGFR inhibitor gefitinib and Syk inhibitor can block Blimp-1 gene expression caused by PMA. Reporter assay of Blimp-1 promoter activity further indicated the involvement of AP-1 in PMA-, TNF-α-, LPS- and EGF-elicited Blimp-1 mRNA expression. Confocal microscopic data indicated the nuclear loclization of Blimp-1, and such localization was not changed by stimuli. Moreover, Blimp-1 silencing enhanced SCC cell migration. Taken together, Blimp-1 can be transcriptionally upregulated by several stimuli in keratinocytes and SCC via EGFR transactivation and AP-1 pathway. These include growth factor PMA, cytokine TNF-α, TLR ligands (LPS and polyIC), and ROS insults (H2O2 and UVB). The function of Blimp-1 as a negative regulator of cell migration in SCC can provide a new therapeutic target in SCC.

TGF-\u03b21 increases viral burden and promotes HIV-1 latency in primary differentiated human bronchial epithelial cells

Combination antiretroviral therapy (cART) has increased the life expectancy of HIV patients. However, the incidence of non-AIDS associated lung comorbidities, such as COPD and asthma, and that of opportunistic lung infections have become more common among this population. HIV proteins secreted by the anatomical HIV reservoirs can have both autocrine and paracrine effects contributing to the HIV-associated comorbidities. HIV has been recovered from cell-free bronchoalveolar lavage fluid, alveolar macrophages, and intrapulmonary lymphocytes. We have recently shown that ex-vivo cultured primary bronchial epithelial cells and the bronchial brushings from human subjects express canonical HIV receptors CD4, CCR5 and CXCR4 and can be infected with HIV. Together these studies suggest that the lung tissue can serve as an important reservoir for HIV. In this report, we show that TGF-β1 promotes HIV latency by upregulating a transcriptional repressor BLIMP-1. Furthermore, we identify miR-9-5p as an important intermediate in TGF-β-mediated BLIMP-1 upregulation and consequent HIV latency. The transcriptionally suppressed HIV can be reactivated by common latency reactivating agents. Together our data suggest that in patients with chronic airway diseases, TGF-β can elevate the HIV viral reservoir load that could further exacerbate the HIV associated lung comorbidities.

Teamwork by IL-10+ and IL-35+ Tregs

Distinct populations of IL-10– and IL-35–producing Treg cells in the tumor microenvironment cooperatively promote upregulation of BLIMP1 and exhaustion of tumor-infiltrating conventional T cells. Distinct populations of IL-10– and IL-35–producing Treg cells in the tumor microenvironment cooperatively promote upregulation of BLIMP1 and exhaustion of tumor-infiltrating conventional T cells. CITATION Sawant DV, Yano H, Chikina M, et al. Adaptive plasticity of IL-10+ and IL-35+ Treg cells cooperatively promotes tumor T cell exhaustion. Nat Immunol. 2019;20(6):724-735. FoxP3+ regulatory T cells (Tregs) are key mediators of self-tolerance and can therapeutically induce tolerance to transplanted organs. Tregs utilize multiple mechanisms to suppress activity of conventional T cells (Tconvs), including metabolic disruption of target cells, surface expression of inhibitory molecules, and secretion of immunosuppressive cytokines, such as interleukin (IL)-10, transforming growth factor (TGF)-β and IL-35. By suppressing self- or donor-reactive Tconvs, Tregs prevent pathological immune responses. However, these suppressive functions become harmful when Tregs inhibit antitumor immunity. Broadly disrupting Treg function in cancer patients to unleash antitumor immunity carries a risk of severe autoimmune complications. Thus, recent work has sought to identify and disrupt suppressive mechanisms employed by Tregs specifically within the tumor microenvironment (TME). Indeed, Tregs have been shown to use specific suppressive mechanisms in a tissue- and context-dependent manner, and, within a particular tissue, Tregs have heterogeneous functional profiles. Sawant and colleagues reveal that within the TME, IL-10 and IL-35 production are segregated into distinct Treg subtypes and the production of both of these cytokines by Tregs is required to maximally promote BLIMP1 expression and exhaustion in tumor-infiltrating Tconvs. Using mice engineered to indicate the expression of FoxP3, IL-10 and a subunit of IL-35 (Ebi3), the authors determined that distinct populations of FoxP3+ cells produced IL-10 and IL-35, with very few cells producing both cytokines simultaneously. This was evident in Tregs isolated from mouse lymph nodes, tumors and lungs as well as human peripheral blood and tumors. While both IL-10– and IL-35–producing Tregs were enriched in the TME compared with tumor-draining lymph nodes, IL-35–producing T cells were the most enriched, suggesting that targeting IL-35–producing Tregs may specifically disrupt immune evasion by the tumor without promoting severe autoimmunity. Both subsets exhibited the ability to interconvert after stimulation in vitro, although more work is needed to determine whether they exhibit plasticity in vivo and which signals direct cells to develop into either subset. To determine the functional role of each Treg subset, the authors used mice with conditional deletion of IL-10 or Ebi3 in FoxP3+ cells. Treg-specific deletion of either cytokine resulted in similarly reduced tumor growth and a decrease in inhibitory receptor (PD-1, TIM3, LAG3, TIGIT and 2B4) expression in tumor-infiltrating Tconvs. Interestingly, the combined conditional deletion of both cytokines did not further reduce tumor growth, indicating that Treg-derived IL-10 and IL-35 play partially overlapping noncompensatory roles in promoting tumor growth. As for cytokine-specific functions, IL-35 better promoted inhibitory receptor expression and reduced differentiation into central memory cells among tumor-infiltrating CD4+ and CD8+ Tconvs, while only IL-10 modestly suppressed effector cytokine production. Both IL-10 and IL-35 contributed to an exhausted transcriptional signature in tumor-infiltrating CD8+ T cells, including upregulation of Prdm1 (BLIMP1) expression in PD1hi TIM3+ tumor-infiltrating cells. The authors found that mediators of IL-35 signaling (STAT1 and STAT4), and to a lesser extent IL-10 signaling (STAT3), exhibited greater binding to the Prdm1 locus in CD8+ T cells after in vitro stimulation, suggesting that these cytokines promote Tconv exhaustion by cooperatively promoting Prdm1 expression. Prdm1 did appear to play a critical role in Tconv exhaustion, because deletion of one Prdm1 allele in CD8+ T cells was associated with reduced expression of inhibitory receptors in tumor-infiltrating cells as well as slower tumor growth. Whether modulation of BLIMP1 expression is the main mechanism of IL-10 and IL-35 promotion of tumor growth and immune evasion remains to be determined. In transplantation, induction of allospecific Tregs and dysfunction of allospecific Tconvs have been associated with a state of donor-specific tolerance. Adoptive transfer of Tregs is under investigation for use as an immunosuppressive therapy in transplant recipients as well as patients with autoimmune diseases, with studies seeking to optimize the suppressive function of transferred cells. The study of Sawant et al. suggests that multiple subsets of Tregs, including both IL-10– and IL-35–producing cells, may play different roles. Likewise, strategies for allospecific Treg induction may be most beneficial if they induce multiple subsets of Tregs.

Toxoplasma gondii: CD8 T Cells Cry for CD4 Help.

Toxoplasma gondii, an apicomplexan parasite, is a pathogenic protozoan that can infect the central nervous system. In pregnant women, infection can result in congenital problems of the fetus, while in immunocompromised individual it can lead to severe neurological consequences. Although CD8 T cells play an important effector role in controlling the chronic infection, their maintenance is dependent on the critical help provided by CD4 T cells. In a recent study, we demonstrated that reactivation of the infection in chronically infected host is a consequence of CD8 T dysfunction caused by CD4 T cell exhaustion. Furthermore, treatment of chronically infected host with antigen-specific non-exhausted CD4 T cells can restore CD8 T cell functionality and prevent reactivation of the latent infection. The exhaustion status of CD4 T cells is mediated by the increased expression of the transcription factor BLIMP-1, and deletion of this molecule led to the restoration of CD4 T cell function, reversal of CD8 exhaustion and prevention of reactivation of the latent infection. In a recent study from our laboratory, we also observed an increased expression of miR146a levels by CD4 T cells from the chronically infected animals. Recent reports have demonstrated that microRNAs (especially miR146a) has a strong impact on the immune system of T. gondii infected host. Whether these molecules have any role in the BLIMP-1 up-regulation and dysfunctionality of these cells needs to be investigated.

T-bet Transcription Factor Promotes Antibody-Secreting Cell Differentiation by Limiting the Inflammatory Effects of IFN-γ on B Cells

Although viral infections elicit robust interferon-γ (IFN-γ) and long-lived antibody-secreting cell (ASC) responses, the roles for IFN-γ and IFN-γ-induced transcription factors (TFs) in ASC development are unclear. We showed that B cell intrinsic expression of IFN-γR and the IFN-γ-induced TF T-bet were required for T-helper 1 cell-induced differentiation of B cells into ASCs. IFN-γR signaling induced Blimp1 expression in B cells but also initiated an inflammatory gene program that, if not restrained, prevented ASC formation. T-bet did not affect Blimp1 upregulation in IFN-γ-activated B cells but instead regulated chromatin accessibility within the Ifng and Ifngr2 loci and repressed the IFN-γ-induced inflammatory gene program. Consistent with this, B cell intrinsic T-bet was required for formation of long-lived ASCs and secondary ASCs following viral, but not nematode, infection. Therefore, T-bet facilitates differentiation of IFN-γ-activated inflammatory effector B cells into ASCs in the setting of IFN-γ-, but not IL-4-, induced inflammatory responses.

Blimp-1-mediated CD4 T cell exhaustion causes CD8 T cell dysfunction during chronic toxoplasmosis.

CD8, but not CD4, T cells are considered critical for control of chronic toxoplasmosis. Although CD8 exhaustion has been previously reported in Toxoplasma encephalitis (TE)-susceptible model, our current work demonstrates that CD4 not only become exhausted during chronic toxoplasmosis but this dysfunction is more pronounced than CD8 T cells. Exhausted CD4 population expressed elevated levels of multiple inhibitory receptors concomitant with the reduced functionality and up-regulation of Blimp-1, a transcription factor. Our data demonstrates for the first time that Blimp-1 is a critical regulator for CD4 T cell exhaustion especially in the CD4 central memory cell subset. Using a tamoxifen-dependent conditional Blimp-1 knockout mixed bone marrow chimera as well as an adoptive transfer approach, we show that CD4 T cell-intrinsic deletion of Blimp-1 reversed CD8 T cell dysfunction and resulted in improved pathogen control. To the best of our knowledge, this is a novel finding, which demonstrates the role of Blimp-1 as a critical regulator of CD4 dysfunction and links it to the CD8 T cell dysfunctionality observed in infected mice. The critical role of CD4-intrinsic Blimp-1 expression in mediating CD4 and CD8 T cell exhaustion may provide a rational basis for designing novel therapeutic approaches.

PAX5 tyrosine phosphorylation by SYK co-operatively functions with its serine phosphorylation to cancel the PAX5-dependent repression of BLIMP1: A mechanism for antigen-triggered plasma cell differentiation

Plasma cell differentiation is initiated by antigen stimulation of the B cell receptor (BCR) and is regulated by BLIMP1. Prior to the stimulation of BCR, BLIMP1 is suppressed by PAX5, which is a key transcriptional repressor that maintains B cell identity. The upregulation of BLIMP1 and subsequent suppression of PAX5 by BLIMP1 are observed after the BCR stimulation. These events are considered to trigger plasma cell differentiation; however, the mechanisms responsible currently remain unclear. We herein demonstrated that the BCR signaling component, SYK, caused PAX5 tyrosine phosphorylation in vitro and in cells. Transcriptional repression on the BLIMP1 promoter by PAX5 was attenuated by this phosphorylation. The BCR stimulation induced the phosphorylation of SYK, tyrosine phosphorylation of PAX5, and up-regulation of BLIMP1 mRNA expression in B cells. The tyrosine phosphorylation of PAX5 co-operatively functioned with PAX5 serine phosphorylation by ERK1/2, which was our previous findings, to cancel the PAX5-dependent repression of BLIMP1. This co-operation may be a trigger for plasma cell differentiation. These results imply that PAX5 phosphorylation by a BCR signal is the initial event in plasma cell differentiation.

Inflammasome-independent role of NLRP12 in suppressing colonic inflammation regulated by Blimp-1.

NLRP12 is a member of the Nod-like receptor (NLR). Previous studies have reported enhanced colitis-associated inflammatory responses in NLRP12-deficient mice. In this study, we sought to investigate the role of NLRP12 in DSS-stimulated proinflammatory response in dendritic cells and mice colitis, and the molecular mechanisms involved in the development of the inflammation. Our results showed that down-regulation of NLRP12 is required for DSS-induced release of proinflammatory cytokines IL-1β and TNF-α; that PR domain zinc finger protein 1 (also known as Blimp-1) induces NLRP12 down-regulation during DSS-induced proinflammatory response and colitis; and that TLR4 is implicated in the up-regulation of Blimp-1 that led to the down-regulation of NLRP12 expression in DSS-induced colitis. Taken together, the results suggest that the TLR4-Blimp-1 axis promotes DSS induced experimental colitis through the down-regulation of NLRP12.

CD28 Promotes Plasma Cell Survival, Sustained Antibody Responses, and BLIMP-1 Upregulation through Its Distal PYAP Proline Motif.

In health, long-lived plasma cells (LLPC) are essential for durable protective humoral immunity, and, conversely, in disease are a major source of pathogenic Abs in autoimmunity, graft rejection, and allergy. However, the molecular basis for their longevity is largely unknown. We have recently found that CD28 signaling in plasma cells (PC) is essential for sustaining Ab titers, by supporting the survival of LLPC, but not short-lived PC (SLPC). We now find that, unlike SLPC, CD28 activation in LLPC induces prosurvival downstream Vav signaling. Knockin mice with CD28 cytoplasmic tail mutations that abrogate Vav signaling (CD28-AYAA) had significantly fewer LLPC but unaffected SLPC numbers, whereas mice with mutations that abrogate PI3K signaling (CD28-Y170F) were indistinguishable from wild-type controls. This was consistent with the loss of CD28's prosurvival effect in LLPC from CD28-AYAA, but not CD28-Y170F, mice. Furthermore, the CD28 Vav motif in the B lineage was essential for the long-term maintenance of Ag-specific LLPC populations and Ab titers in vivo. Signaling downstream of the CD28 Vav motif induced previously undescribed transcriptional regulation of B lymphocyte-induced maturation protein-1, a key mediator of PC differentiation and maintenance. These findings suggest CD28 signaling in LLPC modulates the central B lymphocyte-induced maturation protein-1 transcriptional nexus involved in long-term survival and function.

PAX5 Tyrosine Phosphorylation By Syk Co-Operatively Works with Its Serine Phosphorylation By ERK1/2 and Cancels PAX5-Dependent Repression of BLIMP1: A Mechanism of Antigen-Triggering Plasma Cell Differentiation through B Cell Receptor Signal

Pax5 is an essential transcription factor to maintain B cell identity. Pax5 is expressed in stages from pro-B to mature B cells and promotes the B cell differentiation program by transcriptional activation of many B cell receptor (BCR)-related genes such as CD19, CD79a, and BLNK. On the contrary, it inhibits plasma cell differentiation by suppressing the expression of BLIMP1 and XBP-1, transcription factors essential for plasma cell differentiation. After BCR stimulation by antigen, upregulation of BLIMP1 and XBP-1 and subsequent suppression of PAX5 by BLIMP1 were observed and thought to be the trigger of plasma cell differentiation. We previously demonstrated that serine phosphorylation of PAX5 by ERK1/2, a main component of BCR signal, attenuated the BLIMP1 suppression by PAX5 and that the PAX5 phosphorylation might be the initial event for plasma cell differentiation (Yasuda T et al, J Immunol. 2012; 188: 6127-34). Here, we investigated additional PAX5 phosphorylation by BCR signal and found that another BCR signal component, Syk, caused PAX5 phosphorylation in vitro (Figure A). We identified the tyrosines that were phosphorylated by Syk in vitro by making phosphorylation-defective mutants, and confirmed that Syk phosphorylated PAX5 at the same sites in vivo (Figure B). In the luciferase reporter assays, PAX5 tyrosine phosphorylation by Syk attenuated the BLIMP1 suppression by PAX5, similarly to its serine phosphorylation by ERK1/2, and both phosphorylations co-operatively worked for it (Figure C). Furthermore, we demonstrated that B cell receptor stimulation with anti-IgM antibody induced Syk and ERK1/2 activation, tyrosine and serine phosphorylation of endogenous Pax5, and upregulation of Blimp1 mRNA. These results suggested that PAX5 phosphorylations by Syk and ERK1/2 co-operatively work for the cancelation of transcriptional repression of Blimp1 by PAX5 after BCR activation by antigen. This might be a trigger of plasma cell differentiation. Our findings give a new insight into the regulation of the terminal differentiation of B cells. [Display omitted] DisclosuresNaoe:Zenyaku Kogyo: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Kyowa Hakko Kirin Co. LTD: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Novartis Pharma,: Research Funding; Bristol-Myers Squibb: Research Funding; Otsuka Pharmaceutical Co. LTD: Research Funding; FUJIFILM Corporation: Research Funding. Kiyoi:Zenyaku Kogyo: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Kyowa Hakko Kirin Co. LTD.: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Bristol-Myers Squibb: Research Funding; FUJIFILM Corporation: Research Funding.

CD28 Regulates Mitochondrial Metabolism in Long Lived Plasma Cells for Survival

Sustained humoral immunity is dependent upon the ability of plasma cells to produce antigen specific antibody titers over a long period of time. Whether fighting against pathogens such as Ebola, Influenza, or the common cold, the continual presence of neutralizing antibodies in circulation is critical for an effective humoral immune response. When activated by antigen, B cells differentiate into a short lived plasma cell (SLPC) pool and reside in secondary lymphoid organs such as the spleen for days to weeks before dying by apoptosis. However, in the absence of constant antigenic presence there are examples of continual production of antigen-specific antibodies in the human body. To explain this, a different subset of long lived plasma cells (LLPC) has been proposed wherein the plasma cells compete for space in specialized bone marrow niches. These cells are not intrinsically long lived, but rather depend upon extrinsic survival factors for their persistence. Many of the survival factors for LLPCs in the bone marrow are shared with their malignant counterpart, e.g. multiple myeloma (MM). Our work centers on the elucidation of mechanisms by which both MM and LLPCs survive in the bone marrow microenvironment.Recently our lab has demonstrated a cell intrinsic role for CD28 signaling in the survival of both LLPCs and MM. CD28 is known as the canonical T cell co-stimulatory molecule and is required for effector T cell metabolic fitness. Under nutrient deprivation and chemotherapeutic challenge, CD28 is able to induce survival of LLPCs and MM cells respectively. However, the molecular and metabolic pathways that govern this prosurvival effect are not well understood.Here we demonstrate that CD28 induces mitochondrial respiration in bone marrow resident LLPCs but not in splenic SLPCs by staining with a mitochondrial-specific dye that is taken up in proportion to the mitochondrial membrane potential. A major byproduct of mitochondrial respiration is the production of reactive oxygen species (ROS). As a result of increased mitochondrial metabolism through the electron transport chain, CD28 is able to induce ROS in LLPCs but not in SLPCs. This is somewhat counterintuitive, in that ROS are well-characterized as cell damaging agents. Fascinatingly, mitochondrial respiration dependent ROS production downstream of CD28 is required for the prosurvival effect seen in LLPCs. Mechanistically, CD28-mediated production of ROS drive NFkB translocation as seen by ImageStream technology, which goes on to drive Blimp1 expression, the master transcriptional regulator of plasma cell identity. Utilizing a luciferase expressing plasmid and including different lengths of the Blimp1 promoter, we show that the CD28 responsive element lies from 4500 to 7500 base pairs from the transcriptional start site. Furthermore, we are able to demonstrate by CHIP that NFkB binds directly to the Blimp1 promoter.In order to understand why this occurs in MM and bone marrow resident LLPCs but not splenic derived SLPCs, we made use of in silico and genetic approaches to discover how the cells differentially signal through CD28. We demonstrate that the Grb2-Vav binding domain in the cytoplasmic tail of CD28 is critical for its prosurvival signal. Vav is known to bind the major adaptor molecule Slp-76. Using transcriptomic analysis we demonstrate that in humans, the major adaptor molecule Slp-76 is highly expressed in LLPCs but not SLPCs. A major downstream target of Slp-76 is PLC-g1 which is phosphorylated by CD28 activation in LLPCs but not SLPCs. To demonstrate that signals emanating from Slp-76 drive LLPC survival, we made use of Slp-76 mutant mice wherein the Vav binding domain is mutated. These mice had lower LLPC numbers and could not transduce a CD28 prosurvival signal. Furthermore, genetic knockdown of Slp-76 diminished the ability of CD28 to induce Blimp1 upregulation. Altogether these data suggest that CD28, through a Grb2-Vav-Slp-76 signal induces mitochondrial respiration dependent production of ROS. These ROS go on to activate NFkB mediated induction of Blimp1, thereby reinforcing the plasma cell phenotype for survival and function. This knowldedge will augment our ability to create effective vaccines as well as disrupt antibody-mediated autoimmunity and multiple myeloma progression in patients. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1691: Synergistic anti-tumor activity of lenalidomide with the BET bromodomain inhibitor CPI203 in bortezomib-resistant mantle cell lymphoma

Abstract Mantle cell lymphoma (MCL) is an aggressive B lymphoid neoplasm genetically characterized by the t(11;14)(q13;q32) leading to the overexpression of cyclin D1. As a consequence of its poor responses to conventional chemotherapy and relatively short patient survival, new therapeutic strategies are required. Despite the promising introduction of the proteasome inhibitor bortezomib in the treatment of MCL, not all the patients respond and relapse frequently occurs. To unravel the factors involved in the acquisition of bortezomib resistance in vivo, immunodeficient mice were engrafted with a set of MCL cell lines with different levels of sensitivity to the drug, followed by gene expression profiling of the tumors and functional validation of the identified gene signatures. We observed an increased tumorigenicity of bortezomib-resistant MCL cells in vivo, that was associated with plasmacytic differentiation features, like IRF4 and Blimp-1 upregulation. As the immunomoduladory drug lenalidomide has been shown to modulate IRF4 expression in various B-cell malignancies, we assessed its activity in in vitro and in vivo settings by means of flow cytometry, western blot, antibody array, real-time PCR, immunofluorescence, in vivo imaging, and immunohistochemistry. In vitro, lenalidomide as single agent was found to exert antitumor activity in 4/11 MCL cell lines, corresponding to those cells with either primary or acquired resistance to bortezomib. Lenalidomide-treated cells showed decreased IRF4 expression, increased cytosolic amounts of p27 and caspase-dependent apoptosis. Accordingly, mice bearing bortezomib-resistant tumors and treated for 3 weeks with a lenalidomide regimen of 10-50 mg/kg/day, showed a 30 to 45% reduction in tumor burden when compared to vehicle-treated mice (p=0.04), with several hallmarks of lenalidomide activity, like downregulation of IRF4 and its target gene MYC, decreased mitotic index, p27 cytosolic accumulation and caspase-3 processing. Importantly, the inhibition of tumor growth induced by the combination of lenalidomide with bortezomib (0.15 mg/kg, twice a week) was 37% and 66% greater than that for lenalidomide alone and vehicle arms, respectively (p=0.02). Moreover, repression of MYC in bortezomib-resistant cells by gene knockdown or treatment with CPI203, a BET bromodomain inhibitor, synergistically induced cell death when combined with lenalidomide therapy. Accordingly, co-treatment of mice with lenalidomide plus CPI203 synergistically reduced MYC and IRF4 expression and tumor burden, and induced caspase processing. Together, these results suggest that exacerbated IRF4/MYC signaling is associated to bortezomib resistance in MCL in vivo and warrant clinical evaluation of lenalidomide plus BET inhibitor combination in MCL cases refractory to proteasome inhibition. Citation Format: Alexandra Moros, Vanina Rodriguez, Ifigenia Saborit-Villarroya, Arnau Montraveta, Patricia Balsas, Peter Sandy, Antonio Martinez, Emmanuel Normant, Patricia Perez-Galan, Elias Campo, Dolors Colomer, Gael Roue. Synergistic anti-tumor activity of lenalidomide with the BET bromodomain inhibitor CPI203 in bortezomib-resistant mantle cell lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1691. doi:10.1158/1538-7445.AM2014-1691

Germinal center B cell maintenance and differentiation are controlled by distinct NF-κB transcription factor subunits.

Germinal centers (GCs) are the sites where memory B cells and plasma cells producing high-affinity antibodies are generated during T cell-dependent immune responses. The molecular control of GC B cell maintenance and differentiation remains incompletely understood. Activation of the NF-κB signaling pathway has been implicated; however, the distinct roles of the individual NF-κB transcription factor subunits are unknown. We report that GC B cell-specific deletion of the NF-κB subunits c-REL or RELA, which are both activated by the canonical NF-κB pathway, abolished the generation of high-affinity B cells via different mechanisms acting at distinct stages during the GC reaction. c-REL deficiency led to the collapse of established GCs immediately after the formation of dark and light zones at day 7 of the GC reaction and was associated with the failure to activate a metabolic program that promotes cell growth. Conversely, RELA was dispensable for GC maintenance but essential for the development of GC-derived plasma cells due to impaired up-regulation of BLIMP1. These results indicate that activation of the canonical NF-κB pathway in GC B cells controls GC maintenance and differentiation through distinct transcription factor subunits. Our findings have implications for the role of NF-κB in GC lymphomagenesis.

CD28 induces mitochondrial respiration in long lived plasma cells for reactive oxygen species dependent survival and NFκB-mediated Blimp1 expression. (LYM6P.771)

Abstract Sustained humoral immunity is dependent upon bone marrow resident long-lived plasma cells (LLPCs). We have shown that CD28 signaling in LLPCs is necessary for their survival and antibody production; however, the signaling pathways that define LLPC survival and function are not completely understood. In T cells, CD28 is known to induce glycolysis at the expense of mitochondrial respiration. To our great surprise, CD28 induces mitochondrial respiration-dependent reactive oxygen species (ROS) through PLCγ1 in LLPCs but not splenic short lived plasma cells (SLPCs). Paradoxically, inhibition of ROS kills LLPCs while promoting survival of SLPCs. In LLPCs, CD28-mitochondrial ROS activates NFκB, which in turn binds directly to the Blimp1 promoter, the master regulator of plasma cell identity. Furthermore, CD28 induces Blimp1 expression in LLPCs but not SLPCs. These data suggest a model wherein CD28-induced mitochondrial respiration drives survival of LLPCs through mitochondrial ROS-dependent activation of NFκB and subsequent Blimp1 upregulation. By understanding CD28 regulation of LLPC metabolism, new therapeutic targets arise for antibody-mediated autoimmune disease and vaccine design.

Synergistic antitumor activity of lenalidomide with the BET bromodomain inhibitor CPI203 in bortezomib-resistant mantle cell lymphoma.

Bortezomib therapy has shown promising clinical activity in mantle cell lymphoma (MCL), but the development of resistance to proteasome inhibition may limit its efficacy. To unravel the factors involved in the acquisition of bortezomib resistance in vivo, immunodeficient mice were engrafted with a set of MCL cell lines with different levels of sensitivity to the drug, followed by gene expression profiling of the tumors and functional validation of the identified gene signatures. We observed an increased tumorigenicity of bortezomib-resistant MCL cells in vivo, which was associated with plasmacytic differentiation features, like interferon regulatory factor 4 (IRF4) and Blimp-1 upregulation. Lenalidomide was particularly active in this subgroup of tumors, targeting IRF4 expression and plasmacytic differentiation program, thus overcoming bortezomib resistance. Moreover, repression of the IRF4 target gene MYC in bortezomib-resistant cells by gene knockdown or treatment with CPI203, a BET (bromodomain and extra terminal) bromodomain inhibitor, synergistically induced cell death when combined with lenalidomide. In mice, addition of CPI203 to lenalidomide therapy further decreased tumor burden, involving simultaneous MYC and IRF4 downregulation and apoptosis induction. Together, these results suggest that exacerbated IRF4/MYC signaling is associated to bortezomib resistance in MCL in vivo and warrant clinical evaluation of lenalidomide plus BET inhibitor combination in MCL cases refractory to proteasome inhibition.

Downregulation Of FOXO1 Prevents Induction Of The Tumor Suppressor BLIMP-1 In Classical Hodgkin Lymphoma

In contrast to other B cell lymphomas, Hodgkin and Reed-Sternberg (HRS) cells, the malignant cells of classical Hodgkin lymphoma (cHL), have mainly lost their B cell identity. Recently, we reported that the transcription factor FOXO1, indispensable for B cell development and differentiation, is downregulated in HRS cells and expression of FOXO1 results in growth arrest and apoptosis in cHL cell lines.To find molecular targets of FOXO1 we performed gene expression profiling of 5 cHL cell lines expressing constitutively active FOXO1 fused to ligand binding domain of estrogen receptor (FOXO1(A3)ER).We found that FOXO1 activation led to downregulation of genes, which are normally upregulated in cHL, such as CD30/TNFRSF8 and the proto-oncogene MYC. On the other hand, FOXO1 activated expression of germinal center-specific genes including BCL6, AICDA, BACH2, and GCET2. The most surprising finding was induction of BLIMP-1/PRDM1, the master regulator of plasma cell differentiation and tumor suppressor in activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL). A positive correlation of FOXO1 and BLIMP-1 expression in microdissected HRS cells further indicated a role for FOXO1 in BLIMP-1 regulation.Of note, HRS cells do not undergo plasma cell differentiation despite constitutive expression of NF-κB, IRF4, and STAT3, known inducers of BLIMP-1. However, BLIMP-1 levels remain low in HRS cells thereby preventing terminal differentiation. The mechanisms leading to BLIMP-1 repression in cHL are poorly understood. We found that in most cHL cell lines, FOXO1 specifically upregulated BLIMP-1α representing the full-length isoform of BLIMP-1. In addition, luciferase reporter assay showed that FOXO1 activates the BLIMP-1α promoter in the cHL cell line L428. Overexpression of BLIMP-1α using a lentiviral vector strongly inhibited growth of cHL cell lines. In line with the fact that BLIMP-1 and MYC form a negative feedback loop, we found that ectopic BLIMP-1α expression resulted in downregulation of MYC protein levels, which most likely contributes to the anti-tumor effect of BLIMP-1α. On the other hand, MYC inhibition in cHL cell lines led to BLIMP-1 upregulation. Thus, our data indicate that FOXO1, BLIMP-1 and MYC constitute a negative feed-forward regulatory loop, which is controlled by FOXO1.Searching for additional mechanisms of BLIMP-1α downregulation in cHL we found that BLIMP-1α promoter was hypermethylated in two cHL cell lines but not in microdissected HRS cells. This indicates that hypermethylation is not critical for BLIMP-1α repression.Interestingly, the functionally impaired BLIMP-1 variant BLIMP-1β, which is normally not detected in normal B cells, was upregulated in cHL cell lines. BLIMP-1β is also expressed in ABC-DLBCL and has been suggested to block BLIMP-1α in a dominant-negative manner. We speculate that BLIMP-1β attenuates function of residual BLIMP-1α in cHL. However, further investigation is required.Taken together, we show for the first time that FOXO1 induces expression of BLIMP-1. We identified BLIMP-1α as a tumor suppressor which downregulates expression of the proto-oncogene MYC in cHL. Therefore, FOXO1 might exert its tumor suppressor function at least in part by upregulation of BLIMP-1α.Further work on BLIMP-1α regulation by FOXO1 and investigation of a potential role of FOXO1 in plasma cell differentiation is warranted. Disclosures:No relevant conflicts of interest to declare.

IL-21 and CD40L Synergistically Promote Plasma Cell Differentiation through Upregulation of Blimp-1 in Human B Cells

After undergoing Ig somatic hypermutation and Ag selection, germinal center (GC) B cells terminally differentiate into either memory or plasma cells (PCs). It is known that the CD40L and IL-21/STAT3 signaling pathways play critical roles in this process, yet it is unclear how the B cell transcription program interprets and integrates these two types of T cell-derived signals. In this study, we characterized the role of STAT3 in the GC-associated PC differentiation using purified human tonsillar GC B cells and a GC B cell-like cell line. When primary GC B cells were cultured under PC differentiation condition, STAT3 inhibition by AG490 prevented the transition from GC centrocytes to preplasmablast, suggesting that STAT3 is required for the initiation of PC development. In a GC B cell-like human B cell line, although IL-21 alone can induce low-level Blimp-1 expression, maximum Blimp-1 upregulation and optimal PC differentiation required both IL-21 and CD40L. CD40L, although having no effect on Blimp-1 as a single agent, greatly augmented the amplitude and duration of IL-21-triggered Jak-STAT3 signaling. In the human PRDM1 locus, CD40L treatment enhanced the ability of STAT3 to upregulate Blimp-1 by removing BCL6, a potent inhibitor of Blimp-1 expression, from a shared BCL6/STAT3 site in intron 3. Thus, IL-21 and CD40L collaborate through at least two distinct mechanisms to synergistically promote Blimp-1 activation and PC differentiation.

Frontiers in B-cell immunology

Frontiers in B-cell immunology

IRF-2 regulates B-cell proliferation and antibody production through distinct mechanisms

Interferon regulatory factor (IRF)-2 is a transcription factor involved in type I (IFN- α/β) signaling. It has been reported that IRF-2 deficiency results in various immune dysfunctions. However, the role of IRF-2 in B-cell functions needs to be elucidated. Unlike wild-type (WT) B cells, IRF-2(-/-) B2 cells were refractory to anti-IgM, but not LPS. Such a defect in proliferation was dependent on IFN- α/β receptor (IFNAR). Marginal zone B cells increased in the proportion relative to B2 cells in IRF-2(-/-) mice produced IgM normally to LPS stimulation. However, IRF-2(-/-) B2 cells were defective in IgM production in an IFNAR-independent manner, although both B-cell subsets differentiated phenotypically to plasma cells at elevated efficiencies. Class switch recombination of IRF-2(-/-) B2 cells by LPS plus IL-4 was also impaired. Their reduced IgM production was conceivably due to an inefficient up-regulation of Blimp-1. Consistent with these in vitro observations, specific antibody production in vivo to a T-dependent antigen by B2 cells was severely impaired in IRF-2(-/- )mice. However, a low, but significant, level of IgG was detected at a late time point, and this IgG exhibited comparable binding affinity to that in WT mice. Follicular helper T-cell development and germinal center formation were normal. A similar tendency was observed when µ chain(-/-) mice were reconstituted with IRF-2(-/- )B cells. These results revealed a multi-faceted role of IRF-2 in the function of B cells, particularly B2 cells, through regulating proliferation in an IFNAR-dependent manner and antibody production via up-regulation of Blimp-1.