The adaptor protein TRAF3 is an immune checkpoint that inhibits myeloid-derived suppressor cell expansion.

Myeloid-derived suppressor cells (MDSCs) are aberrantly expanded in cancer patients and under other pathological conditions. These cells orchestrate the immunosuppressive and inflammatory network to facilitate cancer metastasis and mediate patient resistance to therapies, and thus are recognized as a prime therapeutic target of human cancers. Here we report the identification of the adaptor protein TRAF3 as a novel immune checkpoint that critically restrains MDSC expansion. We found that myeloid cell-specific Traf3-deficient (M-Traf3 -/-) mice exhibited MDSC hyperexpansion during chronic inflammation. Interestingly, MDSC hyperexpansion in M-Traf3 -/- mice led to accelerated growth and metastasis of transplanted tumors associated with an altered phenotype of T cells and NK cells. Using mixed bone marrow chimeras, we demonstrated that TRAF3 inhibited MDSC expansion via both cell-intrinsic and cell-extrinsic mechanisms. Furthermore, we elucidated a GM-CSF-STAT3-TRAF3-PTP1B signaling axis in MDSCs and a novel TLR4-TRAF3-CCL22-CCR4-G-CSF axis acting in inflammatory macrophages and monocytes that coordinately control MDSC expansion during chronic inflammation. Taken together, our findings provide novel insights into the complex regulatory mechanisms of MDSC expansion and open up unique perspectives for the design of new therapeutic strategies that aim to target MDSCs in cancer patients.

A Posttranscriptional Pathway of CD40 Ligand mRNA Stability Is Required for the Development of an Optimal Humoral Immune Response.

CD40 ligand (CD40L) mRNA stability is dependent on an activation-induced pathway that is mediated by the binding complexes containing the multifunctional RNA-binding protein, polypyrimidine tract-binding protein 1 (PTBP1) to a 3' untranslated region of the transcript. To understand the relationship between regulated CD40L and the requirement for variegated expression during a T-dependent response, we engineered a mouse lacking the CD40L stability element (CD40LΔ5) and asked how this mutation altered multiple aspects of the humoral immunity. We found that CD40LΔ5 mice expressed CD40L at 60% wildtype levels, and lowered expression corresponded to significantly decreased levels of T-dependent Abs, loss of germinal center (GC) B cells and a disorganized GC structure. Gene expression analysis of B cells from CD40LΔ5 mice revealed that genes associated with cell cycle and DNA replication were significantly downregulated and genes linked to apoptosis upregulated. Importantly, somatic hypermutation was relatively unaffected although the number of cells expressing high-affinity Abs was greatly reduced. Additionally, a significant loss of plasmablasts and early memory B cell precursors as a percentage of total GL7+ B cells was observed, indicating that differentiation cues leading to the development of post-GC subsets was highly dependent on a threshold level of CD40L. Thus, regulated mRNA stability plays an integral role in the optimization of humoral immunity by allowing for a dynamic level of CD40L expression on CD4 T cells that results in the proliferation and differentiation of pre-GC and GC B cells into functional subsets.

The RNA-Binding Protein, Polypyrimidine Tract-Binding Protein 1 (PTBP1) Is a Key Regulator of CD4 T Cell Activation.

We have previously shown that the RNA binding protein, polypyrimidine tract-binding protein (PTBP1) plays a critical role in regulating the expression of CD40L in activated CD4 T cells. This is achieved mechanistically through message stabilization at late times of activation as well as by altered distribution of CD40L mRNA within distinct cellular compartments. PTBP1 has been implicated in many different processes, however whether PTBP1 plays a broader role in CD4 T cell activation is not known. To examine this question, experiments were designed to introduce shRNA into primary human CD4 T cells to achieve decreased, but not complete ablation of PTBP1 expression. Analyses of shPTB-expressing CD4 T cells revealed multiple processes including cell proliferation, activation-induced cell death and expression of activation markers and cytokines that were regulated in part by PTBP1 expression. Although there was an overall decrease in the steady-state level of several activation genes, only IL-2 and CD40L appeared to be regulated by PTBP1 at the level of RNA decay suggesting that PTBP1 is critical at different regulatory steps of expression that is gene-specific. Importantly, even though the IL-2 protein levels were reduced in cells with lowered PTBP1, the steady-state level of IL-2 mRNA was significantly higher in these cells suggesting a block at the translational level. Evaluation of T cell activation in shPTB-expressing T cells revealed that PTBP1 was linked primarily to the activation of the PLCγ1/ERK1/2 and the NF-κB pathways. Overall, our results reveal the importance of this critical RNA binding protein in multiple steps of T cell activation.

N-benzyladriamycin-14-valerate (AD 198) exhibits potent anti-tumor activity on TRAF3-deficient mouse B lymphoma and human multiple myeloma.

BACKGROUND: TRAF3, a new tumor suppressor identified in human non-Hodgkin lymphoma (NHL) and multiple myeloma (MM), induces PKCδ nuclear translocation in B cells. The present study aimed to evaluate the therapeutic potential of two PKCδ activators, N-Benzyladriamycin-14-valerate (AD 198) and ingenol-3-angelate (PEP005), on NHL and MM. METHODS: In vitro anti-tumor activities of AD 198 and PEP005 were determined using TRAF3-/- mouse B lymphoma and human patient-derived MM cell lines as model systems. In vivo therapeutic effects of AD 198 were assessed using NOD SCID mice transplanted with TRAF3-/- mouse B lymphoma cells. Biochemical studies were performed to investigate signaling mechanisms induced by AD 198 or PEP005, including subcellular translocation of PKCδ. RESULTS: We found that AD 198 exhibited potent in vitro and in vivo anti-tumor activity on TRAF3-/- tumor B cells, while PEP005 displayed contradictory anti- or pro-tumor activities on different cell lines. Detailed mechanistic investigation revealed that AD 198 did not affect PKCδ nuclear translocation, but strikingly suppressed c-Myc expression and inhibited the phosphorylation of ERK, p38 and JNK in TRAF3-/- tumor B cells. In contrast, PEP005 activated multiple signaling pathways in these cells, including PKCδ, PKCα, PKCε, NF-κB1, ERK, JNK, and Akt. Additionally, AD198 also potently inhibited the proliferation/survival and suppressed c-Myc expression in TRAF3-sufficient mouse and human B lymphoma cell lines. Furthermore, we found that reconstitution of c-Myc expression conferred partial resistance to the anti-proliferative/apoptosis-inducing effects of AD198 in human MM cells. CONCLUSIONS: AD 198 and PEP005 have differential effects on malignant B cells through distinct biochemical mechanisms. Our findings uncovered a novel, PKCδ-independent mechanism of the anti-tumor effects of AD 198, and suggest that AD 198 has therapeutic potential for the treatment of NHL and MM involving TRAF3 inactivation or c-Myc up-regulation.

c-Rel deficiency increases caspase-4 expression and leads to ER stress and necrosis in EBV-transformed cells.

LMP1-mediated activation of nuclear factor of kappaB (NF-κB) is critical for the ligand independent proliferation and cell survival of in vitro EBV-transformed lymphoblastoid cell lines (LCLs). Previous experiments revealed that a majority of LMP1-dependent responses are regulated by NF-κB. However, the extent that individual NF-κB family members are required for these responses, in particular, c-Rel, whose expression is restricted to mature hematopoietic cells, remains unclear. Here we report that low c-Rel expression in LCLs derived from a patient with hyper-IgM syndrome (Pt1), resulted in defects in proliferation and cell survival. In contrast to studies that associated loss of NF-κB with increased apoptosis, Pt1 LCLs failed to initiate apoptosis and alternatively underwent autophagy and necrotic cell death. Whereas the proliferation defect appeared linked to a c-Rel-associated decrease in c-myc expression, identified pro-survival and pro-apoptotic targets were expressed at or near control levels consistent with the absence of apoptosis. Ultrastructural examination of Pt1 LCLs revealed a high level of cellular and ER stress that was further supported by gene expression profiling showing the upregulation of several genes involved in stress and inflammation. Apoptosis-independent cell death was accompanied by increased expression of the inflammatory marker, caspase-4. Using gene overexpression and siRNA knockdown we demonstrated that levels of c-Rel directly modulated expression of caspase-4 as well as other ER stress genes. Overall, these findings reveal the importance of c-Rel in maintaining LCL viability and that decreased expression results in ER stress and a default response leading to necrotic cell death.

Polypyrimidine tract-binding protein is critical for the turnover and subcellular distribution of CD40 ligand mRNA in CD4+ T cells.

CD40L (CD154) is regulated at the posttranscriptional level by an activation-induced process that results in a highly stable transcript at extended times of T cell activation. Transcript stability is mediated by polypyrimidine tract-binding protein (PTB)-containing complexes (complex I and II) that bind to three adjacent CU-rich sequences within the 3' untranslated region. To assess the role of PTB in the expression and distribution of CD40L mRNA, PTB was targeted using short hairpin RNA in both primary T cells and a T cell line that recapitulates the stability phase of regulated CD40L mRNA decay. PTB knockdown resulted in a marked decrease in the mRNA stability that resulted in lowered CD40L surface expression. PTB was also critical for appropriate distribution of CD40L mRNA between the nucleus and cytoplasm and in the cytoplasm between the cytosol and the translating polysomes. The activation-induced formation of PTB-specific ribonucleoprotein complexes was observed only with cytoplasmic and not nuclear PTB indicating functional differences in the protein defined by cellular localization. Finally, we observed that cytoplasmic and nuclear PTB isoforms were differentially modified relative to each other and that the changes in cytoplasmic PTB were consistent with activation-induced phosphorylation. Together this work suggests that differentially modified PTB regulates CD40L expression at multiple steps by 1) retaining CD40L mRNA in the nucleus, 2) directly regulating mRNA stability at late times of activation, and 3) forming a ribonuclear complex that preferentially associates with translating ribosomes thus leading to an enhanced level of CD40L protein.

In vivo post-transcriptional regulation of CD154 in mouse CD4+ T cells.

Interactions between CD40 and its ligand CD154 are involved in the progression of both cell mediated and innate immunity. These interactions are brought about by the transient expression of CD154 on activated CD4(+) T cells, which is regulated, in part, at the level of mRNA turnover. Here we have focused on analyzing the pattern of post-transcriptional regulation in mouse CD4(+) T cells in response to activation. Initial experiments identify a region of the murine CD154 mRNA that binds a polypyrimidine tract-binding protein-containing complex (mComplex I), which is activation-dependent and binds to a single CU-rich site within the 3' uTR Subsequent findings demonstrate that in vivo polyclonal activation of T cells leads to a pattern of differential CD154 mRNA stability that is directly dependent on extent of activation. Furthermore, in vitro activation of antigen-primed T cells shows that the CD154 mRNA half-life increases relative to that of unprimed cells. Importantly, this is the first report demonstrating that the regulation of CD154 in vivo is connected to an activation-induced program of mRNA decay and thus provides strong evidence for post-transcriptional mechanisms having a physiological role in regulating CD154 expression during an ongoing immune response.

Post-transcriptional regulation in lymphocytes: the case of CD154.

The control of mRNA decay is emerging as an important control point and a major contributor to gene expression in both immune and non-immune cells. The identification of protein factors and cis-acting elements responsible for transcript degradation has illuminated a comprehensive picture of precisely orchestrated events required to both regulate and establish the decay process. One gene that is highly regulated at the post-transcriptional level is CD40 ligand (CD154 or CD40L). CD154 on CD4(+) T cells is tightly controlled by an interacting network of transcriptional and post-transcriptional processes that result in precise surface levels of protein throughout an extended time course of antigen stimulation. The activation-induced stabilization of the CD154 transcript by a polypyrimidine tract-binding protein (PTB)-complex is a key event that corresponds to the temporal expression of CD154. In this review, we discuss known and potential roles of major mRNA decay pathways in lymphocytes and focus on the unique post-transcriptional mechanisms leading to CD154 expression by activated CD4(+) T cells.

Single nucleotide changes in the human Igamma1 and Igamma4 promoters underlie different transcriptional responses to CD40.

Analysis of subclass-specific germline transcription in activated peripheral B cells revealed a highly biased expression pattern of the four Igamma transcripts to signals through CD40 and IL-4. This difference was most pronounced when comparing the profile of Igamma1 and Igamma4 transcripts and was not expected given the very high degree of sequence conservation between promoters. In this report, the influence of sequence differences on the regulation of the Igamma1 and Igamma4 promoters has been investigated given the highly muted transcriptional activity of the Igamma4 promoter. Two regions were analyzed where single nucleotide differences corresponded to major changes in transcriptional activity. These regions were the previously defined CD40 response region containing three putative NF-kappaB-binding sites and the downstream 36-bp region containing CREB/activating transcription factor and kappaB6 sites. Mutation of a single nucleotide at position 6 within the Igamma4 kappaB6 site increased promoter activity to approximately 50% of the activity of the Igamma1 promoter. Furthermore, elevated promoter strength corresponded with increased binding of p50, p65, c-Rel, RelB, and p300 proteins to a level comparable with that of Igamma1. Minor nucleotide changes to both the Igamma4 CD40 response region and the 36-bp element resulted in a response undistinguishable from an Igamma1 response, suggesting cooperation between the two regulatory regions for optimal transcriptional activity. Collectively, these mutational analyses suggest that minor sequence differences contribute to the composition and affinity of transcriptional protein complexes regulating subclass-specific germline transcription, which in part impacts the overall level of class switch recombination to targeted C(H) regions.

A polypyrimidine tract-binding protein-dependent pathway of mRNA stability initiates with CpG activation of primary B cells.

The mRNA encoding CD154, a critical protein involved in both humoral and cell-mediated immune responses, is regulated at the posttranscriptional level by the binding of complex I, a polypyrimidine tract-binding (PTB) protein-containing complex, which acts to increase message stability at late times of activation. Our current work focuses on analyzing a similar complex in B cells, designated B-cpx I, which is increased in B cells activated by CpG engagement of the TLR9 receptor but not by activation through CD40. Expression profiling of transcripts from primary B cells identified 31 mRNA transcripts with elevated PTB binding upon activation. Two of these transcripts, Rab8A and cyclin D(2), contained binding sites for B-cpx I in their 3' untranslated regions (UTRs). Analysis of turnover of endogenous Rab8A transcript in B cells revealed that like CD154, the mRNA half-life increased following activation and insertion of the Rab8A B-cpx I binding site into a heterologous transcript led to a 3-fold increase in stability. Also, short hairpin RNA down-regulation of PTB resulted in a corresponding decrease in Rab8A mRNA half-life. Overall these data strongly support a novel pathway of mRNA turnover that is expressed both in T cells and B cells and depends on the formation of a PTB-containing stability complex in response to cellular activation.

Functional analysis of a tripartite stability element within the CD40 ligand 3' untranslated region.

We previously identified a cis-acting element within the 3' untranslated region of CD40 ligand messenger RNA (mRNA) that is composed of three complex binding sites and acts to increase mRNA stability in both in vitro and in vivo systems. We now demonstrate the functional consequences of the three binding sites with respect to increasing both luciferase activity and mRNA stability in a heterologous transcript expressed in a T-cell line. The internal region B was shown to be a bona fide stability element because its presence increased luciferase activity fourfold over the unmodified transcript and its removal from the XbaI-HaeIII region resulted in rapid degradation of the transcript. Region A contained both a binding site for a polypyrimidine-tract-binding protein (PTB)-mediated complex (Complex I) and an upstream, adjacent sequence that was a negative regulator of mRNA stability. Region C bound Complex II, which contained both PTB and heterogeneous nuclear ribonucleoproteinL (hnRNPL), and was less effective as a stability element on its own compared to region B. Our findings demonstrate differential levels of activity for the three binding sites relative to the turnover of CD40 ligand mRNA, suggesting that the lack of binding of Complex I/II during the early stages of T-cell activation contributes to the rapid degradation of the CD40 ligand mRNA transcript.

c-Rel plays a key role in deficient activation of B cells from a non-X-linked hyper-IgM patient.

Our previous results demonstrated that B cells from a patient (pt1) with non-X-linked hyper-IgM syndrome (HIGM) possess an atypical CD23(lo) phenotype that is unaffected by CD40-mediated activation. To investigate the molecular mechanism underlying defective CD23 expression in pt1 B cells, we used lymphoblastoid cell lines that express LMP1 under the control of a tetracycline-inducible promoter (LCL(tet)). Our analysis revealed that the CD23(lo) phenotype in the pt1-LCL(tet) cells is a direct consequence of diminished CD23 transcription. We demonstrate a marked decrease in c-Rel-containing complexes that bind to the proximal CD23a/b promoters in pt1-LCL(tet) extracts, resulting from an overall lower expression of c-Rel in pt1-LCL(tet) cells. Analysis of c-Rel mRNA revealed relatively equal amounts in pt1-LCL(tet) and control LCL(tet) cells, indicating that diminished c-Rel protein expression is unrelated to decreased transcription. Finally, a critical role for c-Rel in CD23 regulation was demonstrated by effectively altering c-Rel expression that resulted in the direct modulation of CD23 surface expression. Collectively, these findings demonstrate that low levels of c-Rel are the underlying cause of aberrant CD23 expression in pt1 B cells and are likely to play a critical role in the pathophysiology of this form of HIGM.

Use of chromatin immunoprecipitation (ChIP) to detect transcription factor binding to highly homologous promoters in chromatin isolated from unstimulated and activated primary human B cells.

The Chromatin Immunoprecipiation (ChIP) provides a powerful technique for identifying the in vivo association of transcription factors with regulatory elements. However, obtaining meaningful information for promoter interactions is extremely challenging when the promoter is a member of a class of highly homologous elements. Use of PCR primers with small numbers of mutations can limit cross-hybridization with non-targeted sequences and distinguish a pattern of binding for factors with the regulatory element of interest. In this report, we demonstrate the selective in vivo association of NF-kappaB, p300 and CREB with the human Igamma1 promoter located in the intronic region upstream of the Cgamma1 exons in the immunoglobulin heavy chain locus. These methods have the ability to extend ChIP analysis to promoters with a high degree of homology.

A novel NF-kappa B-regulated site within the human I gamma 1 promoter requires p300 for optimal transcriptional activity.

Transcriptional activation of germline (GL) promoters occurs through binding of NF-kappaB to three evolutionarily conserved sites within a CD40 response region in the human and mouse GL Igamma and Iepsilon promoters. Here we identify and characterize a novel NF-kappaB binding site (kappaB6) within the human GL Igamma1 promoter that plays an essential role in basal- and CD40-induced transcription. This site is adjacent to identified CREB/activating transcription factor (ATF) sites, present in the Igamma1 but not the Igamma3 promoter, which are important for the amplification of transcription. Our data suggest a cohesive protein complex regulating Igamma1 promoter activity because disruption of any individual NF-kappaB or CREB/ATF site markedly lowers the overall inducible activity of the promoter. In addition, alteration of helical phasing within the promoter indicates spatial orientation of CREB/ATF and NF-kappaB, proteins contributes directly to promoter activity. We found that CREB and p50 transactivators, as well as coactivator p300, interact in vivo with the Igamma1 promoter in the presence and absence of CD40 signaling in Ramos and primary B cells. However, the level of CREB and p300 binding differs as a consequence of activation in primary B cells. Furthermore, overexpression of p300, and not a mutant lacking acetyltransferase activity, significantly increases Igamma1 construct-specific transcription. Together these data support a model whereby CREB and multiple NF-kappaB complexes bind to the Igamma1 promoter and recruit p300. CD40 signals induce p300-dependent changes that result in optimal Igamma1 promoter activity.

Maintenance of the CD40-related immunodeficient response in hyper-IgM B cells immortalized with a LMP1-regulated mini-EBV.

Our previous investigation of a patient (pt1) with non-X-linked hyper-immunoglobulin M syndrome revealed a CD40-mediated defect in B cell activation that resulted in low CD23 expression and absence of germ-line transcription and class-switch recombination. These deficiencies were complemented in vitro by a high threshold of sustained signaling through CD40. To further analyze the signaling defect in pt1 B cells, two types of Epstein-Barr virus lymphoblastoid cell lines (LCLs) were generated that either constitutively expressed the viral transforming protein latent membrane protein-1 (LMP1; pt1-LCL) or expressed it under the control of a tet-inducible promoter (pt1-LCL(tet)). Because LMP1 signals through the CD40 pathway, the pt1-LCL and pt1-LCL(tet) lines allow comparison of downstream functions in response to either constitutive LMP1 signals or regulated LMP1 and CD40 signals. Immortalized pt1-LCLs were initially CD23(lo)/CD38(hi) and reverted to a CD23(hi)/CD38(lo) phenotype upon extended growth in culture, suggesting that the CD40 defect was reversed by selection and/or constitutive expression of LMP1. In contrast, pt1-LCL(tet) cells retained the CD23(lo)/CD38(hi) phenotype after extended periods of culture and failed to up-regulate CD23 in response to CD40 signals. Analysis of pt1-LCL(tet) cells in response to the CD40 signals in the presence or absence of LMP1 revealed that mitogenic activation resulted only from LMP1 and not CD40, indicating a difference in the response of pt1 B cells to these two distinct signals. Together, these data demonstrate that the pt1-LCL(tet) cells maintain the CD40-related defect and provide a unique approach to study the independent effects of LMP1- and CD40-directed signals.

Nucleolin is a second component of the CD154 mRNA stability complex that regulates mRNA turnover in activated T cells.

CD154 (CD40L) mRNA turnover is regulated in part at the posttranscriptional level by a protein complex (termed Complex I) that binds to a highly CU-rich region of the 3'UTR. Polypyrimidine tract-binding protein (PTB) has previously been identified as a major RNA-binding protein in Complex I. Nondenaturing gel filtration of total extract from Jurkat T cells demonstrated that the CD154 mRNA-binding activity migrates as a approximately 200-kDa complex, indicating the presence of multiple complex-associated proteins. We have currently undertaken a biochemical approach to further characterize Complex I and observed that it segregates over DEAE-Sepharose into two subcomplexes (termed I-L and I-U). Furthermore, nucleolin was identified as a component of both subcomplexes and was shown that it is the major RNA-binding protein in I-U. To directly demonstrate the biological significance of Complex I binding to the CD154 transcript, cytoplasm from human Jurkat cells was fractionated over a sucrose gradient and the different cellular fractions subjected to immunoprecipitation with anti-PTB and anti-nucleolin Abs. RT-PCR of the immunoprecipitated products using CD154-specific primers clearly demonstrated that nucleolin and PTB are associated with CD154 mRNA in both the ribonucleoprotein and polysome fractions. These data strongly support a model whereby nucleolin and PTB are integral to the stability of CD154 mRNA and are components of the CD154 ribonucleoprotein particle associated with actively translating ribosomes.

A complex containing polypyrimidine tract-binding protein is involved in regulating the stability of CD40 ligand (CD154) mRNA.

CD40 ligand (CD154) expression has been shown to be regulated, in part, at the posttranscriptional level by a pathway of "regulated instability" of mRNA decay throughout a time course of T cell activation. This pathway is modulated at late times of activation by the binding of a stability complex (termed complex I) to a CU-rich region in the 3' untranslated region of the CD154 message. We have undertaken experiments to extend these findings and to analyze the cis-acting elements and trans-acting factors involved in this regulation. We have previously shown that the minimal binding sequence for complex I is a 63 nt CU-rich motif. However, our current study shows that when this site was deleted additional complex binding was observed upstream and downstream of the minimal binding region. Only after deletion of an extended region (termed Delta1515) was complex binding completely abolished. Analysis of complex binding using competition experiments revealed that the three adjacent regions bound related but not identical complexes. However, all three sites appeared to have a 55-kDa protein as the RNA-binding protein. Deletion of the Delta1515 region resulted in reduced transcript stability as measured by both in vitro and in vivo decay assays. Finally, using Abs against known RNA-binding proteins, we identified the polypyrimidine tract-binding protein (or heterogeneous nuclear ribonucleoprotein I) as a candidate RNA-binding component of complex I.