Christopher Roman

Sphingomyelin Synthase 2 Deficiency Attenuates NFκB Activation

Background—NF&kgr;B has long been regarded as a proatherogenic factor, mainly because of its regulation of many of the proinflammatory genes linked to atherosclerosis. Metabolism of sphingomyelin (SM) has been suggested to affect NF&kgr;B activation, but the mechanism is largely unknown. SMS2 regulates SM levels in cell plasma membrane and lipid rafts and has a potential to regulate NF&kgr;B activation. Methods and Results—To investigate the role of SMS2 in NF&kgr;B activation we used macrophages from SMS2 knockout (KO) mice and SMS2 siRNA-treated HEK 293 cells. We found that NF&kgr;B activation and its target gene expression are attenuated in macrophages from SMS2 KO mice in response to lipopolysaccharide (LPS) stimulation and in SMS2 siRNA- treated HEK 293 cells after tumor necrosis factor (TNF)–&agr; simulation. In line with attenuated NF&kgr;B activation, we found that SMS2 deficiency substantially diminished the abundance of toll like receptor 4 (TLR4)-MD2 complex levels on the surface of macrophages after LPS stimulation, and SMS2 siRNA treatment reduced TNF-&agr;-stimulated lipid raft recruitment of TNF receptor-1 (TNFR1) in HEK293 cells. SMS2 deficiency decreased the relative amounts of SM and diacylglycerol (DAG) and increased ceramide, suggesting multiple mechanisms for the decrease in NF&kgr;B activation. Conclusions—SMS2 is a modulator of NF&kgr;B activation, and thus it could play an important role in NF&kgr;B-mediated proatherogenic process.

Transcription factors TFE3 and TFEB are critical for CD40 ligand expression and thymus-dependent humoral immunity

TFE3 and TFEB are broadly expressed transcription factors related to the transcription factor Mitf. Although they have been linked to cytokine signaling pathways in nonlymphoid cells, their function in T cells is unknown. TFE3-deficient mice are phenotypically normal, whereas TFEB deficiency causes early embryonic death. We now show that combined inactivation of TFE3 and TFEB in T cells resulted in a hyper–immunoglobulin M syndrome due to impaired expression of CD40 ligand by CD4+ T cells. Native TFE3 and TFEB bound to multiple cognate sites in the promoter of the gene encoding CD40 ligand (Cd40lg), and maximum Cd40lg promoter activity and gene expression required TFE3 or TFEB. Thus, TFE3 and TFEB are direct, physiological and mutually redundant activators of Cd40lg expression in activated CD4+ T cells critical for T cell–dependent antibody responses.

Semliki Forest virus-induced demyelination and remyelination--involvement of B cells and anti-myelin antibodies.

Semliki Forest virus (SFV) infection induces a demyelinating encephalomyelitis in the central nervous system (CNS) of mice and serves as a model for multiple sclerosis (MS). This study investigated CNS immune responses at different stages of infection and during SFV-induced demyelination and remyelination. Following the initial CNS inflammation, pathology and viral clearance on days 6-10 post-infection (pi), primary demyelination was observed in cerebellar, brainstem and corpus collosal white matter by days 15-21 pi, with plasma cells and microglia as main participants, and this was followed by remyelination. By day 35 pi, the tissue appeared almost normal. Fluorescent antibody cell sorter (FACS) analysis showed that brain CD8(+) T cells increased during the initial inflammatory response and gradually decreased thereafter. Brain B cell (B220(+)CD19(+)) numbers did not change significantly during the course of infection; however, from days 14 to 35 pi, they matured and produced antibodies to viral and myelin proteins (and peptides) during the period of demyelination and remyelination. The proportion of CD3(-)B220(-)CD11b(+) cells also progressively increased throughout the periods of de- and remyelination. Our results suggest that CD8(+) T cells are involved in the initial destruction of CNS tissue during the first weeks of SFV infection, while B cells, antibodies and microglia may contribute to the myelin pathology seen after recovery.

Conventional and Surrogate Light Chains Differentially Regulate Ig μ and Dμ Heavy Chain Maturation and Surface Expression

Positive selection of precursor (pre-) B cells by Ig membrane μ H chains (μm HC) and counterselection mediated by the truncated HC Dμ depend on the ability of each HC to form a pre-B cell receptor (pre-BCR) signaling complex with the surrogate L chain (SLC) components λ5 and Vpre-B. To better understand how pre-BCR signaling output is determined by its Ig components and the SLC, we investigated the regulation of pre-BCR surface expression and HC secretory maturation in a new nonlymphoid system. We took this approach as a means to distinguish B-lineage-specific effects from pre-BCR-intrinsic properties that may influence these aspects of pre-BCR homeostasis necessary for signaling. As in pre-B cells, the SLC in nonlymphoid cells supported only a limited degree of μm HC maturation and low pre-BCR surface expression levels compared with conventional LCs, indicating that this was due to an intrinsic property of the SLC. We identified the non-Ig region of λ5 as harboring the restrictive activity responsible for this phenotype. This property of λ5 was also evident with Dμ, but the overall SLC- and L chain-dependent requirements for Dμ maturation and surface expression were markedly different from those for μm. Surprisingly, Dμ was modified in an unusual manner that was only dependent on Vpre-B. These results establish a novel function of λ5 in limiting surface pre-BCR levels and reveal biochemical properties of Ig molecules that may underlie the diverse consequences of pre-BCR signaling in vivo by different HCs.

The unique and immunoglobulin-like regions of surrogate light chain component λ5 differentially interrogate immunoglobulin heavy-chain structure

PreBCR signaling is critical for B cell development and normally depends on the association of a nascent, component Ig H chain with the surrogate L chain (SLC), which helps ensure that only B cells that synthesize structurally sound antibody can develop. How the invariant and lambda-like SLC vets billions of unique V(H) domains for compatibility with polymorphic kappa and lambda L chains is unclear, because the SLC is composed of not only the Ig domains of VpreB and lambda5, but also the unique regions (URs) that reside at what would be the L chain CDR3. We evaluated the contribution of the Ig and UR domains of lambda5 to H chain screening by evaluating the preBCR-forming capability of lambda5 mutants with a diverse panel of H chains. Using transformed mouse B cells, we demonstrate that the Ig domain of lambda5 was sufficient and its UR dispensable for the rejection of V(H)Q52 and V(H)10 SLC-incompatible H chains. In contrast, the lambda5 UR was necessary to discriminate between SLC-incompatible and -compatible V(H)81X H chains. Substituting the Ig domains of lambda5 with equivalent kappa sequences impaired the SLCs ability to escort all H chains to the surface. Two SLC-incompatible H chains were able to form surface BCRs with two kappa L chains, indicating that the SLCs ability to predict the L chain compatibility of a H chain is not absolute. In sum, lambda5 differentially relies on the lambda-like Ig and UR to probe H chain structure to best accommodate diversity among H chains.

Precursor B Cell Receptor Signaling Activity Can Be Uncoupled from Surface Expression

Signals from the precursor BCR (preBCR) cause proliferation and differentiation of progenitor (pro-) B cells into pre-B cells. Given the very low amounts of surface preBCRs and the demonstrated cell autonomy of preBCR signaling, we examined the possible occurrence of preBCR signal propagation from intracellular membranes such as the endoplasmic reticulum (ER) and the trans-Golgi network (TGN) in transformed and primary pro-B cells. PreBCRs composed of normal Ig μ or truncated Dμ heavy chains (HCs) were redirected to intracellular sites via localization sequences appended to the HC cytoplasmic tail. PreBCR complexes retained in the TGN or shunted from the TGN to lysosomes were as or 50% as active as the corresponding wild-type preBCRs in directing preBCR-dependent events, including CD2 and CD22 expression and proliferation in primary pro-B cells. This occurred despite their low to undetectable surface expression in transformed cells, which otherwise allowed significant surface accumulation of wild-type preBCRs. In contrast, ER-retained preBCRs were inactive. These results suggest that preBCR signaling is remarkably tolerant of dramatic changes in its subcellular distribution within post-ER compartments and support the possibility that the preBCR can activate signaling pathways in the TGN as well as the plasma membrane.

A Novel Isoform of Microphthalmia-Associated Transcription Factor Inhibits IL-8 Gene Expression in Human Cervical Stromal Cells

Cervical ripening during pregnancy is a profound change in cervix structure and function characterized by increases in the proinflammatory cytokine IL-8 and dissolution of the cervical extracellular matrix. Relatively little is known about the molecular mechanisms that underlie these events. Here, we report identification of a novel isoform of micropthalmia-associated transcription factor in human cervical stromal cells (MiTF-CX) that is down-regulated 12-fold during cervical ripening and that represses expression of IL-8. Ectopic expression of MiTF-CX in human cervical stromal cells resulted in substantial suppression of endogenous IL-8 mRNA and protein expression, whereas expression of dominant negative MiTF-CX mutants with impaired DNA binding resulted in dramatic increases in IL-8 production. Gel shift, reporter gene, and chromatin immunoprecipitation assays revealed one strong binding site (E-box (-397) CACATG(-391)) in the human IL-8 promoter that was crucial for mediating transcriptional repression by MiTF-CX. Moreover, we show that MiTF-CX expression in the cervix was itself positively autoregulated via two E-box motifs within a 2.1-kb promoter fragment. We therefore propose that maintenance of cervical competency during pregnancy is an active process maintained through suppression of IL-8 by the transcription factor MiTF-CX. During cervical ripening, loss of MiTF-CX would result in significant up-regulation of IL-8 mRNA and protein synthesis, thereby leading to recruitment and activation of leukocytes within the cervix and dissolution of the extracellular matrix.

The Unique Region of Surrogate Light Chain Component λ5 Is a Heavy Chain-Specific Regulator of Precursor B Cell Receptor Signaling

Signals transduced by precursor-BCRs (pre-BCRs) composed of Ig μ heavy chains (HCs) and the surrogate L chain components λ5 and VpreB are critical for B cell development. A conserved unique region (UR) of λ5 was shown to activate pre-BCR complexes in transformed cells and to engage putative ligands, but its contribution to pre-B cell development is not known. It is also not clear why the λ-like sequences in λ5 are used to select HCs that will associate mainly with κ L chains. In this study, we show that, in transformed and primary mouse B cell progenitors, receptors containing full-length HCs and lacking the λ5UR were expressed at higher surface levels, but exhibited reduced activity compared with normal pre-BCRs in supporting developmental changes that accompany the progenitor to pre-B cell transition in primary cell culture systems and in the bone marrow in vivo. In contrast, deletion of the λ5UR did not change net signaling output by the Dμ-pre-BCR, a developmentally defective receptor that exhibited impaired activity in the primary cell culture system. Moreover, the λ-like sequences in λ5 were more accommodating than κ in supporting surface expression and signaling by the different HCs. These results show that the λ5UR is important, although not essential, for surrogate L chain-dependent receptor signaling in primary cells, and furthermore may help allow discrimination of signaling competency between normal and Dμ-pre-BCRs. That the λ-like portion of λ5 in the absence of the UR was nondiscriminatory suggests that the λ5UR focuses pre-BCR-dependent selection on the HC V region.

Defective Thymocyte Maturation by Transgenic Expression of a Truncated Form of the T Lymphocyte Adapter Molecule and Fyn Substrate, Sin

Adapter molecules that promote protein-protein interactions play a central role in T lymphocyte differentiation and activation. In this study, we examined the role of the T lymphocyte-expressed adapter protein and Src kinase substrate, Sin, on thymocyte function using transgenic mice expressing an activated, truncated allele of Sin (SinΔC). We found that SinΔC expression led to reduced numbers of CD4+ and CD8+ single-positive cells and reduced thymic cellularity due to increased thymocyte apoptosis. Because the adapter properties of Sin are mediated by tyrosine-based motifs and given that Sin is a substrate for Src tyrosine kinases, we examined the involvement of these kinases in the inhibitory effects of SinΔC. We found that in transgenic thymocytes, SinΔC was constitutively phosphorylated by the Src kinase Fyn, but not by the related kinase Lck. Using SinΔC and fyn−/− animals, we also found that the expression of Fyn was required for the inhibitory effect of SinΔC on thymocyte apoptosis but not for SinΔC-mediated inhibition of T cell maturation. The inhibitory effect of SinΔC on thymocyte maturation correlated with defective activation of the mitogen-activated protein kinase extracellular signal-regulated kinase. Our results suggest that the Sin mutant inhibits thymocyte differentiation through Fyn-dependent and -independent mechanisms and that endogenous Sin may be an important regulator of thymocyte development.

Immune checkpoint inhibitors in malignancies with mismatch repair deficiency: a review of the state of the current knowledge

The use of immune checkpoint inhibitors to treat malignant tumors with microsatellite instability is an emerging new modality. This is based on the observations that these tumors may have a high mutation rate—thus a potential source of tumor-specific neoantigens—and harbor infiltrating cytotoxic T cells in response, suggesting that they may be particularly susceptible to immune checkpoint therapy. PUBMED and ASCO library were systematically reviewed to identify all relevant data that involved the use of immune checkpoint inhibitors in the treatment of cancers with microsatellite instability. The manual search retrieved a total of 3 relevant articles and 1 abstract published between 2015 and 2016. A total of 61 patients with colorectal, 3 with ampullary/cholangiocarcinoma, 2 with endometrial carcinomas, 3 with small bowel cancers, 2 with glioblastoma multiforme, and 1 with bladder cancer with reported efficacy results were reviewed. All the patients had stage IV cancer and were treated with immune checkpoint inhibitors until progression of disease or intolerable side effects emerged. The range of objective response rate was 25–71%. Responses were also durable with progression-free survival at 20 weeks of around 67–78% and to 46% at 1 year. The use of immune checkpoint inhibitors is effective in cancers that express microsatellite instability.