Michael P. Bell

Polyquarternary amines prevent peptide loss from sequenators

Abstract Automatic spinning-cup sequenators are often incapable of approaching the carboxy terminus of a peptide because of the mechanical loss of material (washout). We report here an adjunct, polyquarternary amines, specifically Polybrene, that greatly reduces or eliminates this problem.

Inhibition of Interleukin-1-stimulated NF-κB RelA/p65 Phosphorylation by Mesalamine Is Accompanied by Decreased Transcriptional Activity

Nuclear factor κB (NF-κB) is an inducible transcription factor that regulates genes important in immunity and inflammation. The activity of NF-κB is highly regulated: transcriptionally active NF-κB proteins are sequestered in the cytoplasm by inhibitory proteins, IκB. A variety of extracellular signals, including interleukin-1 (IL-1), activate NF-κB by inducing phosphorylation and degradation of IκB, allowing nuclear translocation and DNA binding of NF-κB. Many of the stimuli that activate NF-κB by inducing IκB degradation also cause phosphorylation of the NF-κB RelA (p65) polypeptide. The transactivating capacity of RelA is positively regulated by phosphorylation, suggesting that in addition to cytosolic sequestration by IκB, phosphorylation represents another mechanism for control of NF-κB activity. In this report, we demonstrate that mesalamine, an anti-inflammatory aminosalicylate, dose-dependently inhibits IL-1-stimulated NF-κB-dependent transcription without preventing IκB degradation or nuclear translocation and DNA binding of the transcriptionally active NF-κB proteins, RelA, c-Rel, or RelB. Mesalamine was found to inhibit IL-1-stimulated RelA phosphorylation. These data suggest that pharmacologic modulation of the phosphorylation status of RelA regulates the transcriptional activity of NF-κB, independent of nuclear translocation and DNA binding. These findings highlight the importance of inducible phosphorylation of RelA in the control of NF-κB activity.

Effects of p56lck deficiency on the growth and cytolytic effector function of an interleukin-2-dependent cytotoxic T-cell line.

The growth, differentiation, and functional activities of antigen-stimulated T lymphocytes are regulated by the interaction of the T-cell-derived cytokine, interleukin-2 (IL-2), with the high-affinity IL-2 receptor (IL-2R). IL-2R occupancy initiates a rapid increase in intracellular protein tyrosine phosphorylation, suggesting that a receptor-coupled protein tyrosine kinase (PTK) serves as a proximal signaling element for the IL-2R. Previous studies implicated the src-family kinase, p56lck, as a potential IL-2R-linked signal transducer. In this study, we have characterized a spontaneous variant of the IL-2-dependent cytotoxic T-cell line, CTLL-2, which contains no detectable lck-derived mRNA transcripts, protein, or PTK activity. The p56lck-deficient CTLL-2 cells retained strict dependence on IL-2 for both viability and growth, indicating that p56lck activity was not required for the transduction of IL-2-mediated mitogenic signals. However, the p56lck-deficient cells exhibited a moderate decrease in their rate of IL-2-dependent proliferation. In contrast to this relatively modest proliferative defect, the p56lck-deficient cell line displayed a profound reduction in T-cell antigen receptor-dependent cytolytic effector functions. Both the proliferative and the cytolytic defects observed in the p56lck-deficient cells were completely reversed by transfection of these cells with a wild-type lck expression vector. These results indicate that p56lck expression is not obligatory for IL-2-mediated T-cell growth stimulation; however, this PTK plays a central role in the generation T-cell-mediated cytotoxic responses.

Reprogrammed FoxP3+ T Regulatory Cells Become IL-17+ Antigen-Specific Autoimmune Effectors In Vitro and In Vivo

In the course of investigating suspicious patterns of experimental results in the laboratory, a systematic and in-depth study of key findings in this article was carried out using blinded protocols. In these repeat studies, no evidence was found to support our original conclusions that B7-DC XAb modulates dendritic cell functions. We do not believe our failure to reproduce our earlier findings is the result of a technical problem. A member of the B7-DC XAb investigative team, Dr. Suresh Radhakrishnan, who was involved in or had access to all the work on this subject, was found in a formal investigation to have engaged in scientific misconduct in unpublished experiments involving the B7-DC XAb reagent. This finding of misconduct and our inability to reproduce key findings using blinded protocols has undermined our confidence in our published reports. We seek, therefore, to retract this body of work.

Ly9 (CD229)-deficient mice exhibit T cell defects yet do not share several phenotypic characteristics associated with SLAM- and SAP-deficient mice.

Signaling lymphocyte activation molecule (SLAM) family receptors are critically involved in modulating innate and adaptive immune responses. Several SLAM family receptors have been shown to interact with the adaptor molecule SAP; however, subsequent intracellular signaling is poorly defined. Notably, mutations in SLAM-associated protein (SAP) lead to X-linked lymphoproliferative disease, a rare but fatal immunodeficiency. Although the SLAM family member Ly9 (CD229) is known to interact with SAP, the functions of this receptor have remained elusive. Therefore, we have generated Ly9−/− mice and compared their phenotype with that of SLAM−/− and SAP−/− mice. We report that Ly9−/− T cells exhibit a mild Th2 defect associated with reduced IL-4 production after stimulation with anti-TCR and anti-CD28 in vitro. This defect is similar in magnitude to the previously reported Th2 defect in SLAM−/− mice but is more subtle than that observed in SAP−/− mice. In contrast to SLAM−/− and SAP−/− mice, T cells from Ly9−/− mice proliferate poorly and produce little IL-2 after suboptimal stimulation with anti-CD3 in vitro. We have also found that Ly9−/− macrophages exhibit no defects in cytokine production or bacterial killing as was observed in SLAM−/− macrophages. Additionally, Ly9−/− mice differ from SAP−/− mice in that they foster normal development of NKT cells and mount appropriate T and B cell responses to lymphocytic choriomeningitis virus. We have identified significant phenotypic differences between Ly-9−/− mice as compared with both SLAM−/− and SAP−/− mice. Although Ly9, SLAM, and SAP play a common role in promoting Th2 polarization, Ly-9 is uniquely involved in enhancing T cell activation.

Mechanism Responsible for T-Cell Antigen Receptor- and CD28- or Interleukin 1 (IL-1) Receptor-Initiated Regulation of IL-2 Gene Expression by NF-κB

ABSTRACT Initiation of the T-helper lymphocyte activation program is regulated through the T-cell receptor (TCR) and costimulatory receptors. Analysis of TCR and either anti-CD28- or interleukin 1 (IL-1)-mediated activation of the IL-2 promoter shows that costimulatory signals augment promoter activity through NF-κB sites. This study comparatively evaluates the mechanisms whereby signals initiated from the TCR and these two costimulatory receptors converge to synergistically increase NF-κB transcriptional activity. IL-1 alone stimulates an acute but transient NF-κB nuclear localization and a suboptimal NF-κB transcriptional response. In contrast, anti-CD3–anti-CD28 or anti-CD3–IL-1 synergistically stimulate prolonged NF-κB nuclear localization and NF-κB-mediated transcription. Both TCR- and costimulatory receptor-initiated synergistic NF-κB responses result from prolonging high rates of cytosolic IκB degradation during the second phase of the biphasic NF-κB nuclear localization. However, in contrast to previous reports, prolonged nuclear localization of NF-κB complexes is not necessarily associated with long-term depletion of IκBβ. In response to either costimulus, c-Rel selectively translocated to the nucleus as a result of induced c-Rel expression and the continued production of c-Rel–IκBα complexes, which turn over rapidly due to the high rate of IκBα degradation in the cytosol during the second phase of the response. In contrast, IκBβ is nearly completely degraded during the acute response to either IL-1 or anti-CD3–IL-1 while anti-CD3–anti-CD28 stimulates only a partial reduction (35 to 40%) in cytosolic IκBβ. Cyclosporine (CsA), which inhibits stimulus-induced NF-κB transcriptional activity, selectively inhibits the stimulus-induced c-Rel nuclear localization and the rapid formation and degradation of c-Rel–IκBα complexes in the cytosol. CsA also inhibits both the prolonged, high rate of IκBα degradation and the lower level of IκBβ turnover during the second phase of the activation response. Together, these results suggest a mechanism by which signals from the T-cell antigen receptor and either CD28 or IL-1 synergistically regulate IL-2 gene transcription by modulating NF-κB nuclear translocation.

The Pathogen Recognition Receptor NOD2 Regulates Human FOXP3+ T Cell Survival

The expression of pathogen recognition receptors in human FOXP3+ T regulatory cells is established, yet the function of these receptors is currently obscure. In the process of studying the function of both peripheral and lamina propria FOXP3+ lymphocytes in patients with the human inflammatory bowel disease Crohn’s disease, we observed a clear deficiency in the quantity of FOXP3+ lymphocytes in patients with disease-associated polymorphisms in the pathogen recognition receptor gene NOD2. Subsequently, we determined that the NOD2 ligand, muramyl dipeptide (MDP), activates NF-κB in primary human FOXP3+ T cells. This activation is functionally relevant, as MDP-stimulated human FOXP3+ T cells are protected from death receptor Fas-mediated apoptosis. Importantly, apoptosis protection was not evident in MDP-stimulated FOXP3+ T cells isolated from a patient with the disease-associated polymorphism. Thus, we propose that one function of pathogen recognition receptors in human T regulatory cells is the protection against death receptor-mediated apoptosis in a Fas ligand-rich environment, such as that of the inflamed intestinal subepithelial space.

FOXP3 regulates TLR10 expression in human T regulatory cells

Although functionally relevant TLRs can be expressed on human T regulatory (Treg) cells, little is known about the transcriptional control of their expression. We hypothesized that the transcription factor forkhead box P3 (FOXP3) regulates the expression of TLR family members in human Treg cells. Using primary human T cells and a reporter assay in Jurkat T cell lines, we dissected the regulation of TLR10, a TLR highly expressed in human Treg cells. We determined that TLR10 was expressed in human Treg cells through quantitative PCR, Western blotting, and flow cytometry. DNA binding of FOXP3 to a suspected cis-regulatory region in proximity to the transcription start site of TLR10 was established through EMSA and chromatin immunoprecipitation. Transcriptional control of TLR10 by FOXP3 was determined through luciferase reporter assays in Jurkat T cell lines. Relevance of FOXP3 to TLR10 gene transcription in primary T cells was established through the transfection of primary CD4+CD25−FOXP3− T cells with a FOXP3 expression vector, which resulted in prompt production of TLR10 mRNA. Enhanced expression of TLR10 protein in primary Treg cells was induced in a calcium-dependent fashion through TCR activation. The suspected promotional cooperation between FOXP3 and NF-AT was established in the abolition of the luciferase signal upon transfection of a mutant FOXP3 devoid of NF-AT-binding activity. These results suggest that human Treg cells express TLR10, and this expression is regulated through a cooperative complex of FOXP3 and NF-AT.

Maturation Versus Death of Developing Double-Positive Thymocytes Reflects Competing Effects on Bcl-2 Expression and Can Be Regulated by the Intensity of CD28 Costimulation

Immature double-positive (DP) thymocytes mature into CD4+CD8− cells in response to coengagement of TCR with any of a variety of cell surface “coinducer” receptors, including CD2. In contrast, DP thymocytes are signaled to undergo apoptosis by coengagement of TCR with CD28 costimulatory receptors, but the molecular basis for DP thymocyte apoptosis by TCR plus CD28 coengagement is not known. In the present study, we report that TCR plus CD28 coengagement does not invariably induce DP thymocyte apoptosis but, depending on the intensity of CD28 costimulation, can induce DP thymocyte maturation. We demonstrate that distinct but interacting signal transduction pathways mediate DP thymocyte maturation signals and DP thymocyte apoptotic signals. Specifically, DP maturation signals are transduced by the extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and up-regulate expression of the antiapoptotic protein Bcl-2. In contrast, the apoptotic response stimulated by CD28 costimulatory signals is mediated by ERK/MAPK-independent pathways. Importantly, when TCR-activated thymocytes are simultaneously coengaged by both CD28 and CD2 receptors, CD28 signals can inhibit ERK/MAPK-dependent Bcl-2 protein up-regulation. Thus, there is cross-talk between the signal transduction pathways that transduce apoptotic and maturation responses, enabling CD28-initiated signal transduction pathways to both stimulate DP thymocyte apoptosis and also negatively regulate maturation responses initiated by TCR plus CD2 coengagement.

The analysis of costimulatory receptor signaling cascades in normal T lymphocytes using in vitro gene transfer and reporter gene analysis

Ligation of the antigen receptor and costimulatory receptors on the surface of T lymphocytes initiates intracellular signals that regulate cell-cycle progression and cell differentiation. To effectively manipulate the activation of T cells for immunotherapeutic applications, it will be important to understand how these signaling pathways are integrated to control specific gene transcription events. Here we describe a novel transient transfection procedure that efficiently introduces DNA into non-dividing normal human and murine T lymphocytes while maintaining high cell viability. Using this technique, reporter genes can be introduced to characterize intracellular signaling pathways that regulate specific gene transcription events in normal T-lymphocyte populations. We show that the CD28 receptor can be differentially coupled to downstream signaling pathways in different T-lymphocyte populations. In addition, we demonstrate that a gene encoding a tagged constitutively active mitogen-activated kinase kinase-1 protein can be transfected and rapidly expressed to regulate the expression of Bcl-2 in normal thymocytes.