bluuebunny/crossref_metadata_2025 huggingface dataset:
Requirement for Nuclear Factor-κB Activation by a Distinct Subset of CD40-Mediated Effector Functions in B Lymphocytes
Authors: Hsing Yina , Bishop Gail A.
Published: December 2022

CD40 stimulation, which is crucial for generating an effective T-dependent humoral response, leads to the activation of transcription factors NF-AT (nuclear factor of activated T cells), AP-1 (activator protein-1), and NF-κB (nuclear factor-κB). However, which CD40-mediated B cell functions actually require activation of specific transcription factors is unknown. We examined the causal relationship between NF-κB activation and CD40 effector functions by evaluating CD40 functions in the presence of an inducible mutant inhibitory κBα (IκBα) superrepressor. IκBαAA inhibited nuclear translocation of multiple NF-κB dimers without the complicating effect of depriving cells of NF-κB during development. This approach complements studies that use mice genetically deficient in single or multiple NF-κB subunits. Interestingly, only a subset of CD40 effector functions was found to require NF-κB activation. Both CD40-induced Ab secretion and B7-1 up-regulation were completely abrogated by expression of IκBαAA. Surprisingly, up-regulation of Fas, CD23, and ICAM-1 was partially independent, and up-regulation of LFA-1 was completely independent, of CD40-induced NF-κB activation. For the first time, it is clear that distinct transcription factors are required for the dynamic regulation of CD40 functions.
Control of Syngeneic Tumor Growth by Activation of CD8+ T Cells: Efficacy Is Limited by Migration Away from the Site and Induction of Nonresponsiveness
Authors: Shrikant Protul , Mescher Matthew F.
Published: December 2022

Activation of Ag-specific CD8+ T cells in response to syngeneic tumor has been visualized by adoptive transfer of CD8+ T cells from OT-I mice, with a transgenic TCR specific for H-2Kb and an OVA peptide, into Thy-1 congenic recipients. Intraperitoneal challenge with E.G7, the EL-4 thymoma transfected with OVA, results in activation and clonal expansion of the OT-I cells in the peritoneal cavity and transient control of tumor growth. However, within 2 days after becoming activated, the OT-I cells migrate out of the peritoneal cavity into the spleen and lymph nodes, and tumor growth resumes in the peritoneal cavity. The OT-I cells in lymph nodes and spleen have lytic effector activity, but exhibit split anergy in that they cannot proliferate in response to Ag unless exogenous IL-2 is provided. The failure to remain at the tumor site and continue to control tumor growth is not due to selection of Ag loss variants or development of suppression. These results suggest that effective CD8-targeted immunotherapy may depend less on enhancing the initial activation and more on sustaining the response at the appropriate location and/or reactivating cells that have left the site of tumor growth and become nonresponsive.
Macrophage Control of Herpes Simplex Virus Type 1 Replication in the Peripheral Nervous System
Authors: Kodukula Padma , Liu Ting , Rooijen Nico Van , Jager Martine J. , Hendricks Robert L.
Published: December 2022

After corneal infection, herpes simplex virus type 1 (HSV-1) invades sensory neurons with cell bodies in the trigeminal ganglion (TG), replicates briefly, and then establishes a latent infection in these neurons. HSV-1 replication in the TG can be detected as early as 2 days after corneal infection, reaches peak titers by 3–5 days after infection, and is undetectable by 7–10 days. During the period of HSV-1 replication, macrophages and γδ TCR+ T lymphocytes infiltrate the TG, and TNF-α, IFN-γ, the inducible nitric oxide synthase (iNOS) enzyme, and IL-12 are expressed. TNF-α, IFN-γ, and the iNOS product nitric oxide (NO) all inhibit HSV-1 replication in vitro. Macrophage and γδ TCR+ T cell depletion studies demonstrated that macrophages are the main source of TNF-α and iNOS, whereas γδ TCR+ T cells produce IFN-γ. Macrophage depletion, aminoguanidine inhibition of iNOS, and neutralization of TNF-α or IFN-γ all individually and synergistically increased HSV-1 titers in the TG after HSV-1 corneal infection. Moreover, individually depleting macrophages or neutralizing TNF-α or IFN-γ markedly reduced the accumulation of both macrophages and γδ TCR+ T cells in the TG. Our findings establish that after primary HSV-1 infection, the bulk of virus replication in the sensory ganglia is controlled by macrophages and γδ TCR+ T lymphocytes through their production of antiviral molecules TNF-α, NO, and IFN-γ. Our findings also strongly suggest that cross-regulation between these two cell types is necessary for their accumulation and function in the infected TG.
Effect of Nitric Oxide Donors on Oxygen-Dependent Cytotoxic Responses Mediated by Neutrophils
Authors: Andonegui G. , Trevani A. S. , Gamberale R. , Carreras M. C. , Poderoso J. J. , Giordano M. , Geffner J. R.
Published: December 2022

We analyzed the effect of nitric oxide (NO) on oxygen-dependent cytotoxic responses mediated by neutrophils against unopsonized erythrocytes using three NO donors: S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP), and sodium nitroprusside (SNP). Neutrophils were treated with these compounds for 1–2 min at 37°C and cytotoxicity was then triggered in the presence of NO donors by precipitating immune complexes, aggregated IgG, the chemotactic peptide FMLP, or opsonized zymosan. GSNO induced, in all cases, a marked increase in cytotoxic responses, while SNAP moderately increased cytotoxicity triggered by immune complexes, aggregated IgG, or Z, opsonized zymosen, without modifying those responses induced by FMLP. By contrast, SNP dramatically suppressed cytotoxicity triggered by all of the stimuli assessed. The enhancing effects mediated by GSNO and SNAP did not depend on the stimulation of guanylyl cyclase and were prevented by the NO scavengers hemoglobin and PTIO (2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide). The inhibitory activity of SNP, on the other hand, was not prevented by NO scavengers, suggesting that it cannot be ascribed to the release of NO. In another set of experiments, neutrophils were pretreated with GSNO or SNAP for different times. Then cells were washed to remove NO donors from the culture medium, and cytotoxicity was triggered by different stimuli. It was found that neutrophils must be pretreated with NO donors for at least 4 h to increase cytotoxic responses, and pretreatment for longer periods (i.e., 8 or 18 h) further increased cytotoxicity. Not only cytotoxic responses, but also the production of O2− and H2O2, and the release of myeloperoxidase were increased under these conditions.
Selective Regulation of Cytokine Induction by Adenoviral Gene Transfer of IκBα into Human Macrophages: Lipopolysaccharide-Induced, But Not Zymosan-Induced, Proinflammatory Cytokines Are Inhibited, But IL-10 Is Nuclear Factor-κB Independent
Authors: Bondeson Jan , Browne Kylie A. , Brennan Fionula M. , Foxwell Brian M. J. , Feldmann Marc
Published: December 2022

Macrophages are the major cytokine producers in chronic inflammatory diseases, but the biochemical pathways regulating cytokine production are poorly understood. This is because genetic tools to dissect signaling pathways cannot be used in macrophages because of difficulties in transfection. We have developed an adenoviral technique to achieve high efficiency gene delivery into macrophages and recently showed that spontaneous TNF-α production in rheumatoid arthritis joint cells, chiefly from macrophages, is 75% blocked by adenoviral transfer of IκBα. In this report we use the same adenovirus to investigate whether the production of a number of proinflammatory cytokines (e.g., TNF-α, IL-1β, IL-6, and IL-8) from human macrophages depends on NF-κB. While the cytokine response to certain inducers, such as LPS, PMA, and UV light, is blocked by overexpression of IκBα, the response to zymosan is not. In contrast, anti-inflammatory mediators (IL-10 and IL-1 receptor antagonist) induced by LPS are only marginally inhibited by IκBα excess. These studies demonstrate several new points about macrophage cytokine production. First, there is heterogeneity of mechanisms regulating both the proinflammatory and anti-inflammatory cytokines within populations of a single cell type. In addition, the results confirm the utility of the adenoviral technique for functional analysis of cytokine induction. The results also confirm that there are autocrine and paracrine interactions regulating cytokine synthesis within a single cell type. The selectivity of NF-κB blockade for proinflammatory but not anti-inflammatory mediators indicates that in macrophages, NF-κB may be a good target for the treatment of chronic inflammatory diseases.
Ethanol-Induced Macrophage Apoptosis: The Role of TGF-β
Authors: Singhal Pravin C. , Reddy Krishna , Ding Guohua , Kapasi Aditi , Franki Nicholas , Ranjan Rajiv , Nwakoby Izuchukwu E. , Gibbons Nora
Published: December 2022

Both clinical and laboratory reports indicate that ethanol addicts are prone to recurrent infections. We hypothesize that ethanol promotes macrophage apoptosis, thus compromising the efficiency of the mononuclear phagocyte system in dealing with infection. We studied the effect of ethanol on macrophage apoptosis. Human monocytes isolated from healthy subjects after an alcohol drinking binge showed enhanced apoptosis (before, 1.2 ± 0.3% vs after, 28.4 ± 3.7% apoptotic cells/field). Peritoneal macrophages harvested from ethanol-treated rats also showed increased (p < 0.0001) apoptosis. DNA isolated from peritoneal macrophages of ethanol-treated rats displayed integer multiples of 200 base pairs (ladder pattern). Furthermore, macrophages harvested from ethanol-treated rats had an enhanced expression as well as accumulation of TGF-β. In in vitro studies, ethanol promoted apoptosis of human monocytes as well as rat peritoneal macrophages. In addition, ethanol enhanced apoptosis of murine macrophages (J774) in a time-dependent manner. The ethanol-induced apoptosis was amplified by LPS and partly attenuated (p < 0.001) by anti-TGF-β Ab. TGF-β also promoted macrophage apoptosis in a dose-dependent manner. Moreover, ethanol enhanced TGF-β protein production by macrophages. These results indicate that ethanol promotes macrophage apoptosis. This effect of ethanol seems to be partly mediated through the generation of TGF-β by macrophages.
EBV Structural Antigens, gp350 and gp85, as Targets for Ex Vivo Virus-Specific CTL During Acute Infectious Mononucleosis: Potential Use of gp350/gp85 CTL Epitopes for Vaccine Design
Authors: Khanna Rajiv , Sherritt Martina , Burrows Scott R.
Published: December 2022

For many years, EBV vaccine development efforts have concentrated on the use of structural Ag, gp350, and have been directed toward Ab-mediated blocking virus attachment to the target cell. There is increasing evidence to suggest that the development of neutralizing Abs in vaccinated animals does not always correlate with protection; nevertheless, it has been postulated that gp350-specific T cell-mediated immune responses may have an effector role in protection. This hypothesis has largely remained untested. In the present study, we demonstrate that CTL from acute infectious mononucleosis patients display strong ex vivo reactivity against the EBV structural Ags, gp85 and gp350. Moreover, long-term follow up studies on infectious mononucleosis-recovered individuals showed that these individuals maintain gp350- and gp85-specific memory CTL, albeit at low levels, in the peripheral blood. These results strongly suggest that CTL specific for EBV structural proteins may play an important role in the control of EBV infection during acute infection. More importantly, we also show that prior immunization of HLA A2/Kb transgenic mice with gp350 and gp85 CTL epitopes induced a strong epitope-specific CTL response and afforded protection against gp85- or gp350-expressing vaccinia virus challenge. These results have important implications for future EBV vaccine design and provides evidence, for the first time, that CTL epitopes from EBV structural proteins may be used for establishing strong antiviral immunity against EBV infection.
Cutting Edge: Protective Response to Pulmonary Injury Requires γδ T Lymphocytes
Authors: King Donald P. , Hyde Dallas M. , Jackson Kenneth A. , Novosad Denise M. , Ellis Terri N. , Putney Lei , Stovall Mary Y. , Van Winkle Laura S. , Beaman Blaine L. , Ferrick David A.
Published: December 2022

γδ intraepithelial lymphocytes are thought to coordinate responses to pathogens that penetrate the epithelial barrier. To directly test this, mice were inoculated with Nocardia asteroides. At doses that were nonlethal for control mice, γδ-deficient mice became severely ill and died within 14 days. Histologic examination of these lungs demonstrated the presence of severe tissue damage and unimpeded bacterial growth in the γδ-deficient mice compared with neutrophilic lesions and clearance of the organism in control mice. Interestingly, ozone exposure that targets a comparable lung region also resulted in diffuse epithelial necrosis associated with a similar lack of neutrophil recruitment in γδ-deficient mice. These data demonstrate that γδ intraepithelial lymphocytes can protect the host from pathogenic and nonpathogenic insults by targeting the inflammatory response to epithelial necrosis.
Cutting Edge: HLA-B27 Can Form a Novel β2-Microglobulin-Free Heavy Chain Homodimer Structure
Authors: Allen Rachel L. , O’Callaghan Chris A. , McMichael Andrew J. , Bowness Paul
Published: December 2022

HLA-B27 has a striking association with inflammatory arthritis. We show that free HLA-B27 heavy chains can form a disulfide-bonded homodimer, dependent on residue Cys67 in their extracellular α1 domain. Despite the absence of β2-microglobulin, HLA-B27 heavy chain homodimers (termed HC-B27) were stabilized by a known peptide epitope. HC-B27 complexes were recognized by the conformation-specific Ab W6/32, but not the ME1 Ab. Surface labeling and immunoprecipitation demonstrated the presence of similar W6/32-reactive free heavy chains at the surface of HLA-B27-transfected T2 cells. HC-B27 homodimer formation might explain the ability of HLA-B27 to induce spondyloarthropathy in β2-microglobulin-deficient mice.
IL-18 Is a Potent Coinducer of IL-13 in NK and T Cells: A New Potential Role for IL-18 in Modulating the Immune Response
Authors: Hoshino Tomoaki , Wiltrout Robert H. , Young Howard A.
Published: December 2022

IL-13 and IL-4 have similar biological activities and are characteristic of cytokines expressed by Th2 cells. In contrast, IL-12 and IL-18 have been shown to be strong cofactors for Th1 cell development. In this study, we found strong induction of IL-13 mRNA and protein by IL-2 + IL-18 in NK and T cells. In contrast, IL-12 did not enhance the IL-13 production induced by IL-2 alone. Moreover, IL-13 mRNA and protein expression induced by IL-2 + IL-18 in purified NK and T cells obtained from IFN-γ knockout (−/−) mice were greater than seen in purified cells from normal controls. In contrast, IL-10 production induced by IL-2 and/or IL-12 was not significantly different in IFN-γ (−/−) mice and normal controls. These results suggest IL-13 expression induced by IL-2 + IL-18 may be regulated by IFN-γ in vivo, while IL-10 expression may be IFN-γ-independent. Thus, depending upon the cell type, IL-18 may act as a strong coinducer of Th1 or Th2 cytokines. Our findings suggest that IL-12 and IL-18 have different roles in the regulation of gene expression in NK and T cells.
T Cell Reconstitution of BB/W Rats After the Initiation of Insulitis Precipitates the Onset of Diabetes
Authors: Ramanathan Sheela , Poussier Philippe
Published: December 2022

One of the diabetes susceptibility genes of the BB/W (Biobreeding/Worcester) rat maps to the lyp locus on chromosome 4. The BB/W lyp allele is responsible for a severe peripheral T lymphopenia. Correction of this lymphopenia by transfer of normal, histocompatible T cells prevents diabetes, providing T cell reconstitution is initiated before insulitis. We have analyzed this time-dependent regulation of the diabetogenic process by normal T cells. We demonstrate that T cell reconstitution after the initiation of insulitis precipitates the onset of diabetes through the recruitment of donor T cells to the autoimmune process. This inability of normal T cells to regulate primed diabetogenic BB/W T cells and their own autoreactive potential were observed when normal T cells outnumbered pathogenic T cells by approximately 1000-fold. Analysis of donor-derived T cells recovered from BB/W rats that were reconstituted before insulitis, and hence protected from diabetes, demonstrates that early T cell reconstitution of BB/W rats does not result in a long term physical or functional depletion of islet cell-specific T cell precursors among donor cells or in the expansion of T cells that can regulate the activation and expansion of diabetogenic T cells.
A Novel Approach to Visualize Polyclonal Virus-Specific CD8 T Cells In Vivo
Authors: Zimmermann Christine , Pircher Hanspeter
Published: December 2022

Recent technical breakthroughs in generating soluble MHC class I-peptide tetramers now allow the direct visualization of virus-specific CD8 T cells after infection in vivo. However, this technique requires the knowledge of the immunodominant viral epitopes recognized by T cells. Here, we describe an alternative approach to visualize polyclonal virus-specific CD8 T cells in vivo using a simple adoptive transfer system. In our approach, C57BL/6 (Thy1.2) mice were infected with lymphocytic choriomeningitis virus, vesicular stomatitis virus, or vaccinia virus to induce virus-specific memory T cells. Tracer T cells (2 × 106) from these virus-immune mice were adoptively transferred into nonirradiated (C57BL/6 × B6.PL-Thy-1a)F1 mice. After infection of the F1-recipient mice with the appropriate virus, the transferred cells expanded vigorously, and on day 8 postinfection 60–80% of total CD8 T cells were of donor T cell origin. Under the same conditions memory CD4 T cells gave rise to at least 10 times less cell numbers than memory CD8 T cells. The transfer system described here not only allows to visualize effector and memory CD8 T cells in vivo but also to isolate them for further in vitro characterization without knowing the epitopes recognized by these Ag-specific CD8 T cells.
Signaling Pathways Activated by Leukocyte Function-Associated Ag-1-Dependent Costimulation
Authors: Ni Hsiao-Tzu , Deeths Matthew J. , Li Wei , Mueller Daniel L. , Mescher Matthew F.
Published: December 2022

LFA-1 binding to ICAM-1 can enhance TCR-dependent proliferation of T cells, but it has been difficult to distinguish contributions from increased adhesion, and thus TCR occupancy, versus costimulatory signaling. Whether LFA-1 ligation results in generation of a unique costimulatory signal(s) distinct from those activated by the TCR has been unclear. Using purified ligands, it is shown that ICAM-1 and B7.1 provide comparable costimulation for proliferation of CD8+ T cells, and that both ligands up-regulate the activities of phosphatidylinositol 3-kinase, sphingomyelinase, and c-Jun NH2-terminal kinase (JNK). These pathways are distinct from those activated by the TCR, and have previously been implicated in up-regulating IL-2 production in response to CD28-B7 interaction. Thus, under conditions in which ICAM-1 provides costimulation of proliferation, LFA-1 ligation activates some of the same signaling pathways as does CD28 ligation. LFA-1 and CD28 do not act identically, however, as indicated by differential sensitivity to inhibitors of phosphatidylinositol 3-kinase; LFA-1-dependent costimulation of proliferation is inhibited, while CD28-dependent costimulation is not. Given the broad distribution of class I and ICAMs on many cell types, the ability of LFA-1 to provide costimulatory signals has implications for where and how CD8+CTL may become activated in response to an antigenic challenge.
Cell Cycle-Dependent Regulation of FLIP Levels and Susceptibility to Fas-Mediated Apoptosis
Authors: Algeciras-Schimnich Alicia , Griffith Thomas S. , Lynch David H. , Paya Carlos V.
Published: December 2022

Activation-induced cell death of peripheral T cells results from the interaction between Fas and Fas ligand. Resting peripheral T cells are resistant to Fas-induced apoptosis and become susceptible only after their activation. We have investigated the molecular mechanism mediating the sensitization of resting peripheral T cells to Fas-mediated apoptosis following TCR stimulation. TCR activation decreases the steady state protein levels of FLIP (FLICE-like inhibitory protein), an inhibitor of the Fas signaling pathway. Reconstitution of intracellular FLIP levels by the addition of a soluble HIV transactivator protein-FLIP chimera completely restores resistance to Fas-mediated apoptosis in TCR primary T cells. Inhibition of IL-2 production by cyclosporin A, or inhibition of IL-2 signaling by rapamycin or anti-IL-2 neutralizing Abs prevents the decrease in FLIP levels and confers resistance to Fas-mediated apoptosis following T cell activation. Using cell cycle-blocking agents, we demonstrate that activated T cells arrested in G1 phase contain high levels of FLIP protein, whereas activated T cells arrested in S phase have decreased FLIP protein levels. These findings link regulation of FLIP protein levels with cell cycle progression and provide an explanation for the increase in TCR-induced apoptosis observed during the S phase of the cell cycle.
NF-κB-Inducing Kinase Is a Common Mediator of IL-17-, TNF-α-, and IL-1β-Induced Chemokine Promoter Activation in Intestinal Epithelial Cells
Authors: Awane Masaaki , Andres Pietro G. , Li Dan Jun , Reinecker Hans-Christian
Published: December 2022

IL-17 expression is restricted to activated T cells, whereas the IL-17R is expressed in a variety of cell types including intestinal epithelial cells. However, the functional responses of intestinal epithelial cells to stimulation with IL-17 are unknown. Moreover, the signal transduction pathways activated by the IL-17R have not been characterized. IL-17 induced NF-κB protein-DNA complexes consisting of p65/p50 heterodimers in the rat intestinal epithelial cell line IEC-6. The induction of NF-κB correlated with the induction of CXC and CC chemokine mRNA expression in IEC-6 cells. IL-17 acted in a synergistic fashion with IL-1β to induce the NF-κB site-dependent CINC promoter. Induction of the CINC promoter by IL-17 in IEC-6 cells was TNF receptor-associated factor-6 (TRAF6), but not TRAF2, dependent. Furthermore, IL-17 induction of the CINC promoter could be inhibited by kinase-negative mutants of NF-κB-inducing kinase and IκB kinase-α. In addition to activation of the NF-κB, IL-17 regulated the activities of extracellular regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases in IEC-6 cells. Whereas the IL-17-mediated activation of extracellular regulated kinase mitogen-activated protein kinases was mediated through ras, c-Jun N-terminal kinase activation was dependent on functional TRAF6. These data suggest that NF-κB-inducing kinase serves as the common mediator in the NF-κB signaling cascades triggered by IL-17, TNF-α, and IL-1β in intestinal epithelial cells.
HLA Class I-Mediated Induction of Cell Proliferation Involves Cyclin E-Mediated Inactivation of Rb Function and Induction of E2F Activity
Authors: Nath Niharika , Bian Hong , Reed Elaine F. , Chellappan Srikumar P.
Published: December 2022

Chronic rejection of transplanted organs is manifested as atherosclerosis of the blood vessels of the allograft. HLA class I Ags have been implicated to play a major role in this process, since signaling via HLA class I molecules can induce the proliferation of aortic endothelial as well as smooth muscle cells. In this study, we show that HLA class I-mediated induction of cell proliferation correlates with inactivation of the Rb protein in the T cell line Jurkat as well as human aortic endothelial cells. HLA class I-mediated inactivation of Rb can be inhibited specifically by neutralizing Abs to basic fibroblast growth factor (bFGF), suggesting a role for FGF receptors in the signaling process. Signaling through HLA class I molecules induced cyclin E-associated kinase activity within 4 h in quiescent endothelial cells, and appeared to mediate the inactivation of Rb. A cdk2 inhibitor, Olomoucine, as well as a dominant-negative cdk2 construct prevented HLA class I-mediated inactivation of Rb; in contrast, dominant-negative cdk4 and cdk6 constructs had no effect. Furthermore, there was no increase in cyclin D-associated kinase activity upon HLA class I ligation, suggesting that cyclin E-dependent kinase activity mediates Rb inactivation, leading to E2F activation and cell proliferation.
The Role of p38 Mitogen-Activated Protein Kinase in IL-1β Transcription
Authors: Baldassare Joseph J. , Bi Yanhua , Bellone Clifford J.
Published: December 2022

Several reports have shown that bicyclic imidazoles, specific inhibitors of the p38 mitogen-activated protein kinase (MAPK), block cytokine synthesis at the translational level. In this study, we examined the role of p38 MAPK in the regulation of the IL-1β cytokine gene in monocytic cell lines using the bicyclic imidazole SB203580. Addition of SB203580 30 min before stimulation of monocytes with LPS inhibited IL-1β protein and steady state message in a dose-dependent manner in both RAW264.7 and J774 cell lines. The loss of IL-1β message was due mainly to inhibition of transcription, since nuclear run-off analysis showed an ∼80% decrease in specific IL-1 RNA synthesis. In contrast, SB203580 had no effect on the synthesis of TNF-α message. LPS-stimulated p38 MAPK activity in the RAW264.7 cells was blocked by SB203580, as measured by the inhibition of MAPKAP2 kinase activity, a downstream target of the p38 MAPK. CCAATT/enhancer binding protein (C/EBP)/NFIL-6-driven chloramphenicol acetyltransferase (CAT) reporter activity was sensitive to SB203580, indicating that C/EBP/NFIL-6 transcription factor(s) are also targets of p38 MAPK. In contrast, transfected CAT constructs containing NF-κB elements were only partially inhibited (∼35%) at the highest concentration of SB203580 after LPS stimulation. As measured by EMSA, LPS-stimulated NF-κB activation was not affected by SB203580. Overall, the results demonstrate, for the first time, a role for p38 MAPK in IL-1β transcription by acting through C/EBP/NFIL-6 transcription factors.
Peptide Recognition by Two HLA-A2/Tax11–19-Specific T Cell Clones in Relationship to Their MHC/Peptide/TCR Crystal Structures
Authors: Hausmann Stefan , Biddison William E. , Smith Kathrine J. , Ding Yuan-Hua , Garboczi David N. , Utz Ursula , Wiley Don C. , Wucherpfennig Kai W.
Published: December 2022

The crystal structures of two human TCRs specific for a HTLV-I Tax peptide bound to HLA-A2 were recently determined, for the first time allowing a functional comparison of TCRs for which the MHC/peptide/TCR structures are known. Extensive amino acid substitutions show that the native Tax residues are optimal at each peptide position. A prominent feature of the TCR contact surface is a deep pocket that accommodates a tyrosine at position 5 of the peptide. For one of these TCRs, this pocket is highly specific for aromatic residues. In the other TCR structure, this pocket is larger, allowing many different residues to be accommodated. The CTL clones also show major differences in the specificity for several other peptide residues, including side chains that are not directly contacted by the TCR. Despite the specificity of these clones, peptides that are distinct at five or six positions from Tax11–19 induce CTL activity, indicating that substantial changes of the peptide surface are tolerated. Human peptides with limited sequence homology to Tax11–19 represent partial TCR agonists for these CTL clones. The distinct functional properties of these CTL clones highlight structural features that determine TCR specificity and cross-reactivity for MHC-bound peptides.
T Cell Responses to Gram-Negative Intracellular Bacterial Pathogens: A Role for CD8+ T Cells in Immunity toSalmonellaInfection and the Involvement of MHC Class Ib Molecules
Authors: Lo Wei-Feng , Ong Helena , Metcalf Eleanor S. , Soloski Mark J.
Published: December 2022

Despite being a major group of intracellular pathogens, the role of class I-restricted T cells in the clearance of Gram-negative bacteria is not resolved. Using a murine typhoid model, a role for class I-restricted T cells in the immune response to the Gram-negative pathogen Salmonella typhimurium is revealed. Class I-deficient β2-microglobulin−/− mice show increased susceptibility to infection with S. typhimurium. Following infection, CD8+ CTLs specific for Salmonella-infected targets can be readily detected. The Salmonella-specific CTLs recognize infected H-2-mismatched targets, suggesting the involvement of shared class Ib molecules. Studies using transfectants expressing defined class Ia and class Ib molecules indicate the involvement of the class Ib molecule, Qa-1. Ab-blocking studies and the measurement of bacteria-specific CTL frequencies identified Qa-1 as a dominant restricting element. The Qa-1-restricted CTL recognition depends on TAP and proteasome functions. Surprisingly, Qa-1-restricted CTLs recognized cells infected with other closely related Gram-negative bacteria. Taken together, these observations indicate that Salmonella-specific CTLs recognize a cross-reactive epitope presented by Qa-1 molecules and, as such, may be novel targets for vaccine development.
CD8+ T Cells Are a Biologically Relevant Source of Macrophage Inflammatory Protein-1α In Vivo
Authors: Cook Donald N. , Smithies Oliver , Strieter Robert M. , Frelinger Jeffrey A. , Serody Jonathan S.
Published: December 2022

Chemokines are small proteins that direct the migration of leukocytes to inflammatory foci. Many cell types, including macrophages, fibroblasts, endothelial cells, and lymphocytes, produce chemokines in vitro, but biologically relevant sources of chemokines in vivo have not been well characterized. To investigate the pertinent sources of macrophage inflammatory protein-1α (MIP-1α) in vivo, we used MIP-1α-deficient (MIP-1α−/−) mice as donors and as recipients in adoptive transfer experiments after a lethal infection with Listeria monocytogenes (LM). Unexpectedly, we found that the production of MIP-1α by CD8+ T cells was critical in this system, as the cells from MIP-1α−/− mice primed with LM were significantly less effective in protecting naive mice against a lethal infection by LM than were the CD8+ T cells from wild-type (wt) mice. This requirement for donor T cell production of MIP-1α was confirmed by the observation that wt donor T cells do not mediate protection when coadministered with an anti-MIP-1α polyclonal antiserum. Production of MIP-1α by the recipient mice was not required for protection, because wt and MIP-1α−/− recipients were equally well protected by wt T cells. A 2- to 3-fold decrease in the number of transferred lymphocytes was seen in the spleens of mice receiving T cells from MIP-1α−/− mice compared with those receiving wt T cells. In addition, CD8+ T cells from MIP-1α−/− mice had a reduced ability to kill LM-infected target cells in vitro. These findings demonstrate that T cell production of MIP-1α is required for clearance of an intracellular pathogen in vivo.
Inhibition of a Membrane Complement Regulatory Protein by a Monoclonal Antibody Induces Acute Lethal Shock in Rats Primed with Lipopolysaccharide
Authors: Mizuno Masashi , Nishikawa Kazuhiro , Okada Noriko , Matsuo Seiichi , Ito Kenichi , Okada Hidechika
Published: December 2022

Rats pretreated with traces of LPS developed acute fatal shock syndrome after i.v. administration of a mAb that inhibits the function of a membrane complement regulatory molecule. Such a shock was not observed after the administration of large amounts of LPS instead of the mAb following LPS pretreatment. The lethal response did not occur in rats depleted of either leukocytes or complement, and a C5a receptor antagonist was found to inhibit the reaction. Furthermore, LPS-treated rats did not suffer fatal shock following the injection of cobra venom factor, which activates complement in the fluid phase so extensively as to exhaust complement capacity. Therefore, complement activation on cell membranes is a requirement for this type of acute reaction.
Ischemia/Reperfusion-Induced IFN-γ Up-Regulation: Involvement of IL-12 and IL-18
Authors: Daemen Marc A. R. C. , Veer Cornelis van′t , Wolfs Tim G. A. M. , Buurman Wim A.
Published: December 2022

Tissue injury as a consequence of ischemia followed by reperfusion is characterized by early as well as late signs of inflammation. The latter, among others, involves IFN-γ-dependent up-regulation of MHC class I and II Ag expression. Employing a murine model of renal ischemia, we show that renal IL-18 mRNA up-regulation coincides with caspase-1 activation at day 1 following ischemia. IFN-γ and IL-12 mRNA are subsequently up-regulated at day 6 following ischemia. Combined, but not separate, in vivo neutralization of the IFN-γ inducing cytokines IL-12 and IL-18 reduces IFN-γ-dependent MHC class I and II up-regulation to a similar extent as IFN-γ neutralization, suggesting the involvement of functional IL-12, IL-18, and IFN-γ protein. These results reveal a novel relationship between tissue injury of nonmicrobial origin and the induction of IL-12 as well as IL-18. The collaboration observed between endogenous IL-12 and IL-18 in the induction of IFN-γ after renal ischemia/reperfusion, resembles the immune response to bacterial infections.
Mast Cell Expression of Gelatinases A and B Is Regulated bykitLigand and TGF-β
Authors: Fang Kenneth C. , Wolters Paul J. , Steinhoff Martin , Bidgol Afshin , Blount John L. , Caughey George H.
Published: December 2022

Our prior work shows that cultured BR cells derived from dog mastocytomas secrete the 92-kDa proenzyme form of gelatinase B. We provided a possible link between mast cell activation and metalloproteinase-mediated matrix degradation by demonstrating that α-chymase, a serine protease released from secretory granules by degranulating mast cells, converts progelatinase B to an enzymatically active form. The current work shows that these cells also secrete gelatinase A. Furthermore, gelatinases A and B both colocalize to α-chymase-expressing cells of canine airway, suggesting that normal mast cells are a source of gelatinases in the lung. In BR cells, gelatinase B and α-chymase expression are regulated, whereas gelatinase A expression is constitutive. Progelatinase B mRNA and enzyme expression are strongly induced by the critical mast cell growth factor, kit ligand, which is produced by fibroblasts and other stromal cells. Induction of progelatinase B is blocked by U-73122, Ro31-8220, and thapsigargin, implicating phospholipase C, protein kinase C, and Ca2+, respectively, in the kit ligand effect. The profibrotic cytokine TGF-β virtually abolishes the gelatinase B mRNA signal and also attenuates kit ligand-mediated induction of gelatinase B expression, suggesting that an excess of TGF-β in inflamed or injured tissues may alter mast cell expression of gelatinase B, which is implicated in extracellular matrix degradation, angiogenesis, and apoptosis. In summary, these data provide the first evidence that normal mast cells express gelatinases A and B and suggest pathways by which their regulated expression by mast cells can influence matrix remodeling and fibrosis.
Decreased IL-12 Production Underlies the Decreased Ability of Male Lymph Node Cells to Induce Experimental Autoimmune Encephalomyelitis
Authors: Kim Sookhyun , Voskuhl Rhonda R.
Published: December 2022

Myelin basic protein (MBP)-specific T lymphocytes from male SJL mice were shown to be less encephalitogenic than MBP-specific T lymphocytes from females. Mechanisms underlying this gender difference in the induction phase of EAE were examined. Following immunization with MBP, draining lymph nodes contained fewer cells, and Ag-specific proliferative responses were decreased in males as compared with females. These gender differences in the proliferative response were not unique to MBP-specific responses since they were also observed after immunization with hen eggwhite lysozyme. Short-term MBP-specific T cell lines derived from females and males mapped with identical specificity, indicating no defect in the ability of male APCs to process Ag. Interestingly, IL-12 and IFN-γ production was decreased following Ag-specific stimulation of draining lymph node cells (LNC) from males as compared with females, but IL-10 and IL-4 were no different. While male-derived LNCs were less encephalitogenic than female derived LNCs, cotransfer and coculture of male LNCs with female LNCs demonstrated that male LNCs were not immunosuppressive. Administration of IL-12 to LNCs from male mice enhanced encephalitogenicity. These data indicate that deficient endogenous IL-12 production within draining LNCs of male SJL mice is central to gender differences in the induction phase of experimental autoimmune encephalomyelitis.
Emergence of Regulatory CD4+ T Cell Response to Repetitive Stimulation with Antigen-Presenting Cells In Vitro: Implications in Designing Antigen-Presenting Cell-Based Tumor Vaccines
Authors: Chakraborty Nitya G. , Li Li , Sporn Jonathan R. , Kurtzman Scott H. , Ergin M. T. , Mukherji Bijay
Published: December 2022

Because APCs play a crucial role in the generation of T cell-mediated immune responses, numerous clinical trials with APC-based vaccines have been initiated in different types of human cancers. Encouraging results have emerged from some of these initial studies. Thus far, APC-based vaccinations usually include multiple rounds of immunization. With this approach, although we and others have detected induction of Ag-specific CTL responses in vaccinated patients after stimulation with the same APC-based immunogen, in vitro we also find that repetitive in vitro stimulation with Ag-loaded APC can, at times, lead to the emergence of noncytolytic CD4+ T cells exhibiting the characteristic phenotype of Th2 cells. These noncytolytic CD4+ T cells synthesize large quantities of type 2 cytokines such as IL-4 and IL-10 on stimulation with the autologous APC or tumor cells in an MHC class II-restricted manner. Further, these CD4+ T cells and a cell-free supernatant factor block the activation of fresh T lymphocytes. The supernatant factor also exhibits a marked inhibitory effect on the expression of the costimulatory molecules, CD80 and CD86, by APC. The inhibitory effect of the supernatant factor can be abrogated by neutralizing IL-10 in the supernatant. These observations therefore have implications in the APC-based tumor vaccine protocol design.
An Adoptive Transfer Model of Allergic Lung Inflammation in Mice Is Mediated by CD4+CD62LlowCD25+ T Cells
Authors: Wise James T. , Baginski Theodore J. , Mobley James L.
Published: December 2022

Animal models of allergic lung inflammation have provided important insight into the cellular and biochemical factors involved in the pathogenesis of human asthma. Herein, we describe an adoptive transfer model of OVA-specific eosinophilic lung inflammation in the mouse that is used to characterize the cells involved in mediating the pulmonary inflammatory response. We report that freshly isolated spleen cells from OVA-sensitized mice are unable to prime naive recipient mice to respond to a subsequent OVA aerosol challenge. Subjecting the spleen cells to short term restimulation with Ag in vitro, however, renders the cells competent to transfer activity. The magnitude and the kinetics of the eosinophilic pulmonary inflammation in the adoptive transfer recipients are nearly identical with those generated by a more conventional active sensitization/challenge protocol, with the notable exception of differential production of plasma IgE in the two models. Extensive negative and positive selection of splenocyte subtypes indicates that the transfer of Ag-primed CD4+ T cells is both necessary and sufficient to establish full responsiveness in the recipient mice. Additional phenotypic characterization of the transfer-reactive CD4+ T cells indicates that they are found within the CD62LlowCD25+ subset and secrete high levels of IL-5 in response to Ag stimulation. Limiting dilution analysis-derived minimal frequency estimates indicate that approximately 1 in 8500 of the sensitized, cultured spleen cells produces IL-5 in response to OVA stimulation in vitro, suggesting that eosinophilic lung inflammation can be induced in naive mice by the transfer of <1200 Ag-specific CD4+ T cells.
Down-Regulation of Human Basophil IgE and FCεRIα Surface Densities and Mediator Release by Anti-IgE-Infusions Is Reversible In Vitro and In Vivo
Authors: Saini Sarbjit S. , MacGlashan Donald W. , Sterbinsky Sherry A. , Togias Alkis , Adelman Daniel C. , Lichtenstein Lawrence M. , Bochner Bruce S.
Published: December 2022

Previously, infusions of an anti-IgE mAb (rhumAb-E25) in subjects decreased serum IgE levels, basophil IgE and FcεRIα surface density, and polyclonal anti-IgE and Ag-induced basophil histamine release responses. We hypothesized that these effects would be reversed in vivo by discontinuation of infusions and in vitro by exposing basophils to IgE. Subjects received rhumAb-E25 biweekly for 46 wk. Blood samples taken 0–52 wk after rhumAb-E25 were analyzed for serum IgE and basophil expression of IgE, FcεRIα, and CD32. Basophil numbers were unaffected by infusions. Eight weeks after infusions, free IgE levels rose in vivo but did not reach baseline. Basophil IgE and FcεRIα rose in parallel with free IgE while CD32 was stable. FcεRI densities, measured by acid elution, returned to 80% of baseline, whereas histamine release responses returned to baseline. Basophils cultured with or without IgE or IgG were analyzed for expression of IgE, FcεRIα, and CD32. By 7 days with IgE, expression of IgE and FcεRIα rose significantly, whereas cultures without IgE declined. IgE culture did not effect CD32. IgG culture did not effect expression of any marker. The present results strongly suggest that free IgE levels regulate FcεRIα expression on basophils.
Differential Modulation of Human Epidermal Langerhans Cell Maturation by Ultraviolet B Radiation
Authors: Nakagawa Satoshi , Koomen Cock W. , Bos Jan D. , Teunissen Marcel B. M.
Published: December 2022

UVB irradiation of the skin causes immunosuppression and Ag-specific tolerance in which Langerhans cells (LC) are involved. We tested the effect of UVB on LC that had migrated out of cultured epidermal sheets derived from the skin that was irradiated ex vivo (200, 400, 800, or 1600 J/m2). Two separate subpopulations of LC were distinguished: large-sized LC with high HLA-DR expression, and HLA-DR-low, small LC. UVB stimulated the maturation of the former LC subset as demonstrated by enhanced up-regulation of CD80, CD86, CD54, CD40, and CD83 and reduced CD1a expression in comparison with unirradiated controls. In contrast, the latter LC exhibited little or no up-regulation of these molecules except for high CD1a expression and high binding of annexin V, indicating that they were apoptotic, although their CD95 expression was relatively low. Stimulation of enriched LC with CD40 ligand-transfected cells and IFN-γ revealed that the release of IL-1β, IL-6, IL-8, and TNF-α was enhanced by UVB. In comparison with HLA-DR-low LC, HLA-DR-high LC were the principal IL-8 producers as demonstrated by intracellular cytokine staining, and they retained more accessory function. There was no detectable secretion of IL-12 p70, and IL-18 production was neither affected by any stimulus nor by UVB. These results suggest a dual action of UVB on LC when irradiated in situ: 1) immunosuppression by preventing maturation and inducing apoptotic cell death in part of LC, and 2) immunopotentiation by enhancing the up-regulation of costimulatory molecules and the production of proinflammatory cytokines in another part.
Induction of Tumor Immunity by Removing CD25+CD4+ T Cells: A Common Basis Between Tumor Immunity and Autoimmunity
Authors: Shimizu Jun , Yamazaki Sayuri , Sakaguchi Shimon
Published: December 2022

This study shows that removal of a T cell subpopulation can evoke effective tumor immunity in otherwise nonresponding animals. Elimination of CD25-expressing T cells, which constitute 5–10% of peripheral CD4+ T cells in normal naive mice, elicited potent immune responses to syngeneic tumors in vivo and eradicated them. The responses were mediated by tumor-specific CD8+ CTLs and tumor-nonspecific CD4−8− cytotoxic cells akin to NK cells. Furthermore, in vitro culture of CD25+4+ T cell-depleted splenic cell suspensions prepared from tumor-unsensitized normal mice led to spontaneous generation of similar CD4−8− cytotoxic cells capable of killing a broad spectrum of tumors; reconstitution of CD25+4+ T cells inhibited the generation. In this culture, self-reactive CD25−4+ T cells responding to self peptides/class II MHC complexes on APCs spontaneously proliferated upon removal of CD25+4+ T cells, secreting large amounts of IL-2. The IL-2 thus produced appeared to be responsible for the generation of CD4−8− NK cells as lymphokine-activated killer cells, because direct addition of an equivalent amount of IL-2 to the culture of CD4−8− cells generated similar lymphokine-activated killer/NK cells, whereas coculture of normal CD4−8− cells with CD25−4+ T cells from IL-2-deficient mice did not. Thus, removal of immunoregulatory CD25+4+ T cells can abrogate immunological unresponsiveness to syngeneic tumors in vivo and in vitro, leading to spontaneous development of tumor-specific effector cells as well as tumor-nonspecific ones. This novel way of evoking tumor immunity would help to devise effective immunotherapy for cancer in humans.
Generation of Mature Dendritic Cells from a CD14+ Cell Line (XS52) by IL-4, TNF-α, IL-1β, and Agonistic Anti-CD40 Monoclonal Antibody
Authors: Yamada Nobuo , Katz Stephen I.
Published: December 2022

We established a model system to generate mature dendritic cells (DC) from a GM-CSF-dependent cell line, XS52, which had been isolated from the epidermis of newborn BALB/c mice. Screening of various soluble factors revealed that IL-4 induces phenotypic maturation of XS52 (as evaluated by enhanced expression of class II, CD40, CD80, CD86, CD11c, and loss of expression of CD14) in a time-dependent manner. The addition of TNF-α, IL-1β, and agonistic anti-CD40 mAb further enhanced expression of these maturation markers. Consistent with their phenotypic maturation, these cells (termed XS-DC) exhibited potent Ag-presenting capacity to both naive and primed T cells. In addition, injection of hapten-conjugated XS-DC induced contact hypersensitivity in vivo, suggesting their potential as tools for vaccination. Expression of CD14 by the starting cell population, the requirement for GM-CSF and IL-4, and the relatively long culture period are the common characteristics shared between our cells and human monocyte-derived DC, whose analogues in mice have not been identified. Because large numbers of skin-associated mature DC devoid of other cell lineages are easily obtained, this model system may facilitate the study of molecular events associated with maturation of DC and the use of DC for immunization.