bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple HLA Class II-Restricted Melanocyte Differentiation Antigens Are Recognized by Tumor-Infiltrating Lymphocytes from a Patient with Melanoma
Authors: Robbins Paul F. , El-Gamil Mona , Li Yong F. , Zeng Gang , Dudley Mark , Rosenberg Steven A.
Published: April 2014

Dramatic clinical responses were observed in patient 888 following the adoptive transfer of autologous tumor-infiltrating lymphocytes (TIL). Previously, extensive analysis of the specificity of class I-restricted T cells from patient 888 TIL has revealed that these T cells recognize a mutated, as well as several nonmutated tumor Ags. Additional studies that were conducted on TIL from patient 888 indicated that they contained CD4-positive T cells that recognized the autologous tumor that had been induced to express HLA class II molecules. Tumor-reactive CD4-positive T cell clones were isolated from TIL and tested for their ability to react with Ags that are recognized by HLA class I-restricted, melanoma-reactive T cells. Using this approach, T cell clones were identified that recognized an epitope expressed in both the tyrosinase-related protein 1 and tyrosinase-related protein 2 Ags in the context of the HLA-DRβ1*1502 class II gene product. Additional clones were found to recognize an epitope of gp100 in the context of the same HLA-DR restriction element. These observations provide an impetus to develop strategies directed toward generating HLA class II-restricted tumor-reactive T cells.
Activation of CD8 T Cells by Antigen Expressed in the Pituitary Gland
Authors: de Jersey James , Carmignac Danielle , Barthlott Thomas , Robinson Iain , Stockinger Brigitta
Published: April 2014

Ag expressed exclusively in the anterior pituitary gland and secreted locally by pituitary somatotrophs can gain access to the MHC class I presentation pathway and activate CD8 T cells. Influenza nucleoprotein (NP) was expressed as a transgene under the control of the human growth hormone (GH) locus control region. Activation of monoclonal F5 CD8 T cells specific for NP resulted in spontaneous autoimmune pathology of the pituitary gland in mice transgenic for both NP and the F5 TCR. Destruction of somatotrophs resulted in drastically reduced GH levels in adult mice and a dwarf phenotype. Adoptive transfer of F5 T cells into NP-transgenic hosts resulted in full T cell activation, first demonstrable in regional lymph nodes, followed by their migration to the pituitary gland. Despite the presence of activated, IFN-γ-producing CD8 T cells in the pituitary gland and a slight reduction in pituitary GH levels, no effect on growth was observed. Thus, CD8 T cells have access to the neuroendocrine system and get fully activated in the absence of CD4 help, but Ag recognition in this location causes autoimmune pathology only in the presence of excessive CD8 T cell numbers.
Naive B Lymphocytes Undergo Homeostatic Proliferation in Response to B Cell Deficit
Authors: Cabatingan Mark S. , Schmidt Madelyn R. , Sen Ranjan , Woodland Robert T.
Published: April 2014

Naive peripheral B cells are maintained in sufficient numbers and diversity to mount effective immune responses against infectious agents. However, the size and repertoire of this B cell pool is constantly diminished by normal cell turnover and Ag activation. Homeostatic (Ag-independent) proliferation in response to B cell depletion is one mechanism to compensate for this cell loss. We have used purified CFSE-labeled B cells and an adoptive transfer model system to show that immature and mature B cells divide in a variety of B cell-deficient (scid, xid, IL-7−/−, and sublethally irradiated) hosts. Homeostatic B cell proliferation is T cell independent, and B cells that have replicated by this mechanism retain the antigenic phenotype of naive B cells. Replication is significantly reduced in B cell-sufficient normal or B cell-reconstituted immunodeficient recipients by the action of competing mature follicular B cells. Using xid mice and transcription factor knockouts, we show that the activation signal(s) that lead to homeostatic B cell proliferation require Bruton’s tyrosine kinase; however, c-Rel, a Bruton’s tyrosine kinase-induced NF-κB/Rel transcription factor critical for Ag and mitogen stimulation, is dispensable, indicating the uniqueness of this activation pathway. Survival and replication signals can also be separated, because the transcription factor p50 (NF-κB1), which is required for the survival of peripheral B cells, is not necessary for homeostatic replication. Homeostatic B cell proliferation provides an Ag-independent mechanism for the maintenance and expansion of naive B cells selected into the mature B cell pool.
A TNF Family Member LIGHT Transduces Costimulatory Signals into Human T Cells
Authors: Wan Xiaochun , Zhang Jun , Luo Hongyu , Shi Guixiu , Kapnik Elena , Kim Sunghee , Kanakaraj Palanisamy , Wu Jiangping
Published: April 2014

DcR3/TR6 is a secreted protein belonging to the TNFR family. It binds to Fas ligand, LIGHT, and TL1A, all of which are TNF family members. LIGHT is expressed on activated T cells. Its known receptors are TR2 and LTβR on the cell surface, and TR6 in solution. In the present study, we report soluble TR6-Fc or solid-phase TR6-Fc costimulated proliferation, lymphokine production, and cytotoxicity of human T cells in the presence of TCR ligation. These costimulating effects were blocked by soluble LIGHT but not by soluble Fas-Fc. TR6-Fc could also effectively costimulate gld/gld mouse T cells. We further demonstrated that TR6 bound to both Th1 and Th2 cells, according to flow cytometry, and that the association was inhibited by soluble LIGHT. Cross-linking Th1 and Th2 cells with solid-phase TR6-Fc along with a suboptimal concentration of anti-CD3 enhanced proliferation of both Th1 and Th2 cells, and augmented Th1 but not Th2 lymphokine production. These data suggest that TR6 delivers costimulation through its ligand(s) on the T cell surface, and at least the major part of such costimulation is via LIGHT.
Antibody Repertoire Development in Fetal and Neonatal Piglets. VIII. Colonization Is Required for Newborn Piglets to Make Serum Antibodies to T-Dependent and Type 2 T-Independent Antigens
Authors: Butler John E. , Weber Patrick , Sinkora Marek , Baker Diane , Schoenherr Amanda , Mayer Balazs , Francis David
Published: April 2014

Cesarean-derived piglets were reared for 5 wk under germfree conditions or monoassociated with a benign Escherichia coli (G58-1) or a enterohemorrhagic strain (933D) derived from O157:H7, and immunized i.p. with the T-dependent (TD) Ags fluorescein-labeled (FL) keyhole limpet hemocyanin or trinitrophenylated (TNP) keyhole limpet hemocyanin and the type 2 T-independent Ags TNP-Ficoll or FL-Ficoll. Only colonized piglets showed an increase in serum IgG, IgA, and IgM and had serum Abs to FL, TNP, and colonizing bacteria. While serum Abs to FL or TNP appeared following colonization alone, secondary responses were restricted to piglets immunized using TD carriers. While animals colonized with 933D had significantly higher total serum IgG and IgM levels and specific IgG Abs than those colonized with G58-1, no differences were seen in serum IgA levels, B cell diversification in the ileal Peyer’s patches, and specific activity (ELISA activity per micrograms of Ig) of pre-boost serum IgG and IgM anti-TNP and anti-FL Abs. Serum IgA Abs to TNP, FL, or bacteria were not detected. Ag-driven responses, as measured by an increase in specific Ab activity, were only observed in secondary responses to TD Ags and to colonizing, pathogenic E. coli. We propose that germline-encoded, isotype-switched B cells in newborn piglets differentiate to Ab-secreting cells 1) after stimulation by bacteria-activated APCs or 2) through direct stimulation by bacterial products. We further propose that Ag-driven systemic responses require both bacterial colonization and TD Ags translocated to the peritoneum.
Mycobacterium tuberculosisAntigens Induce the Differentiation of Dendritic Cells from Bone Marrow
Authors: Latchumanan Vinoth K. , Singh Balwan , Sharma Pawan , Natarajan Krishnamurthy
Published: April 2014

We show in this study that incubation of freshly isolated bone marrow cells with Mycobacterium tuberculosis (M. tb) secretory Ag (MTSA), in the absence of any growth or differentiation-inducing factor, differentiates them into dendritic cell (DC)-like APCs. These DCs expressed moderate to high levels of various markers typical of DCs. These included T cell costimulatory molecules CD80, CD86, CD40, and CD54 and high levels of surface MHC class I and II on CD11c+ cells. The levels and the kinetics of up-regulation of these molecules were comparable with those of GM-CSF-differentiated DCs. Furthermore, these DCs exhibited morphology characteristics to DCs like the presence of dendritic processes. These DCs were also potent stimulators of allogeneic T cells and preferentially induced the secretion of IFN-γ over IL-10 from the interacting T cells. Interestingly, the differentiation of bone marrow cells into DC-like APCs was obtained with many other M. tb Ags, including whole cell extract of M. tb. Further characterization of MTSA-differentiated DCs showed that they were immature in nature, as stimulation of these DCs with TNF-α, anti-CD40, or LPS further up-regulated the surface levels of various molecules together with an increase in their T cell stimulatory capacity. The Ag-specific T cell responses of MTSA-differentiated DCs were mainly contributed by the CD4+ subset, indicating that MTSA was largely MHC II restricted. Furthermore, stimulation of bone marrow cells with MTSA induced the nuclear translocation of the transcription factor NF-κB, thereby indicating its role during MTSA-induced differentiation of DCs.
B Cell Development Arrest Upon Insertion of a neo Gene Between JH and Eμ: Promoter Competition Results in Transcriptional Silencing of Germline JH and Complete V(D)J Rearrangements
Authors: Delpy Laurent , Decourt Catherine , Le Bert Marc , Cogné Michel
Published: April 2014

Previous targeting experiments within the IgH locus have shown that V(D)J recombination was affected by an insertion of a neo gene within Eμ upstream of the core enhancer, but not by insertions downstream of the enhancer. Similarly, class switch recombination to a given (C) gene was affected only by interposition of neo in between that gene and the 3′ IgH enhancers. Here we show that insertion of neo upstream Eμ only marginally impairs V(D)J recombination, but results in an altered D and JH gene usage and completely blocks transcription of the germline JH region and the rearranged VDJ segments. Although transcriptional silencing of JH occurs upstream of the insertion and results in the lack of mature B cells in homozygous mutant animals, IgH transcription is maintained downstream of the insertion together with neo transcription and can be up-regulated by LPS stimulation or upon fusion with plasmacytoma cells. Altogether these data argue for a polarized “neo effect” involving promoter competition and further show that V(D)J rearrangement can be uncoupled from transcription.
TNFR-Associated Factor-3 Is Associated With BAFF-R and Negatively Regulates BAFF-R-Mediated NF-κB Activation and IL-10 Production
Authors: Xu Liang-Guo , Shu Hong-Bing
Published: April 2014

TALL-1 is a member of the TNF family that is critically involved in B cell survival, maturation, and progression of lupus-like autoimmune diseases. TALL-1 has three receptors, including BCMA, TACI, and BAFF-R, which are mostly expressed by B lymphocytes. Gene knockout studies have indicated that BAFF-R is the major stimulatory receptor for TALL-1 signaling and is required for normal B cell development. The intracellular signaling mechanisms of BAFF-R are not known. In this report, we attempted to identify BAFF-R-associated downstream proteins by yeast two-hybrid screening. This effort identified TNFR-associated factor (TRAF)3 as a protein specifically interacting with BAFF-R in yeast two-hybrid assays. Coimmunoprecipitation experiments indicated that BAFF-R interacts with TRAF3 in B lymphoma cells and this interaction is stimulated by TALL-1 treatment. Domain mapping experiments indicated that both a 6-aa membrane proximal region and the C-terminal 35 aa of BAFF-R are required for its interaction with TRAF3. Moreover, overexpression of TRAF3 inhibits BAFF-R-mediated NF-κB activation and IL-10 production. Taken together, our findings suggest that TRAF3 is a negative regulator of BAFF-R-mediated NF-κB activation and IL-10 production.
Defective Thymocyte Maturation by Transgenic Expression of a Truncated Form of the T Lymphocyte Adapter Molecule and Fyn Substrate, Sin
Authors: Donlin Laura T. , Roman Christopher A. , Adlam Matthew , Regelmann Adam G. , Alexandropoulos Konstantina
Published: April 2014

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.
Positive and Negative Transcriptional States of a Variegating Immunoglobulin Heavy Chain (IgH) Locus Are Maintained by a cis-Acting Epigenetic Mechanism
Authors: Ronai Diana , Berru Maribel , Shulman Marc J.
Published: April 2014

Analyses of transgene expression have defined essential components of a locus control region (LCR) in the JH-Cμ intron of the IgH locus. Targeted deletion of this LCR from the endogenous IgH locus of hybridoma cells results in variegated expression, i.e., cells can exist in two epigenetically inherited states in which the Igμ H chain gene is either active or silent; the active or silent state is typically transmitted to progeny cells through many cell divisions. In principle, cells in the two states might differ either in their content of specific transcription factors or in a cis-acting feature of the IgH locus. To distinguish between these mechanisms, we generated LCR-deficient, recombinant cell lines in which the Igμ H chain genes were distinguished by a silent mutation and fused cells in which the μ gene was active with cells in which μ was silent. Our analysis showed that both parental active and silent transcriptional states were preserved in the hybrid cell, i.e., that two alleles of the same gene in the same nucleus can exist in two different states of expression through many cell divisions. These results indicate that the expression of the LCR-deficient IgH locus is not fully determined by the cellular complement of transcription factors, but is also subject to a cis-acting, self-propagating, epigenetic mark. The methylation inhibitor, 5-azacytidine, reactivated IgH in cells in which this gene was silent, suggesting that methylation is part of the epigenetic mark that distinguishes silent from active transcriptional states.
Complement C3b/C3d and Cell Surface Polyanions Are Recognized by Overlapping Binding Sites on the Most Carboxyl-Terminal Domain of Complement Factor H
Authors: Hellwage Jens , Jokiranta T. Sakari , Friese Manuel A. , Wolk Tobias U. , Kampen Eva , Zipfel Peter F. , Meri Seppo
Published: April 2014

Factor H (FH) is a potent suppressor of the alternative pathway of C in plasma and when bound to sialic acid- or glycosaminoglycan-rich surfaces. Of the three interaction sites on FH for C3b, one interacts with the C3d part of C3b. In this study, we generated recombinant constructs of FH and FH-related proteins (FHR) to define the sites required for binding to C3d. In FH, the C3d-binding site was localized by surface plasmon resonance analysis to the most C-terminal short consensus repeat domain (SCR) 20. To identify amino acids of FH involved in binding to C3d and heparin, we compared the sequences of FH and FHRs and constructed a homology-based molecular model of SCR19–20 of FH. Subsequently, we created an SCR15–20 mutant with substitutions in five amino acids that were predicted to be involved in the binding interactions. These mutations reduced binding of the SCR15–20 construct to both C3b/C3d and heparin. Binding of the wild-type SCR15–20, but not the residual binding of the mutated SCR15–20, to C3d was inhibited by heparin. This indicates that the heparin- and C3d-binding sites are overlapping. Our results suggest that a region in the most C-terminal domain of FH is involved in target recognition by binding to C3b and surface polyanions. Mutations in this region, as recently reported in patients with familial hemolytic uremic syndrome, may lead to indiscriminatory C attack against self cells.
Nippostrongylus brasiliensisCan Induce B7-Independent Antigen-Specific Development of IL-4-Producing T Cells from Naive CD4 T Cells In Vivo
Authors: Liu Zhugong , Liu Qian , Pesce John , Whitmire Jeannette , Ekkens Melinda J. , Foster Anthony , VanNoy Jansie , Sharpe Arlene H. , Urban Joseph F. , Gause William C.
Published: April 2014

Th2 immune responses to a number of infectious pathogens are dependent on B7-1/B7-2 costimulatory molecule interactions. We have now examined the Th2 immune response to Nippostrongylus brasiliensis (Nb) in B7-1/B7-2−/− mice and show that Th2 effector cells develop that can mediate worm expulsion and produce substantial Th2 cytokines comparable with wild-type infected mice; however, in marked contrast, B cell Ag-specific Ab production is abrogated after B7 blockade. To examine the mechanism of T cell activation, OVA-specific DO11.10 T cells were transferred to recipient mice, which were then immunized with a combination of Nb plus OVA or either alone. Only the combination of Nb plus OVA triggered T cell differentiation to OVA-specific Th2 cells, suggesting that Nb acts as an adjuvant to stimulate Ag-specific naive T cells to differentiate to effector Th2 cells. Furthermore, using the DO11.10 TCR-transgenic T cell adoptive transfer model, we show that blocking B7-1/B7-2 interactions does not impair nonparasite Ag-specific DO11.10 Th2 cell differentiation; however, DO11.10 T cell cycle progression and migration to the B cell zone are inhibited.
Requisite Elements in Vaccine Immunity to Blastomyces dermatitidis: Plasticity Uncovers Vaccine Potential in Immune-Deficient Hosts
Authors: Wüthrich Marcel , Filutowicz Hanna I. , Warner Tom , Klein Bruce S.
Published: April 2014

Understanding fundamental mechanisms of vaccine immunity will allow proper use and optimization of vaccines. Vaccination with a genetically engineered, live, attenuated strain of Blastomyces dermatitidis carrying a targeted deletion at the BAD1 locus confers sterilizing immunity against experimental lethal pulmonary infection. We found in this study that αβ T cells are requisite for durable vaccine immunity, whereas other T and B cells are dispensable. In immune-competent animals, CD4+ T-cell derived cytokines TNF-α and IFN-γ mediate vaccine immunity. Surprisingly, these factors are dispensable in immune-deficient animals, which rely on alternate mechanisms for robust vaccine immunity, yet still require O2− production rather than generation of NO. Our results clarify the cellular and molecular bases behind the first genetically engineered fungal vaccine. They also illustrate a sharp difference in vaccine mechanisms between immune-competent and immune-deficient hosts, which underscores the plasticity of residual immune elements in compromised hosts, and points to the feasibility of developing vaccines against invasive fungal infection in this fast growing patient population.
C-Reactive Protein Mediates Protection from Lipopolysaccharide Through Interactions With FcγR
Authors: Mold Carolyn , Rodriguez Wilfredo , Rodic-Polic Bojana , Du Clos Terry W.
Published: April 2014

C-reactive protein (CRP) is a component of the acute phase response to infection, inflammation, and trauma. A major activity of acute phase proteins is to limit the inflammatory response. It has been demonstrated that CRP protects mice from lethal doses of LPS. In the mouse, CRP binds to the regulatory receptor, FcγRIIb, and to the γ-chain-associated receptor, FcγRI. The goal ofthis study was to determine whether FcγRs are necessary for the protective effect of CRP. The ability of CRP to protect mice from a lethal dose of LPS was confirmed using injections of 500 and 250 μg of CRP at 0 and 12 h. CRP treatment of FcγRIIb-deficient mice increased mortality after LPS challenge and increased serum levels of TNF and IL-12 in response to LPS. CRP did not protect FcR γ-chain-deficient mice from LPS-induced mortality. Treatment of normal mice, but not γ-chain-deficient mice, with CRP increased IL-10 levels following LPS injection. In vitro, in the presence of LPS, CRP enhanced IL-10 synthesis and inhibited IL-12 synthesis by bone marrow macrophages from normal, but not γ-chain-deficient mice. The protective effect of CRP appears to be mediated by binding to FcγRI and FcγRII resulting in enhanced secretion of the anti-inflammatory cytokine IL-10 and the down-regulation of IL-12. These results suggest that CRP can alter the cytokine profile of mouse macrophages by acting through FcγR leading to a down-regulation of the inflammatory response.
Hydrogen Peroxide Induces Murine Macrophage Chemokine Gene Transcription Via Extracellular Signal-Regulated Kinase- and Cyclic Adenosine 5′-Monophosphate (cAMP)-Dependent Pathways: Involvement of NF-κB, Activator Protein 1, and cAMP Response Element Binding Protein
Authors: Jaramillo Maritza , Olivier Martin
Published: April 2014

Hydrogen peroxide (H2O2) has been shown to act as a second messenger that activates chemokine expression. In the present study, we investigated the mechanisms underlying this cellular regulation in the murine macrophage cell line B10R. We report that H2O2 increases mRNA expression of various chemokines, macrophage-inflammatory protein (MIP)-1α/CC chemokine ligand (CCL)3, MIP-1β/CCL4, MIP-2/CXC chemokine ligand 2, and monocyte chemoattractant protein-1/CCL2, by activating the extracellular signal-regulated kinase (ERK) pathway and the nuclear translocation of the transcription factors NF-κB, AP-1, and CREB. Blockage of the ERK pathway with specific inhibitors against mitogen-activated protein kinase kinase 1/2 and ERK1/ERK2 completely abolished both the H2O2-mediated chemokine up-regulation and the activation of all NF studied. Similarly, selective inhibition of cAMP and NF-κB strongly down-regulated the induction of all chemokine transcripts as well as CREB and NF-κB activation, respectively. Of interest, we detected a significant decrease of NF-κB, AP-1, and CREB DNA binding activities by reciprocal competition for these binding sites when either specific cold oligonucleotides (NF-κB, AP-1, and CREB) or Abs against various transcription factor subunits (p50, p65, c-Fos, Jun B, c-Jun, and CREB-1) were added. These findings indicate that cooperation between ERK- and cAMP-dependent pathways seems to be required to achieve the formation of an essential transcriptional factor complex for maximal H2O2-dependent chemokine modulation. Finally, experiments performed with actinomycin D suggest that H2O2-mediated MIP-1β mRNA up-regulation results from transcriptional control, whereas that of MIP-1α, MIP-2, and monocyte chemoattractant protein-1 is due to both gene transcription activation and mRNA posttranscriptional stabilization.
Differential Role of IFN-γ-Inducible Protein 10 kDa in a Cockroach Antigen-Induced Model of Allergic Airway Hyperreactivity: Systemic Versus Local Effects
Authors: Thomas Molly S. , Kunkel Steven L. , Lukacs Nicholas W.
Published: April 2014

The ability of IFN-γ to antagonize established Th2 type allergic responses is well documented. To investigate the role of IFN-γ-inducible protein 10 kDa (IP10) in the allergic response, we chose to investigate the effect of IP10 neutralization on an established Th2 response. Systemic neutralization of IP10 at the time of allergen challenge increased airway hyperreactivity as well as airway eosinophil accumulation. Interestingly, IFN-γ levels were markedly reduced in both the lung and peripheral lymph node following IP10 neutralization. Furthermore, the number of CXCR3+CD4+ T cells was decreased in the peripheral lymph node following neutralization of IP10. Introduction of exogenous IP10 into the airway at the time of allergen challenge also dramatically increased eosinophil accumulation in the airway. Protein levels of IL-4, IL-5, and IL-13 were significantly increased in the lung following exogenous airway administration of IP10 with allergen. Interestingly, airway hyperreactivity was significantly decreased at early time points following concurrent IP10 and allergen challenge but rebounded at 24 and 48 h post allergen challenge. Although IP10 may initially be acting locally to dampen the allergic response, its ability to recruit eosinophils may ultimately supersede any immunomodulatory effect it may have in an established allergic response. These results suggest that while systemic levels of IP10 are beneficial in controlling the allergic response, possibly by regulating cellular trafficking in the lymph node, local administration of exogenous IP10 into an established allergic response may be detrimental.
Interactions Between Hemopoietically Derived TNF and Central Nervous System-Resident Glial Chemokines Underlie Initiation of Autoimmune Inflammation in the Brain
Authors: Murphy Craig A. , Hoek Robert M. , Wiekowski Maria T. , Lira Sergio A. , Sedgwick Jonathon D.
Published: April 2014

Tumor necrosis factor is a proinflammatory cytokine that induces directly many of the components required for inflammation to proceed rapidly. We show in this study that the interplay between TNF and chemokines, now recognized to be essential for normal secondary lymphoid tissue development, is also a feature of CNS inflammation, and that the two apparently dissimilar biological processes share many properties. Thus, induction of seven chemokines, including T cell activation gene 3 (TCA3), monocyte chemoattractant protein-1, and IFN-γ-inducible protein-10 within the CNS during experimental autoimmune encephalomyelitis fails to occur early in the inflammatory process in TNF-deficient mice, despite local expression of monokines and IFN-γ. The critical source of TNF in CNS inflammation is the infiltrating hemopoietic cell, and, in its absence, chemokine expression by irradiation-resistant CNS-resident cells fails. The CCR8 ligand, TCA3, is shown to be produced predominantly by resident microglia of the CNS in response to TNF. Using CCR8−/− mice, evidence is provided that TCA3-CCR8 interactions contribute to rapid-onset CNS inflammation. Thus, through TNF production, the hemopoietic compartment initiates the signals for its own movement into tissues, although the tissue ultimately defines the nature of that movement. Chemokines are a major, although not exclusive, mechanism by which tissues regulate leukocyte movement in response to TNF.
An Aspirin-Triggered Lipoxin A4 Stable Analog Displays a Unique Topical Anti-Inflammatory Profile
Authors: Schottelius Arndt J. , Giesen Claudia , Asadullah Khusru , Fierro Iolanda M. , Colgan Sean P. , Bauman John , Guilford William , Perez Hector D. , Parkinson John F.
Published: April 2014

Lipoxins and 15-epi-lipoxins are counter-regulatory lipid mediators that modulate leukocyte trafficking and promote the resolution of inflammation. To assess the potential of lipoxins as novel anti-inflammatory agents, a stable 15-epi-lipoxin A4 analog, 15-epi-16-p-fluorophenoxy-lipoxin A4 methyl ester (ATLa), was synthesized by total organic synthesis and examined for efficacy relative to a potent leukotriene B4 (LTB4) receptor antagonist (LTB4R-Ant) and the clinically used topical glucocorticoid methylprednisolone aceponate. In vitro, ATLa was 100-fold more potent than LTB4R-Ant for inhibiting neutrophil chemotaxis and trans-epithelial cell migration induced by fMLP, but was ∼10-fold less potent than the LTB4R-Ant in blocking responses to LTB4. A broad panel of cutaneous inflammation models that display pathological aspects of psoriasis, atopic dermatitis, and allergic contact dermatitis was used to directly compare the topical efficacy of ATLa with that of LTB4R-Ant and methylprednisolone aceponate. ATLa was efficacious in all models tested: LTB4/Iloprost-, calcium ionophore-, croton oil-, and mezerein-induced inflammation and trimellitic anhydride-induced allergic delayed-type hypersensitivity. ATLa was efficacious in mouse and guinea pig skin inflammation models, exhibiting dose-dependent effects on edema, neutrophil or eosinophil infiltration, and epidermal hyperproliferation. We conclude that the LXA4 and aspirin-triggered LXA4 pathways play key anti-inflammatory roles in vivo. Moreover, these results suggest that ATLa and related LXA4 analogs may have broad therapeutic potential in inflammatory disorders and could provide an alternative to corticosteroids in certain clinical settings.
Mitochondrial Reactive Oxygen Species Regulate Spatial Profile of Proinflammatory Responses in Lung Venular Capillaries
Authors: Parthasarathi Kaushik , Ichimura Hideo , Quadri Sadiqa , Issekutz Andrew , Bhattacharya Jahar
Published: April 2014

Cytokine-induced lung expression of the endothelial cell (EC) leukocyte receptor P-selectin initiates leukocyte rolling. To understand the early EC signaling that induces the expression, we conducted real-time digital imaging studies in lung venular capillaries. To compare receptor- vs nonreceptor-mediated effects, we infused capillaries with respectively, TNF-α and arachidonate. At concentrations adjusted to give equipotent increases in the cytosolic Ca2+, both agents increased reactive oxygen species (ROS) production and EC P-selectin expression. Blocking the cytosolic Ca2+ increases abolished ROS production; blocking ROS production abrogated P-selectin expression. TNF-α, but not arachidonate, released Ca2+ from endoplasmic stores and increased mitochondrial Ca2+. Furthermore, Ca2+ depletion abrogated TNF-α responses partially, but arachidonate responses completely. These differences in Ca2+ mobilization by TNF-α and arachidonate were reflected in spatial patterning in the capillary in that the TNF-α effects were localized at branch points, while the arachidonate effects were nonlocalized and extensive. Furthermore, mitochondrial blockers inhibited the TNF-α- but not the arachidonate-induced responses. These findings indicate that the different modes of Ca2+ mobilization determined the spatial patterning of the proinflammatory response in lung capillaries. Responses to TNF-α revealed that EC mitochondria regulate the proinflammatory process by generating ROS that activate P-selectin expression.
Essential Role of the Adhesion Receptor LFA-1 for T Cell-Dependent Fulminant Hepatitis
Authors: Matsumoto Goichi , Tsunematsu Satoshi , Tsukinoki Kei-ichi , Ohmi Yasushi , Iwamiya Mariko , Oliveira-dos-Santos Antonio , Tone Daisuke , Shindo Junichi , Penninger Josef M.
Published: April 2014

Viral hepatitis affects more than 2 billion people worldwide. In particular, no effective treatment exists to abrogate death and liver damage in fulminant hepatitis. Activation of T cells is an initial and critical event in the pathogenesis of liver damage in autoimmune and viral hepatitis. The precise molecular mechanisms that induce T cell-mediated hepatocyte injury remain largely unclear. In mice, T cell-dependent hepatitis and acute liver damage can be modeled using ConA. In this study, we examined the role of the adhesion receptor LFA-1 in ConA-induced acute hepatic damage using LFA-1−/− (CD11a) mice. Massive liver cell apoptosis and metabolic liver damage were observed in LFA-1+/+ mice following ConA injection. By contrast, LFA-1−/− mice were completely resistant to ConA-induced hepatitis and none of the LFA-1−/− mice showed any hepatic damage. Whereas activated hepatic T cells remained in the liver in LFA-1+/+ mice, activated T cells were rapidly cleared from the livers of LFA-1−/− mice. Mechanistically, T cells from LFA-1−/− mice showed markedly reduced cytotoxicity toward liver cells as a result of impaired, activation-dependent adhesion. Importantly, adoptive transfer of hepatic T cells from LFA-1+/+ mice, but not from LFA-1−/− mice, sensitized LFA-1−/− mice to ConA-induced hepatitis. Thus, LFA-1 expression on T cells is necessary and sufficient for T cell-mediated liver damage in vivo. These results provide the first genetic evidence on an adhesion receptor, LFA-1, that has a crucial role in fulminant hepatitis. These genetic data identify LFA-1 as a potential key target for the treatment of T cell-mediated hepatitis and the prevention of liver damage.
IL-13 Receptor-Targeted Cytotoxin Cancer Therapy Leads to Complete Eradication of Tumors with the Aid of Phagocytic Cells in Nude Mice Model of Human Cancer
Authors: Kawakami Koji , Kawakami Mariko , Puri Raj K.
Published: April 2014

Tumor-directed therapeutic approaches require unique or overexpressed specific Ag or receptor as a target to achieve selective tumor killing. However, heterogeneous expression of these targets on tumor cells limits the efficacy of this form of therapy. In this study, we forced abundant expression of IL-13Rα2 chain by plasmid-mediated gene transfer in head and neck, as well as prostate tumors to provide a potential target. This was followed by successfully treating xenograft tumor-bearing nude mice with IL-13R-directed cytotoxin (IL13-PE38QQR). Although we did not observe an indirect cytotoxic bystander effect conveyed to nontransduced tumor cells in vitro, our approach in vivo led to a complete regression of established tumors transfected with IL-13Rα2 chain in most animals. We found that the tumor eradication was achieved in part by infiltration of macrophages and NK cells, assessed by immunohistochemistry. Moreover, head and neck tumors xenografted in macrophage-depleted nude mice were less sensitive to the antitumor effect of IL-13 cytotoxin. Because we did not observe vector-related toxicity in any vital organs, our novel combination strategy of gene transfer of IL-13Rα2 chain and receptor-directed cytotoxin therapy may be a useful approach for the treatment of localized cancer.
TCR-Like Human Antibodies Expressed on Human CTLs Mediate Antibody Affinity-Dependent Cytolytic Activity
Authors: Chames Patrick , Willemsen Ralph A. , Rojas Gertrudis , Dieckmann Detlef , Rem Louise , Schuler Gerold , Bolhuis Reinder L. , Hoogenboom Hennie R.
Published: April 2014

The permanent genetic programming via gene transfer of autologous T cells with cell surface receptors directed toward tumor-related Ags holds great promise for the development of more-specific tumor therapies. In this study we have explored the use of Abs directed to MHC-peptide complexes (or TCR-like Abs) to engraft CTLs with exquisite specificity for cancer cells. First, we affinity matured in vitro a previously selected TCR-like Ab, Fab-G8, which is highly specific for the peptide melanoma-associated Ag-A1 presented by the HLA-A1 molecule. A combination of L chain shuffling, H chain-targeted mutagenesis, and in vitro selection of phage display libraries yielded a Fab-G8 Ab derivative, Fab-Hyb3, with an 18-fold improved affinity yet identical peptide fine specificity. Fab-G8 and Fab-Hyb3 were expressed on primary human T lymphocytes as cell surface-anchored Fab, demonstrating that T cells expressing the high-affinity Fab-Hyb3 molecule eradicate tumor cells much more effectively. Furthermore, the gain in ligand-binding affinity resulted in a 2-log improvement in the detection of peptide/MHC complexes on melanoma-associated Ag-A1 peptide-loaded cells. In summary, an affinity-matured Ab specifically recognizing a cancer-related peptide/MHC complex was generated and used to improve the tumor cell killing capacity of human T cells. This strategy, based on engraftment of T cells with in vitro engineered Abs, is an attractive alternative to the laborious, and in many cases unsuccessful, generation of highly potent tumor-specific T lymphocytes.
Histone Peptide-Induced Nasal Tolerance: Suppression of Murine Lupus
Authors: Wu Henry Yim , Ward Frank J. , Staines Norman A.
Published: April 2014

Induced mucosal tolerance has been shown to be beneficial in preventing or treating a number of murine and human autoimmune disorders. However, this particular form of therapy has not been thoroughly tested in systemic lupus erythematosus. In this study, we investigated the conditions for induction of nasal tolerance using a histone peptide named H471 expressing a dominant T cell epitope in the histone protein H4 of mononucleosome in lupus-prone SNF1 female mice. We also tested the effect of chronic peptide nasal treatment on the development of autoimmune reactivities in these mice. Results demonstrated that a dose-dependent nasal tolerance to peptide H471 can be achieved before or after peptide sensitization in SNF1 mice. In addition, tolerance to mononucleosomes was induced by nasal instillation of SNF1 mice with H471. This was accompanied by an increase in IL-10 and suppression of IFN-γ production by lymph node cells. Suppression of Th1-type cytokines was also observed in SNF1 mice that were nasally administered with H471 before intradermal injection with the peptide. Finally, chronic nasal instillation of mice with the H471 peptide not only suppressed the development of autoantibodies, but also altered the severity of glomerulonephritis in lupus-prone SNF1 mice.
Cutting Edge: Targeted Ligation of CTLA-4 In Vivo by Membrane-Bound Anti-CTLA-4 Antibody Prevents Rejection of Allogeneic Cells
Authors: Hwang Kwang Woo , Sweatt William B. , Brown Ian E. , Blank Christian , Gajewski Thomas F. , Bluestone Jeffrey A. , Alegre Maria-Luisa
Published: April 2014

Natural engagement of CTLA-4 on host B7 limits T cell activation. We hypothesized that therapeutic cross-linking of CTLA-4 in vivo may further inhibit T cell function and prevent allograft rejection. However, none of the currently available CTLA-4-binding reagents have ligating properties when injected in vivo. The observation that surface-immobilized anti-CTLA-4 mAb inhibits T cell activation in vitro prompted us to develop a membrane-bound single-chain anti-CTLA-4 Ab (7M). To model whether tissue expression of 7M could suppress allograft rejection, we examined the ability of H-2Ld-specific TCR-transgenic T cells to reject 7M-expressing allogeneic tumor cells injected s.c. Expression of 7M significantly inhibited allogeneic rejection in mice that received CTLA-4+/+ but not CTLA-4−/− T cells. Furthermore, CTLA-4+/+ T cells that had encountered 7M-expressing tumors in vivo acquired defects in cytokine production and cytotoxicity. Thus, deliberate ligation of CTLA-4 in vivo potently inhibits allogeneic T cell responses.
Cutting Edge: Changes in Histone Acetylation at the IL-4 and IFN-γ Loci Accompany Th1/Th2 Differentiation
Authors: Fields Patrick E. , Kim Sean T. , Flavell Richard A.
Published: April 2014

Peripheral T cell differentiation is accompanied by chromatin changes at the signature cytokine loci. Using chromatin immunoprecipitation we demonstrate that profound increases in histone acetylation occur at the IFN-γ and IL-4 loci during Th1/Th2 differentiation. These changes in histone acetylation status are locus and lineage specific, and are maintained by the transcription factors Tbet and GATA3 in a STAT-dependent manner. Our results suggest a model of cytokine locus activation in which TCR signals initiate chromatin remodeling and locus opening in a cytokine-independent fashion. Subsequently, cytokine signaling reinforces polarization by expanding and maintaining the accessible state at the relevant cytokine locus (IL-4 or IFN-γ). In this model, GATA3 and Tbet serve as transcriptional maintenance factors, which keep the locus accessible to the transcriptional machinery.
A Soluble Isoform of the Rhesus Monkey Nonclassical MHC Class I Molecule Mamu-AG Is Expressed in the Placenta and the Testis
Authors: Ryan Andy F. , Grendell Richard L. , Geraghty Daniel E. , Golos Thaddeus G.
Published: April 2014

The nonclassical MHC class I locus HLA-G is expressed primarily in the placenta, although other sites of expression have been noted in normal and pathological situations. In addition, soluble HLA-G isoforms have been detected in the serum of pregnant and nonpregnant women as well as men. The rhesus monkey placenta expresses a novel nonclassical MHC class I molecule Mamu-AG, which has features remarkably similar to those of HLA-G. We determined that the rhesus placenta expresses Mamu-AG mRNA (Mamu-AG5), retaining intron 4 as previously noted in HLA-G5. Immunostaining experiments with Ab 16G1 against the soluble HLA-G5 intron 4 peptide demonstrated that an immunoreactive protein(s) was present in the syncytiotrophoblasts of the chorionic villi of the rhesus placenta, within villous cytotrophoblasts, and occasionally within cells of the villous stroma. The Mamu-AG5 mRNA was readily detected in rhesus testis (although not in ejaculated sperm). Whereas an Ab against membrane-bound Mamu-AG stained few cells, primarily in the interstitium of the testis, there was consistent immunostaining for Mamu-AG5 in cells within the seminiferous tubules, which was corroborated by localization of Mamu-AG mRNA by in situ hybridization. While primary spermatocytes were negative, Sertoli cells, spermatocytes, and spermatids were consistently positive for 16G1 immunostaining. The specific recognition of the soluble Mamu-AG isoform was confirmed by Western blotting of Mamu-AG5 expressed in heterologous cells. The results demonstrate that a soluble nonclassical MHC class I molecule is expressed in the rhesus monkey placenta and testis, and confirm and extend the unique homology between HLA-G and the rhesus nonclassical molecule Mamu-AG.
CD94/NKG2 Expression Does Not Inhibit Cytotoxic Function of Lymphocytic Choriomeningitis Virus-Specific CD8+ T Cells
Authors: Miller Joseph D. , Peters Michael , Oran Alp E. , Beresford Guy W. , Harrington Laurie , Boss Jeremy M. , Altman John D.
Published: April 2014

Murine Ag-specific CD8+ T cells express various NK markers and NK inhibitory receptors that have been proposed to modulate immune responses. Following acute infection of C57BL/6 and BALB/cJ mice with lymphocytic choriomeningitis virus (LCMV), we observed that Ag-specific CD8+ T cells expressed CD94/NKG2. Only slight expression of Ly49A and Ly49C receptors was observed on NP396-specific T cells, while all NP396-specific T cells expressed the NKT cell marker U5A2-13 Ag. Expression of CD94/NKG2 was maintained for at least 1 year following LCMV infection, as was the NKT cell marker. By means of cell sorting and quantitative PCR, we found that NP118-specific CD8+ T cells primarily express transcripts for inhibitory NKG2 receptor isoforms. CD94/NKG2 expression was also observed on Ag-specific CD8+ T cells following infection with polyoma virus, influenza virus, and Listeria monocytogenes, suggesting that it may be a common characteristic of Ag-specific CD8+ T cells following infection with viral or bacterial pathogens. Expression of CD94/NKG2 on memory-specific CD8+ T cells did not change following secondary challenge with LCMV clone 13 and did not inhibit viral clearance. Furthermore, we found no evidence that CD94/NKG2 inhibits either the lytic function of LCMV-specific T cells or their capacity to produce effector cytokines upon peptide stimulation. Finally, down-regulation of CD94/NKG2 was found to occur only during chronic LCMV infection. Altogether, this study suggests that CD94/NKG2 expression is not necessarily correlated with inhibition of T cell function.
The Homeostasis But Not the Differentiation of T Cells Is Regulated by p27Kip1
Authors: Shen Randy , Kaplan Mark H.
Published: April 2014

The cyclin-dependent kinase inhibitor p27Kip1 is a critical regulator of T cell proliferation. To further examine the relationship of T cell proliferation and differentiation, we examined the ability of T cells deficient in p27Kip1 to differentiate into Th subsets. We observed increased Th2 differentiation in p27Kip1-deficient cultures. In addition to increases in CD4+ and CD8+ T cells, there is a similar increase in γδ T cells in p27Kip1-deficient mice compared with wild-type mice. The increase in Th2 differentiation is correlated to an increase of IL-4 secretion by CD4+DX5+TCRαβ+CD62Llow T cells but not to increased expansion of differentiating Th2 cells. While STAT4- and STAT6-deficient T cells have diminished proliferative responses to IL-12 and IL-4, respectively, proliferative responses are increased in T cells doubly deficient in p27Kip1 and STAT4 or STAT6. In contrast, the increased proliferation and differentiative capacity of p27Kip1-deficient T cells has no effect on the ability of STAT4/p27Kip1- or STAT6/p27Kip1-deficient CD4+ cells to differentiate into Th1 or Th2 cells, respectively. Thus, while p27Kip1 regulates the expansion and homeostasis of several T cell subsets, it does not affect the differentiation of Th subsets.
Src Homology Region 2 Domain-Containing Phosphatase 1 Positively Regulates B Cell Receptor-Induced Apoptosis by Modulating Association of B Cell Linker Protein with Nck and Activation of c-Jun NH2-Terminal Kinase
Authors: Mizuno Kazuya , Tagawa Yuko , Mitomo Katsuyuki , Watanabe Noriyuki , Katagiri Tatsuo , Ogimoto Mami , Yakura Hidetaka
Published: April 2014

Src homology region 2 domain-containing phosphatase 1 (SHP-1) is a key mediator in lymphocyte differentiation, proliferation, and activation. We previously showed that B cell linker protein (BLNK) is a physiological substrate of SHP-1 and that B cell receptor (BCR)-induced activation of c-Jun NH2-terminal kinase (JNK) is significantly enhanced in cells expressing a form of SHP-1 lacking phosphatase activity (SHP-1-C/S). In this study, we confirmed that SHP-1 also exerts negative regulatory effects on JNK activation in splenic B cells. To further clarify the role of SHP-1 in B cells, we examined how dephosphorylation of BLNK by SHP-1 affects downstream signaling events. When a BLNK mutant (BLNKΔN) lacking the NH2-terminal region, which contains four tyrosine residues, was introduced in SHP-1-C/S-expressing WEHI-231 cells, the enhanced JNK activation was inhibited. Among candidate proteins likely to regulate JNK activation through BLNK, Nck adaptor protein was found to associate with tyrosine-phosphorylated BLNK and this association was more pronounced in SHP-1-C/S-expressing cells. Furthermore, expression of dominant-negative forms of Nck inhibited BCR-induced JNK activation. Finally, BCR-induced apoptosis was suppressed in SHP-1-C/S-expressing cells and coexpression of Nck SH2 mutants or a dominant-negative form of SEK1 reversed this phenotype. Collectively, these results suggest that SHP-1 acts on BLNK, modulating its association with Nck, which in turn negatively regulates JNK activation but exerts a positive effect on apoptosis.