bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Memory B Cells Transferred by Allogenic Bone Marrow Transplantation Contribute Significantly to the Antibody Repertoire of the Recipient
Authors: Lausen Birgitte F. , Hougs Lotte , Schejbel Lone , Heilmann Carsten , Barington Torben
Published: April 2014

The bone marrow is an important source of Abs involved in long-term protection from recurrence of infections. Allogenic bone marrow transplantation (BMT) fails to restore this working memory. Attempts to overcome this immunodeficiency by immunization of the donor have not been very successful. More needs to be known about transfer of B cell memory by BMT. We tracked memory B cells from the donor to the recipient during BMT of a girl with leukocyte adhesion deficiency. Vaccination of her HLA-identical sibling donor 7 days before harvest induced Haemophilus influenzae type b (Hib) capsular polysaccharide (HibCP)-specific B cells readily detectable in marrow and blood. BMT did not lead to spontaneous production of HibCP Abs, but the recipient responded well to booster immunizations 9 and 11 mo after BMT. HibCP-specific B cells were obtained 7 days after the vaccinations, and their VH genes were sequenced and analyzed for rearrangements and unique patterns of somatic hypermutations identifying clonally related cells. Ninety (74%) of 121 sequences were derived from only 16 precursors. Twelve clones were identified in the donor, and representatives from all of them were detected in the recipient where they constituted 61 and 68% of the responding B cells after the first and second vaccinations, respectively. No evidence for re-entry of memory clones into the process of somatic hypermutation was seen in the recipient. Thus, memory B cells were transferred from the donor, persisted for at least 9 mo in the recipient, and constituted the major part of the HibCP-specific repertoire.
Cutting Edge: DGYW/WRCH Is a Better Predictor of Mutability at G:C Bases in Ig Hypermutation Than the Widely Accepted RGYW/WRCY Motif and Probably Reflects a Two-Step Activation-Induced Cytidine Deaminase-Triggered Process
Authors: Rogozin Igor B. , Diaz Marilyn
Published: April 2014

A feature of Ig hypermutation is the presence of hypermutable DNA sequences that are preferentially found in the V regions of Ig genes. Among these, RGYW/WRCY is the most pronounced motif (G:C is a mutable position; R = A/G, Y = C/T, and W = A/T). However, a molecular basis for the high mutability of RGYW was not known until recently. The discovery that activation-induced cytidine deaminase targets the DNA encoding V regions, has enabled the analysis of its targeting properties when expressed outside of the context of hypermutation. We analyzed these data and found evidence that activation-induced cytidine deaminase is the major source of the RGYW mutable motif, but with a new twist: DGYW/WRCH (G:C is the mutable position; D = A/G/T, H = T/C/A) is a better descriptor of the Ig mutation hotspot than RGYW/WRCY. We also found evidence that a DNA repair enzyme may play a role in modifying the sequence of hypermutation hotspots.
Indirect Minor Histocompatibility Antigen Presentation by Allograft Recipient Cells in the Draining Lymph Node Leads to the Activation and Clonal Expansion of CD4+ T Cells That Cause Obliterative Airways Disease
Authors: Richards David M. , Dalheimer Stacy L. , Ehst Benjamin D. , Vanasek Tracy L. , Jenkins Marc K. , Hertz Marshall I. , Mueller Daniel L.
Published: April 2014

Ag recognition by OVA-reactive OT-II (I-Ab restricted) and DO11.10 (I-Ad restricted) TCR-Tg CD4+ T cells after heterotopic transplantation of OVA transgene-expressing tracheal grafts was examined as a model of minor histocompatibility Ag (mHAg)-induced chronic allograft rejection. In response to airway allotransplantation with grafts expressing the OVA transgene, these TCR-Tg CD4+ T cells expressed the activation markers CD69 and CD44, demonstrated evidence of blastogenesis, underwent multiple rounds of cell division leading to their clonal expansion in the draining lymph node, and proceeded to differentiate to a effector/memory T cell phenotype based on a reduction in the expression of CD45RB. These mHAg-specific TCR-Tg CD4+ T cells responded equally well to fully MHC-mismatched tracheas and to class II-deficient allografts, demonstrating that donor mHAg recognition by recipient CD4+ T cells does not rely on Ag presentation by donor-derived APC. The activation of mHAg-specific TCR-Tg CD4+ T cells after their adoptive transfer into recipient mice given MHC-matched, but mHAg-disparate, airway allografts was associated with their movement into the allograft and the near uniform destruction of the transplanted airway tissue secondary to the development of obliterative airways disease. These results demonstrate that an activation of mHAg-reactive CD4+ T cells in the draining lymph node by recipient APC that indirectly express graft mHAg-derived peptide/class II MHC complexes precedes responder T cell proliferation and differentiation, and leads to the eventual migration of these alloreactive T cells to the transplanted airway tissue and the promotion of chronic graft rejection.
Nasal Flt3 Ligand cDNA Elicits CD11c+CD8+ Dendritic Cells for Enhanced Mucosal Immunity
Authors: Kataoka Kosuke , McGhee Jerry R. , Kobayashi Ryoki , Fujihashi Keiko , Shizukuishi Satoshi , Fujihashi Kohtaro
Published: April 2014

Nasal immunization is an effective way to induce both mucosal and systemic immune responses. In this study, we assessed a cDNA vector for Flt3 ligand (FL) for its potential to enhance mucosal immunity or tolerance. Interestingly, tolerance was avoided and elevated levels of OVA-specific Ab responses were induced in nasal washes, fecal extracts, and saliva as well as in plasma when compared with mice given nasal OVA plus DNA plasmid without the FL gene. In addition, significant levels of OVA-specific CD4+ T cell proliferative responses and OVA-induced IL-4 and IL-2 production were noted in spleen and cervical lymph nodes. Further, marked increases in FL protein occurred in the nasal lamina propria and submandibular glands and the frequencies of CD11c+CD8+ dendritic cells (DCs) significantly increased in the mucosal tissues. Moreover, these DCs expressed high levels of CD40, CD80, CD86, and MHC class II molecules. Nasal delivery of plasmid FL with OVA resulted in FL expression in both mucosal inductive and effector sites and resulted in expanded activated lymphoid DCs. Thus, nasal plasmid FL prevents mucosal tolerance and enhances active immunity when given by a mucosal route.
A Major Allergen from Pollen Defines a Novel Family of Plant Proteins and Shows Intra- and Interspecie Cross-Reactivity
Authors: Barral Patricia , Batanero Eva , Palomares Oscar , Quiralte Joaquín , Villalba Mayte , Rodríguez Rosalía
Published: April 2014

Olive tree (Olea europaea) pollen is a main cause of allergy associated with extensive areas of Europe and North America. Ole e 10, a small (10.8 kDa) and acidic (pI 5.8) protein, has been identified as a major allergen from the olive pollen, isolated, and characterized. Circular dichroism analysis gave 17% α helix, 33% β sheet, and 21% β turn for its secondary structure. Based on amino acid sequences of tryptic peptides, the protein was cloned and sequenced. The allergen consists of a single polypeptide chain of 102 aa, with a signal peptide of 21 residues. Ole e 10 showed homology with the C-terminal domain of another olive allergen, Ole e 9 (1,3-β-glucanase, 53% identity), with deduced sequences from Arabidopsis thaliana genes (42–46% identity) and with polypeptide segments (Cys boxes) of proteins involved in yeast development (Epd1/Gas-1p/Phr2 families; 42–43% similarity). Ole e 10 showed 55% prevalence for olive-allergic patients and exhibited an IgE response dependent on its conformation. Remarkable IgE cross-reactivity was detected with Ole e 9, but no correlation was observed between the individual IgE responses to both allergens. Ole e 10 shares IgE B cell epitopes with proteins from Oleaceae, Gramineae, Betulaceae, Chenopodiaceae, Cupressaceae, Ambrosia, and Parietaria pollens, latex, and vegetable foods, such as tomato, kiwi, potato, and peach. These data indicate that Ole e 10 is a new pan-allergenic plant protein that shows notable intra- and interspecie IgE cross-reactivity and is a powerful candidate to be involved in pollen-latex-fruit syndrome.
Toll-Like Receptor 2 Suppresses Immunity against Candida albicans through Induction of IL-10 and Regulatory T Cells
Authors: Netea Mihai G. , Sutmuller Roger , Hermann Corinna , Van der Graaf Chantal A. A. , Van der Meer Jos W. M. , van Krieken Johan H. , Hartung Thomas , Adema Gosse , Kullberg Bart Jan
Published: April 2014

Toll-like receptor (TLR) 2 and TLR4 play a pivotal role in recognition of Candida albicans. We demonstrate that TLR2−/− mice are more resistant to disseminated Candida infection, and this is associated with increased chemotaxis and enhanced candidacidal capacity of TLR2−/− macrophages. Although production of the proinflammatory cytokines TNF, IL-1α, and IL-1β is normal, IL-10 release is severely impaired in the TLR2−/− mice. This is accompanied by a 50% decrease in the CD4+CD25+ regulatory T (Treg) cell population in TLR2−/− mice. In vitro studies confirmed that enhanced survival of Treg cells was induced by TLR2 agonists. The deleterious role of Treg cells on the innate immune response during disseminated candidiasis was underscored by the improved resistance to this infection after depletion of Treg cells. In conclusion, C. albicans induces immunosuppression through TLR2-derived signals that mediate increased IL-10 production and survival of Treg cells. This represents a novel mechanism in the pathogenesis of fungal infections.
Activation of the D Prostanoid Receptor 1 Regulates Immune and Skin Allergic Responses
Authors: Angeli Véronique , Staumont Delphine , Charbonnier Anne-Sophie , Hammad Hamida , Gosset Philippe , Pichavant Muriel , Lambrecht Bart N. , Capron Monique , Dombrowicz David , Trottein François
Published: April 2014

The mobilization of Langerhans cells (LCs) from epithelia to the draining lymph nodes is an essential process to initiate primary immune responses. We have recently shown that in mice, PGD2 is a potent inhibitor of epidermal LC emigration. In this study, we demonstrate that activation of the D prostanoid receptor 1 (DP1) impedes the TNF-α-induced migration of human LCs from skin explants and strongly inhibits the chemotactic responses of human LC precursors and of maturing LCs to CC chemokine ligands 20 and 19, respectively. Using a murine model of atopic dermatitis, a chronic Th2-type allergic inflammatory disease, we demonstrate that the potent DP1 agonist BW245C dramatically decreases the Ag-specific T cell activation in the skin draining lymph nodes and markedly prevents the skin lesions following repeated epicutaneous sensitization with OVA. Interestingly, analysis of the local response indicates that BW245C treatment strongly reduces the recruitment of inflammatory cells into the dermis and disrupts the Th1/Th2 balance, probably through the increased production of the immunoregulatory cytokine IL-10, in the skin of sensitized mice. Taken together, our results suggest a new function for DP1 in the regulation of the immune and inflammatory responses. We propose that DP1 activation by specific agonists may represent a strategy to control cutaneous inflammatory Th2-associated diseases.
CXCR2 Is Critical to Hyperoxia-Induced Lung Injury
Authors: Sue Richard D. , Belperio John A. , Burdick Marie D. , Murray Lynne A. , Xue Ying Ying , Dy Maria C. , Kwon Jeffery J. , Keane Michael P. , Strieter Robert M.
Published: April 2014

Hyperoxia-induced lung injury is characterized by infiltration of activated neutrophils in conjunction with endothelial and epithelial cell injury, followed by fibrogenesis. Specific mechanisms recruiting neutrophils to the lung during hyperoxia-induced lung injury have not been fully elucidated. Because CXCL1 and CXCL2/3, acting through CXCR2, are potent neutrophil chemoattractants, we investigated their role in mediating hyperoxia-induced lung injury. Under variable concentrations of oxygen, murine survival during hyperoxia-induced lung injury was dose dependent. Eighty percent oxygen was associated with 50% mortality at 6 days, while greater oxygen concentrations were more lethal. Using 80% oxygen, we found that lungs harvested at day 6 demonstrated markedly increased neutrophil sequestration and lung injury. Expression of CXCR2 ligands paralleled neutrophil recruitment to the lung and CXCR2 mRNA expression. Inhibition of CXC chemokine ligands/CXCR2 interaction using CXCR2−/− mice exposed to hyperoxia significantly reduced neutrophil sequestration and lung injury, and led to a significant survival advantage as compared with CXCR2+/+ mice. These findings demonstrate that CXC chemokine ligand/CXCR2 biological axis is critical during the pathogenesis of hyperoxia-induced lung injury.
Critical Protection from Renal Ischemia Reperfusion Injury by CD55 and CD59
Authors: Yamada Koei , Miwa Takashi , Liu Jianuo , Nangaku Masaomi , Song Wen-Chao
Published: April 2014

Renal ischemia-reperfusion injury (IRI) is a feature of ischemic acute renal failure and it impacts both short- and long-term graft survival after kidney transplantation. Complement activation has been implicated in renal IRI, but its mechanism of action is uncertain and the determinants of complement activation during IRI remain poorly understood. We engineered mice deficient in two membrane complement regulatory proteins, CD55 and CD59, and used them to investigate the role of these endogenous complement inhibitors in renal IRI. CD55-deficient (CD55−/−), but not CD59-deficient (CD59−/−), mice exhibited increased renal IRI as indicated by significantly elevated blood urea nitrogen levels, histological scores, and neutrophil infiltration. Remarkably, although CD59 deficiency alone was inconsequential, CD55/CD59 double deficiency greatly exacerbated IRI. Severe IRI in CD55−/−CD59−/− mice was accompanied by endothelial deposition of C3 and the membrane attack complex (MAC) and medullary capillary thrombosis. Complement depletion in CD55−/−CD59−/− mice with cobra venom factor prevented these effects. Thus, CD55 and CD59 act synergistically to inhibit complement-mediated renal IRI, and abrogation of their function leads to MAC-induced microvascular injury and dysfunction that may exacerbate the initial ischemic assault. Our findings suggest a rationale for anti-complement therapies aimed at preventing microvascular injury during ischemia reperfusion, and the CD55−/−CD59−/− mouse provides a useful animal model in this regard.
IL-2 Production by Virus- and Tumor-Specific Human CD8 T Cells Is Determined by Their Fine Specificity
Authors: Mallard Eric , Vernel-Pauillac Frédérique , Velu Thierry , Lehmann Frédéric , Abastado Jean-Pierre , Salcedo Margarita , Bercovici Nadège
Published: April 2014

Memory CD8 T cells mediate rapid and effective immune responses against previously encountered Ags. However, these cells display considerable phenotypic and functional heterogeneity. In an effort to identify parameters that correlate with immune protection, we compared cell surface markers, proliferation, and cytokine production of distinct virus- and tumor-specific human CD8 populations. Phenotypic analysis of epitope-specific CD8 T cells showed that Ag specificity is associated with distinct CCR7/CD45RA expression profiles, suggesting that Ag recognition drives the expression of these molecules on effector/memory T cells. Moreover, the majority of central memory T cells (CD45RAlowCCR7dull) secreting cytokines in response to an EBV epitope produces both IL-2 and IFN-γ, whereas effector memory CD8 cells (CD45RAdullCCR7−) found in EBV, CMV, or Melan-A memory pools are mostly composed of cells secreting exclusively IFN-γ. However, these various subsets, including Melan-A-specific effector memory cells differentiated in cancer patients, display similar Ag-driven proliferation in vitro. Our findings show for the first time that human epitope-specific CD8 memory pools differ in IL-2 production after antigenic stimulation, although they display similar intrinsic proliferation capacity. These results provide new insights in the characterization of human virus- and tumor-specific CD8 lymphocytes.
Establishment of Early Lymphoid Organ Infrastructure in Transplanted Tumors Mediated by Local Production of Lymphotoxin α and in the Combined Absence of Functional B and T Cells
Authors: Kim Hye-Jung , Kammertoens Thomas , Janke Marko , Schmetzer Oliver , Qin Zhihai , Berek Claudia , Blankenstein Thomas
Published: April 2014

Lymphoid organogenesis is a highly coordinated process involving orchestrated expression of a number of genes. Although the essential role of lymphotoxin α (LTα) for the normal development of secondary lymphoid organs is well established, it is not clear to which extent it depends upon cooperation with T and B lymphocytes for lymphoid neo-organogenesis. To determine whether LTα is sufficient to mediate recruitment of basic elements needed for lymphoid organogenesis, we made use of a LTα-transfected cell line as an experimental tool and established tumors in nude and SCID mice. Our data showed that high endothelial venules formed and follicular dendritic cells accumulated and differentiated in response to LTα in the absence of lymphocytes. A CD4+CD3−CD11c+ cell population that is found in the secondary lymphoid organ was also recruited into tumors expressing LTα. Furthermore, in nude mice, B cells migrated in response to LTα and formed intratumoral follicles. These B cell follicles were structurally well equipped with follicular dendritic cell networks and high endothelial venules; however, they were not functionally active; e.g., those B cells specific for a surrogate Ag expressed by the tumor were found in the spleen, but not in the tumor. We show that, even in the absence of functional T and B lymphocytes, local expression of LTα in transplanted tumors induced typical stromal characteristics of lymphoid tissue, emphasizing that LTα is a critically important cytokine for formation of lymphoid organ infrastructure.
Separation of the New Zealand Black Genetic Contribution to Lupus from New Zealand Black Determined Expansions of Marginal Zone B and B1a Cells
Authors: Atencio Stephanie , Amano Hirofumi , Izui Shozo , Kotzin Brian L.
Published: April 2014

The F1 hybrid of New Zealand Black (NZB) and New Zealand White (NZW) mice develop an autoimmune disease similar to human systemic lupus erythematosus. Because NZB and (NZB × NZW)F1 mice manifest expansions of marginal zone (MZ) B and B1a cells, it has been postulated that these B cell abnormalities are central to the NZB genetic contribution to lupus. Our previous studies have shown that a major NZB contribution comes from the Nba2 locus on chromosome 1. C57BL/6 (B6) mice congenic for Nba2 produce antinuclear Abs, and (B6.Nba2 × NZW)F1 mice develop elevated autoantibodies and nephritis similar to (NZB × NZW)F1 mice. We studied B cell populations of B6.Nba2 mice to better understand the mechanism by which Nba2 leads to disease. The results showed evidence of B cell activation early in life, including increased levels of serum IgM, CD69+ B cells, and spontaneous IgM production in culture. However, B6.Nba2 compared with B6 mice had a decreased percentage of MZ B cells in spleen, and no increase of B1a cells in the spleen or peritoneum. Expansions of these B cell subsets were also absent in (B6.Nba2 × NZW)F1 mice. Among the strains studied, B cell expression of β1 integrin correlated with differences in MZ B cell development. These results show that expansions of MZ B and B1a cells are not necessary for the NZB contribution to lupus and argue against a major role for these subsets in disease pathogenesis. The data also provide additional insight into how Nba2 contributes to lupus.
Detection of Cell Surface Ligands for the γδ TCR Using Soluble TCRs
Authors: Aydintug M. Kemal , Roark Christina L. , Yin Xiang , Wands J. M. , Born Willi K. , O’Brien Rebecca L.
Published: April 2014

The natural ligands recognized by γδ TCRs are still largely unknown, in part because immunization does not normally result in Ag-specific γδ T cell responses. Taking advantage of an established ligand for a particular γδ TCR, we demonstrated that a multimerized recombinant form of this γδ TCR can be used like a mAb to specifically detect its own ligand. Using the same approach for more common γδ TCRs whose ligands remain unknown, we detected on certain cell lines molecules that appear to be ligands for three additional γδ TCRs. One of these represents the mouse Vγ6/Vδ1 invariant γδ TCR, which predominates in the female reproductive tract, the tongue, and the lung, and other tissues during inflammation. The second represents the closely related Vγ5/Vδ1 invariant γδ TCR expressed by most epidermal T cells. The third is a Vγ1/Vδ6.3 TCR, representative of a variable type frequently found on lymphoid γδ T cells. We found evidence that ligands for multiple γδ TCRs may be simultaneously expressed on a single cell line, and that at least some of the putative ligands are protease sensitive. This study suggests that soluble versions of γδ TCRs can be as tools to identify and characterize the natural ligands of γδ T cells.
CD4-Directed Peptide Vaccination Augments an Antitumor Response, but Efficacy Is Limited by the Number of CD8+ T Cell Precursors
Authors: Hanson Holly L. , Kang Silvia S. , Norian Lyse A. , Matsui Ken , O’Mara Leigh A. , Allen Paul M.
Published: April 2014

Peptide vaccination is an immunotherapeutic strategy being pursued as a method of enhancing Ag-specific antitumor responses. To date, most studies have focused on the use of MHC class I-restricted peptides, and have not shown a correlation between Ag-specific CD8+ T cell expansion and the generation of protective immune responses. We investigated the effects of CD4-directed peptide vaccination on the ability of CD8+ T cells to mount protective antitumor responses in the DUC18/CMS5 tumor model system. To accomplish this, we extended the amino acid sequence of the known MHC class I-restricted DUC18 rejection epitope from CMS5 to allow binding to MHC class II molecules. Immunization with this peptide (tumor-derived extracellular signal-regulated kinase-II (tERK-II)) induced Ag-specific CD4+ T cell effector function, but did not directly prime CD8+ T cells. Approximately 31% of BALB/c mice immunized with tERK-II were protected from subsequent tumor challenge in a CD40-dependent manner. Priming of endogenous CD8+ T cells in immunized mice was detected only after CMS5 challenge. Heightened CD4+ Th cell function in response to tERK II vaccination allowed a 12-fold reduction in the number of adoptively transferred CD8+ DUC18 T cells needed to protect recipients against tumor challenge as compared with previous studies using unimmunized mice. Furthermore, tERK-II immunization led to a more rapid and transient expansion of transferred DUC18 T cells than was seen in unimmunized mice. These findings illustrate that CD4-directed peptide vaccination augments antitumor immunity, but that the number of tumor-specific precursor CD8+ T cells will ultimately dictate the success of immunotherapy.
Enrichment of Lck in Lipid Rafts Regulates Colocalized Fyn Activation and the Initiation of Proximal Signals through TCRαβ
Authors: Filipp Dominik , Leung Bernadine L. , Zhang Jenny , Veillette André , Julius Michael
Published: April 2014

Recent results provide insight into the temporal and spatial relationship governing lck-dependent fyn activation and demonstrate TCR/CD4-induced activation and translocation of lck into lipid rafts and the ensuing activation of colocalized fyn. The prediction follows that directly targeting lck to lipid rafts will bypass the requirement for juxtaposing TCR and CD4-lck, and rescue cellular activation mediated by Ab specific for the constant region of TCRβ chain. The present study uses a family of murine IL-2-dependent CD4+ T cell clonal variants in which anti-TCRCβ signaling is impaired in an lck-dependent fashion. Importantly, these variants respond to Ag- and mAb-mediated TCR-CD4 coaggregation, both of which enable the coordinated interaction of CD4-associated lck with the TCR/CD3 complex. We have previously demonstrated that anti-TCRCβ responsiveness in this system correlates with the presence of kinase-active, membrane-associated lck and preformed hypophosphorylated TCRζ:ζ-associated protein of 70 kDa complexes, a phenotype recapitulated in primary resting CD4+ T cells. We show in this study that forced expression of wild-type lck achieved the same basal composition of the TCR/CD3 complex and yet did not rescue anti-TCRCβ signaling. In contrast, forced expression of C20S/C23S-mutated lck (double-cysteine lck), unable to bind CD4, rescues anti-TCRCβ proximal signaling and cellular growth. Double-cysteine lck targets lipid rafts, colocalizes with >98% of cellular fyn, and results in a 7-fold increase in basal fyn kinase activity. Coaggregation of CD4 and TCR achieves the same outcome. These results underscore the critical role of lipid rafts in spatially coordinating the interaction between lck and fyn that predicates proximal TCR/CD3 signaling.
Immune Regulation by Self-Reactive T Cells is Antigen Specific
Authors: Tanchot Corinne , Vasseur Florence , Pontoux Christiane , Garcia Corinne , Sarukhan Adelaida
Published: April 2014

Immune regulation plays an important role in the establishment and maintenance of self-tolerance. Nevertheless, it has been difficult to conclude whether regulation is Ag specific because studies have focused on polyclonal populations of regulatory T cells. We have used in this study a murine transgenic model that generates self-reactive, regulatory T cells of known Ag specificity to determine their capacity to suppress naive T cells specific for other Ags. We show that these regulatory cells can regulate the responses of naive T cells with the same TCR specificity, but do not inhibit T cell proliferation or differentiation of naive T cells specific for other Ags. These results demonstrate that immune regulation may be more Ag specific than previously proposed.
TNF Receptor-Associated Factor 5 Limits the Induction of Th2 Immune Responses
Authors: So Takanori , Salek-Ardakani Shahram , Nakano Hiroyasu , Ware Carl F. , Croft Michael
Published: April 2014

The TNF receptor-associated factor (TRAF) family of molecules acts as adapter proteins for signaling pathways initiated by several members of the TNF receptor (TNFR) superfamily. TRAF5−/− animals are viable and have normal development of the immune system despite interacting with several TNFR family members. A clear role for TRAF5 has yet to emerge. OX40 (CD134) interacts with TRAF5, suggesting that this pathway could be involved in regulating T cell differentiation into Th1 or Th2 cells. In tissue culture, OX40 stimulation of TRAF5−/− T cells resulted in a pronounced Th2 phenotype with elevated levels of IL-4 and IL-5. Similarly, in vivo immunization with protein in adjuvant in the presence of an agonist anti-OX40 Ab resulted in enhanced Th2 development in TRAF5−/− mice. Additionally, lung inflammation induced by T cells, which is critically controlled by OX40, was more pronounced in TRAF5−/− mice, characterized by higher levels of Th2 cytokines. These results suggest that TRAF5 can limit the induction of Th2 responses, and that TRAF5 can play a role in modulating responses driven by OX40 costimulation.
Characterization of Recombinant Mannan-Binding Lectin-Associated Serine Protease (MASP)-3 Suggests an Activation Mechanism Different from That of MASP-1 and MASP-2
Authors: Zundel Stéphanie , Cseh Sandor , Lacroix Monique , Dahl Mads R. , Matsushita Misao , Andrieu Jean-Pierre , Schwaeble Wilhelm J. , Jensenius Jens C. , Fujita Teizo , Arlaud Gérard J. , Thielens Nicole M.
Published: April 2014

Mannan-binding lectin (MBL)-associated serine proteases (MASP-1, -2, and -3) are homologous modular proteases that each associate with MBL and L- and H-ficolins, which are oligomeric serum lectins involved in innate immunity. To investigate its physicochemical, interaction, and enzymatic properties, human MASP-3 was expressed in insect cells. Ultracentrifugation analysis indicated that rMASP-3 sedimented as a homodimer (s20,w = 6.2 ± 0.1 S) in the presence of Ca2+, and as a monomer (s20,w = 4.6 ± 0.1 S) in EDTA. As shown by surface plasmon resonance spectroscopy, it associated with both MBL (KD = 2.6 nM) and L-ficolin (KD = 7.2 nM). The protease was produced in a single-chain, proenzyme form, but underwent slow activation upon prolonged storage at 4°C, resulting from cleavage at the Arg430-Ile431 activation site. Activation was prevented in the presence of protease inhibitors iodoacetamide and 1,10-phenanthroline but was not abolished upon substitution of Ala for the active site Ser645 of MASP-3, indicating extrinsic proteolysis. In contrast, the corresponding mutations Ser627→Ala in MASP-1 and Ser618→Ala in MASP-2 stabilized the latter in their proenzyme form. Likewise, the MASP-1 and MASP-2 mutants were each activated by their active counterparts, but MASP-3 S645A was not. Activated MASP-3 did not react with C1 inhibitor; had no activity on complement proteins C2, C4, and C3; and only cleaved the N-carboxybenzyloxyglycine-l-arginine thiobenzyl ester substrate to a significant extent. Based on these observations, it is postulated that MASP-3 activation and control involve mechanisms that are different from those of MASP-1 and -2.
Identification and Molecular Cloning of Functional Chicken IL-12
Authors: Degen Winfried G. J. , van Daal Nancy , van Zuilekom Hanneke I. , Burnside Joan , Schijns Virgil E. J. C.
Published: April 2014

By a combination of large-scale sequencing, bioinformatics, and traditional molecular biology, we identified the long-searched-for cDNA sequences encoding the homologues of the chicken IL-12p35 and IL-12p40 chains. These molecules are the first discovered nonmammalian IL-12 subunits. The homologies of the chicken IL-12p35 and IL-12p40 proteins to the corresponding known subunits of various species, i.e., humans, sheep, horse, cat, bovine, mouse, and woodchuck, ranged between 21 and 42%, respectively. The expression of IL-12 subunits was observed in lymphoid cells and proved to be dependent on the cell type and stimulus, while expression was not detected in stimulated primary chicken embryo fibroblast cells. Following transient expression of both molecules in COS-7 cells, we confirmed the necessity of heterodimerization into IL-12p70 to yield bioactivity as was also shown for its mammalian counterparts. The chicken IL-12p70 molecule, generated either by transient coexpression of monomeric IL-12p35 and monomeric IL-12p40 or as a fusion protein (as in a fusion linker construct), induced IFN-γ synthesis and proliferative activity of freshly exposed chicken splenocytes. The high degree of functional similarity between chicken IL-12 and IL-12 of higher mammalian vertebrates, despite their poor sequence homology, illustrates the conservation and vital importance of the IL-12 molecule since the evolutionary dichotomy of birds and mammals >300 million years ago. In this article, we describe the first nonmammalian IL-12 molecule and show that this chicken IL-12 molecule is bioactive.
Aerobic Exercise Attenuates Airway Inflammatory Responses in a Mouse Model of Atopic Asthma
Authors: Pastva Amy , Estell Kim , Schoeb Trenton R. , Atkinson T. Prescott , Schwiebert Lisa M.
Published: April 2014

Recent reports indicate that aerobic exercise improves the overall physical fitness and health of asthmatic patients. The specific exercise-induced improvements in the pathology of asthma and the mechanisms by which these improvements occur, however, are ill-defined; thus, the therapeutic potential of exercise in the treatment of asthma remains unappreciated. Using an OVA-driven mouse model, we examined the role of aerobic exercise in modulating inflammatory responses associated with atopic asthma. Data demonstrate that moderate intensity aerobic exercise training decreased leukocyte infiltration, cytokine production, adhesion molecule expression, and structural remodeling within the lungs of OVA-sensitized mice (n = 6–10; p < 0.05). Because the transcription factor NF-κB regulates the expression of a variety of genes that encode inflammatory mediators, we monitored changes in NF-κB activation in the lungs of exercised/sensitized mice. Results show that exercise decreased NF-κB nuclear translocation and IκBα phosphorylation, indicating that exercise decreased NF-κB activation in the lungs of sensitized mice (n = 6). Taken together, these results suggest that aerobic exercise attenuates airway inflammation in a mouse model of atopic asthma via modulation of NF-κB activation. Potential exists, therefore, for the amelioration of asthma-associated chronic airway inflammation through the use of aerobic exercise training as a non-drug therapeutic modality.
A Self T Cell Epitope Induces Autoantibody Response: Mechanism for Production of Antibodies to Diverse Glomerular Basement Membrane Antigens
Authors: Wu Jean , Arends Jon , Borillo Jason , Zhou Cindy , Merszei Justin , McMahon John , Lou Ya-Huan
Published: April 2014

The anti-glomerular basement membrane (GBM) Ab has been regarded as a prototypical example of pathogenic autoantibodies. However, the mechanism for elicitation of this Ab remains unknown. In the present paper, we report that the Ab to diverse GBM Ags was induced by a single nephritogenic T cell epitope in a rat model. The T cell epitope pCol28–40 of noncollagen domain 1 of collagen type IV α3 chain not only uniformly induced severe glomerulonephritis but also elicited anti-GBM Ab in 76% of the immunized rats after prominent glomerular injury. Furthermore, we demonstrated that the anti-GBM Ab was not related to the peptidic B cell epitope nested in pCol28–40; that is, 1) elimination of the B cell epitope, either by substitution of the critical residues of the B cell epitope or by truncation, failed to abrogate anti-GBM Ab production, and 2) the anti-GBM Ab, eluted from the diseased kidneys, reacted only with native GBM, but not with pCol28–40. Confocal microscopy and immunoprecipitation further demonstrated that the eluted anti-GBM Ab recognized conformational B cell epitope(s) of multiple native GBM proteins. We conclude that autoantibody response to diverse native GBM Ags was induced by a single nephritogenic T cell epitope. Thus, anti-GBM Ab may actually be a consequence of T cell-mediated glomerulonephritis.
New Loci from New Zealand Black and New Zealand White on Chromosomes 4 and 12 Contribute to Lupus-Like Disease in the Context of BALB/c
Authors: Rigby Robert J. , Rozzo Stephen J. , Boyle Joseph J. , Lewis Margarita , Kotzin Brian L. , Vyse Timothy J.
Published: April 2014

New Zealand Black (NZB) and New Zealand White (NZW) mice are genetically predisposed to a lupus-like autoimmune syndrome. To further define the loci linked to disease traits in NZB and NZW mice in the context of the BALB/c genetic background, linkage analyses were conducted in two crosses: (NZW × BALB/c.H2z)F1 × NZB and (NZB × BALB/c)F2. Novel loci linked to autoantibody production and glomerulonephritis, present in both NZB and NZW mice, were identified on proximal chromosomes 12 and 4. The chromosome 12 locus showed the strongest linkage to anti-nuclear Ab production. Additionally, a number of other novel loci linked to lupus traits derived from both the New Zealand and non-autoimmune BALB/c genomes were identified. Furthermore, we confirm the linkage of disease to a number of previously described lupus-associated loci, demonstrating that they are relatively background independent. These data provide a number of additional candidate gene regions in murine lupus, and highlight the powerful effect the non-autoimmune background strain has in influencing the genetic loci linked to disease.
STAT5 Activation Underlies IL7 Receptor-Dependent B Cell Development
Authors: Goetz Christine A. , Harmon Ian R. , O’Neil Jennifer J. , Burchill Matthew A. , Farrar Michael A.
Published: April 2014

Signals initiated by the IL7R are required for B cell development. However, the roles that distinct IL7R-induced signaling pathways play in this process remains unclear. To identify the function of the Raf and STAT5 pathways in IL7R-dependent B cell development, we used transgenic mice that express constitutively active forms of Raf (Raf-CAAX) or STAT5 (STAT5b-CA) throughout lymphocyte development. Both Raf-CAAX and STAT5b-CA mice exhibit large increases in pro-B cells. However, crossing the Raf-CAAX transgene onto the IL7R−/− background fails to rescue B cell development. In contrast, STAT5 activation selectively restores B cell expansion in IL7R−/− mice. Notably, the expansion of pro-B cells in STAT5b-CA mice correlated with an increase in cyclin D2, pim-1, and bcl-xL expression, suggesting that STAT5 directly affects pro-B cell proliferation and survival. In addition, STAT5 activation also restored B cell differentiation in IL7R−/− mice as determined by 1) the restoration of VH Ig gene rearrangement and 2) the appearance of immature and mature B cell subsets. These findings establish STAT5 as the key player entraining B cell development downstream of the IL7R.
The Novel Cyclophilin Binding Compound, Sanglifehrin A, Disassociates G1 Cell Cycle Arrest from Tolerance Induction
Authors: Allen Amy , Zheng Yan , Gardner Lawrence , Safford Meredith , Horton Maureen R. , Powell Jonathan D.
Published: April 2014

T cell anergy has been demonstrated to play a role in maintaining peripheral tolerance to self Ags as well as a means by which tumors can evade immune destruction. Although the precise pathways involved in anergy induction have yet to be elucidated, it has been linked to TCR engagement in the setting of cell cycle arrest. Indeed, rapamycin, which inhibits T cell proliferation in G1, has the ability to promote tolerance even in the presence of costimulation. To better define the role of the cell cycle in regulating anergy induction, we used the novel cyclophilin-binding ligand, sanglifehrin A (SFA). We demonstrate that SFA can inhibit TCR-induced cytokine and chemokine production without preventing TCR-induced anergy. Our data also indicate that despite its ability to induce G1 arrest, SFA does not induce anergy in the presence of costimulation. Furthermore, although SFA blocks proliferation to exogenous IL-2, it does not prevent IL-2-induced reversal of anergy. When we examined the phosphorylation of 4EBP-1, a downstream substrate of the mammalian target of rapamycin, we found that rapamycin, but not SFA, inhibited the mammalian target of rapamycin activity. Based on these data, we propose that the decision as to whether TCR engagement will lead to productive activation or tolerance is dictated by a rapamycin -inhibitable pathway, independent of the G1→S phase cell cycle progression.
Simultaneous Cellular and Humoral Immune Response against Mutated p53 in a Patient with Lung Cancer
Authors: Ichiki Yoshinobu , Takenoyama Mitsuhiro , Mizukami Makiko , So Tetsuya , Sugaya Masakazu , Yasuda Manabu , So Tomoko , Hanagiri Takeshi , Sugio Kenji , Yasumoto Kosei
Published: April 2014

We recently identified several Ags recognized by tumor-infiltrating B lymphocyte-derived Ab using SCID mice and a xenografted non-small cell lung cancer system. One of these identified Ags was mutated p53 with a point mutation resulting in the alteration of codon 158 from Arg to Leu. The aim of this study was to ascertain whether cellular immunity against mutated p53 exists in the same patient together with humoral immunity. Two different nona peptides (mutated p53150 and p53155 peptides), including a mutated amino acid derived from p53, were synthesized according to the binding motif of HLA class I of the established cancer cell line A904L from the patient. Mediastinal lymph node lymphocytes of the patient were stimulated weekly with the peptides. The mutated p53155 peptide-stimulated lymphocytes showed specific cytotoxicity against both autologous EBV-transformed B cells pulsed with mutated p53155 peptide and A904L. The mutated p53155 peptide-specific CTL clone in an HLA-Cw*0702 restriction was established and analyzed for its TCR usage. Clonotypic PCR using CDR3-specific primers was applied to the tumor tissue containing the tumor-infiltrating lymphocytes. The specific amplification of PCR was found in the tumor tissue. These results demonstrated that not only B lymphocytes producing specific Ab against the p53 protein, but also CTL against mutated p53, expressed in autologous lung cancer cells exist in the tumor tissue. This approach may allow us to better understand the mechanisms of T and B cell immunity against the same tumor Ag in cancer patients.
A Leucine-Rich Repeat Motif of Leishmania Parasite Surface Antigen 2 Binds to Macrophages through the Complement Receptor 3
Authors: Kedzierski Lukasz , Montgomery Jacqui , Bullen Denise , Curtis Joan , Gardiner Elizabeth , Jimenez-Ruiz Antonio , Handman Emanuela
Published: April 2014

Membrane glycoconjugates on the Leishmania parasites, notably leishmanolysin and lipophosphoglycan, have been implicated in attachment and invasion of host macrophages. However, the function of parasite surface Ag 2 (PSA-2) and membrane proteophosphoglycan (PPG) has not been elucidated. In this study we demonstrate that native and recombinant Leishmania infantum PSA-2, which consists predominantly of 15 leucine-rich repeats (LRR) and a recombinant LRR domain derived from L. major PPG, bind to macrophages. The interaction is restricted to macrophages and appears to be calcium independent. We have investigated the PSA-2-macrophage interaction to identify the host receptor involved in binding and we show that binding of PSA-2 to macrophages can be blocked by Abs to the complement receptor 3 (CR3, Mac-1). Data derived from mouse macrophage studies were further confirmed using cell lines expressing human CR3, and showed that PSA-2 also binds to the human receptor. This is the first demonstration of a functional role for PSA-2. Our data indicate that in addition to leishmanolysin and lipophosphoglycan, parasite attachment and invasion of macrophages involve a third ligand comprising the LRRs shared by PSA-2 and PPG and that these interactions occur via the CR3.
Transient Corticosteroid Treatment Permanently Amplifies the Th2 Response in a Murine Model of Asthma
Authors: Wiley Ryan E. , Cwiartka Monika , Alvarez David , Mackenzie David C. , Johnson Jill R. , Goncharova Susanna , Lundblad Lennart , Jordana Manel
Published: April 2014

Corticosteroids (CS) remain the most efficacious pharmacotherapeutic option for the management of asthma. Although the acute anti-inflammatory effects of CS treatment have been amply documented both clinically and experimentally, recent human data intimate that exposure to CS may be associated with retrograde immune phenomena, including enhanced synthesis of IgE in vivo and elevated Th2 cytokine production in vitro. We have investigated the long-term immunologic effects of CS treatment in a murine model of allergic airway inflammation. CS treatment during initial exposure to OVA or upon long-term Ag rechallenge remarkably attenuated eosinophilic airway inflammation and airway hyperresponsiveness. Interestingly, however, Th2 cytokine production by cultured splenocytes from CS-treated mice was significantly elevated, while IFN-γ synthesis was depressed. Moreover, mice rechallenged with OVA several weeks after CS intervention during allergic sensitization not only developed airway inflammation, but also exhibited enhanced Th2 cytokine production in lymphoid tissues and OVA-specific IgE in serum. This amplification of the systemic immune response was associated with an intact APC compartment during CS-conditioned sensitization to OVA. These data indicate that immune processes underlying the allergic phenotype remain impervious to CS treatment and raise the possibility that treatment with CS during sensitization may amplify elements of the allergen-specific immune response.
Thymocyte-Intrinsic Genetic Factors Influence CD8 T Cell Lineage Commitment and Affect Selection of a Tumor-Reactive TCR
Authors: Shanker Anil , Auphan-Anezin Nathalie , Chomez Patrick , Giraudo Laurent , Eynde Benoı̂t Van den , Schmitt-Verhulst Anne-Marie
Published: April 2014

Selection of immature CD4CD8 double-positive (DP) thymocytes for CD4 or CD8-lineage commitment is controlled by the interaction of the TCR with stromal cell-expressed peptide/MHC. We show that thymocyte-intrinsic genes influence the pattern of expression of a MHC class I-restricted transgenic (tg) TCR so that in DBA/2 mice, DP thymocytes with a characteristically high expression of tg TCR, infrequently transit to CD8 single-positive thymocytes. In contrast, in B10.D2 mice, the same tg TCR is expressed at lower levels on a subpopulation of DP thymocytes that more frequently transit to CD8 single-positive thymocytes. These characteristics were not influenced by thymic stromal components that control positive selection. Radiation chimeras reconstituted with a mixture of BM from tg TCR mice of the two genetic backgrounds revealed that the relative frequency of transit to the CD8 lineage remained thymocyte-intrinsic. Identifying the gene products whose polymorphism controls CD8 T cell development may shed new light on the mechanisms controlling T cell commitment/selection in mice other than the most studied “C57BL/6”-based strains.
Bacterial Heat Shock Proteins Promote CD91-Dependent Class I MHC Cross-Presentation of Chaperoned Peptide to CD8+ T Cells by Cytosolic Mechanisms in Dendritic Cells versus Vacuolar Mechanisms in Macrophages
Authors: Tobian Aaron A. R. , Canaday David H. , Boom W. Henry , Harding Clifford V.
Published: April 2014

APCs process mammalian heat shock protein (HSP):peptide complexes to present HSP-chaperoned peptides on class I MHC (MHC-I) molecules to CD8+ T cells. HSPs are also expressed in prokaryotes and chaperone microbial peptides, but the ability of prokaryotic HSPs to contribute chaperoned peptides for Ag presentation is unknown. Our studies revealed that exogenous bacterial HSPs (Escherichia coli DnaK and Mycobacterium tuberculosis HSP70) delivered an extended OVA peptide for processing and MHC-I presentation by both murine macrophages and dendritic cells. HSP-enhanced MHC-I peptide presentation occurred only if peptide was complexed to the prokaryotic HSP and was dependent on CD91, establishing CD91 as a receptor for prokaryotic as well as mammalian HSPs. Inhibition of cytosolic processing mechanisms (e.g., by transporter for Ag presentation deficiency or brefeldin A) blocked HSP-enhanced peptide presentation in dendritic cells but not macrophages. Thus, prokaryotic HSPs deliver chaperoned peptide for alternate MHC-I Ag processing and cross-presentation via cytosolic mechanisms in dendritic cells and vacuolar mechanisms in macrophages. Prokaryotic HSPs are a potential source of microbial peptide Ags during phagocytic processing of bacteria during infection and could potentially be incorporated in vaccines to enhance presentation of peptides to CD8+ T cells.