bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vivo Antigen Stability Affects DNA Vaccine Immunogenicity
Authors: Bins Adriaan D. , Wolkers Monika C. , van den Boom Marly D. , Haanen John B. A. G. , Schumacher Ton N. M.
Published: April 2014

The factors that determine the immunogenicity of Ags encoded by viral vaccines or DNA vaccines in vivo are largely unknown. Depending on whether T cell induction occurs via direct presentation of vaccine-encoded epitopes or via one of the different proposed pathways for Ag cross-presentation, the effect of intracellular Ag stability on immunogenicity may possibly vary. However, the influence of Ag stability on CD8+ T cell induction has not been addressed in clinically relevant vaccine models, nor has the accumulation of vaccine-encoded Ags been monitored in vivo. In this study, we describe the relationship between in vivo Ag stability and immunogenicity of DNA vaccine-encoded Ags. We show that in vivo accumulation of DNA vaccine-encoded Ags is required for the efficient induction of CD8+ T cell responses. These data suggest that many of the currently used transgene designs in DNA vaccination trials may be suboptimal, and that one should either use pathogen-derived or tumor-associated Ags that are intrinsically stable, or should increase the stability of vaccine-encoded Ags by genetic engineering.
The Context of Epitope Presentation Can Influence Functional Quality of Recalled Influenza A Virus-Specific Memory CD8+ T Cells
Authors: Day E. Bridie , Zeng Weiguang , Doherty Peter C. , Jackson David C. , Kedzierska Katherine , Turner Stephen J.
Published: April 2014

Lipopeptide constructs offer a novel strategy for eliciting effective cellular and humoral immunity by directly targeting the vaccine Ag to dendritic cells. Importantly, it is not known how closely immunity generated after lipopeptide vaccination mimics that generated after natural infection. We have used a novel lipopeptide vaccine strategy to analyze both the quantity and quality of CD8+ T cell immunity to an influenza A virus epitope derived from the acidic polymerase protein (PA224) in B6 mice. Vaccination with the PA224 lipopeptide resulted in accelerated viral clearance after subsequent influenza virus infection. The lipopeptide was also effective at recalling secondary DbPA224 responses in the lung. Lipopeptide recalled DbPA224-specific CTL produced lower levels of IFN-γ and TNF-α, but produced similar levels of IL-2 when compared with DbPA224-specific CTL recalled after virus infection. Furthermore, lipopeptide- and virus-recalled CTL demonstrated similar TCR avidity. Interestingly, lipopeptide administration resulted in expansion of DbPA224-specific CTL using a normally subdominant TCRBV gene segment. Overall, these results demonstrate that protective CTL responses elicited by lipopeptide vaccines can be correlated with TCR avidity, IL-2 production, and broad TCR repertoire diversity. Furthermore, factors that impact the quality of immunity are discussed. These factors are important considerations when evaluating the efficacy of novel vaccine strategies that target dendritic cells for eliciting cellular immunity.
CD134 Costimulation Couples the CD137 Pathway to Induce Production of Supereffector CD8 T Cells That Become IL-7 Dependent
Authors: Lee Seung-Joo , Rossi Robert J. , Lee Sun-Kyeong , Croft Michael , Kwon Byoung S. , Mittler Robert S. , Vella Anthony T.
Published: April 2014

The TNFR superfamily members 4-1BB (CD137) and OX40 (CD134) are costimulatory molecules that potently boost CD8 and CD4 T cell responses. Concomitant therapeutic administration of agonist anti-CD137 and -CD134 mAbs mediates rejection of established tumors and fosters powerful CD8 T cell responses. To reveal the mechanism, the role of CD137 expression by specific CD8 T cells was determined to be essential for optimal clonal expansion and accumulation of effector cells. Nonetheless, dual costimulation induced production of supereffector CD8 T cells when either the specific T cells or the host alone bore CD137. Perhaps surprisingly, the total absence of CD137 prevented anti-CD134 augmentation of supereffector differentiation demonstrating an unappreciated link between these related pathways. Ultimately, it was reasoned that these powerful dual costimulatory responses involved common γ family members, and we show substantial increases of CD25 and IL-7Rα-chain expression by the specific CD8 T cells. To investigate this further, it was shown that IL-7 mediated T cell accumulation, but importantly, a gradual and preferential effect of survival was directed toward supereffector CD8 T cells. In fact, a clear enhancement of effector differentiation was demonstrated to be proportional to the increasing amount of IL-7Rα expression by the specific CD8 T cells. Therefore, dual costimulation through CD137 and CD134 drives production and survival of supereffector CD8 T cells through a distinct IL-7-dependent pathway.
MHC Class II+ Exosomes in Plasma Suppress Inflammation in an Antigen-Specific and Fas Ligand/Fas-Dependent Manner
Authors: Kim Seon Hee , Bianco Nicole R. , Shufesky William J. , Morelli Adrian E. , Robbins Paul D.
Published: April 2014

Exosomes are 50- to 100-nm vesicles that are formed within the late endocytic compartment and released from a variety of cell types. Previously, we demonstrated that exosomes derived from dendritic cells transduced with adenoviral vectors expressing IL-10, IL-4, or Fas ligand (FasL) produce anti-inflammatory exosomes able to reduce inflammation in a murine paw delayed-type hypersensitivity model, suppress the onset on murine collagen-induced arthritis, and reduce the severity of established collagen-induce arthritis. In this study, we examined the ability of endogenous, blood-borne exosomes to regulate the immune response. Exosomes isolated from plasma of mice immunized to keyhole limpet hemocyanin, but not from naive or OVA-immunized mice, were able to suppress the keyhole limpet hemocyanin-specific delayed-type hypersensitivity inflammatory response. The anti-inflammatory effect was mediated by MHC class II+ plasma exosomes that were also FasL+ and CD11b+, but CD11c−. Moreover, the anti-inflammatory effect of the MHC class II+ plasma-derived exosomes was, in part, dependent upon the presence of FasL in the exosomes and Fas in the recipient mouse. These results suggest that exosomes in the plasma, produced by MHC class II+ and CD11b+ cells, have the ability to suppress the immune response in an Ag-specific manner in part through a Fas/FasL-dependent manner.
TACI Is Required for Efficient Plasma Cell Differentiation in Response to T-Independent Type 2 Antigens
Authors: Mantchev George T. , Cortesão Catarina S. , Rebrovich Michelle , Cascalho Marilia , Bram Richard J.
Published: April 2014

The control of systemic infection by encapsulated microorganisms requires T-independent type II (TI-2) Ab responses to bacterial polysaccharides. To understand how such responses evolve, we explored the function of transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI), a member of the TNFR family, required for TI-2 Ab production. Quasimonoclonal (QM) mice produce robust TI-2 responses to 4-hydroxy-3-nitrophenylacetate (NP)-Ficoll, owing to the high precursor frequency of NP-specific B cells in the marginal zone of the spleen. QM mice that lack TACI produce decreased numbers of IgM (2-fold) and IgG (1.6-fold) NP-specific ASCs, compared with TACI-positive QM mice in response to immunization with NP-Ficoll. Our studies indicate that TACI acts at a remote time from activation because TACI is not necessary for activation and proliferation of B cells both in vitro and in vivo. Instead, TACI-deficient QM B cells remained in the cell cycle longer than TACI-proficient QM cells and had impaired plasma cell differentiation in response to NP-Ficoll. We conclude that TACI has dual B cell-autonomous functions, inhibiting prolonged B cell proliferation and stimulating plasma cell differentiation, thus resolving the longstanding paradox that TACI may have both B cell-inhibitory and -stimulatory functions. By promoting plasma cell differentiation earlier during clonal expansion, TACI may decrease the chances of autoantibody production by somatic hypermutation of Ig genes in response to T-independent Ags.
CCR5 Blockade Modulates Inflammation and Alloimmunity in Primates
Authors: Schröder Carsten , Pierson Richard N. , Nguyen Bao-Ngoc H. , Kawka Douglas W. , Peterson Laurence B. , Wu Guosheng , Zhang Tianshu , Springer Martin S. , Siciliano Sal J. , Iliff Susan , Ayala Julia M. , Lu Min , Mudgett John S. , Lyons Kathy , Mills Sander G. , Miller Geraldine G. , Singer Irwin I. , Azimzadeh Agnes M. , DeMartino Julie A.
Published: April 2014

Pharmacologic antagonism of CCR5, a chemokine receptor expressed on macrophages and activated T cells, is an effective antiviral therapy in patients with macrophage-tropic HIV infection, but its efficacy in modulating inflammation and immunity is only just beginning to be investigated. In this regard, the recruitment of CCR5-bearing cells into clinical allografts is a hallmark of acute rejection and may anticipate chronic rejection, whereas conventionally immunosuppressed renal transplant patients homozygous for a nonfunctional Δ32 CCR5 receptor rarely exhibit late graft loss. Therefore, we explored the effects of a potent, highly selective CCR5 antagonist, Merck’s compound 167 (CMPD 167), in an established cynomolgus monkey cardiac allograft model. Although perioperative stress responses (fever, diminished activity) and the recruitment of CCR5-bearing leukocytes into the graft were markedly attenuated, anti-CCR5 monotherapy only marginally prolonged allograft survival. In contrast, relative to cyclosporine A monotherapy, CMPD 167 with cyclosporine A delayed alloantibody production, suppressed cardiac allograft vasculopathy, and tended to further prolong graft survival. CCR5 therefore represents an attractive therapeutic target for attenuating postsurgical stress responses and favorably modulating pathogenic alloimmunity in primates, including man.
Molecular and Cellular Requirements for Enhanced Antigen Cross-Presentation to CD8 Cytotoxic T Lymphocytes
Authors: Oizumi Satoshi , Strbo Natasa , Pahwa Savita , Deyev Vadim , Podack Eckhard R.
Published: April 2014

MHC class I-mediated cross-priming of CD8 T cells by APCs is critical for CTL-based immunity to viral infections and tumors. We have shown previously that tumor-secreted heat shock protein gp96-chaperoned peptides cross prime CD8 CTL that are specific for genuine tumor Ags and for the surrogate Ag OVA. We now show that tumor-secreted heat shock protein gp96-chaperoned peptides enhance the efficiency of Ag cross-priming of CD8 CTL by several million-fold over the cross-priming activity of unchaperoned protein alone. Gp96 also acts as adjuvant for cross-priming by unchaperoned proteins, but in this capacity gp96 is 1000-fold less active than as a peptide chaperone. Mechanistically, the in situ secretion of gp96-Ig by transfected tumor cells recruits and activates dendritic cells and NK cells to the site of gp96 release and promotes CD8 CTL expansion locally. Gp96-mediated cross-priming of CD8 T cells requires B7.1/2 costimulation but proceeds unimpeded in lymph node-deficient mice, in the absence of NKT and CD4 cells and without CD40L. Gp96-driven MHC I cross-priming of CD8 CTL in the absence of lymph nodes provides a novel mechanism for local, tissue-based CTL generation at the site of gp96 release. This pathway may constitute a critically important, early detection, and rapid response mechanism that is operative in parenchymal tissues for effective defense against tissue damaging antigenic agents.
O-Glycosylated Human MUC1 Repeats Are Processed In Vitro by Immunoproteasomes
Authors: Ninkovic Tanja , Hanisch Franz-Georg
Published: April 2014

The targeting of epitopes on tumor-associated glycoforms of human MUC1 represents a primary goal in immunotherapeutic anticancer strategies. Effective immune responses to cancer cells certainly require the activation of specific cytotoxic T cell repertoires by cross-priming of dendritic cells either via immunoproteasomal or by endosomal processing of ectodomain epitopes on MUC1-positive carcinomas. Because no evidence is currently available on the capacities of human immunoproteasomes to cleave mucin-type O-glycosylated peptides, we performed in vitro studies to address the questions of whether glycosylated MUC1 repeats are cleaved by immunoproteasomes and in which way O-linked glycans control the site specificity of peptide cleavage via their localization and structures. We show for the first time that mucin-type O-glycosylated peptides are effective substrates of immunoproteasomes, however, the patterns of cleavage are qualitatively and quantitatively influenced by O-glycosylation. The nonglycosylated MUC1 repeat peptide (clusters of oligorepeats AHGVTSAPDTRPAPGSTAPP or AHGVTSAPESRPAPGSTAPA) is cleaved preferentially within or adjacent to the SAP and GST motifs with formation of a complex fragment pattern that includes major nona- and decapeptides. O-GalNAc modified peptides are largely resistant to proteolysis if these preferred cleavage sites are located adjacent to O-glycosylation, whereas peptides even with elongated glycans at more distant sites can form effective substrates yielding major glycopeptide fragments in the class I size range.
IL-10 Is Excluded from the Functional Cytokine Memory of Human CD4+ Memory T Lymphocytes
Authors: Dong Jun , Ivascu Claudia , Chang Hyun-Dong , Wu Peihua , Angeli Roberta , Maggi Laura , Eckhardt Florian , Tykocinski Lars , Haefliger Carolina , Möwes Beate , Sieper Jochen , Radbruch Andreas , Annunziato Francesco , Thiel Andreas
Published: April 2014

Epigenetic modifications, including DNA methylation, profoundly influence gene expression of CD4+ Th-specific cells thereby shaping memory Th cell function. We demonstrate here a correlation between a lacking fixed potential of human memory Th cells to re-express the immunoregulatory cytokine gene IL10 and its DNA methylation status. Memory Th cells secreting IL-10 or IFN-γ were directly isolated ex vivo from peripheral blood of healthy volunteers, and the DNA methylation status of IL10 and IFNG was assessed. Limited difference in methylation was found for the IL10 gene locus in IL-10-secreting Th cells, as compared with Th cells not secreting IL-10 isolated directly ex vivo or from in vitro-established human Th1 and Th2 clones. In contrast, in IFN-γ+ memory Th cells the promoter of the IFNG gene was hypomethylated, as compared with IFN-γ-nonsecreting memory Th cells. In accordance with the lack of epigenetic memory, almost 90% of ex vivo-isolated IL-10-secreting Th cells lacked a functional memory for IL-10 re-expression after restimulation. Our data indicate that IL10 does not become epigenetically marked in human memory Th cells unlike effector cytokine genes such as IFNG. The exclusion of IL-10, but not effector cytokines, from the functional memory of human CD4+ T lymphocytes ex vivo may reflect the need for appropriate regulation of IL-10 secretion, due to its potent immunoregulatory potential.
Human Anti-CXCR4 Antibodies Undergo VH Replacement, Exhibit Functional V-Region Sulfation, and Define CXCR4 Antigenic Heterogeneity
Authors: Xu Chen , Sui Jianhua , Tao Hong , Zhu Quan , Marasco Wayne A.
Published: April 2014

The chemokine receptor CXCR4 and its ligand stromal-derived factor-1 (SDF-1/CXCL12) are essential for many biological processes and various pathological conditions. However, the relationship between CXCR4 antigenic structure and SDF-1-mediated biological responses is poorly understood. In this report, a panel of human anti-CXCR4 Abs were isolated and used to explore CXCR4 antigenic heterogeneity and function. Multiple fixed CXCR4 antigenic isoforms were detected on the surface of hemopoietic cells. Epitope mapping studies demonstrated the complex nature of the surface-exposed CXCR4 epitopes. Ab-mediated inhibition of chemotaxis correlated strongly with binding affinity, epitope recognition, as well as the level of CXCR4 isoform expression. In addition, detailed genetic analyses of these Abs showed evidence of VH replacement. Importantly, structural and biochemical studies demonstrated tyrosine sulfation in novel regions of the V genes that contributed bidirectionally to the binding activity of the Abs. These data provide the first evidence that functional tyrosine sulfation occurs in self-reactive Abs and suggest a potential new mechanism that may contribute to the pathogenesis of Ab-mediated autoimmune disease. These Abs also provide valuable tools to explore the selective in vivo targeting of CXCR4 isoforms that may be preferentially expressed in certain disease states and involved in steady-state CXCR4-SDF-1 homeostasis.
Sequence and Characterization of the Ig Heavy Chain Constant and Partial Variable Region of the Mouse Strain 129S1
Authors: Retter Ida , Chevillard Christophe , Scharfe Maren , Conrad Ansgar , Hafner Martin , Im Tschong-Hun , Ludewig Monika , Nordsiek Gabriele , Severitt Simone , Thies Stephanie , Mauhar America , Blöcker Helmut , Müller Werner , Riblet Roy
Published: April 2014

Although the entire mouse genome has been sequenced, there remain challenges concerning the elucidation of particular complex and polymorphic genomic loci. In the murine Igh locus, different haplotypes exist in different inbred mouse strains. For example, the Ighb haplotype sequence of the Mouse Genome Project strain C57BL/6 differs considerably from the Igha haplotype of BALB/c, which has been widely used in the analyses of Ab responses. We have sequenced and annotated the 3′ half of the Igha locus of 129S1/SvImJ, covering the CH region and approximately half of the VH region. This sequence comprises 128 VH genes, of which 49 are judged to be functional. The comparison of the Igha sequence with the homologous Ighb region from C57BL/6 revealed two major expansions in the germline repertoire of Igha. In addition, we found smaller haplotype-specific differences like the duplication of five VH genes in the Igha locus. We generated a VH allele table by comparing the individual VH genes of both haplotypes. Surprisingly, the number and position of DH genes in the 129S1 strain differs not only from the sequence of C57BL/6 but also from the map published for BALB/c. Taken together, the contiguous genomic sequence of the 3′ part of the Igha locus allows a detailed view of the recent evolution of this highly dynamic locus in the mouse.
BXSB/long-livedIs a Recombinant Inbred Strain Containing Powerful Disease Suppressor Loci
Authors: Haywood Michelle E. K. , Gabriel Luisa , Rose S. Jane , Rogers Nicola J. , Izui Shozo , Morley Bernard J.
Published: April 2014

The BXSB strain of recombinant inbred mice develops a spontaneous pathology that closely resembles the human disease systemic lupus erythematosus. Six non-MHC loci, Yaa, Bxs1–4, and Bxs6, have been linked to the development of aspects of the disease while a further locus, Bxs5, may be a BXSB-derived disease suppressor. Disease development is delayed in a substrain of BXSB, BXSB/MpJScr-long-lived (BXSB/ll). We compared the genetic derivation of BXSB/ll mice to the original strain, BXSB/MpJ, using microsatellite markers and single nucleotide polymorphisms across the genome. These differences were clustered and included two regions known to be important in the disease-susceptibility of these mice, Bxs5 and 6, as well as regions on chromosomes 5, 6, 9, 11, 12, and 13. We compared BXSB/ll to >20 strains including the BXSB parental SB/Le and C57BL/6 strains. This revealed that BXSB/ll is a separate recombinant inbred line derived from SB/Le and C57BL/6, but distinctly different from BXSB, that most likely arose due to residual heterozygosity in the BXSB stock. Despite the continued presence of the powerful disease-susceptibility locus Bxs3, BXSB/ll mice do not develop disease. We propose that the disappearance of the disease phenotype in the BXSB/ll mice is due to the inheritance of one or more suppressor loci in the differentially inherited intervals between the BXSB/ll and BXSB strains.
Filarial Parasites Induce NK Cell Activation, Type 1 and Type 2 Cytokine Secretion, and Subsequent Apoptotic Cell Death
Authors: Babu Subash , Blauvelt Carla P. , Nutman Thomas B.
Published: April 2014

NK cells are an important source of early cytokine production in a variety of intracellular viral, bacterial, and protozoan infections; however, the role of NK cells in extracellular parasitic infections such as filarial infections is not well-defined. To investigate the role of NK cells in filarial infections, we have used an in vitro model system of culturing live infective-stage larvae (L3) or live microfilariae (Mf) of Brugia malayi, a causative agent of human lymphatic filariasis, with PBMC of normal individuals. We found that NK cells undergo early cell activation and produce IFN-γ and TNF-α within 24 h after stimulation with both live L3 and Mf. Interestingly, NK cells also express IL-4 and IL-5 at this time point in response to live Mf but not L3. This is accompanied by significant alterations in NK cell expression of costimulatory molecules and natural cytotoxicity receptors. This activation is dependent on the presence of monocytes in the culture, IL-12, and direct contact with live parasites. The early activation event is subsequently followed by apoptosis of NK cells involving a caspase-dependent mechanism in response to live L3 but not live Mf. Thus, the NK cell-parasite interaction is complex, with filarial parasites inducing NK cell activation and cytokine secretion and finally NK cell apoptosis, which may provide an additional mechanism of down-regulating the host immune response.
A Role for IFN-γ from Antigen-Specific CD8+ T Cells in Protective Immunity toListeria monocytogenes
Authors: Messingham Kelly A. N. , Badovinac Vladimir P. , Jabbari Ali , Harty John T.
Published: April 2014

Whether IFN-γ contributes to the per-cell protective capacity of memory CD8+ T cells against Listeria monocytogenes (LM) has not been formally tested. In this study, we generated LM Ag-specific memory CD8+ T cells via immunization of wild-type (WT) and IFN-γ-deficient (gamma knockout (GKO)) mice with LM peptide-coated dendritic cells and compared them phenotypically and functionally. Immunization of WT and GKO mice resulted in memory CD8+ T cells that were similar in number, functional avidity, TCR repertoire use, and memory phenotype. The protective capacity of memory CD8+ T cells from immunized WT and GKO mice was evaluated after adoptive transfer of equal numbers of WT or GKO cells into naive BALB/c mice followed by LM challenge. The adoptively transferred CD8+ T cells from GKO donors exhibited a decreased ability to reduce bacterial numbers in the organs of recipient mice when compared with an equivalent number of Ag-matched WT CD8+ T cells. This deficiency was most evident early (day 3) after infection if a relatively low infectious dose was used; however, transferring fewer memory CD8+ T cells or increasing the LM challenge dose revealed a more pronounced defect in protective immunity mediated by the CD8+ T cells from GKO mice. Our studies identified a decrease in Ag-specific target cell lysis in vivo by CD8+ T cells from GKO mice as the mechanism for the decreased protective immunity after LM challenge. Further studies suggest that the lack of IFN-γ production by the Ag-specific CD8 T cells themselves diminishes target cell sensitivity to cytolysis, thereby reducing the lytic potency of IFN-γ-deficient LM-specific memory CD8+ T cells.
Icsbp1/IRF-8 Is Required for Innate and Adaptive Immune Responses against Intracellular Pathogens
Authors: Turcotte Karine , Gauthier Susan , Malo Danielle , Tam Mifong , Stevenson Mary M. , Gros Philippe
Published: April 2014

The chronic myeloid leukemia syndrome of the BXH-2 mouse strain (Mus musculus) is caused by a recessive mutation (R294C) in the transcriptional regulator Icsbp1/IRF-8. In trans activation assays using an IL-12p40 gene reporter construct introduced in RAW 264.7 mouse macrophages, we show that the Icsbp1C294 isoform behaves as a partial loss-of-function. The Icsbp1C294 hypomorph allele appears to have a threshold effect on IL-12 production, with pleiotropic consequences on resistance to different types of infections in vivo. Despite the presence of a resistance Nramp1G169 allele, BXH-2 mice (Icsbp1C294) show impaired control of Mycobacterium bovis (bacille Calmette-Guérin) multiplication both early and late during infection, with uncontrolled replication linked to inability to form granulomas in infected liver and spleen. Studies in informative (BXH-2 × BALB/cJ)F2 mice show that homozygosity for Icsbp1C294 causes susceptibility to Salmonella enterica serovar Typhimurium to a level comparable to that seen for mice lacking functional Nramp1 or TLR4. Finally, impaired Icsbp1C294 function is associated with the following: 1) increased replication of the Plasmodium chabaudi AS malarial parasite during the first burst of blood parasitemia, and 2) recurring waves of high blood parasitemia late during infection. These results show that Icsbp1 is required for orchestrating early innate responses and also long-term immune protection against unrelated intracellular pathogens.
A Novel Role for the Bactericidal/Permeability Increasing Protein in Interactions of Gram-Negative Bacterial Outer Membrane Blebs with Dendritic Cells
Authors: Schultz Hendrik , Hume Janet , Zhang De Sheng , Gioannini Theresa L. , Weiss Jerrold P.
Published: April 2014

The bactericidal/permeability-increasing protein (BPI) is thought to play an important role in killing and clearance of Gram-negative bacteria and the neutralization of endotoxin. A possible role for BPI in clearance of cell-free endotoxin has also been suggested based on studies with purified endotoxin aggregates and blood monocytes. Because the interaction of BPI with cell-free endotoxin, during infection, occurs mainly in tissue and most likely in the form of shed bacterial outer membrane vesicles (“blebs”), we examined the effect of BPI on interactions of metabolically labeled ([14C]-acetate) blebs purified from Neisseria meningitidis serogroup B with either human monocyte-derived macrophages or monocyte-derived dendritic cells (MDDC). BPI produced a dose-dependent increase (up to 3-fold) in delivery of 14C-labeled blebs to MDDC, but not to monocyte-derived macrophages in the presence or absence of serum. Both, fluorescently labeled blebs and BPI were internalized by MDDC under these conditions. The closely related LPS-binding protein, in contrast to BPI, did not increase association of the blebs with MDDC. BPI-enhanced delivery of the blebs to MDDC did not increase cell activation but permitted CD14-dependent signaling by the blebs as measured by changes in MDDC morphology, surface expression of CD80, CD83, CD86, and MHC class II and secretion of IL-8, RANTES, and IP-10. These findings suggest a novel role of BPI in the interaction of bacterial outer membrane vesicles with dendritic cells that may help link innate immune recognition of endotoxin to Ag delivery and presentation.
IFN-γ-Producing Dendritic Cells Are Important for Priming of Gut Intraepithelial Lymphocyte Response Against Intracellular Parasitic Infection
Authors: Moretto Magali M. , Weiss Louis M. , Combe Crescent L. , Khan Imtiaz A.
Published: April 2014

The importance of intraepithelial lymphocytes (IEL) in immunoprotection against orally acquired pathogens is being increasingly recognized. Recent studies have demonstrated that Ag-specific IEL can be generated and can provide an important first line of defense against pathogens acquired via oral route. However, the mechanism involved in priming of IEL remains elusive. Our current study, using a microsporidial model of infection, demonstrates that priming of IEL is dependent on IFN-γ-producing dendritic cells (DC) from mucosal sites. DC from mice lacking the IFN-γ gene are unable to prime IEL, resulting in failure of these cells to proliferate and lyse pathogen-infected targets. Also, treatment of wild-type DC from Peyer’s patches with Ab to IFN-γ abrogates their ability to prime an IEL response against Encephalitozoon cuniculi in vitro. Moreover, when incubated with activated DC from IFN-γ knockout mice, splenic CD8+ T cells are not primed efficiently and exhibit reduced ability to home to the gut compartment. These data strongly suggest that IFN-γ-producing DC from mucosal sites play an important role in the generation of an Ag-specific IEL response in the small intestine. To our knowledge, this report is the first demonstrating a role for IFN-γ-producing DC from Peyer’s patches in the development of Ag-specific IEL population and their trafficking to the gut epithelium.
Lymphoma Immunotherapy with CpG Oligodeoxynucleotides Requires TLR9 Either in the Host or in the Tumor Itself
Authors: Li Jiali , Song Wenru , Czerwinski Debra K. , Varghese Bindu , Uematsu Satoshi , Akira Shizuo , Krieg Arthur M. , Levy Ronald
Published: April 2014

Established widely metastatic tumor was cured in a transplanted mouse B cell lymphoma model, by the combination of chemotherapy plus intratumoral injection of oligodeoxynucleotides containing unmethylated C-G motifs (CpG). This therapeutic effect required that the CpG be injected directly into the tumor and was dependent on CD8 T cells. Although the efficacy of CpG oligodeoxynucleotides has been thought to depend on the expression of TLR9, we unexpectedly found that tumor rejection did not require host expression of TLR9. By using a TLR9-deficient tumor and a TLR9KO host, we demonstrate that TLR9 expression either by the host or the tumor is required. These results indicate that activation of Ag presentation by cells within the tumor via TLR9 stimulation can be an effective form of immunotherapy. This study forms the basis of an ongoing clinical trial in patients with lymphoma.
C-Terminal Tail Phosphorylation of N-Formyl Peptide Receptor: Differential Recognition of Two Neutrophil Chemoattractant Receptors by Monoclonal Antibodies NFPR1 and NFPR2
Authors: Riesselman Marcia , Miettinen Heini M. , Gripentrog Jeannie M. , Lord Connie I. , Mumey Brendan , Dratz Edward A. , Stie Jamal , Taylor Ross M. , Jesaitis Algirdas J.
Published: April 2014

The N-formyl peptide receptor (FPR), a G protein-coupled receptor that binds proinflammatory chemoattractant peptides, serves as a model receptor for leukocyte chemotaxis. Recombinant histidine-tagged FPR (rHis-FPR) was purified in lysophosphatidyl glycerol (LPG) by Ni2+-NTA agarose chromatography to >95% purity with high yield. MALDI-TOF mass analysis (>36% sequence coverage) and immunoblotting confirmed the identity as FPR. The rHis-FPR served as an immunogen for the production of 2 mAbs, NFPR1 and NFPR2, that epitope map to the FPR C-terminal tail sequences, 305-GQDFRERLI-313 and 337-NSTLPSAEVE-346, respectively. Both mAbs specifically immunoblotted rHis-FPR and recombinant FPR (rFPR) expressed in Chinese hamster ovary cells. NFPR1 also recognized recombinant FPRL1, specifically expressed in mouse L fibroblasts. In human neutrophil membranes, both Abs labeled a 45–75 kDa species (peak Mr ∼60 kDa) localized primarily in the plasma membrane with a minor component in the lactoferrin-enriched intracellular fractions, consistent with FPR size and localization. NFPR1 also recognized a band of Mr ∼40 kDa localized, in equal proportions to the plasma membrane and lactoferrin-enriched fractions, consistent with FPRL1 size and localization. Only NFPR2 was capable of immunoprecipitation of rFPR in detergent extracts. The recognition of rFPR by NFPR2 is lost after exposure of cellular rFPR to f-Met-Leu-Phe (fMLF) and regained after alkaline phosphatase treatment of rFPR-bearing membranes. In neutrophils, NFPR2 immunofluorescence was lost upon fMLF stimulation. Immunoblotting ∼60 kDa species, after phosphatase treatment of fMLF-stimulated neutrophil membranes, was also enhanced. We conclude that the region 337–346 of FPR becomes phosphorylated after fMLF activation of rFPR-expressing Chinese hamster ovary cells and neutrophils.
The Cellular Immune Response to Mycobacterium tuberculosis Infection in the Guinea Pig
Authors: Ordway Diane , Palanisamy Gopinath , Henao-Tamayo Marcela , Smith Erin E. , Shanley Crystal , Orme Ian M. , Basaraba Randall J.
Published: April 2014

Pulmonary tuberculosis in guinea pigs is an extremely useful model for drug and vaccine testing due to the fact that its pathological disease process is similar to that present in humans. Progress in this field has been hindered because the tools necessary to undertake a complete immunological analysis of the guinea pig cellular immune response against Mycobacterium tuberculosis have been lacking. In this study, we combined a new flow cytometric gating strategy with immunohistochemistry to track T cells, B cells, and the MIL4 Ab, which detects both guinea pig heterophils (neutrophils) and eosinophils, to provide the first documentation of the kinetics of influx and positioning of these cell populations. The results show that the responding T cells are mostly CD4 cells and that after day 30 of the infection numbers of these cells in the lungs drops dramatically. These appear to be replaced by a steady increase in B cells and granulocytes which was associated with worsening lung pathology. These data reveal new information about the cellular phenotypes which mediate protective immunity or host immunopathogenesis during M. tuberculosis infection in this key animal model.
IL-1 Receptor Accessory Protein and ST2 Comprise the IL-33 Receptor Complex
Authors: Chackerian Alissa A. , Oldham Elizabeth R. , Murphy Erin E. , Schmitz Jochen , Pflanz Stefan , Kastelein Robert A.
Published: April 2014

IL-33 (IL-1F11) is a recently described member of the IL-1 family of cytokines that stimulates the generation of cells, cytokines, and Igs characteristic of a type 2 immune response. IL-33 mediates signal transduction through ST2, a receptor expressed on Th2 and mast cells. In this study, we demonstrate that IL-33 and ST2 form a complex with IL-1R accessory protein (IL-1RAcP), a signaling receptor subunit that is also a member of the IL-1R complex. Additionally, IL-1RAcP is required for IL-33-induced in vivo effects, and IL-33-mediated signal transduction can be inhibited by dominant-negative IL-1RAcP. The implications of this shared usage of IL-1RAcP by IL-1(α and β) and IL-33 are discussed.
Inhibition of Experimental Allergic Airways Disease by Local Application of a Cell-Penetrating Dominant-Negative STAT-6 Peptide
Authors: McCusker Christine T. , Wang Yufa , Shan Jichuan , Kinyanjui Margaret W. , Villeneuve Annie , Michael Husheem , Fixman Elizabeth D.
Published: April 2014

Allergic airways disease is initiated and perpetuated by an aberrant Th2 inflammatory response regulated in part by the cytokines IL-4 and IL-13, each of which induces activation of the STAT-6 transcription factor. Data from murine models indicate that the clinical manifestations of acute asthma are STAT-6 dependent, and thus, STAT-6 is a target for drug development in allergic airways disease. We designed a novel chimeric peptide (STAT-6 inhibitory peptide (STAT-6-IP)) comprised of a sequence predicted to bind to and inhibit STAT-6, fused to a protein transduction domain, to facilitate cellular uptake of the STAT-6-binding peptide. Our data demonstrate that the STAT-6-IP inhibited OVA-induced production of Th2 cytokines IL-4 and IL-13 in vitro. In contrast, the STAT-6-IP did not affect production of IFN-γ, demonstrating specificity for Th2 cytokine inhibition. Following intranasal administration, the STAT-6-IP was localized to epithelial cells in the airways. Finally, in in vivo murine models of allergic rhinitis and asthma, intranasal delivery of the STAT-6-IP inhibited OVA-induced lung inflammation and mucus production as well as accumulation of eosinophils and IL-13 in bronchoalveolar lavage fluid and OVA-dependent airway hyperresponsiveness. Together these data show that local application of cell-penetrating peptide inhibitors of STAT-6 has significant potential for the treatment of allergic rhinitis and asthma.
Platelet Factor 4 (CXC Chemokine Ligand 4) Differentially Regulates Respiratory Burst, Survival, and Cytokine Expression of Human Monocytes by Using Distinct Signaling Pathways
Authors: Kasper Brigitte , Brandt Ernst , Brandau Sven , Petersen Frank
Published: April 2014

Platelet factor 4 (PF4; CXCL4) is an abundant platelet α-granule CXC chemokine with unique functions. Although lacking a chemotactic activity, PF4 initiates a signal transduction cascade in human monocytes leading to the induction of a broad spectrum of acute and delayed functions including phagocytosis, respiratory burst, survival, and the secretion of cytokines. Surprisingly, although these monocyte functions are well defined, only very limited information exists on the specific signaling pathways that are involved in the regulation of these biological responses. By using specific inhibitors and direct phosphorylation/activation studies, we show in the present study that PF4-mediated respiratory burst is dependent on a very rapid activation of PI3K, Syk, and p38 MAPK. Moreover, monocyte survival and differentiation instead is controlled by a delayed activation of Erk, with an activity peak after 6 h of stimulation. The inhibition of Erk completely reverted PF4-mediated protection against apoptosis. Finally, even though JNK is rapidly activated in PF4-treated monocytes, it is dispensable for the regulation of survival and respiratory burst. However, PF4-induced up-regulation of chemokine and cytokine mRNA and protein requires a sustained activation of JNK and Erk. Taken together, PF4-stimulated immediate monocyte functions (oxygen radical formation) are regulated by p38 MAPK, Syk, and PI3K, whereas delayed functions (survival and cytokine expression) are controlled by Erk and JNK.
Properdin Can Initiate Complement Activation by Binding Specific Target Surfaces and Providing a Platform for De Novo Convertase Assembly
Authors: Spitzer Dirk , Mitchell Lynne M. , Atkinson John P. , Hourcade Dennis E.
Published: April 2014

Complement promotes the rapid recognition and elimination of pathogens, infected cells, and immune complexes. The biochemical basis for its target specificity is incompletely understood. In this report, we demonstrate that properdin can directly bind to microbial targets and provide a platform for the in situ assembly and function of the alternative pathway C3 convertases. This mechanism differs from the standard model wherein nascent C3b generated in the fluid phase attaches nonspecifically to its targets. Properdin-directed complement activation occurred on yeast cell walls (zymosan) and Neisseria gonorrhoeae. Properdin did not bind wild-type Escherichia coli, but it readily bound E. coli LPS mutants, and the properdin-binding capacity of each strain correlated with its respective serum-dependent AP activation rate. Moreover, properdin:single-chain Ab constructs were used to direct serum-dependent complement activation to novel targets. We conclude properdin participates in two distinct complement activation pathways: one that occurs by the standard model and one that proceeds by the properdin-directed model. The properdin-directed model is consistent with a proposal made by Pillemer and his colleagues >50 years ago.
Unchecked CD70 Expression on T Cells Lowers Threshold for T Cell Activation in Rheumatoid Arthritis
Authors: Lee Won-Woo , Yang Zhi-Zhang , Li Guangjin , Weyand Cornelia M. , Goronzy Jörg J.
Published: April 2014

Rheumatoid arthritis (RA) is characterized by premature immune aging with accumulation of degenerate T cells deficient for CD28. Gene expression profiling of CD4+CD28− and CD4+CD28+ T cells to discover disease-promoting activities of CD28− T cells identified expression of CD70 as a most striking difference. Hence, CD70 was significantly more expressed in CD4 T cells from RA patients compared with age-matched controls (p < 0.006). The underlying mechanism was a failure to repress CD70 expression after activation-dependent induction. This defect in RA was not related to differential promoter demethylation. CD70 on bystander CD4+CD28− T cells functioned by lowering the threshold for T cell activation; admixture of CD4+CD28− T cells augmented TCR-induced responses of autologous naive CD4+CD28+ T cells, particularly of low-avidity T cells. The data support a model in which CD70 expressed on T cells causes degeneracy in T cell responses and undermines tolerance mechanisms that normally control T cell autoreactivity.
Functional T Cell Responses to Tumor Antigens in Breast Cancer Patients Have a Distinct Phenotype and Cytokine Signature
Authors: Inokuma Margaret , dela Rosa Corazon , Schmitt Charles , Haaland Perry , Siebert Janet , Petry Douglas , Tang MengXiang , Suni Maria A. , Ghanekar Smita A. , Gladding Daiva , Dunne John F. , Maino Vernon C. , Disis Mary L. , Maecker Holden T.
Published: April 2014

The overall prevalence with which endogenous tumor Ags induce host T cell responses is unclear. Even when such responses are detected, they do not usually result in spontaneous remission of the cancer. We hypothesized that this might be associated with a predominant phenotype and/or cytokine profile of tumor-specific responses that is different from protective T cell responses to other chronic Ags, such as CMV. We detected significant T cell responses to CEA, HER-2/neu, and/or MAGE-A3 in 17 of 21 breast cancer patients naive to immunotherapy. The pattern of T cell cytokines produced in response to tumor-associated Ags (TAAs) in breast cancer patients was significantly different from that produced in response to CMV or influenza in the same patients. Specifically, there was a higher proportion of IL-2-producing CD8+ T cells, and a lower proportion of IFN-γ-producing CD4+ and/or CD8+ T cells responding to TAAs compared with CMV or influenza Ags. Finally, the phenotype of TAA-responsive CD8+ T cells in breast cancer patients was almost completely CD28+CD45RA− (memory phenotype). CMV-responsive CD8+ T cells in the same patients were broadly distributed among phenotypes, and contained a high proportion of terminal effector cells (CD27−CD28−CD45RA+) that were absent in the TAA responses. Taken together, these results suggest that TAA-responsive T cells are induced in breast cancer patients, but those T cells are phenotypically and functionally different from CMV- or influenza-responsive T cells. Immunotherapies directed against TAAs may need to alter these T cell signatures to be effective.
Neutrophils as a Novel Source of Eosinophil Cationic Protein in IgE-Mediated Processes
Authors: Monteseirín Javier , Vega Antonio , Chacón Pedro , Camacho M. Jesús , El Bekay Rajaa , Asturias Juan A. , Martínez Alberto , Guardia Pedro , Pérez-Cano Ramón , Conde José
Published: April 2014

The production of eosinophil cationic protein (ECP) in IgE-mediated diseases has been associated mainly with eosinophils, although no IgE-dependent ECP release has been observed in these cells. Because there is increasing evidence of neutrophil participation in allergic processes, we have examined whether human neutrophils from allergic patients were able to produce ECP by an IgE-dependent mechanism. After challenge with specific Ags to which the patients were sensitized, ECP release was detected in the culture medium. Furthermore, intracellular protein was detected by flow cytometry, immunofluorescence staining, and Western blotting. Expression at both mRNA and de novo protein synthesis were detected, respectively, by RT-PCR and radiolabeling with 35S. Ag effect was mimicked by cell treatment with anti-IgE Abs or Abs against FcεRI and galectin-3 (FcεRI>galectin-3), but not against FcεRII. These observations represent a novel view of neutrophils as possible source of ECP in IgE-dependent diseases.
The Multitasking Mast Cell: Positive and Negative Roles in the Progression of Autoimmunity
Authors: Christy Alison L. , Brown Melissa A.
Published: April 2014

Among the potential outcomes of an aberrantly functioning immune system are allergic disease and autoimmunity. Although it has been assumed that the underlying mechanisms mediating these conditions are completely different, recent evidence shows that mast cells provide a common link. Mast cells reside in most tissues, are particularly prevalent at sites of Ag entry, and act as sentinel cells of the immune system. They express many inflammatory mediators that affect both innate and adaptive cellular function. They contribute to pathologic allergic inflammation but also serve an important protective role in bacterial and parasite infections. Given the proinflammatory nature of autoimmune responses, it is not surprising that studies using murine models of autoimmunity clearly implicate mast cells in the initiation and/or progression of autoimmune disease. In this review, we discuss the defined and hypothesized mechanisms of mast cell influence on autoimmune diseases, including their surprising and newly discovered role as anti-inflammatory cells.
PDGF Synergistically Enhances IFN-γ-Induced Expression of CXCL10 in Blood-Derived Macrophages: Implications for HIV Dementia
Authors: Dhillon Navneet Kaur , Peng Fuwang , Ransohoff Richard M. , Buch Shilpa
Published: April 2014

There is increasing cumulative evidence that activated mononuclear phagocytes (macrophages/microglia) releasing inflammatory mediators in the CNS are a better correlate of HIV-associated dementia (HAD) than the actual viral load in the brain. Earlier studies on simian HIV/rhesus macaque model of NeuroAIDS confirmed that pathological changes in brains of macaques with encephalitis were associated with up-regulation of platelet-derived growth factor (PDGF) and the chemokine, CXCL10. Because the complex interplay of inflammatory mediators released by macrophages often leads to the induction of neurotoxins in HAD, we hypothesized that PDGF could interact with IFN-γ to modulate the expression of CXCL10 in these primary virus target cells. Although PDGF alone had no effect on the induction of CXCL10 in human macrophages, in conjunction with IFN-γ, it significantly augmented the expression of CXCL10 RNA & protein through transcriptional and posttranscriptional mechanisms. Signaling molecules, such as JAK and STATs, PI3K, MAPK, and NF-κB were found to play a role in the synergistic induction of CXCL10. Furthermore, PDGF via its activation of p38 MAPK was able to increase the stability of IFN-γ-induced CXCL10 mRNA. Understanding the mechanisms involved in the synergistic up-regulation of CXCL10 could aid in the development of therapeutic modalities for HAD.
Novel Exosome-Targeted CD4+ T Cell Vaccine Counteracting CD4+25+ Regulatory T Cell-Mediated Immune Suppression and Stimulating Efficient Central Memory CD8+ CTL Responses
Authors: Hao Siguo , Liu Yongqing , Yuan Jinying , Zhang Xueshu , He Tianpei , Wu Xiaochu , Wei Yangdou , Sun Deming , Xiang Jim
Published: April 2014

T cell-to-T cell Ag presentation is increasingly attracting attention. In this study, we demonstrated that active CD4+ T (aT) cells with uptake of OVA-pulsed dendritic cell-derived exosome (EXOOVA) express exosomal peptide/MHC class I and costimulatory molecules. These EXOOVA-uptaken (targeted) CD4+ aT cells can stimulate CD8+ T cell proliferation and differentiation into central memory CD8+ CTLs and induce more efficient in vivo antitumor immunity and long-term CD8+ T cell memory responses than OVA-pulsed dendritic cells. They can also counteract CD4+25+ regulatory T cell-mediated suppression of in vitro CD8+ T cell proliferation and in vivo CD8+ CTL responses and antitumor immunity. We further elucidate that the EXOOVA-uptaken (targeted)CD4+ aT cell’s stimulatory effect is mediated via its IL-2 secretion and acquired exosomal CD80 costimulation and is specifically delivered to CD8+ T cells in vivo via acquired exosomal peptide/MHC class I complexes. Therefore, EXO-targeted active CD4+ T cell vaccine may represent a novel and highly effective vaccine strategy for inducing immune responses against not only tumors, but also other infectious diseases.