bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulatory CD4 T Cells Control the Size of the Peripheral Activated/Memory CD4 T Cell Compartment
Authors: Annacker Oliver , Burlen-Defranoux Odile , Pimenta-Araujo Ricardo , Cumano Ana , Bandeira Antonio
Published: April 2014

The mechanisms leading to stable T cell numbers in the periphery of a healthy animal are, to date, not well understood. We followed the expansion of CD45RBhigh (naive) and CD45RBlow (activated/memory) CD4 T cells transferred from normal mice into syngeneic Rag-20/0 recipients and the dynamics of peripheral reconstitution when both populations were coinjected. Naive cells acquired an activated phenotype and showed a high proliferative capacity that was dependent on the environment in which the recipients were kept (specific pathogen-free vs conventional housing conditions), the age of the recipients, and the presence of CD45RBlow T cells in the injected cohort. CD45RBlow CD4 T cells protected the host from CD45RBhigh CD4 T cell-induced inflammatory bowel disease and showed a limited degree of expansion. CD45RBlow CD4 T cells isolated from GF mice also showed the ability to prevent inflammatory bowel disease, indicating that at least part of the natural regulatory T cells are self-reactive. The results indicate that 1) peripheral T cell expansion in lymphocyte-deficient recipients represent classical immune responses, which are mainly promoted by exogenous Ags and 2) natural regulatory T cells control the size of the activated/memory peripheral CD4 T cell compartment.
Targeting Rare Populations of Murine Antigen-Specific T Lymphocytes by Retroviral Transduction for Potential Application in Gene Therapy for Autoimmune Disease
Authors: Costa Gina L. , Benson Jacqueline M. , Seroogy Christine M. , Achacoso Philip , Fathman C. Garrison , Nolan Garry P.
Published: April 2014

CD4+ T cells are important mediators in the pathogenesis of autoimmunity and would therefore provide ideal candidates for lymphocyte-based gene therapy. However, the number of Ag-specific T cells in any single lesion of autoimmunity may be quite low. Successful gene transfer into autoantigen-specific CD4+ T cells would serve as an ideal vehicle for site-targeted gene therapy if it were possible to transduce preferentially the small number of autoantigen-specific T cells. In this study we have demonstrated that retroviral infection of CD4+ lymphocytes from either autoantigen-stimulated TCR transgenic mice, or Ag-activated immunized nontransgenic mice, with a retroviral vector (pGCIRES), resulted in the transduction of only the limited number of Ag-reactive CD4+ T cells. In contrast, polyclonal activation of the same cultures resulted in transduction of non-antigen-specific lymphocytes. Transduction of Ag-reactive CD4+ T cells with pGCIRES retrovirus encoding the regulatory genes IL-4 (IL4) and soluble TNF receptor (STNFR) resulted in stable integration and long-term expression of recombinant gene products. Moreover, expression of the pGCIRES marker protein, GFP, directly correlated with the expression of the upstream regulatory gene. Retroviral transduction of CD4+ T cells targeted specifically Ag-reactive cells and was cell cycle-dependent and evident only during the mitosis phase. These studies suggest that retroviral transduction of autoantigen-specific murine CD4+ T cells, using the pGCIRES retroviral vector, may provide a potential method to target and isolate the low frequency of autoantigen-specific murine CD4+ T cells, and provides a rational approach to gene therapy in animal models of autoimmunity.
Coordinate Regulation of T Cell Activation by CD2 and CD28
Authors: Green Jonathan M. , Karpitskiy Vladimir , Kimzey Stephanie L. , Shaw Andrey S.
Published: April 2014

T cell activation requires co-engagement of the TCR with accessory and costimulatory molecules. However, the exact mechanism of costimulatory function is unknown. Mice lacking CD2 or CD28 show only mild deficits, demonstrating that neither protein is essential for T cell activation. In this paper we have generated mice lacking both CD2 and CD28. T cells from the double-deficient mice have a profound defect in activation by soluble anti-CD3 Ab and Ag, yet remain responsive to immobilized anti-CD3. This suggests that CD2 and CD28 may function together to facilitate interactions of the T cell and APC, allowing for efficient signal transduction through the TCR.
Indoleamine 2,3-Dioxygenase Production by Human Dendritic Cells Results in the Inhibition of T Cell Proliferation
Authors: Hwu Patrick , Du Mark X. , Lapointe Réjean , Do My , Taylor Milton W. , Young Howard A.
Published: April 2014

Dendritic cells (DCs) play a key role in the activation and regulation of B and T lymphocytes. Production of indoleamine 2,3-dioxygenase (IDO) by macrophages has recently been described to result in inhibition of T cell proliferation through tryptophan degradation. Since DCs can be derived from monocytes, we sought to determine whether DCs could produce IDO which could potentially regulate T cell proliferation. Northern blot analysis of RNA from cultured monocyte-derived human DC revealed that IDO mRNA was induced upon activation with CD40 ligand and IFN-γ. IDO produced from activated DCs was functionally active and capable of metabolizing tryptophan to kynurenine. Activated T cells were also capable of inducing IDO production by DCs, which was inhibited by a neutralizing Ab against IFN-γ. DC production of IDO resulted in inhibition of T cell proliferation, which could be prevented using the IDO inhibitor 1-methyl-dl-tryptophan. These results suggest that activation of DCs induces the production of functional IDO, which causes depletion of tryptophan and subsequent inhibition of T cell proliferation. This may represent a potential mechanism for DCs to regulate the immune response.
Large-Scale Culture and Selective Maturation of Human Langerhans Cells from Granulocyte Colony-Stimulating Factor-Mobilized CD34+ Progenitors
Authors: Gatti Evelina , Velleca Mark A. , Biedermann Barbara C. , Ma Weilie , Unternaehrer Juli , Ebersold Melanie W. , Medzhitov Ruslan , Pober Jordan S. , Mellman Ira
Published: April 2014

Dendritic cells (DCs) play a critical role as APCs in the induction of the primary immune response. Their capacity for Ag processing and presentation is tightly regulated, controlled by a terminal developmental sequence accompanied by striking changes in morphology, organization, and function. The maturation process, which converts DCs from cells adapted for Ag accumulation to cells adapted for T cell stimulation, remains poorly understood due in part to difficulties in the culture and manipulation of DCs of defined lineages. To address these issues, we have devised conditions for the culture of a single DC type, Langerhans cells (LCs), using CD34+ cells from G-CSF-mobilized patients. Homogenous populations of LCs, replete with abundant immunocytochemically demonstrable Birbeck granules, could be stably maintained as immature DCs for long periods in culture. Unlike other human DC preparations, the LCs remained fully differentiated after cytokine removal. Following exposure to TNF-α, LPS, or CD40 ligand, the LCs could be synchronously induced to mature. Depending on the agent used, distinct types of LCs emerged differing in their capacity for T cell stimulation, IL-12 production, intracellular localization of MHC products, and overall morphology. Most interestingly, the expression of different sets of Toll family receptors is induced or down-regulated according to the maturation stimulus provided. These results strongly suggest that different proinflammatory stimuli might drive distinct developmental events.
IL-15 Is Highly Expressed in Inflammatory Bowel Disease and Regulates Local T Cell-Dependent Cytokine Production
Authors: Liu Zhanju , Geboes Karel , Colpaert Stefaan , D’Haens Geert R. , Rutgeerts Paul , Ceuppens Jan L.
Published: April 2014

IL-15 shares biological activities but no significant sequence homology with IL-2. It induces T cell recruitment to sites of inflammation, T cell proliferation, and cytokine production and rescue from apoptosis. The aim of this study was to investigate expression of IL-15 and its effects on proinflammatory cytokine production in inflammatory bowel disease (IBD). Immunohistochemistry demonstrated local IL-15 production by macrophages in inflamed mucosa from IBD patients. Isolated lamina propria mononuclear cells from these patients but not from controls produced IL-15 when stimulated with LPS or IFN-γ. Moreover, lamina propria T cells (LP-T) from IBD patients were more responsive to IL-15 as compared with controls, and IL-15 alone without a primary T cell stimulus induced IFN-γ and TNF production by isolated IBD LP-T cells, especially by LP-T cells from patients with Crohn’s disease. LP-T cells from IBD patients could induce CD40-CD40 ligand (CD40L) interaction-dependent TNF and IL-12 production by monocytes in a coculture system. This capacity of LP-T cells was strongly enhanced by preincubation in IL-15 and was the result of higher CD40L expression after culture in IL-15. These data indicate that IL-15 is overexpressed in the inflamed mucosa in IBD and that IL-15 enhances local T cell activation, proliferation, and proinflammatory cytokine production by both T cells and macrophages, the latter via a CD40-CD40L interaction-dependent mechanism. Treatment directed against IL-15 may have therapeutic potential in IBD.
Role of Gut Cryptopatches in Early Extrathymic Maturation of Intestinal Intraepithelial T Cells
Authors: Oida Takatoku , Suzuki Kenji , Nanno Masanobu , Kanamori Yutaka , Saito Hisashi , Kubota Eiro , Kato Shingo , Itoh Mamoru , Kaminogawa Shuichi , Ishikawa Hiromichi
Published: April 2014

Lympho-hemopoietic progenitors residing in murine gut cryptopatches (CP) have been shown to generate intestinal intraepithelial T cells (IEL). To investigate the role of CP in progenitor maturation, we analyzed IEL in male mice with a truncated mutation of common cytokine receptor γ-chain (CRγ−/Y) in which CP were undetectable. IEL-expressing TCR-γδ (γδ-IEL) were absent, and a drastically reduced number of Thy-1highCD4+ and Thy-1highCD8αβ+ αβ-IEL were present in CRγ−/Y mice, whereas these αβ-IEL disappeared from athymic CRγ−/Y littermate mice. Athymic CRγ−/Y mice possessed a small TCR- and αEβ7 integrin-negative IEL population, characterized by the disappearance of the extrathymic CD8αα+ subset, that expressed pre-Tα, RAG-2, and TCR-Cβ but not CD3ε transcripts. These TCR− IEL from athymic CRγ−/Y mice did not undergo Dβ-Jβ and Vδ-Jδ joinings, despite normal rearrangements at the TCR-β and -δ loci in thymocytes from euthymic CRγ−/Y mice. In contrast, athymic severe combined immunodeficient mice in which CP developed normally possessed two major TCR−αEβ7+ CD8αα+ and CD8− IEL populations that expressed pre-Tα, RAG-2, TCR-Cβ, and CD3ε transcripts. These findings underscore the role of gut CP in the early extrathymic maturation of CD8αα+ IEL, including cell-surface expression of αEβ7 integrin, CD3ε gene transcription, and TCR gene rearrangements.
Anti-LFA-1 Therapy Induces Long-Term Islet Allograft Acceptance in the Absence of IFN-γ or IL-4
Authors: Nicolls Mark R. , Coulombe Marilyne , Yang Huan , Bolwerk Amy , Gill Ronald G.
Published: April 2014

mAb therapy directed against a variety of cell surface accessory molecules has been effectively utilized to prolong allograft acceptance in various models of tissue and organ transplantation. The purpose of this study was to determine whether transient therapy directed against the adhesion molecule LFA-1 (CD11a) was sufficient to induce donor-specific tolerance to pancreatic islet allografts. Anti-LFA-1 monotherapy was found to be efficacious in inducing long-term islet allograft acceptance in multiple donor-recipient strain combinations. Graft acceptance following anti-LFA-1 therapy was not simply due to clonal ignorance of donor Ags in that the majority of recipients bearing established islet allografts resisted rejection induced by immunization with donor-type APCs. Furthermore, donor-specific tolerance from anti-LFA-1-treated animals could be transferred to secondary immune-deficient animals. Taken together, these results indicated that transient anti-LFA-1 monotherapy resulted in donor-specific tolerance. In vitro, functionally tolerant animals retained normal anti-donor reactivity as assessed by proliferative, cytotoxic, and cytokine release assays that demonstrated that tolerance was not secondary to general clonal deletion or anergy of donor-reactive T cells. Finally, anti-LFA-1 treatment was effective in both IL-4-deficient and IFN-γ-deficient recipients, indicating that neither of these cytokines are universally required for allograft acceptance. These results suggest that anti-adhesion-based therapy can induce a nondeletional form of tolerance that is not overtly dependent on the prototypic Th1 and Th2 cytokines, IFN-γ and IL-4, respectively, in contrast to results in other transplantation models.
Analysis of Signals and Functions of the Chimeric Human Granuloctye-Macrophage Colony-Stimulating Factor Receptor in BA/F3 Cells and Transgenic Mice
Authors: Watanabe Sumiko , Aoki Yutaka , Nishijima Ichiko , Xu Ming-jiang , Arai Ken-ichi
Published: April 2014

Receptors for GM-CSF, IL-3, and IL-5 are composed of two subunits: α, which is specific for each cytokine, and βc, which is shared by all. Although the role of βc in signal transduction has been extensively studied, the role of the α subunit has remained to be clarified. To analyze the role of the human (h) GM-CSF receptor α subunit, we constructed a chimeric receptor subunit composed of extracellular and transmembrane regions of α fused with the cytoplasmic region of βc, designated α/β. In BA/F3 cells, chimeric receptor composed of α/β,β can transduce signals for mitogen-activated protein kinase cascade activation and proliferation in response to hGM-CSF. Although phosphorylation of Jak1 but not of Jak2 occurred with stimulation of hGM-CSF, the dominant-negative Jak2 but not the dominant-negative Jak1 suppresses c-fos promoter activation. To determine whether the chimeric receptor α/β,β is functional in vivo, we developed transgenic mice expressing the chimeric receptor α/β,β. Bone marrow cells from the transgenic mice expressing the α/β,β receptor form not only GM colonies but also various lineages of colonies in response to GM-CSF. In addition, mast cells were produced when bone marrow cells of the transgenic mouse were cultured with hGM-CSF. Thus, it appears that the cytoplasmic region of the α subunit is not required for hGM-CSF promoting activities, even in bone marrow cells.
Crucial Role of TNF-α in CD8 T Cell-Mediated Elimination of 3LL-A9 Lewis Lung Carcinoma Cells In Vivo
Authors: Prévost-Blondel Armelle , Roth Evelyn , Rosenthal Felicia M. , Pircher Hanspeter
Published: April 2014

The role of perforin, IFN-γ, and TNF-α in anti-tumor CD8 T cell immunity was examined in a new tumor model using a CD8 T cell epitope (GP33) derived from lymphocytic choriomeningitis virus as a tumor-associated Ag. In contrast with parental 3LL-A9 (A9) Lewis lung carcinoma cells that progressively grow in C57BL/6 mice, s.c. injection of GP33-transfected A9GP33 tumor cells induced a protective GP33-specific CD8 T cell response that led to complete tumor cell elimination. Tumor regression was dependent on perforin, IFN-γ, or TNF-α, because A9GP33 tumors developed in mice deficient in one of these genes. A9GP33 tumors arising in perforin- and IFN-γ-deficient mice represented GP33 Ag-loss variants, demonstrating that GP33-specific CD8 T cells from these mice were able to exert an Ag selection pressure. In contrast, tumor cells growing in TNF-α knock-out mice still expressed the tumor-associated GP33 peptide despite the presence of activated GP33-specific CD8 T cells. These findings provide evidence for a crucial role of TNF-α in A9 tumor cell elimination by CD8 T cells in vivo.
SHPS-1 Induces Aggregation of Ba/F3 Pro-B Cells Via an Interaction with CD47
Authors: Babic Ivan , Schallhorn Annette , Lindberg Frederik P. , Jirik Frank R.
Published: April 2014

SHPS-1 (SH2-domain bearing protein tyrosine phosphatase (SHP) substrate-1), a member of the inhibitory-receptor superfamily that is abundantly expressed in macrophages and neural tissue, appears to regulate intracellular signaling events downstream of receptor protein-tyrosine kinases and integrin-extracellular matrix molecule interactions. To investigate the function of SHPS-1 in a hematopoietic cell line, SHPS-1 was expressed in Ba/F3 cells, an IL-3-dependent pro-B-cell line that lacks endogenous SHPS-1 protein. Interestingly, expression of either SHPS-1, or a mutant lacking the intracellular domain of SHPS-1 (ΔCT SHPS-1), resulted in the rapid formation of macroscopic Ba/F3 cell aggregates. As the integrin-associated protein/CD47 was shown to be a SHPS-1 ligand in neural cells, we investigated whether CD47 played a role in the aggregation of SHPS-1-expressing Ba/F3 cells. In support of this idea, aggregate formation was inhibited by an anti-CD47 Ab. Furthermore, erythrocytes from control, but not from CD47-deficient mice, were able to form rosettes on SHPS-1-expressing Ba/F3 cells. Because erythrocytes do not express integrins, this result suggested that SHPS-1-CD47 interactions can take place in the absence of a CD47-integrin association. We also present evidence that the amino-terminal Ig domain of SHPS-1 mediates the interaction with CD47. Although SHPS-1-CD47 binding likely triggers bidirectional intracellular signaling processes, these results demonstrate that this interaction can also mediate cell-cell adhesion.
Correct Immunoglobulin α mRNA Processing Depends on Specific Sequence in the Cα3-αM Intron
Authors: Coyle John H. , Lebman Deborah A.
Published: April 2014

The maturation of IgM-expressing B cells to IgM-secreting plasma cells is associated with both an increase in μ mRNA and the ratio of secreted to membrane forms of μ mRNA which differ at the 3′ termini. In contrast, both in vitro and in vivo the secreted form of α mRNA is predominant at all stages in the development of a secretory IgA response. Previous studies demonstrated that preferential usage of the αs poly(A) site does not result from transcription termination and is independent of either the poly(A) sites or the 3′ splice site associated with the exon encoding the membrane exon of IgA (αM). The present study demonstrates that a 349-bp region located 774 bp 3′ to the αs poly(A) site is required for the preferential usage of the αs terminus. This region, which is the first isotype-specific cis-acting regulatory sequence not immediately adjacent to a secretory poly(A) site to be identified, contains regulatory elements that increase the efficiency of polyadenylation/cleavage. A ubiquitous, ∼58-kDa RNA-binding protein interacts specifically with this regulatory region. These studies support the premise that cis-acting elements unique to each CH gene can impinge upon a common mechanism regulating Ig mRNA processing.
Novel Mutations Within the RFX-B Gene and Partial Rescue of MHC and Related Genes Through Exogenous Class II Transactivator in RFX-B-Deficient Cells
Authors: Nagarajan Uma M. , Peijnenburg Ad , Gobin Sam J. P. , Boss Jeremy M. , van den Elsen Peter J.
Published: April 2014

MHC class II deficiency or bare lymphocyte syndrome is a severe combined immunodeficiency caused by defects in MHC-specific regulatory factors. Fibroblasts derived from two recently identified bare lymphocyte syndrome patients, EBA and FZA, were found to contain novel mutations in the RFX-B gene. RFX-B encodes a component of the RFX transcription factor that functions in the assembly of multiple transcription factors on MHC class II promoters. Unlike RFX5- and RFXAP-deficient cells, transfection of exogenous class II transactivator (CIITA) into these RFX-B-deficient fibroblasts resulted in the induction of HLA-DR and HLA-DP and, to a lesser extent, HLA-DQ. Similarly, CIITA-mediated induction of MHC class I, β2-microglobulin, and invariant chain genes was also found in these RFX-B-deficient fibroblasts. Expression of wild-type RFX-B completely reverted the noted deficiencies in these cells. Transfection of CIITA into Ramia cells, a B cell line that does not produce a stable RFX-B mRNA, resulted in induction of an MHC class II reporter, suggesting that CIITA overexpression may partially override the RFX-B defect.
p53-Dependent and -Independent Pathways of Apoptotic Cell Death in Sepsis
Authors: Hotchkiss Richard S. , Tinsley Kevin W. , Hui Jia-Ji , Chang Katherine C. , Swanson Paul E. , Drewry Anne M. , Buchman Timothy G. , Karl Irene E.
Published: April 2014

Sepsis induces extensive apoptosis of lymphocytes, which may be responsible for the profound immune suppression of the disorder. Two potential pathways of sepsis-induced lymphocyte apoptosis, Fas and p53, were investigated. Lymphocyte apoptosis was evaluated 20–22 h after sepsis by annexin V or DNA nick-end labeling. Fas receptor-deficient mice had no protection against sepsis-induced apoptosis in thymocytes or splenocytes. p53 knockout mice (p53−/−) had complete protection against thymocyte apoptosis but, surprisingly, had no protection in splenocytes. p53−/− mice had no improvement in sepsis survival compared with appropriately matched control mice with sepsis. We conclude that both p53-dependent and p53-independent pathways of cell death exist in sepsis. This differential apoptotic response of thymocytes vs splenocytes in p53−/− mice suggests that either the cellular response or the death-inducing signal is cell-type specific in sepsis. The fact that p53−/− lymphocytes of an identical subtype (CD8−CD4+) were protected in thymi but not in spleens indicates that cell susceptibility to apoptosis differs depending upon other unidentified factors.
B Cell-Deficient Mice Are Highly Resistant to Leishmania donovani Infection, but Develop Neutrophil-Mediated Tissue Pathology
Authors: Smelt Sara C. , Cotterell Sara E. J. , Engwerda Christian R. , Kaye Paul M.
Published: April 2014

Resolution of Leishmania infection is T cell-dependent, and B lymphocytes have been considered to play a minimal role in host defense. In this study, the contribution of B lymphocytes to the response against Leishmania donovani was investigated using genetically modified IgM transmembrane domain (μMT) mutant mice, which lack mature B lymphocytes. When compared with wild-type mice, μMT mice cleared parasites more rapidly from the liver, and infection failed to establish in the spleen. The rapid clearance of parasites in μMT mice was associated with accelerated and more extensive hepatic granuloma formation compared with wild-type mice. However, the liver of infected μMT mice also showed signs of destructive pathology, associated with the presence of increased numbers of neutrophils. The role of neutrophils in controlling parasite growth in the viscera was determined by depletion with the mAb RB6-8C5. This treatment led to a dramatic enhancement of parasite growth in both the liver and spleen of μMT and wild-type mice. As assessed by transfer of both normal and chronic-infection serum, Ig protects μMT mice from destructive hepatic pathology, but minimally alters their resistance compared with wild-type mice. However, adoptive transfer of CD4+ and CD8+ T cells into recombinase activating gene 1 (RAG1−/−) recipients, suggested that T cell function was not altered by maturation in a B cell-deficient environment. Taken together, these data suggest an inhibitory role for B lymphocytes in resistance to L. donovani unrelated to the presence or absence of Ig. However, Ig protects μMT mice from the exaggerated pathology that occurs during infection.
Role of CD40 Ligand and CD28 in Induction and Maintenance of Antiviral CD8+ Effector T Cell Responses
Authors: Andreasen Susanne Ørding , Christensen Jeanette Erbo , Marker Ole , Thomsen Allan Randrup
Published: April 2014

The primary aim of this report was to evaluate the immune responses of CD40 ligand-deficient (CD40L−/−) mice infected with two viruses known to differ markedly in their capacity to replicate in the host. Lymphocytic choriomeningitis virus (LCMV) is a natural mouse pathogen that replicates widely and extensively, whereas vesicular stomatitis virus (VSV) spreads poorly. We found that the primary response of CD40L−/− mice toward VSV is significantly impaired; proliferation of both CD4+ and CD8+ cells is reduced 2- to 3-fold, few CD8+ cells acquire an activated phenotype, and little functional activity is induced. Very similar results were obtained in VSV-infected, CD28-deficient mice. In contrast, neither CD40L nor CD28 was required for induction of a primary CD8+ response toward LCMV. Surprisingly, lack of CD4+ T cells had no impact on the primary immune response toward any of the viruses, even though the CD40 ligand dependence demonstrated for VSV would be expected to be associated with CD4 dependence. Upon coinfection of VSV-infected mice with LCMV, the requirement for CD40 ligand (but not CD28) could be partially bypassed, as evidenced by a 3-fold increase in the frequency of VSV-specific CD8+ T cells on day 6 postinfection. Finally, despite the fact that the primary LCMV-specific CD8+ response is virtually unimpaired in CD40L−/− mice, their capacity to maintain CD8+ effector activity and to permanently control the infection is significantly reduced. Thus, our results demonstrate that the importance of CD40/CD40L interaction for activation of CD8+ T cells varies between viruses and over time.
Neonatal Administration of IL-12 Enhances the Protective Efficacy of Antiviral Vaccines
Authors: Arulanandam Bernard P. , Mittler James N. , Lee William T. , O’Toole Margot , Metzger Dennis W.
Published: April 2014

Neonates are highly susceptible to infectious agents and are known to display polarized expression of Th2-like cytokines and Abs. This neonatal immune bias has important implications for the development of vaccine strategies, particularly against viral infections. We now report that coadministration of IL-12 and an influenza subunit vaccine at birth enhances the protective efficacy of antiviral vaccination. Immunization and treatment with IL-12 within 24 h of birth resulted in elevated expression of IFN-γ, IL-10, and IL-15 mRNA in the spleens of newborn mice compared with animals exposed to vaccine only. In addition, these animals showed dramatic increases in IFN-γ-, IL-2-, and IL-4-secreting cells, and in IgG2a Ab levels upon adult challenge compared with mice primed with vaccine alone. Most importantly, animals vaccinated and simultaneously treated with IL-12 at birth displayed enhanced survival after lethal challenge with infectious influenza virus as adults compared with infected animals that had been primed with vaccine alone. This augmented protection required B cells and could be transferred to naive mice by immune serum. Collectively, these results provide evidence that administration of IL-12 to neonates induces a Th1-like response in newborns and elicits protective antiviral immune memory.
Redirected Perforin-Dependent Lysis of Colon Carcinoma by Ex Vivo Genetically Engineered CTL
Authors: Darcy Phillip K. , Haynes Nicole M. , Snook Marie B. , Trapani Joseph A. , Cerruti Loretta , Jane Stephen M. , Smyth Mark J.
Published: April 2014

The redirection of autologous lymphocytes to predefined tumor target Ags has considerable potential for the immunotherapeutic treatment of cancer; however, robust experimental systems for comparing various approaches have not been developed. Herein, we have generated a single chain variable domain anti-carcinoembryonic Ag (CEA) Fcε receptor I γ-chain fusion (scFv anti-CEA) receptor and demonstrated high-level expression of this chimeric receptor in naive mouse T lymphocytes by retroviral gene transduction. These gene-modified CTL were able to lyse CEA+ targets and secrete high levels of IFN-γ following Ag stimulation. Depletion studies demonstrated that specific tumor cell cytotoxicity was mediated by gene-modified CD8+ T cells. Importantly, in increasingly stringent tests of efficacy in vivo, transduced CTL were sequentially shown to reject CEA+ colon carcinoma cells in a Winn assay and then reject established s.c. colon carcinoma in scid or syngeneic mice. Furthermore, using gene-targeted and scFv anti-CEA receptor-transduced donor CTL, perforin and IFN-γ were demonstrated to be absolutely critical for the eradication of colon carcinoma in mice. In summary, we have developed a highly efficient gene transfer system for evaluating chimeric receptor expression in cytotoxic lymphocytes. This series of experiments has revealed the utility of scFv anti-CEA chimeras in providing mouse T cells the capacity to reject colon carcinoma in an Ag- and perforin-specific manner.
Survival of Staphylococcus aureus Inside Neutrophils Contributes to Infection
Authors: Gresham Hattie D. , Lowrance Jon H. , Caver Tony E. , Wilson Bridget S. , Cheung Ambrose L. , Lindberg Frederik P.
Published: April 2014

Neutrophils have long been regarded as essential for host defense against Staphylococcus aureus infection. However, survival of the pathogen inside various cells, including phagocytes, has been proposed as a mechanism for persistence of this microorganism in certain infections. Therefore, we investigated whether survival of the pathogen inside polymorphonuclear neutrophils (PMN) contributes to the pathogenesis of S. aureus infection. Our data demonstrate that PMN isolated from the site of infection contain viable intracellular organisms and that these infected PMN are sufficient to establish infection in a naive animal. In addition, we show that limiting, but not ablating, PMN migration into the site of infection enhances host defense and that repletion of PMN, as well as promoting PMN influx by CXC chemokine administration, leads to decreased survival of the mice and an increased bacterial burden. Moreover, a global regulator mutant of S. aureus (sar−) that lacks the expression of several virulence factors is less able to survive and/or avoid clearance in the presence of PMN. These data suggest that the ability of S. aureus to exploit the inflammatory response of the host by surviving inside PMN is a virulence mechanism for this pathogen and that modulation of the inflammatory response is sufficient to significantly alter morbidity and mortality induced by S. aureus infection.
Direct Visualization of Cytokine-Producing Recall Antigen-Specific CD4 Memory T Cells in Healthy Individuals and HIV Patients
Authors: Helms Thomas , Boehm Bernhard O. , Asaad Robert J. , Trezza R. P. , Lehmann Paul V. , Tary-Lehmann Magdalena
Published: April 2014

We have used computer-assisted cytokine ELISA spot analysis to measure the frequencies, the type of cytokine, and the amount of cytokine produced by individual recall Ag-specific CD4 memory cells in freshly isolated blood. We studied the memory cells specific for tetanus toxoid and purified protein derivative in 18 healthy individuals and in 22 HIV-infected patients on highly active antiretroviral therapy (HAART). In healthy individuals, the frequency, cytokine signature, and cytokine production per cell of these memory cells were stable over time. Although it is presently unclear whether the maintenance of the memory T cell pool depends upon Ag persistence, cross-reactive Ag stimulation, or cytokine-driven bystander stimulations and expansions, our data strongly argue for a stable memory cell pool in healthy individuals. In HIV patients, however, the frequency of these memory cells was a function of the viral load. The decreased numbers of functional memory cells in patients with high viral loads might provide one mechanism behind the immunodeficient state. Although the cytokine output per cell was unaffected in most patients (20 of 24), in some patients (4 of 24) it was >100-fold reduced, which might provide an additional mechanism to account for the reduced immunocompetence of these patients. The ability to visualize directly and quantify the cytokine produced by the low frequency memory cells in freshly isolated blood that have been physiologically stimulated by Ag should aid comprehensive studies of the Ag-specific memory cell pool in vivo, in health and disease.
Lactobacilli and Streptococci Activate NF-κB and STAT Signaling Pathways in Human Macrophages
Authors: Miettinen Minja , Lehtonen Anne , Julkunen Ilkka , Matikainen Sampsa
Published: April 2014

Gram-positive bacteria induce the production of several cytokines in human leukocytes. The molecular mechanisms involved in Gram-positive bacteria-induced cytokine production have been poorly characterized. In this work we demonstrate that both nonpathogenic Lactobacillus rhamnosus GG and pathogenic Streptococcus pyogenes (group A streptococci) induce NF-κB and STAT DNA-binding activity in human primary macrophages as analyzed by EMSA. NF-κB activation was rapid and was not inhibited by a protein synthesis inhibitor cycloheximide, suggesting that these bacteria could directly activate NF-κB. STAT1, STAT3, and IFN regulatory factor-1 DNA binding was induced by both bacteria with delayed kinetics compared with NF-κB. In addition, streptococci induced the formation of IFN-α-specific transcription factor complex and IFN-stimulated gene factor-3 (ISGF3). STAT1 and STAT3 activation and ISGF3 complex formation were inhibited by cycloheximide or by neutralization with IFN-α/β-specific Abs. Streptococci were more potent than lactobacilli in inducing STAT1, ISGF3, and IFN regulatory factor-1 DNA binding. Accordingly, only streptococci induced IFN-α production. The activation of the IFN-α signaling pathway by streptococci could play a role in the pathogenesis of these bacteria. These results indicate that extracellular Gram-positive bacteria activate transcription factors involved in cytokine signaling by two mechanisms: directly, leading to NF-κB activation, and indirectly via cytokines, leading to STAT activation.
T Cell Clones Raised from Chronically Infected Healthy Humans by Stimulation with Toxoplasma gondii Excretory-Secretory Antigens Cross-React with Live Tachyzoites: Characterization of the Fine Antigenic Specificity of the Clones and Implications for Vaccine Development
Authors: Prigione Ignazia , Facchetti Paola , Lecordier Laurence , Deslée Didier , Chiesa Sabrina , Cesbron-Delauw Marie-France , Pistoia Vito
Published: April 2014

Excreted-secreted Ags (ESA) of Toxoplasma gondii (Tg) play an important role in the stimulation of the host immune system in both acute and chronic infections. To identify the parasite Ag(s) involved in the maintenance of T cell-mediated long term immunity, 40 ESA-specific T cell clones were derived from three chronically infected healthy subjects. All the clones were CD4+ and recognized both ESA and live tachyzoites in a HLA-DR-restricted manner. Conversely, CD4+ tachyzoite-specific T cell clones from the same subjects proliferated in response to ESA, pointing to shared immunodominant Ags between ESA and Tg tachyzoites. By T cell blot analysis using SDS-PAGE-fractionated parasite extracts, the following patterns of reactivity were detected. Of 25 clones, 6 recognized Tg fractions in the 24- to 28-kDa range and proliferated to purified GRA2, 5 reacted with Tg fractions in the 30- to 33-kDa range; and 4 of them proved to be specific for rSAg1. Although surface Ag (SAg1) is not a member of ESA, small amounts of this protein were present in ESA preparation by Western blot. Of 25 clones, 8 responded to Tg fractions in the 50- to 60-kDa range but not to the 55-kDa recombinant rhoptries-2 parasite Ag, and 6 did not react with any Tg fraction but proliferated in response to either ESA or total parasite extracts. In conclusion, CD4+ T cells specific for either ESA (GRA2) or SAg1 may be involved in the maintenance of long term immunity to Tg in healthy chronically infected individuals.
A Role of Mast Cell Glycosaminoglycans for the Immunological Expulsion of Intestinal Nematode, Strongyloides venezuelensis
Authors: Maruyama Haruhiko , Yabu Yoshisada , Yoshida Ayako , Nawa Yukifumi , Ohta Nobuo
Published: April 2014

We examined effects of mast cell glycosaminoglycans on the establishment of the intestinal nematode, Strongyloides venezuelensis, in the mouse small intestine. When intestinal mastocytosis occurred, surgically implanted adult worms could not invade and establish in the intestinal mucosa. In mast cell-deficient W/Wv mice, inhibition of adult worm invasion was not evident as compared with littermate +/+ control mice. Mucosal mastocytosis and inhibition of S. venezuelensis adult worm mucosal invasion was tightly correlated. To determine effector molecules for the invasion inhibition, adult worms were implanted with various sulfated carbohydrates including mast cell glycosaminoglycans. Among sulfated carbohydrates tested, chondroitin sulfate (ChS)-A, ChS-E, heparin, and dextran sulfate inhibited invasion of adult worms into intestinal mucosa in vivo. No significant inhibition was observed with ChS-C, desulfated chondroitin, and dextran. ChS-E, heparin, and dextran sulfate inhibited adhesion of S. venezuelensis adult worms to plastic surfaces in vitro. Furthermore, binding of intestinal epithelial cells to adhesion substances of S. venezuelensis, which have been implicated in mucosal invasion, was inhibited by ChS-E, heparin, and dextran sulfate. Because adult worms of S. venezuelensis were actively moving in the intestinal mucosa, probably exiting and reentering during infection, the possible expulsion mechanism for S. venezuelensis is inhibition by mast cell glycosaminoglycans of attachment and subsequent invasion of adult worms into intestinal epithelium.
Isolated Pneumocystis carinii Cell Wall Glucan Provokes Lower Respiratory Tract Inflammatory Responses
Authors: Vassallo Robert , Standing Joseph E. , Limper Andrew H.
Published: April 2014

Macrophage-induced lung inflammation contributes substantially to respiratory failure during Pneumocystis carinii pneumonia. We isolated a P. carinii cell wall fraction rich in glucan carbohydrate, which potently induces TNF-α and macrophage-inflammatory protein-2 generation from alveolar macrophages. Instillation of this purified P. carinii carbohydrate cell wall fraction into healthy rodents is accompanied by substantial increases in whole lung TNF-α generation and is associated with neutrophilic infiltration of the lungs. Digestion of the P. carinii cell wall isolate with zymolyase, a preparation containing predominantly β-1,3 glucanase, substantially reduces the ability of this P. carinii cell wall fraction to activate alveolar macrophages, thus suggesting that β-glucan components of the P. carinii cell wall largely mediate TNF-α release. Furthermore, the soluble carbohydrate β-glucan receptor antagonists laminariheptaose and laminarin also substantially reduce the ability of the P. carinii cell wall isolate to stimulate macrophage-inflammatory activation. In contrast, soluble α-mannan, a preparation that antagonizes macrophage mannose receptors, had minimal effect on TNF-α release induced by the P. carinii cell wall fraction. P. carinii β-glucan-induced TNF-α release from alveolar macrophages was also inhibited by both dexamethasone and pentoxifylline, two pharmacological agents with potential activity in controlling P. carinii-induced lung inflammation. These data demonstrate that P. carinii β-glucan cell wall components can directly stimulate alveolar macrophages to release proinflammatory cytokines mainly through interaction with cognate β-glucan receptors on the phagocyte.
Resistance Against the Membrane Attack Complex of Complement Induced in Porcine Endothelial Cells with a Galα(1–3)Gal Binding Lectin: Up-Regulation of CD59 Expression
Authors: Dalmasso Agustin P. , Benson Barbara A. , Johnson Jason S. , Lancto Cheryl , Abrahamsen Mitchell S.
Published: April 2014

Endothelial cells (EC) play central roles in vascular physiology and pathophysiology. EC activation results in proinflammatory activities with production of cytokines and expression of adhesion molecules. However, we have shown before in a model of xenotransplantation that prolonged stimulation of porcine EC with human anti-porcine IgM natural Abs can activate the cells to become resistant against cytotoxicity by the membrane attack complex of complement (MAC). Now we report the major characteristics of induction and maintenance of resistance elicited in porcine EC with Bandeiraea simplicifolia lectin that binds terminal galα(1–3)gal. Lectin-treated cells underwent little or no cytotoxicity and PGI2 release when exposed to MAC. Induction of resistance required incubation of the EC with lectin for 4 h but was not fully manifested until 16 h later. Most of the initially bound lectin remained on the cell surface for >60 h. EC-bound lectin did not inhibit binding of IgM natural Abs or activation and binding of C components, including C9, but a C-induced permeability channel of reduced size was present. Induction of resistance required protein synthesis, developed slowly, and was associated with up-regulation of expression of mRNA for the MAC inhibitor CD59 and membrane-associated CD59 protein. Resistance lasted at least 3 days, and the cells regained normal morphology and were metabolically active. This induced resistance may have a physiologic counterpart that might be amenable to pharmacologic manipulation in vascular endothelium pathophysiology.
Identification of Unique Truncated KC/GROβ Chemokines with Potent Hematopoietic and Anti-Infective Activities
Authors: King Andrew G. , Johanson Kyung , Frey Carrie L. , DeMarsh Peter L. , White John R. , McDevitt Patrick , McNulty Dean , Balcarek Joanna , Jonak Zdenka L. , Bhatnagar Pradip K. , Pelus Louis M.
Published: April 2014

SK&F 107647, a previously described synthetic immunomodulatory peptide, indirectly stimulates bone marrow progenitor cells and phagocytic cells, and enhances host defense effector mechanisms in bacterial and fungal infection models in vivo. In vitro, SK&F 107647 induces the production of a soluble mediator that augments colony forming cell (CFU-GM) formation in the presence of CSFs. In this paper we purified and sequenced the stromal cell-derived hematopoietic synergistic factors (HSF) secreted from both murine and human cell lines stimulated with SK&F 107647. Murine HSF is an N-terminal 4-aa truncated form of the CXC chemokine, KC, while human HSF was identified as an N-terminal 4-aa truncated form of the CXC chemokine, GROβ. In comparison to their full-length forms, truncated KC and truncated GROβ were 10 million times more potent as synergistic growth stimulants for CFU-GM. Enhanced potency of these novel truncated chemokines relative to their full-length forms was also demonstrated in respiratory burst assays, CD11b Ag expression, and intracellular killing of the opportunistic pathogen, Candida albicans. Administration of truncated KC significantly enhanced survival of mice lethally infected with C. albicans. The results reported herein delineate the biological mechanism of action of SK&F 107647, which functions via the induction of unique specific truncated forms of the chemokines KC and GROβ. To our knowledge, this represents the first example where any form of KC or GROβ were purified from marrow stromal cells. Additionally, this is the first demonstration of in vivo efficacy of a CXC chemokine in an animal infectious fungal disease model.
Characterization of Synthetic C3a Analog Peptides on Human Eosinophils in Comparison to the Native Complement Component C3a
Authors: Petering Holger , Köhl Jörg , Weyergraf Ansgar , Dulkys Yasmin , Kimmig Daniela , Smolarski Regina , Kapp Alexander , Elsner Jörn
Published: April 2014

The C3a anaphylatoxin is a potent proinflammatory mediator derived from the complement system inducing biologic effects of human eosinophils like Ca2+ transients and the activation of the respiratory burst. These findings support an important role for C3a in diseases typically associated with a peripheral blood or tissue eosinophilia. Synthetic human C3a analogue peptides with variations at the C-terminal effector domain have been evaluated with respect to their binding affinity and signaling potency on human eosinophils. Flow cytometrical analysis and RT-PCR revealed that the C3a receptor is constitutively expressed on human eosinophils. Peptides bearing an N-terminal 9-fluorenylmethoxycarbonyl and the 6-aminohexanoyl motif were the most powerful peptides tested. Amino acid replacements in the conserved C-terminal pentapeptide decreased binding affinity and functional potency substantially. In addition, synthetic C3a analogue peptides induced C3aR internalization, led to transient changes of intracellular Ca2+ concentration, and did release reactive oxygen species in human eosinophils indicating the in vivo relevance of C3a-related sequences. The tripeptide LAR was found to be essential for C3a receptor binding on human eosinophils. Moreover, the putative binding motif of C3a anaphylatoxin is also crucial for the induction of biologic effects in the human system such as changes of intracellular Ca2+ concentration and the release of reactive oxygen species. This study demonstrates that the carboxyl terminus is important for the interaction with the C3aR and the biologic potency of C3a anaphylatoxin in the human system and plays a key role in the activation process of human eosinophils.
Aerosolized Syk Antisense Suppresses Syk Expression, Mediator Release from Macrophages, and Pulmonary Inflammation
Authors: Stenton Grant R. , Kim Moo-Kyung , Nohara Osamu , Chen Chin-Fu , Hirji Nadir , Wills Fiona L. , Gilchrist Mark , Hwang Pyoung-Han , Park Jong-Gu , Finlay Warren , Jones Richard L. , Befus A. Dean , Schreiber Alan D.
Published: April 2014

Syk protein tyrosine kinase (PTK) is involved in signaling in leukocytes. In macrophages, Fcγ-receptor cross-linking induces Syk PTK phosphorylation and activation, resulting in Syk-dependent events required for phagocytosis and mediator release. We hypothesized that Syk antisense oligodeoxynucleotides (ASO) delivered by aerosol to rat lungs in vivo would depress Syk PTK expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation. RT-PCR and RT-in situ PCR demonstrated that aerosolized Syk ASO administration reduced Syk mRNA expression from alveolar macrophages compared with cells isolated from sham-treated rats. Western blot analysis confirmed that Syk PTK expression was reduced after Syk ASO treatment. Compared with sham-treated rats (scrambled oligodeoxynucleotide), Syk ASO treatment suppressed Fcγ-receptor-mediated nitric oxide (86.0 ± 8.3%) and TNF (73.1 ± 3.1%) production by alveolar macrophages stimulated with IgG-anti-IgG complexes. In contrast, Fcγ-receptor-induced IL-1β release was unaffected by Syk ASO treatment. Additionally, Syk ASO suppressed Ag-induced pulmonary inflammation, suggesting that Syk ASO may prove useful as an anti-inflammatory therapy in disorders such as asthma.