bluuebunny/crossref_metadata_2025 huggingface dataset:
Variegation of the Immune Response with Dendritic Cells and Pathogen Recognition Receptors
Authors: Pulendran Bali
Published: April 2014

One of the most fundamental questions in biology is: “How do cells differentiate in the right place, at the right time, into the right kinds?” Understanding the phenomenon of cell differentiation in its spatial and temporal framework is a prelude to understanding the development and physiology of all multicellular systems, including the immune system. Insights over the past 2300 years, since Aristotle, suggest that biological differentiation is guided by the interplay between genetic programs and specific environmental signals. This is exemplified by the mammalian immune response to pathogens, where qualitatively different types can emerge. Although it is appreciated that this type immunity is critical for optimal defense against different pathogens, the early “decision-making mechanisms” are largely obscure. Recent developments in innate immunity and genomics, especially in the biology of dendritic cells (DCs) and pathogen recognition receptors, have stimulated intense research in understanding the mechanisms guiding the differentiation of Th1, Th2, and T regulatory responses. In this study, I summarize recent findings which suggest that activation of DCs via distinct pathogen recognition receptors stimulate different gene expression programs and signaling networks in DCs that guide the variegation of immune responses.
Cutting Edge: All-trans Retinoic Acid Down-Regulates TLR2 Expression and Function
Authors: Liu Philip T. , Krutzik Stephan R. , Kim Jenny , Modlin Robert L.
Published: April 2014

A major consequence of microbial infection is the tissue injury that results from the host inflammatory response. In acne, inflammation is due in part to the ability of Propionibacterium acnes to activate TLR2. Because all-trans retinoic acid (ATRA) decreases inflammation in acne, we investigated whether it regulates TLR2 expression and function. Treatment of primary human monocytes with ATRA led to the down-regulation of TLR2 as well as it’s coreceptor CD14, but not TLR1 or TLR4. The ability of a TLR2/1 ligand to trigger monocyte cytokine release was inhibited by pre- and cotreatment with ATRA; however, TLR4 activation was affected by cotreatment only. ATRA also down-regulated monocyte cytokine induction by P. acnes. These data indicate that ATRA exerts an anti-inflammatory effect on monocytes via two pathways, one specifically affecting TLR2/1 and CD14 expression and one independent of TLR expression. Agents that target TLR expression and function represent a novel strategy to treat inflammation in humans.
Cutting Edge: Microbial Products Elicit Formation of Dendritic Cell Aggresome-Like Induced Structures in Macrophages
Authors: Canadien Veronica , Tan Tracy , Zilber Rachelle , Szeto Jason , Perrin Andrew J. , Brumell John H.
Published: April 2014

In response to a maturation stimulus, dendritic cells undergo the formation of ubiquitinated protein aggregates known as dendritic cell aggresome-like induced structures (DALIS). DALIS are thought to act as Ag storage structures, allowing for the prioritized degradation of proteins during infection. In this study, we demonstrate that murine macrophages can also form ubiquitinated protein aggregates that are indistinguishable from DALIS. These were formed in a dose- and time-dependent manner, and in response to a variety of microbial products. Surprisingly, the proteasome did not accumulate on these ubiquitinated protein structures, further underlining the difference between DALIS and aggresomes. Our studies suggest that DALIS formation is important for the function of Ag-presenting immune cells during infection.
Cutting Edge: Priming of CTL by Transcutaneous Peptide Immunization with Imiquimod
Authors: Rechtsteiner Gerd , Warger Tobias , Osterloh Philipp , Schild Hansjörg , Radsak Markus P.
Published: April 2014

CTL are important in combating cancer and viruses. Therefore, triggering the complete potential of CTL effector functions by new vaccination strategies will not only improve prophylaxis of tumor or virus-related diseases, but also open opportunities for effective therapeutic immunizations. Using transcutaneous immunization, we show that epicutaneous (e.c.)4 application of an ointment containing a CTL epitope and the TLR7 ligand imiquimod is highly effective in activating T cells in mice using TCR-transgenic CTL or in wild-type mice. Transcutaneous immunization-activated CTL mount a full-blown immune response against the target epitope characterized by proliferation, cytolytic activity, and the production of IFN-γ that is completely restricted to the epitope used for vaccination. Our results obtained by simple e.c. application of an ointment, without further skin irritating procedures, provide the basis for the development of new, easy to use vaccines against cancer or virus-associated diseases.
Cutting Edge: p27Kip1 Deficiency Reduces the Requirement for CD28-Mediated Costimulation in Naive CD8+ but Not CD4+ T Lymphocytes
Authors: Wolfraim Lawrence A. , Letterio John J.
Published: April 2014

Cell cycle re-entry of quiescent T cells is dependent upon cyclin-dependent kinase 2. Inhibition of cyclin-dependent kinase 2 by p27Kip1 is believed to be the principal constraint on S-phase entry in T cells. We report that deficiency for p27Kip1 has a more pronounced effect on the expansion of murine naive CD8+ T cells and that this disparity is due to a reduced requirement for CD28-mediated costimulation in CD8+ but not CD4+ T cells lacking p27Kip1. These data highlight a previously unappreciated difference in the way CD28 signaling is coupled to the core cell cycle machinery in these two T cell subsets.
Cutting Edge: Regulation of T Cell Trafficking and Primary Immune Responses by Sphingosine 1-Phosphate Receptor 1
Authors: Chi Hongbo , Flavell Richard A.
Published: April 2014

Signaling by sphingosine 1-phosphate (S1P) through its receptor S1P1 has recently been shown to promote thymocyte egress. In the periphery, S1P1 is expressed on naive T cells but lost upon T cell activation. To determine the significance of S1P1 down-regulation and function of S1P1 in peripheral T cells, we developed transgenic mice that constitutively express S1P1 in T cells. Mature T cells from these mice exhibited enhanced chemotactic response toward S1P, and preferentially distributed to the blood rather than secondary lymphoid organs. S1P1-transgenic mice showed significant delay in the onset of experimental autoimmune encephalomyelitis, and had defective contact hypersensitivity reaction and local Ag-induced responses. These impairments were associated with reduced numbers of Ag-activated T cells in the draining lymph nodes. Our studies demonstrate that S1P1 signaling affects systemic trafficking of peripheral T cells and immune responses and highlight that levels of S1P1 expression represent an important mechanism of immune regulation.
Cutting Edge: Back to “One-Way” Germinal Centers
Authors: Meyer-Hermann Michael E. , Maini Philip K.
Published: April 2014

The present status of germinal center (GC) research is revisited using in silico simulations based on recent lymphocyte motility data in mice. The generally adopted view of several rounds of somatic hypermutations and positive selection is analyzed with special emphasis on the spatial organization of the GC reaction. We claim that the development of dark zones is not necessary for successful GC reactions to develop. We find that a recirculation of positively selected centrocytes to the dark zone is rather unlikely. Instead we propose a scenario that combines a multiple-step mutation and selection concept with a “one-way” GC in the sense of cell migration.
Cutting Edge: IL-4 Induces Suppressor of Cytokine Signaling-3 Expression in B Cells by a Mechanism Dependent on Activation of p38 MAPK
Authors: Canfield Stephen , Lee Youngnam , Schröder Andreas , Rothman Paul
Published: April 2014

The signaling cascade initiated by IL-4 is classically divisible into two major pathways: one mediated by STAT6, and the other by insulin receptor substrates-1 and -2 via activation of PI3K. In murine splenic B cells, the suppressor of cytokine signaling (SOCS)3 is inducible by IL-4 via a mechanism independent of STAT6 and PI3K. SOCS3 expression increases 9-fold within 5 h of IL-4 treatment. This induction occurs normally in B cells deficient in STAT6 and is unaffected by pretreatment with the PI3K inhibitor wortmannin, or with the ERK pathway inhibitor, PD98059. However, the IL-4 induction of SOCS3 is blocked by inhibitors of either the JNK or p38 MAPK pathways (SP600125 and SB203580, respectively). Direct examination of these pathways reveals rapid, IL-4-directed activation of p38 MAPK, uncovering a previously unappreciated pathway mediating IL-4 signal transduction.
IFN-α Induces Early Lethal Lupus in Preautoimmune (New Zealand Black × New Zealand White)F1 but Not in BALB/c Mice
Authors: Mathian Alexis , Weinberg Arthur , Gallegos Mike , Banchereau Jacques , Koutouzov Sophie
Published: April 2014

Recent studies indicate that IFN-α is involved in pathogenesis of systemic lupus erythematosus. However, direct proof that IFN-α is not only necessary, but also sufficient to induce lupus pathogenicity is lacking. In this study, we show that in vivo adenovector-mediated delivery of murine IFN-α results in preautoimmune (New Zealand Black (NZB) × New Zealand White (NZW))F1, but not in normal, mice, in a rapid and severe disease with all characteristics of systemic lupus erythematosus. Anti-dsDNA Abs appeared as soon as day 10 after initiation of IFN-α treatment. Proteinuria and death caused by glomerulonephritis occurred in all treated mice within, respectively, ∼9 and ∼18 wk, at a time when all untreated (NZB × NZW)F1 did not show any sign of disease. IFN-α in vivo induced an overexpression of B lymphocyte stimulator in circulation at similar levels in both the preautoimmune and the normal mouse strains. All effects elicited by IFN-α were dose dependent. (NZB × NZW)F1 infused with purified murine IFN-α also showed acceleration of lupus. Thus, prolonged expression of IFN-α in vivo induces early lethal lupus in susceptible animals.
PolycombGroup Genemel-18Regulates Early T Progenitor Expansion by Maintaining the Expression ofHes-1, a Target of the Notch Pathway
Authors: Miyazaki Masaki , Kawamoto Hiroshi , Kato Yuko , Itoi Manami , Miyazaki Kazuko , Masuda Kyoko , Tashiro Satoshi , Ishihara Hiroto , Igarashi Kazuhiko , Amagai Takashi , Kanno Rieko , Kanno Masamoto
Published: April 2014

Polycomb group (PcG) proteins play a role in the maintenance of cellular identity throughout many rounds of cell division through the regulation of gene expression. In this report we demonstrate that the loss of the PcG gene mel-18 impairs the expansion of the most immature T progenitor cells at a stage before the rearrangement of the TCR β-chain gene in vivo and in vitro. This impairment of these T progenitors appears to be associated with increased susceptibility to cell death. We also show that the expression of Hes-1, one of the target genes of the Notch signaling pathway, is drastically down-regulated in early T progenitors isolated from mel-18−/− mice. In addition, mel-18−/− T precursors could not maintain the Hes-1 expression induced by Delta-like-1 in monolayer culture. Collectively, these data indicate that mel-18 contributes to the maintenance of the active state of the Hes-1 gene as a cellular memory system, thereby supporting the expansion of early T progenitors.
Naive, Effector, and Memory T Lymphocytes Efficiently Scan Dendritic Cells In Vivo: Contact Frequency in T Cell Zones of Secondary Lymphoid Organs Does Not Depend on LFA-1 Expression and Facilitates Survival of Effector T Cells
Authors: Westermann Jürgen , Bode Ulrike , Sahle Andrea , Speck Uwe , Karin Nathan , Bell Eric B. , Kalies Kathrin , Gebert Andreas
Published: April 2014

Contact between T cells and dendritic cells (DCs) is required for their subsequent interaction leading to the induction of adaptive immune responses. Quantitative data regarding the contact frequencies of T cell subsets in different lymphoid organs and species are lacking. Therefore, naive, effector, and memory CD4 T cells were injected into rats in absence of the cognate Ag, and 0.5–96 h later, spleen, lymph nodes, and Peyer’s patches were removed. Cryosections were analyzed for contact between donor T cells and endogenous DCs in the T cell zone, and donor cell proliferation. More than 60% of injected naive CD4 T cells were in contact with endogenous DCs at all time points and in all organs analyzed. Surprisingly, we were unable to detect any differences between naive, effector, and memory CD4 T cells despite different expression levels of surface molecules. In addition, contact frequency was similar for T cells in lymphoid organs of rats, mice, and humans; it was unaffected by the absence of LFA-1 (CD11a/CD18), and sustained effector T cells in an activated state. Thus, the architecture of the T cell zone rather than expression patterns of surface molecules determines the contact efficiency between T cells and DCs in vivo.
Thymic Anlage Is Colonized by Progenitors Restricted to T, NK, and Dendritic Cell Lineages
Authors: Masuda Kyoko , Itoi Manami , Amagai Takashi , Minato Nagahiro , Katsura Yoshimoto , Kawamoto Hiroshi
Published: April 2014

It remains controversial whether the thymus-colonizing progenitors are committed to the T cell lineage. A major problem that has impeded the characterization of thymic immigrants has been that the earliest intrathymic progenitors thus far identified do not necessarily represent the genuine thymic immigrants, because their developmental potential should have been influenced by contact with the thymic microenvironment. In the present study, we examined the developmental potential of the ontogenically earliest thymic progenitors of day 11 murine fetus. These cells reside in the surrounding mesenchymal region and have not encountered thymic epithelial components. Flow cytometric and immunohistochemical analyses demonstrated that these cells are exclusively Lin−c-kit+IL-7R+. Limiting dilution analyses disclosed that the progenitors with T cell potential were abundant, while those with B cell potential were virtually absent in the region of day 11 thymic anlage. Clonal analyses reveled that they are restricted to T, NK, and dendritic cell lineages. Each progenitor was capable of forming a large number of precursors that may clonally accommodate highly diverse TCRβ chains. These results provide direct evidence that the progenitors restricted to the T/NK/dendritic cell lineage selectively immigrate into the thymus.
Antiangiogenic Drugs Synergize with a Membrane Macrophage Colony-Stimulating Factor-Based Tumor Vaccine to Therapeutically Treat Rats with an Established Malignant Intracranial Glioma
Authors: Jeffes Edward W. B. , Zhang Jian Gang , Hoa Neil , Petkar Animesh , Delgado Christina , Chong Samuel , Obenaus Andre , Sanchez Ramon , Khalaghizadeh Sakineh , Khomenko Tetyana , Knight Brandon A. , Alipanah Reza , Nguyen Tuong-Vi , Shah Chirag , Vohra Seema , Zhuang Jing-Li , Liu Jessie , Wepsic H. Terry , Jadus Martin R.
Published: April 2014

Combining a T9/9L glioma vaccine, expressing the membrane form of M-CSF, with a systemic antiangiogenic drug-based therapy theoretically targeted toward growth factor receptors within the tumor’s vasculature successfully treated >90% of the rats bearing 7-day-old intracranial T9/9L gliomas. The antiangiogenic drugs included (Z)-3-[4-(dimethylamino)benzylidenyl]indolin-2-one (a platelet-derived growth factor receptor β and a fibroblast growth factor receptor 1 kinase inhibitor) and oxindole (a vascular endothelial growth factor receptor 2 kinase inhibitor). A total of 20–40% of the animals treated with the antiangiogenic drugs alone survived, while all nontreated controls and tumor vaccine-treated rats died within 40 days. In vitro, these drugs inhibited endothelial cells from proliferating in response to the angiogenic factors produced by T9/9L glioma cells and prevented endothelial cell tubulogenesis. FITC-labeled tomato lectin staining demonstrated fewer and constricted blood vessels within the intracranial tumor after drug therapy. Magnetic resonance imaging demonstrated that the intracranial T9 glioma grew much slower in the presence of these antiangiogenic drugs. These drugs did not affect in vitro glioma cell growth nor T cell mitogenesis. Histological analysis revealed that the tumor destruction occurred at the margins of the tumor, where there was a heavy lymphocytic infiltrate. Real-time PCR showed more IL-2-specific mRNA was present within the gliomas in the vaccinated rats treated with the drugs. Animals that rejected the established T9/9L glioma by the combination therapy proved immune against an intracranial rechallenge by T9/9L glioma, but showed no resistance to an unrelated MADB106 breast cancer.
Regulated Folding of Tyrosinase in the Endoplasmic Reticulum Demonstrates That Misfolded Full-Length Proteins Are Efficient Substrates for Class I Processing and Presentation
Authors: Ostankovitch Marina , Robila Valentina , Engelhard Victor H.
Published: April 2014

Short-lived protein translation products have been proposed to be the principal substrates that enter the class I MHC processing and presentation pathway. However, the biochemical nature of these substrates is poorly defined. Whether the major processing substrates are misfolded full-length proteins, or alternatively, aberrantly initiated or truncated polypeptides still remains to be addressed. To examine this, we used melanoma in which one-third of wild-type tyrosinase molecules were correctly folded and localized beyond the Golgi, while the remainder were present in the endoplasmic reticulum in an unfolded/misfolded state. Increasing the efficiency of tyrosinase folding using chemical chaperones led to a reduction in the level of substrate available to the proteasome and decreased the expression of a tyrosinase-derived epitope. Conversely, in transfectants expressing tyrosinase mutants that are completely misfolded, both proteasome substrate and epitope presentation were significantly enhanced. Proteasome substrate availability was a consequence of misfolding and not simply due to retention in the endoplasmic reticulum. Thus, the extent of folding/misfolding of a full-length protein is an important determinant of the level of epitope presentation.
Progressive and Controlled Development of Mouse Dendritic Cells from Flt3+CD11b+ Progenitors In Vitro
Authors: Hieronymus Thomas , Gust Tatjana C. , Kirsch Ralf D. , Jorgas Thorsten , Blendinger Gitta , Goncharenko Mykola , Supplitt Kamilla , Rose-John Stefan , Müller Albrecht M. , Zenke Martin
Published: April 2014

Dendritic cells (DC) represent key regulators of the immune system, yet their development from hemopoietic precursors is poorly defined. In this study, we describe an in vitro system for amplification of a Flt3+CD11b+ progenitor from mouse bone marrow with specific cytokines. Such progenitor cells develop into both CD11b+ and CD11b− DC, and CD8α+ and CD8α− DC in vivo. Furthermore, with GM-CSF, these progenitors synchronously differentiated into fully functional DC in vitro. This two-step culture system yields homogeneous populations of Flt3+CD11b+ progenitor cells in high numbers and allows monitoring the consecutive steps of DC development in vitro under well-defined conditions. We used phenotypic and functional markers and transcriptional profiling by DNA microarrays to study the Flt3+CD11b+ progenitor and differentiated DC. We report here on an extensive analysis of the surface Ag expression of Flt3+CD11b+ progenitor cells and relate that to surface Ag expression of hemopoietic stem cells. Flt3+CD11b+ progenitors studied exhibit a broad overlap of surface Ags with stem cells and express several stem cell Ags such as Flt3, IL-6R, c-kit/SCF receptor, and CD93/AA4.1, CD133/AC133, and CD49f/integrin α6. Thus, Flt3+CD11b+ progenitors express several stem cell surface Ags and develop into both CD11b+ and CD11b− DC, and CD8α+ and CD8α− DC in vivo, and thus into both of the main conventional DC subtypes.
The Fate of Low Affinity Tumor-Specific CD8+ T Cells in Tumor-Bearing Mice
Authors: Lyman Michael A. , Nugent C. Thomas , Marquardt Kristi L. , Biggs Judith A. , Pamer Eric G. , Sherman Linda A.
Published: April 2014

A major challenge in tumor immunology is how best to activate the relatively low avidity self-specific and tumor-specific T cells that are available in the self-tolerant repertoire. To address this issue, we produced a TCR transgenic mouse expressing a class I-restricted hemagglutinin (HA)-specific TCR (clone 1 TCR) derived from a mouse that expressed HA as a self-Ag in the insulin-producing β cells of the pancreatic islets (InsHA) mice. Upon transfer of clone 1 TCR CD8+ T cells into InsHA mice, very few cells were activated by cross-presented HA, indicating that the cells were retained in InsHA mice because they ignored the presence of Ag, and not because they were functionally inactivated by anergy or tuning. Upon transfer into recipient mice in which HA is expressed at high concentrations as a tumor-associated Ag in spontaneously arising insulinomas (RIP-Tag2-HA mice), a high proportion of clone 1 cells were activated when they encountered cross-presented tumor Ag in the pancreatic lymph nodes. However, the activated cells exhibited very weak effector function and were soon tolerized. The few activated cells that did migrate to the tumor were unable to delay tumor progression. However, when HA-specific CD4 helper cells were cotransferred with clone 1 cells into RIP-Tag2-HA recipients and the mice were vaccinated with influenza, clone 1 cells were found to exert a significant level of effector function and could delay tumor growth. This tumor model should prove of great value in identifying protocols that can optimize the function of low avidity tumor-specific T cells.
IFN Regulatory Factor-4 and -8 Govern Dendritic Cell Subset Development and Their Functional Diversity
Authors: Tamura Tomohiko , Tailor Prafullakumar , Yamaoka Kunihiro , Kong Hee Jeong , Tsujimura Hideki , O’Shea John J. , Singh Harinder , Ozato Keiko
Published: April 2014

Dendritic cells (DCs) are bone marrow (BM)-derived APCs central to both innate and adaptive immunity. DCs are a heterogeneous cell population composed of multiple subsets with diverse functions. The mechanism governing the generation of multiple DC subsets is, however, poorly understood. In this study we investigated the roles of closely related transcription factors, IFN regulatory factor (IRF)-4 and IRF-8, in DC development by analyzing IRF-4−/−, IRF-8−/−, and IRF-4−/−IRF-8−/− (double-knockout) mice. We found that IRF-4 is required for the generation of CD4+ DCs, whereas IRF-8 is, as reported previously, essential for CD8α+ DCs. Both IRFs support the development of CD4−CD8α− DCs. IRF-8 and, to a lesser degree, IRF-4 contribute to plasmacytoid DC (PDC) development. Thus, the two IRFs together regulate the development of all conventional DCs as well as PDCs. Consistent with these findings, IRF-4, but not IRF-8, was expressed in CD4+ DCs, whereas only IRF-8 was expressed in CD8α+ DCs. CD4−CD8α− DCs and PDCs expressed both IRFs. We also demonstrate in vitro that GM-CSF-mediated DC differentiation depends on IRF-4, whereas Fms-like tyrosine kinase 3 ligand-mediated differentiation depends mainly on IRF-8. Gene transfer experiments with double-knockout BM cells showed that both IRFs have an overlapping activity and stimulate a common process of DC development. Nonetheless, each IRF also possesses a distinct activity to stimulate subset-specific gene expression, leading to the generation of functionally divergent DCs. Together, IRF-4 and IRF-8 serve as a backbone of the molecular program regulating DC subset development and their functional diversity.
CXC Chemokine Ligand 12-Induced Focal Adhesion Kinase Activation and Segregation into Membrane Domains Is Modulated by Regulator of G Protein Signaling 1 in Pro-B Cells
Authors: Le Yi , Honczarenko Marek , Glodek Aleksandra M. , Ho Daniel K. , Silberstein Leslie E.
Published: April 2014

CXCL12-induced chemotaxis and adhesion to VCAM-1 decrease as B cells differentiate in the bone marrow. However, the mechanisms that regulate CXCL12/CXCR4-mediated signaling are poorly understood. We report that after CXCL12 stimulation of progenitor B cells, focal adhesion kinase (FAK) and PI3K are inducibly recruited to raft-associated membrane domains. After CXCL12 stimulation, phosphorylated FAK is also localized in membrane domains. The CXCL12/CXCR4-FAK pathway is membrane cholesterol dependent and impaired by metabolic inhibitors of Gi, Src family, and the GTPase-activating protein, regulator of G protein signaling 1 (RGS1). In the bone marrow, RGS1 mRNA expression is low in progenitor B cells and high in mature B cells, implying developmental regulation of CXCL12/CXCR4 signaling by RGS1. CXCL12-induced chemotaxis and adhesion are impaired when FAK recruitment and phosphorylation are inhibited by either membrane cholesterol depletion or overexpression of RGS1 in progenitor B cells. We conclude that the recruitment of signaling molecules to specific membrane domains plays an important role in CXCL12/CXCR4-induced cellular responses.
CD8+ T Cell Immunity Against a Tumor/Self-Antigen Is Augmented by CD4+ T Helper Cells and Hindered by Naturally Occurring T Regulatory Cells
Authors: Antony Paul A. , Piccirillo Ciriaco A. , Akpinarli Akgül , Finkelstein Steven E. , Speiss Paul J. , Surman Deborah R. , Palmer Douglas C. , Chan Chi-Chao , Klebanoff Christopher A. , Overwijk Willem W. , Rosenberg Steven A. , Restifo Nicholas P.
Published: April 2014

CD4+ T cells control the effector function, memory, and maintenance of CD8+ T cells. Paradoxically, we found that absence of CD4+ T cells enhanced adoptive immunotherapy of cancer when using CD8+ T cells directed against a persisting tumor/self-Ag. However, adoptive transfer of CD4+CD25− Th cells (Th cells) with tumor/self-reactive CD8+ T cells and vaccination into CD4+ T cell-deficient hosts induced autoimmunity and regression of established melanoma. Transfer of CD4+ T cells that contained a mixture of Th and CD4+CD25+ T regulatory cells (Treg cells) or Treg cells alone prevented effective adoptive immunotherapy. Maintenance of CD8+ T cell numbers and function was dependent on Th cells that were capable of IL-2 production because therapy failed when Th cells were derived from IL-2−/− mice. These findings reveal that Th cells can help break tolerance to a persisting self-Ag and treat established tumors through an IL-2-dependent mechanism, but requires simultaneous absence of naturally occurring Treg cells to be effective.
The Wiskott-Aldrich Syndrome Protein Regulates Nuclear Translocation of NFAT2 and NF-κB (RelA) Independently of Its Role in Filamentous Actin Polymerization and Actin Cytoskeletal Rearrangement
Authors: Huang Winifred , Ochs Hans D. , Dupont Bo , Vyas Yatin M.
Published: April 2014

Effector functions mediated by NK cells involve cytotoxicity and transcription-dependent production and release of cytokines and chemokines. Although the JAK/STAT pathway mediates lymphokine-induced transcriptional regulation in NK cells, very little is known about transcriptional regulation induced during cell-cell contact. We demonstrate that the Wiskott-Aldrich syndrome protein (WASp) is an important component for integration of signals leading to nuclear translocation of NFAT2 and NF-κB (RelA) during cell-cell contact and NKp46-dependent signaling. This WASp function is independent of its known role in F-actin polymerization and cytoskeletal rearrangement. Absence of WASp results in decreased accumulation of calcineurin, WASp-interacting protein, and molecules upstream of calcium mobilization, i.e., activated ZAP70 and phospholipase C-γ1, in the disorganized NK cell immune synapse. Production of GM-CSF, but not IFN-γ, is decreased, while natural cytotoxicity of Wiskott-Aldrich syndrome-NK cells is maintained. Our results indicate that WASp independently regulates its dual functions, i.e., actin cytoskeletal remodeling and transcription in NK cells.
Natural Killer Dendritic Cells Have Both Antigen Presenting and Lytic Function and in Response to CpG Produce IFN-γ via Autocrine IL-12
Authors: Pillarisetty Venu G. , Katz Steven C. , Bleier Joshua I. , Shah Alaap B. , DeMatteo Ronald P.
Published: April 2014

We have isolated rare cells bearing the NK cell surface marker NK1.1, as well as the dendritic cell (DC) marker CD11c, from the spleen, liver, lymph nodes, and thymus of normal mice. These cells possess both NK cell and DC function because they can lyse tumor cells and subsequently present Ags to naive Ag-specific T cells. Interestingly, in response to IL-4 plus either IL-2 or CpG, NKDC produce more IFN-γ than do DC, or even NK cells. We determined that CpG, but not IL-2, induces NKDC to secrete IFN-γ via the autocrine effects of IL-12. In vivo, CpG dramatically increases the number of NKDC. Furthermore, NKDC induce greater Ag-specific T cell activation than do DC after adoptive transfer. Their unique ability to lyse tumor cells, present Ags, and secrete inflammatory cytokines suggests that NKDC may play a crucial role in linking innate and adaptive immunity.
Activated Human B Lymphocytes Express Cyclooxygenase-2 and Cyclooxygenase Inhibitors Attenuate Antibody Production
Authors: Ryan Elizabeth P. , Pollack Stephen J. , Murant Thomas I. , Bernstein Steven H. , Felgar Raymond E. , Phipps Richard P.
Published: April 2014

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of inflammatory diseases and target cyclooxygenases 1 and 2 (Cox-1, Cox-2) that are responsible for PG production. Newer Cox-2-selective drugs have been heavily prescribed to quench inflammation. Little is known about whether or not these drugs influence human B lymphocytes and their ability to produce Ab. We report herein that activated human B cells not only highly express Cox-2 and produce PGs, but that the NSAID indomethacin and Cox-2-selective drugs profoundly inhibit the ability of human B cells to produce IgG and IgM in vitro. Human blood B cells highly express Cox-2 mRNA and protein and produce PGs after activation with CD40L, pansorbin, or CD40L plus BCR engagement. Cox-2 is also highly expressed by human tonsil B cells, as shown by immunohistochemistry. Cox-inhibiting drugs modestly affect purified B cell proliferation but profoundly reduce Ab production. The ability of whole blood to produce IgM and IgG following stimulation is also strongly inhibited. In support that Cox-2 plays a seminal role in B lymphocyte Ab production, Cox-2 knockout mice have 64% less IgM and 35% less IgG than normal littermate controls. These findings support that NSAIDs and the new Cox-2-selective drugs have an unsuspected target, the B cell, and attenuate Ab production in humans. Use of NSAIDs may therefore influence autoantibody production in autoimmune diseases and may dampen humoral immunity in response to antigenic challenge/vaccination.
Overexpression of the Runx3 Transcription Factor Increases the Proportion of Mature Thymocytes of the CD8 Single-Positive Lineage
Authors: Kohu Kazuyoshi , Sato Takehito , Ohno Shin-ichiro , Hayashi Keitaro , Uchino Ryuji , Abe Natsumi , Nakazato Megumi , Yoshida Naomi , Kikuchi Toshiaki , Iwakura Yoichiro , Inoue Yoshihiro , Watanabe Toshio , Habu Sonoko , Satake Masanobu
Published: April 2014

The Runx family of transcription factors is thought to regulate the differentiation of thymocytes. Runx3 protein is detected mainly in the CD4−8+ subset of T lymphocytes. In the thymus of Runx3-deficient mice, CD4 expression is de-repressed and CD4−8+ thymocytes do not develop. This clearly implicates Runx3 in CD4 silencing, but does not necessarily prove its role in the differentiation of CD4−8+ thymocytes per se. In the present study, we created transgenic mice that overexpress Runx3 and analyzed the development of thymocytes in these animals. In the Runx3-transgenic thymus, the number of CD4−8+ cells was greatly increased, whereas the numbers of CD4+8+ and CD4+8− cells were reduced. The CD4−8+ transgenic thymocytes contained mature cells with a TCRhighHSAlow phenotype. These cells were released from the thymus and contributed to the elevated level of CD4−8+ cells relative to CD4+8− cells in the spleen. Runx3 overexpression also increased the number of mature CD4−8+ thymocytes in mice with class II-restricted, transgenic TCR and in mice with a class I-deficient background, both of which are favorable for CD4+8− lineage selection. Thus, Runx3 can drive thymocytes to select the CD4−8+ lineage. This activity is likely to be due to more than a simple silencing of CD4 gene expression.
The Human CD1-Restricted T Cell Repertoire Is Limited to Cross-Reactive Antigens: Implications for Host Responses against Immunologically Related Pathogens
Authors: Sieling Peter A. , Torrelles Jordi B. , Stenger Steffen , Chung Woosin , Burdick Anne E. , Rea Thomas H. , Brennan Patrick J. , Belisle John T. , Porcelli Steven A. , Modlin Robert L.
Published: April 2014

The repertoires of CD1- and MHC-restricted T cells are complementary, permitting the immune recognition of both lipid and peptide Ags, respectively. To compare the breadth of the CD1-restricted and MHC-restricted T cell repertoires, we evaluated T cell responses against lipid and peptide Ags of mycobacteria in leprosy, comparing tuberculoid patients, who are able to restrict the pathogen, and lepromatous patients, who have disseminated infection. The striking finding was that in lepromatous leprosy, T cells did not efficiently recognize lipid Ags from the leprosy pathogen, Mycobacterium leprae, or the related species, Mycobacterium tuberculosis, yet were able to efficiently recognize peptide Ags from M. tuberculosis, but not M. leprae. To identify a mechanism for T cell unresponsiveness against mycobacterial lipid Ags in lepromatous patients, we used T cell clones to probe the species specificity of the Ags recognized. We found that the majority of M. leprae-reactive CD1-restricted T cell clones (92%) were cross-reactive for multiple mycobacterial species, whereas the majority of M. leprae-reactive MHC-restricted T cells were species specific (66%), with a limited number of T cell clones cross-reactive (34%) with M. tuberculosis. In comparison with the MHC class II-restricted T cell repertoire, the CD1-restricted T cell repertoire is limited to recognition of cross-reactive Ags, imparting a distinct role in the host response to immunologically related pathogens.
In Vivo Manipulation of Dendritic Cells Overcomes Tolerance to Unmodified Tumor-Associated Self Antigens and Induces Potent Antitumor Immunity
Authors: Okano Fumiyoshi , Merad Miriam , Furumoto Katsuyoshi , Engleman Edgar G.
Published: April 2014

Most tumor-associated Ags are self proteins that fail to elicit a T cell response as a consequence of immune tolerance. Dendritic cells (DCs) generated ex vivo have been used to break tolerance against such self Ags; however, in vitro manipulation of DCs is cumbersome and difficult to control, resulting in vaccines of variable potency. To address this problem we developed a method for loading and activating DCs, in situ, by first directing sufficient numbers of DCs to peripheral tissues using Flt3 ligand and then delivering a tumor-associated Ag and oligonucleotide containing unmethylated CG motifs to these tissues. In this study, we show in three different tumor models that this method can overcome tolerance and induce effective antitumor immunity. Vaccination resulted in the generation of CD8+ T and NK cell effectors that mediated durable tumor responses without attacking normal tissues. These findings demonstrate that unmodified tumor-associated self Ags can be targeted to DCs in vivo to induce potent systemic antitumor immunity.
Tumor-Induced Apoptosis of Human IL-2-Activated NK Cells: Role of Natural Cytotoxicity Receptors
Authors: Poggi Alessandro , Massaro Anna-Maria , Negrini Simone , Contini Paola , Zocchi Maria Raffaella
Published: April 2014

We provide evidence that tumor cells can induce apoptosis of NK cells by engaging the natural cytotoxicity receptors (NCR) NKp30, NKp44, and NKp46. Indeed, the binding between NCR on NK cells and their putative ligands on tumor target cells led to NK cell apoptosis, and this event was abolished by blocking NCR/NCR-ligand interaction by anti-NCR-specific mAbs. The engagement of NCR induced up-regulation of Fas ligand (FasL) mRNA, FasL protein synthesis, and release. In turn, FasL interacting with Fas at NK cell surface causes NK cell suicide, as apoptosis of NK cells was inhibited by blocking FasL/Fas interaction with specific mAbs. Interestingly, NK cell apoptosis, but not killing of tumor target cells, is inhibited by cyclosporin A, suggesting that apoptosis and cytolysis are regulated by different biochemical pathways. These findings indicate that NCR are not only triggering molecules essential for antitumor activity, but also surface receptors involved in NK cell suicide.
Recognition of a New ARTC1 Peptide Ligand Uniquely Expressed in Tumor Cells by Antigen-Specific CD4+ Regulatory T Cells
Authors: Wang Helen Y. , Peng Guangyong , Guo Zhong , Shevach Ethan M. , Wang Rong-Fu
Published: April 2014

CD4+ regulatory T (Treg) cells play an important role in the maintenance of immunological self-tolerance by suppressing immune responses against autoimmune diseases and cancer. Yet very little is known about the natural antigenic ligands that preferentially activate CD4+ Treg cells. Here we report the establishment of tumor-specific CD4+ Treg cell clones from tumor-infiltrating lymphocytes (TILs) of cancer patients, and the identification of an Ag recognized by Treg cells (ARTC1) gene encoding a peptide ligand recognized by tumor-specific TIL164 CD4+ Treg cells. The mutations in a gene encoding an ARTC1 in 164mel tumor cells resulted in the translation of a gene product containing the peptide ligand recognized by CD4+ Treg cells. ARTC1 peptide-activated CD4+ Treg cells suppress the physiological function (proliferation and IL-2 secretion) of melanoma-reactive T cells. Furthermore, 164mel tumor cells, but not tumor lysates pulsed on B cells, were capable of activating TIL164 CD4+ Treg cells. These results suggest that tumor cells may uniquely present an array of peptide ligands that preferentially recruit and activate CD4+ Treg cells in sites where tumor-specific self-peptide is expressed, leading to the induction of local and tumor-specific immune suppression.
Mismatched Antigen Prepares γδ T Cells for Suppression of Airway Hyperresponsiveness
Authors: Jin Niyun , Taube Christian , Sharp Laura , Hahn Youn-Soo , Yin Xiang , Wands J. M. , Roark Christina L. , O’Brien Rebecca L. , Gelfand Erwin W. , Born Willi K.
Published: April 2014

γδ T cells suppress airway hyperresponsiveness (AHR) induced in allergen-challenged mice but it is not clear whether the suppression is allergen specific. The AHR-suppressive cells express TCR-Vγ4. To test whether the suppressive function must be induced, we adoptively transferred purified Vγ4+ cells into γδ T cell-deficient and OVA-sensitized and -challenged recipients (B6.TCR-Vγ4−/−/6−/−) and measured the effect on AHR. Vγ4+ γδ T cells isolated from naive donors were not AHR-suppressive, but Vγ4+ cells from OVA-stimulated donors suppressed AHR. Suppressive Vγ4+ cells could be isolated from lung and spleen. Their induction in the spleen required sensitization and challenge. In the lung, their function was induced by airway challenge alone. Induction of the suppressors was associated with their activation but it did not alter their ability to accumulate in the lung. Vγ4+ γδ T cells preferentially express Vδ4 and -5 but their AHR-suppressive function was not dependent on these Vδs. Donor sensitization and challenge not only with OVA but also with two unrelated allergens (ragweed and BSA) induced Vγ4+ cells capable of suppressing AHR in the OVA-hyperresponsive recipients, but the process of sensitization and challenge alone (adjuvant and saline only) was not sufficient to induce suppressor function, and LPS as a component of the allergen was not essential. We conclude that AHR-suppressive Vγ4+ γδ T cells require induction. They are induced by allergen stimulation, but AHR suppression by these cells does not require their restimulation with the same allergen.
Platelet Factor 4 Differentially Modulates CD4+CD25+ (Regulatory) versus CD4+CD25− (Nonregulatory) T Cells
Authors: Liu Chao Yan , Battaglia Manuela , Lee Seon Ho , Sun Qi-Hong , Aster Richard H. , Visentin Gian Paolo
Published: April 2014

Active suppression mediated by CD4+CD25+ T regulatory (Tr) cells plays an important role in the down-regulation of T cell responses to both foreign and self-Ags. Platelet factor 4 (PF4), a platelet-derived CXC chemokine, has been shown to strongly inhibit T cell proliferation as well as IFN-γ and IL-2 release by isolated T cells. In this report we show that human PF4 stimulates proliferation of the naturally anergic human CD4+CD25+ Tr cells while inhibiting proliferation of CD4+CD25− T cells. In coculture experiments we found that CD4+CD25+ Tr cells exposed to PF4 lose the ability to inhibit the proliferative response of CD4+CD25− T cells. Our findings suggest that human PF4, by inducing Tr cell proliferation while impairing Tr cell function, may play a previously unrecognized role in the regulation of human immune responses. Because platelets are the sole source of PF4 in the circulation, these findings may be relevant to the pathogenesis of certain immune-mediated disorders associated with platelet activation, such as heparin-induced thrombocytopenia and autoimmune thrombocytopenic purpura.
CD38 Signaling Regulates B Lymphocyte Activation via a Phospholipase C (PLC)-γ2-Independent, Protein Kinase C, Phosphatidylcholine-PLC, and Phospholipase D-Dependent Signaling Cascade
Authors: Moreno-García Miguel E. , López-Bojórques Lucia N. , Zentella Alejandro , Humphries Lisa A. , Rawlings David J. , Santos-Argumedo Leopoldo
Published: April 2014

The CD38 cell surface receptor is a potent activator for splenic, B lymphocytes. The molecular mechanisms regulating this response, however, remain incompletely characterized. Activation of the nonreceptor tyrosine kinase, Btk, is essential for CD38 downstream signaling function. The major Btk-dependent substrate in B cells, phospholipase C-γ2 (PLC-γ2), functions to generate the key secondary messengers, inositol-1,4,5 trisphosphate and diacylglycerol. Surprisingly, CD38 ligation results in no detectable increase in phosphoinositide metabolism and only a minimal increase in cytosolic calcium. We hypothesized that Btk functioned independently of PLC-γ2 in the CD38 signaling pathway. Accordingly, we demonstrate that CD38 cross-linking does not result in the functional phosphorylation of PLC-γ2 nor an increase in inositol-1,4,5 trisphosphate production. Furthermore, splenic B cells exhibit a normal CD38-mediated, proliferative response in the presence of the phosphoinositide-PLC inhibitor, U73122. Conversely, protein kinase C (PKC) β-deficient mice, or PKC inhibitors, indicated the requirement for diacylglycerol-dependent PKC isoforms in this pathway. Loss of PKC activity blocked CD38-dependent, B cell proliferation, NF-κB activation, and subsequent expression of cyclin-D2. These results suggested that an alternate diacylglycerol-producing phospholipase must participate in CD38 signaling. Consistent with this idea, CD38 increased the enzymatic activity of the phosphatidylcholine (PC)-metabolizing enzymes, PC-PLC and phospholipase D. The PC-PLC inhibitor, D609, completely blocked CD38-dependent B cell proliferation, IκB-α degradation, and cyclin-D2 expression. Analysis of Btk mutant B cells demonstrated a partial requirement for Btk in the activation of both enzymes. Taken together, these data demonstrate that CD38 initiates a novel signaling cascade leading to Btk-, PC-PLC-, and phospholipase D-dependent, PLC-γ2-independent, B lymphocyte activation.
Differential Effects of IL-21 during Initiation and Progression of Autoimmunity against Neuroantigen
Authors: Vollmer Timothy L. , Liu Ruolan , Price Mary , Rhodes Susan , La Cava Antonio , Shi Fu-Dong
Published: April 2014

The cytokine IL-21 is closely related to IL-2 and IL-15, a cytokine family that uses the common γ-chain for signaling. IL-21 is expressed by activated CD4+ T cells. We examined the role of IL-21 in the autoimmune disease experimental autoimmune encephalomyelitis (EAE), an animal model for human multiple sclerosis. IL-21 administration before induction of EAE with a neuroantigen, myelin oligodendrocyte glycoprotein peptide 35-55, and adjuvant enhanced the inflammatory influx into the CNS, as well as the severity of EAE. Autoreactive T cells purified from IL-21-treated mice transferred more severe EAE than did the control encephalitogenic T cells. No such effects were observed when IL-21 was administered after EAE progressed. Additional studies demonstrated that IL-21 given before the induction of EAE boosted NK cell function, including secretion of IFN-γ. Depletion of NK cells abrogated the effect of IL-21. Therefore, IL-21, by affecting NK cells, has differential effects during the initiation and progression of autoimmune responses against neuroantigens.
Two Structurally Related Rat Ly49 Receptors with Opposing Functions (Ly49 Stimulatory Receptor 5 and Ly49 Inhibitory Receptor 5) Recognize Nonclassical MHC Class Ib-Encoded Target Ligands
Authors: Naper Christian , Dai Ke-Zheng , Kveberg Lise , Rolstad Bent , Niemi Eréne C. , Vaage John T. , Ryan James C.
Published: April 2014

The Ly49 family of lectin-like receptors in rodents includes both stimulatory and inhibitory members. Although NK alloreactivity in mice is regulated primarily by inhibitory Ly49 receptors, in rats activating Ly49 receptors are equally important. Previous studies have suggested that activating rat Ly49 receptors are triggered by polymorphic ligands encoded within the nonclassical class Ib region of the rat MHC, RT1-CE/N/M, while inhibitory Ly49 receptors bind to widely expressed classical class Ia molecules encoded from the RT1-A region. To further investigate rat Ly49-mediated regulation of NK alloreactivity, we report in this study the identification and characterization of two novel paired Ly49 receptors that we have termed Ly49 inhibitory receptor 5 (Ly49i5) and Ly49 stimulatory receptor 5 (Ly49s5). Using a new mAb (mAb Fly5), we showed that Ly49i5 is an inhibitory receptor that recognizes ligands encoded within the class Ib region of the u and l haplotypes, while the structurally related Ly49s5 is an activating receptor that recognizes class Ib ligands of the u haplotype. Ly49s5 is functionally expressed in the high NK-alloresponder PVG strain, but not in the low alloresponder BN strain, in which it is a pseudogene. Ly49s5 is hence not responsible for the striking anti-u NK alloresponse previously described in BN rats (haplotype n), which results from repeated alloimmunizations with u haplotype cells. The present studies support the notion of a complex regulation of rat NK alloreactivity by activating and inhibitory Ly49 members, which may be highly homologous in the extracellular region and bind similar class Ib-encoded target ligands.