bluuebunny/crossref_metadata_2025 huggingface dataset:
CD4+CD25+ Regulatory T Cells Specific for a Thymus-Expressed Antigen Prevent the Development of Anaphylaxis to Self
Authors: Scabeni Stefano , Lapilla Marilena , Musio Silvia , Gallo Barbara , Ciusani Emilio , Steinman Lawrence , Mantegazza Renato , Pedotti Rosetta
Published: April 2014

A role for CD4+CD25+ regulatory T cells (Tregs) in the control of allergic diseases has been postulated. We developed a mouse model in which anaphylaxis is induced in SJL mice by immunization and challenge with the fragment of self myelin proteolipid protein (PLP)139–151, that is not expressed in the thymus, but not with fragment 178–191 of the same protein, that is expressed in the thymus. In this study, we show that resistance to anaphylaxis is associated with naturally occurring CD4+CD25+ Tregs specific for the self peptide expressed in the thymus. These cells increase Foxp3 expression upon Ag stimulation and suppress peptide-induced proliferation of CD4+CD25− effector T cells. Depletion of Tregs with anti-CD25 in vivo significantly diminished resistance to anaphylaxis to PLP178–191, suggesting an important role for CD4+CD25+ Tregs in preventing the development of allergic responses to this thymus-expressed peptide. These data indicate that naturally occurring CD4+CD25+ Tregs specific for a peptide expressed under physiological conditions in the thymus are able to suppress the development of a systemic allergic reaction to self.
Autoimmunity to Both Proinsulin and IGRP Is Required for Diabetes in Nonobese Diabetic 8.3 TCR Transgenic Mice
Authors: Krishnamurthy Balasubramanian , Mariana Lina , Gellert Shane A. , Colman Peter G. , Harrison Leonard C. , Lew Andrew M. , Santamaria Pere , Thomas Helen E. , Kay Thomas W. H.
Published: April 2014

T cells specific for proinsulin and islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) induce diabetes in nonobese diabetic (NOD) mice. TCR transgenic mice with CD8+ T cells specific for IGRP206–214 (NOD8.3 mice) develop accelerated diabetes that requires CD4+ T cell help. We previously showed that immune responses against proinsulin are necessary for IGRP206–214-specific CD8+ T cells to expand. In this study, we show that diabetes development is dramatically reduced in NOD8.3 mice crossed to NOD mice tolerant to proinsulin (NOD-PI mice). This indicates that immunity to proinsulin is even required in the great majority of NOD8.3 mice that have a pre-existing repertoire of IGRP206–214-specific cells. However, protection from diabetes could be overcome by inducing islet inflammation either by a single dose of streptozotocin or anti-CD40 agonist Ab treatment. This suggests that islet inflammation can substitute for proinsulin-specific CD4+ T cell help to activate IGRP206–214-specific T cells.
Impact of Post-Thymic Cellular Longevity on the Development of Age-Associated CD4+ T Cell Defects
Authors: Jones Stephen C. , Clise-Dwyer Karen , Huston Gail , Dibble John , Eaton Sheri , Haynes Laura , Swain Susan L.
Published: April 2014

Elderly people are at higher risk for infections due to declining cellular and humoral immune responses. Central to this dysfunction is the reduced responsiveness of the naive CD4+ T cell compartment. Previous data from our laboratory suggest that although defects in the aged naive CD4+ T cell response are apparent in recent thymic emigrant populations, additional defects develop during extended post-thymic longevity in the periphery. To further investigate the factors that lead to aging defects, we took advantage of the OT-II TCR-transgenic (Tg) mouse model. We show that because of an apparent superantigen-mediated loss of naive Vβ5+ Tg CD4+ T cells from the periphery of aging OT-II mice, this compartment becomes enriched for cells of reduced post-thymic longevity, resulting in a frequency of recent thymic emigrants in aged mice that is similar to that of young mice. Purification and functional analysis of aged OT-II cells with reduced post-thymic longevity reveal that they have an age-associated decrease in expansion and IL-2 production in response to Ag in vitro. However, the in vivo expansion, IL-2 production, and cognate B cell helper ability of these cells are similar to those of cells from young mice. In contrast, T cells from aged HNT Tg mice demonstrate extended post-thymic longevity and exhibit severe defects in the same in vitro and in vivo models. These data support a correlation between the requirement for increased post-thymic longevity and the development of the most severe naive CD4+ T cell-aging defects.
Selective Roles of MAPKs during the Macrophage Response to IFN-γ
Authors: Valledor Annabel F. , Sánchez-Tilló Ester , Arpa Luis , Park Jin Mo , Caelles Carme , Lloberas Jorge , Celada Antonio
Published: April 2014

Macrophages perform essential functions in the infection and resolution of inflammation. IFN-γ is the main endogenous macrophage Th1 type activator. The classical IFN-γ signaling pathway involves activation of Stat-1. However, IFN-γ has also the capability to activate members of the MAPK family. In primary bone marrow-derived macrophages, we have observed strong activation of p38 at early time points of IFN-γ stimulation, whereas weak activation of ERK-1/2 and JNK-1 was detected at a more delayed stage. In parallel, IFN-γ exerted repressive effects on the expression of a number of MAPK phosphatases. By using selective inhibitors and knockout models, we have explored the contributions of MAPK activation to the macrophage response to IFN-γ. Our findings indicate that these kinases regulate IFN-γ-mediated gene expression in a rather selective way: p38 participates mainly in the regulation of the expression of genes required for the innate immune response, including chemokines such as CCL5, CXCL9, and CXCL10; cytokines such as TNF-α; and inducible NO synthase, whereas JNK-1 acts on genes involved in Ag presentation, including CIITA and genes encoding MHC class II molecules. Modest effects were observed for ERK-1/2 in these studies. Interestingly, some of the MAPK-dependent changes in gene expression observed in these studies are based on posttranscriptional regulation of mRNA stability.
Inducing Antitumor T Cell Immunity: Comparative Functional Analysis of Interstitial Versus Langerhans Dendritic Cells in a Human Cell Line Model
Authors: Santegoets Saskia J. A. M. , Bontkes Hetty J. , Stam Anita G. M. , Bhoelan Farien , Ruizendaal Janneke J. , van den Eertwegh Alfons J. M. , Hooijberg Erik , Scheper Rik J. , de Gruijl Tanja D.
Published: April 2014

Dendritic cells (DC) are increasingly applied as a cellular adjuvant in immunotherapy of cancer. Two major myeloid DC subsets are recognized: interstitial DC (IDC) that infiltrate connective tissues and Langerhans cells (LC) that line epithelial surfaces. Yet, functional differences between IDC and LC remain to be defined. We recently showed that the CD34+ acute myeloid leukemia cell line MUTZ-3 supports differentiation of both DC-SIGN+ IDC and Langerin-positive Birbeck granule-expressing LC. By comparative functional characterization of MUTZ-3 IDC and MUTZ-3 LC, we aimed to elucidate the relative abilities of these two DC subsets to induce a specific T cell response and reveal the more suitable candidate for use as a clinical vehicle of tumor vaccines. Although mature LC and IDC displayed comparable lymph node-homing potential, mature LC showed higher allogeneic T cell stimulatory capacity. Nevertheless, IDC supported the induction of tumor Ag-specific CD8+ T cells at an overall higher efficiency. This might be related to the observed inability of LC to release T cell stimulatory cytokines such as IL-12p70, IL-23, and IL-15. Although this inability did not result in a detectable deviation in the cytokine expression profile of primed T cells, transduction with IL-12p70 significantly improved priming efficiency of LC, and ensured a functional equivalence with IDC in this regard. In conclusion, except for the inability of LC to release distinct type 1 T cell stimulatory cytokines, in vitro function of LC and IDC suggests comparable abilities of both subsets for the in vivo induction of antitumor T cells.
Dendritic Cell-Dependent Inhibition of B Cell Proliferation Requires CD22
Authors: Santos Lorna , Draves Kevin E. , Boton Mark , Grewal Prabhjit K. , Marth Jamey D. , Clark Edward A.
Published: April 2014

Recent studies have shown that dendritic cells (DCs) regulate B cell functions. In this study, we report that bone marrow (BM)-derived immature DCs, but not mature DCs, can inhibit BCR-induced proliferation of B cells in a contact-dependent manner. This inhibition is overcome by treatment with BAFF and is dependent on the BCR coreceptor CD22; however, it is not dependent on expression of the CD22 glycan ligand(s) produced by ST6Gal-I sialyltransferase. We found that a second CD22 ligand (CD22L) is expressed on CD11c+ splenic and BM-derived DCs, which does not contain ST6Gal-I-generated sialic acids and which, unlike the B cell-associated CD22L, is resistant to neuraminidase treatment and sodium metaperiodate oxidation. Examination of splenic and BM B cell subsets in CD22 and ST6Gal-I knockout mice revealed that ST6Gal-I-generated B cell CD22L plays a role in splenic B cell development, whereas the maintenance of long-lived mature BM B cells depends only on CD22 and not on α2,6-sialic acids produced by ST6Gal-I. We propose that the two distinct CD22L have different functions. The α2,6-sialic acid-containing glycoprotein is important for splenic B cell subset development, whereas the DC-associated ST6Gal-I-independent CD22L may be required for the maintenance of long-lived mature B cells in the BM.
TRAIL Deficiency Does Not Rescue Impaired CD8+ T Cell Memory Generated in the Absence of CD4+ T Cell Help
Authors: Sacks Jilian A. , Bevan Michael J.
Published: April 2014

Ag-specific CD8+ T cells immunized in the absence of CD4+ T cell help, so-called “unhelped” CD8+ T cells, are defective in function and survival. We investigated the role of the proapoptotic molecule TRAIL in this defect. We first demonstrate that TRAIL does not contribute to the CD8+ T cell response to Listeria monocytogenes strain expressing OVA (LmOVA) in the presence of CD4+ T cells. Secondly, we generated mice doubly deficient in CD4+ T cells and TRAIL and analyzed their CD8+ T cell response to LmOVA. Memory CD8+ T cells in double-deficient mice waned over time and were not protective against rechallenge, similar to their TRAIL-sufficient unhelped counterparts. To avoid the effects of CD4+ T cell deficiency during memory maintenance, and to address whether TRAIL plays a role in the early programming of the CD8+ T cell response, we performed experiments using heterologous prime and early boost immunizations. We did not observe activation-induced cell death of unhelped CD8+ T cells when mice were infected with followed vaccinia virus expressing OVA 9 days later by LmOVA infection. Furthermore, primary immunization of CD4+ T cell-deficient mice with cell-associated Ag followed by LmOVA infection did not reveal a role for TRAIL-mediated activation-induced cell death. Overall, our results suggest that CD4+ T cell help for the CD8+ T cell response is not contingent on the silencing of TRAIL expression and prevention of TRAIL-mediated apoptosis.
Immunodominance in the TCR Repertoire of αTCR Peptide-Specific CD4+ Treg Population That Controls Experimental Autoimmune Encephalomyelitis
Authors: Madakamutil Loui Thomas , Maricic Igor , Sercarz Eli E. , Kumar Vipin
Published: April 2014

Immunodominance in self-Ag-reactive pathogenic CD4+ T cells has been well established in several experimental models. Although it is clear that regulatory lymphocytes (Treg) play a crucial role in the control of autoreactive cells, it is still not clear whether immunodominant CD4+ Treg clones are also involved in control of autoreactivity. We have shown that TCR-peptide-reactive CD4+ and CD8+ Treg play an important role in the spontaneous recovery and resistance from reinduction of experimental autoimmune encephalomyelitis in B10.PL mice. We report, by sequencing of the TCR α- and β-chain associated with CD4+ Treg, that the TCR repertoire is limited and the majority of CD4+ Treg use the TCR Vβ14 and Vα4 gene segments. Interestingly, sequencing and spectratyping data of cloned and polyclonal Treg populations revealed that a dominant public CD4+ Treg clonotype expressing Vβ14-Jβ1.2 with a CDR3 length of 7 aa exists in the naive peripheral repertoire and is expanded during the course of recovery from experimental autoimmune encephalomyelitis. Furthermore, a higher frequency of CD4+ Treg clones in the naive repertoire correlates with less severity and more rapid spontaneous recovery from disease in parental B10.PL or PL/J and (B10.PL × PL/J)F1 mice. These findings suggest that unlike the Ag-nonspecific, diverse TCR repertoire among the CD25+CD4+ Treg population, TCR-peptide-reactive CD4+ Treg involved in negative feedback regulation of autoimmunity use a highly limited TCR V-gene repertoire. Thus, a selective set of immunodominant Treg as well as pathogenic T cell clones can be targeted for potential intervention in autoimmune disease conditions.
Induction of Antitumor Immune Response by Homeostatic Proliferation and CD28 Signaling
Authors: Suzuki Toshihiro , Ogawa Shuhei , Tanabe Kazunari , Tahara Hideaki , Abe Ryo , Kishimoto Hidehiro
Published: April 2014

Inducing lymphopenia before adoptive cell transfer can improve the antitumor effect of donor immune cells. It was recently reported that lymphopenic conditions can initiate the differentiation of naive T cells into effector cells. Although T cells require a specific “strong” signal via TCR as well as costimulatory signals during Ag-driven differentiation, there has been little evidence to suggest any requirement for costimulatory signaling for the differentiation of naive T cells in a lymphopenic host. In this study, we demonstrate that naive CD8+ T cells are indispensable for induction of antitumor effect, and, in addition to Ag-driven differentiation, CD28 signaling is essential for the differentiation of naive CD8+ T cells into functional effector CTLs during homeostatic proliferation (HP). The systemic administration of IL-2 did not restore the antitumor effect induced by HP in the absence of CD28 signaling. These results suggest that homeostatic cytokines enable CD8+ T cells to expand and survive, and that TCR and the CD28 signal initiate the differentiation of effector functions. A deeper understanding of the mechanisms underlying enhanced induction of the antitumor immune response with accompanying HP may allow us to more precisely induce enhanced immunity with costimulation signaling and the administration of common γ-chain cytokines.
Invariant NKT Cells Promote CD8+ Cytotoxic T Cell Responses by Inducing CD70 Expression on Dendritic Cells
Authors: Taraban Vadim Y. , Martin Sonya , Attfield Kathrine E. , Glennie Martin J. , Elliott Tim , Elewaut Dirk , Van Calenbergh Serge , Linclau Bruno , Al-Shamkhani Aymen
Published: April 2014

Activation of invariant NK T (iNKT) cells with the glycolipid α-galactosylceramide promotes CD8+ cytotoxic T cell responses, a property that has been used to enhance the efficacy of antitumor vaccines. Using chimeric mice, we now show that the adjuvant properties of iNKT cells require that CD40 triggering and Ag presentation to CD8+ T cells occur on the same APCs. We demonstrate that injection of α-galactosylceramide triggers CD70 expression on splenic T cell zone dendritic cells and that this is dependent on CD40 signaling. Importantly, we show that blocking the interaction between CD70 and CD27, its costimulatory receptor on T cells, abrogates the ability of iNKT cells to promote a CD8+ T cell response and abolishes the efficacy of α-GalCer as an adjuvant for antitumor vaccines. These results define a key role for CD70 in linking the innate response of iNKT cells to the activation of CD8+ T cells.
Multivectorial Abnormal Cell Migration in the NOD Mouse Thymus
Authors: Mendes-da-Cruz Daniella A. , Smaniotto Salete , Keller Alexandre C. , Dardenne Mireille , Savino Wilson
Published: April 2014

We previously described a fibronectin/VLA-5-dependent impairment of NOD thymocyte migration, correlated with partial thymocyte arrest within thymic perivascular spaces. Yet, NOD thymocytes still emigrate, suggesting the involvement of other cell migration-related alterations. In this context, the aim of this work was to study the role of extracellular matrix ligands, alone or in combination with the chemokine CXCL12, in NOD thymocyte migration. Intrathymic contents of CXCL12, fibronectin, and laminin were evaluated by immunohistochemistry while the expression of corresponding receptors was ascertained by flow cytometry. Thymocyte migration was measured using Transwell chambers and transendothelial migration was evaluated in the same system, but using an endothelial cell monolayer within the chambers. NOD thymocytes express much lower VLA-5 than C57BL/6 thymocytes. This defect was particularly severe in CD4+ thymocytes expressing Foxp3, thus in keeping with the arrest of Foxp3+ cells within the NOD giant perivascular spaces. We observed an enhancement in CXCL12, laminin, and fibronectin deposition and colocalization in the NOD thymus. Furthermore, we detected altered expression of the CXCL12 receptor CXCR4 and the laminin receptor VLA-6, as well as enhanced migratory capacity of NOD thymocytes toward these molecules, combined or alone. Moreover, transendothelial migration of NOD thymocytes was diminished in the presence of exogenous fibronectin. Our data unravel the existence of multiple cell migration-related abnormalities in NOD thymocytes, comprising both down- and up-regulation of specific responses. Although remaining to be experimentally demonstrated, these events may have consequences on the appearance of autoimmunity in NOD mice.
Protein Kinase Cα and ζ Differentially Regulate Death-Inducing Signaling Complex Formation in Cigarette Smoke Extract-Induced Apoptosis
Authors: Park Jeong-Woong , Kim Hong Pyo , Lee Seon-Jin , Wang Xue , Wang Yong , Ifedigbo Emeka , Watkins Simon C. , Ohba Motoi , Ryter Stefan W. , Vyas Yatin M. , Choi Augustine M. K.
Published: April 2014

Cigarette smoke, a major risk factor in emphysema, causes cell death by incompletely understood mechanisms. Death-inducing signaling complex (DISC) formation is an initial event in Fas-mediated apoptosis. We demonstrate that cigarette smoke extract (CSE) induces DISC formation in human lung fibroblasts (MRC-5) and promotes DISC trafficking from the Golgi complex to membrane lipid rafts. We demonstrate a novel role of protein kinase C (PKC) in the regulation of DISC formation and trafficking. The PKC isoforms, PKCα, ζ, ε, and η, were activated by CSE exposure. Overexpression of wild-type PKCα inhibited, while PKCζ promoted, CSE-induced cell death. Dominant-negative (dn)PKCζ protected against CSE-induced cell death by suppressing DISC formation and caspase-3 activation, while dnPKCα enhanced cell death by promoting these events. DISC formation was augmented by wortmannin, an inhibitor of PI3K. CSE-induced Akt phosphorylation was reduced by dnPKCα, but it was increased by dnPKCζ. Expression of PKCα in vivo inhibited DISC formation, caspase-3/8 activation, lung injury, and cell death after prolonged cigarette smoke exposure, whereas expression of PKCζ promoted caspase-3 activation. In conclusion, CSE-induced DISC formation is differentially regulated by PKCα and PKCζ via the PI3K/Akt pathway. These results suggest that modulation of PKC may have therapeutic potential in the prevention of smoke-related lung injury.
Regulatory T Cells Control Dendritic Cell/NK Cell Cross-Talk in Lymph Nodes at the Steady State by Inhibiting CD4+ Self-Reactive T Cells
Authors: Terme Magali , Chaput Nathalie , Combadiere Behazine , Ma Averil , Ohteki Toshiaki , Zitvogel Laurence
Published: April 2014

The CD4+CD25+Foxp3+ regulatory T cells (Treg) play an important role in the control of peripheral tolerance by directly inhibiting conventional T cell proliferative and effector functions. However, the mechanisms by which Treg regulate the homeostasis of lymph nodes remain unclear. In this study, we show in a mouse model that Treg control two major checkpoints dictated by the interaction between self-reactive CD4+ T cells and resident dendritic cell (DC) in secondary lymphoid organs. First, Treg inhibit the production of CCR5 ligands, limiting the CCR5-dependent recruitment of DC in the lymph nodes. Second, Treg prevent the DC exposure of IL-15Rα, markedly interfering in the DC-mediated NK cell proliferation in vivo. Therefore, the DC/T cell autoreactivity leading to NK cell triggering could potentially be controlled by the coinhibition of both IL-15Rα and CCR5 in autoimmune disorders in which NK cells play a deleterious role.
Renal Cell Carcinoma Tumors Induce T Cell Apoptosis through Receptor-Dependent and Receptor-Independent Pathways
Authors: Das Tanya , Sa Gaurisankar , Paszkiewicz-Kozik Ewa , Hilston Cynthia , Molto Luis , Rayman Patricia , Kudo Daisuke , Biswas Kaushik , Bukowski Ronald M. , Finke James H. , Tannenbaum Charles S.
Published: April 2014

Tumors can promote their own progressive growth by inducing T cell apoptosis. Though previous studies suggested that tumor-mediated T cell killing is receptor dependent, we recently showed that tumor gangliosides also participate, a notion consistent with reports indicating that, in some cell types, gangliosides can activate the intrinsic apoptotic pathway by stimulating reactive oxygen species production, cytochrome c release, and caspase-9 activation. In this study, we used normal peripheral blood T cells, as well as caspase-8-, caspase-9-, and Fas-associated death domain protein-deficient Jurkat cells, to assess whether the death ligands and gangliosides overexpressed by the renal cell carcinoma (RCC) cell line SK-RC-45 can independently stimulate T cell apoptosis as a mechanism of immune escape. Anti-FasL Abs and the glycosylceramide synthase inhibitor 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP) each partially inhibited the ability of SK-RC-45 to kill cocultured activated T cells; together, as purified molecules, RCC gangliosides and rFasL induced a more extensive mitochondrial permeability transition and greater levels of apoptosis than either agent alone, equivalent to that induced by the FasL- and ganglioside-expressing RCC line itself. rFasL-mediated apoptosis was completely inhibited in caspase-8- and Fas-associated death domain protein-negative Jurkat cells, though apoptosis induced by purified gangliosides remained intact, findings that correlate with the observed partial inhibition of SK-RC-45-induced apoptosis in the Jurkat lines with defective death receptor signaling. Western blot analysis performed on lysates made from wild-type and mutant Jurkat cells cocultured with SK-RC-45 revealed caspase activation patterns and other biochemical correlates which additionally supported the concept that tumor-associated gangliosides and FasL independently activate the caspase cascade in T cells through the intrinsic and extrinsic pathways, respectively.
Hypoxia and Hypoxia-Inducible Factor-1α Modulate Lipopolysaccharide-Induced Dendritic Cell Activation and Function
Authors: Jantsch Jonathan , Chakravortty Dipshikha , Turza Nadine , Prechtel Alexander T. , Buchholz Björn , Gerlach Roman G. , Volke Melanie , Gläsner Joachim , Warnecke Christina , Wiesener Michael S. , Eckardt Kai-Uwe , Steinkasserer Alexander , Hensel Michael , Willam Carsten
Published: April 2014

Dendritic cells (DC) play a key role in linking innate and adaptive immunity. In inflamed tissues, where DC become activated, oxygen tensions are usually low. Although hypoxia is increasingly recognized as an important determinant of cellular functions, the consequences of hypoxia and the role of one of the key players in hypoxic gene regulation, the transcription factor hypoxia inducible factor 1α (HIF-1α), are largely unknown. Thus, we investigated the effects of hypoxia and HIF-1α on murine DC activation and function in the presence or absence of an exogenous inflammatory stimulus. Hypoxia alone did not activate murine DC, but hypoxia combined with LPS led to marked increases in expression of costimulatory molecules, proinflammatory cytokine synthesis, and induction of allogeneic lymphocyte proliferation compared with LPS alone. This DC activation was accompanied by accumulation of HIF-1α protein levels, induction of glycolytic HIF target genes, and enhanced glycolytic activity. Using RNA interference techniques, knockdown of HIF-1α significantly reduced glucose use in DC, inhibited maturation, and led to an impaired capability to stimulate allogeneic T cells. Alltogether, our data indicate that HIF-1α and hypoxia play a crucial role for DC activation in inflammatory states, which is highly dependent on glycolysis even in the presence of oxygen.
Langerin Expressing Cells Promote Skin Immune Responses under Defined Conditions
Authors: Wang Liangchun , Bursch Laura S. , Kissenpfennig Adrien , Malissen Bernard , Jameson Stephen C. , Hogquist Kristin A.
Published: April 2014

There are conflicting data in the literature regarding the role of epidermal Langerhans cells (LC) in promoting skin immune responses. On one hand, LC can be extremely potent APCs in vitro, and are thought to be involved in contact hypersensitivity (CHS). On the other hand, it seems counterintuitive that a cell type continually exposed to pathogens at the organism’s barrier surfaces should readily trigger potent T cell responses. Indeed, LC depletion in one model led to enhanced contact hypersensitivity, suggesting they play a negative regulatory role. However, apparently similar LC depletion models did not show enhanced CHS, and in one case showed reduced CHS. In this study we found that acute depletion of mouse LC reduced CHS, but the timing of toxin administration was critical: toxin administration 3 days before priming did not impair CHS, whereas toxin administration 1 day before priming did. We also show that LC elimination reduced the T cell response to epicutaneous immunization with OVA protein Ag. However, this reduction was only observed when OVA was applied on the flank skin, and not on the ear. Additionally, peptide immunization was not blocked by depletion, regardless of the site. Finally we show that conditions which eliminate epidermal LC but spare other Langerin+ DC do not impair the epicutaneous immunization response to OVA. Overall, our results reconcile previous conflicting data in the literature, and suggest that Langerin+ cells do promote T cell responses to skin Ags, but only under defined conditions.
Homeostasis and Effector Function of Lymphopenia-Induced “Memory-Like” T Cells in Constitutively T Cell-Depleted Mice
Authors: Voehringer David , Liang Hong-Erh , Locksley Richard M.
Published: April 2014

Naive T lymphocytes acquire a phenotype similar to Ag-experienced memory T cells as a result of proliferation under lymphopenic conditions. Such “memory-like” T (TML) cells constitute a large fraction of the peripheral T cell pool in patients recovering from T cell ablative therapies, HIV patients under highly active antiretroviral therapy, and in the elderly population. To generate a model that allows characterization of TML cells without adoptive transfer, irradiation, or thymectomy, we developed genetically modified mice that express diphtheria toxin A under control of a loxP-flanked stop cassette (R-DTA mice). Crossing these mice to CD4Cre mice resulted in efficient ablation of CD4 single-positive thymocytes, whereas double-positive and CD8 single-positive thymocytes were only partially affected. In the periphery the pool of naive (CD44lowCD62Lhigh) T cells was depleted. However, some T cells were resistant to Cre activity, escaped deletion in the thymus, and underwent lymphopenia-induced proliferation resulting in a pool of TML cells that was similar in size and turnover to the pool of CD44highCD62Llow “memory phenotype” T cells in control mice. CD4Cre/R-DTA mice remained lymphopenic despite the large available immunological “space” and normal Ag-induced T cell proliferation. CD4Cre/R-DTA mice showed a biased TCR repertoire indicating oligoclonal T cell expansion. Infection with the helminth Nippostrongylus brasiliensis resulted in diminished effector cell recruitment and impaired worm expulsion, demonstrating that TML cells are not sufficient to mediate an effective immune response.
Doc2α and Munc13-4 Regulate Ca2+-Dependent Secretory Lysosome Exocytosis in Mast Cells
Authors: Higashio Hironori , Nishimura Noriyuki , Ishizaki Hiroyoshi , Miyoshi Jun , Orita Satoshi , Sakane Ayuko , Sasaki Takuya
Published: April 2014

The Doc2 family comprises the brain-specific Doc2α and the ubiquitous Doc2β and Doc2γ. With the exception of Doc2γ, these proteins exhibit Ca2+-dependent phospholipid-binding activity in their Ca2+-binding C2A domain and are thought to be important for Ca2+-dependent regulated exocytosis. In excitatory neurons, Doc2α interacts with Munc13-1, a member of the Munc13 family, through its N-terminal Munc13-1-interacting domain and the Doc2α-Munc13-1 system is implicated in Ca2+-dependent synaptic vesicle exocytosis. The Munc13 family comprises the brain-specific Munc13-1, Munc13-2, and Munc13-3, and the non-neuronal Munc13-4. We previously showed that Munc13-4 is involved in Ca2+-dependent secretory lysosome exocytosis in mast cells, but the involvement of Doc2 in this process is not determined. In the present study, we found that Doc2α but not Doc2β was endogenously expressed in the RBL-2H3 mast cell line. Doc2α colocalized with Munc13-4 on secretory lysosomes, and interacted with Munc13-4 through its two regions, the N terminus containing the Munc13-1-interacting domain and the C terminus containing the Ca2+-binding C2B domain. In RBL-2H3 cells, Ca2+-dependent secretory lysosome exocytosis was inhibited by expression of the Doc2α mutant lacking either of the Munc13-4-binding regions and the inhibition was suppressed by coexpression of Munc13-4. Knockdown of endogenous Doc2α also reduced Ca2+-dependent secretory lysosome exocytosis, which was rescued by re-expression of human Doc2α but not by its mutant that could not bind to Munc13-4. Moreover, Ca2+-dependent secretory lysosome exocytosis was severely reduced in bone marrow-derived mast cells from Doc2α knockout mice. These results suggest that the Doc2α-Μunc13-4 system regulates Ca2+-dependent secretory lysosome exocytosis in mast cells.
STAT3-Mediated Up-Regulation of BLIMP1 Is Coordinated with BCL6 Down-Regulation to Control Human Plasma Cell Differentiation
Authors: Diehl Sean A. , Schmidlin Heike , Nagasawa Maho , van Haren Simon D. , Kwakkenbos Mark J. , Yasuda Etsuko , Beaumont Tim , Scheeren Ferenc A. , Spits Hergen
Published: April 2014

STAT family members have been implicated in regulating the balance between B cell lymphoma (BCL)6 and B lymphocyte induced maturation protein (BLIMP)1 to control plasma cell differentiation. We previously showed that STAT5 induces BCL6 to block plasma cell differentiation and extend the life span of human B cells. The heterogeneity in STAT activation by cytokines and their effects on B cell differentiation prompted us to investigate the effect of STAT3 activation in plasma cell differentiation. First stimulation with IL-21, which promotes plasma cell differentiation, induced robust and prolonged STAT3 activation in primary human B cells. We then investigated effects of direct STAT3 activation on regulation of plasma cell genes, cellular phenotype, and Ig production. Activation of a tamoxifen-regulated STAT3-estrogen receptor fusion protein triggered BLIMP1 mRNA and protein up-regulation, plasma cell phenotypic features, and Ig secretion. When STAT3 was activated by IL-21 in B cells ectopically expressing BCL6, BLIMP1 was up-regulated, but only partial plasma cell differentiation was achieved. Lastly, through coexpression of BCL6 and STAT3-ER, we verified that STAT3 activation functionally mimicked IL-21 treatment and that STAT3-mediated BLIMP1 up-regulation occurred despite high BCL6 expression levels indicating that BCL6 is not the dominant repressor of BLIMP1. Thus, up-regulation of BLIMP1 alone is not sufficient for differentiation of primary human B cells into plasma cells; concomitant down-regulation of BCL6 is absolutely required for completion of the plasma cell differentiation program.
Nonhuman Primate IgA: Genetic Heterogeneity and Interactions with CD89
Authors: Rogers Kenneth A. , Jayashankar Lakshmi , Scinicariello Franco , Attanasio Roberta
Published: April 2014

Nonhuman primates are extremely valuable animal models for a variety of human diseases. However, it is now becoming evident that these models, although widely used, are still uncharacterized. The major role that nonhuman primate species play in AIDS research as well as in the testing of Ab-based therapeutics requires the full characterization of structure and function of their Ab molecules. IgA is the Ab class mostly involved in protection at mucosal surfaces. By binding to its specific Fc receptor CD89, IgA plays additional and poorly understood roles in immunity. Therefore, Ig heavy α (IGHA) constant (C) genes were cloned and sequenced in four different species (rhesus macaques, pig-tailed macaques, baboons, and sooty mangabeys). Sequence analysis confirmed the high degree of intraspecies polymorphism present in nonhuman primates. Individual animals were either homozygous or heterozygous for IGHA genes. Highly variable hinge regions were shared by animals of different geographic origins and were present in different combinations in heterozygous animals. Therefore, it appears that although highly heterogeneous, hinge sequences are present only in limited numbers in various nonhuman primate populations. A macaque recombinant IgA molecule was generated and used to assess its interaction with a recombinant macaque CD89. Macaque CD89 was able to bind its native ligand as well as human IgA1 and IgA2. Presence of Ag enhanced macaque IgA binding and blocking of macaque CD89 N-glycosylation reduced CD89 expression. Together, our results suggest that, despite the presence of IgA polymorphism, nonhuman primates appear suitable for studies that involve the IgA/CD89 system.
Dendritic Cell Programming by Cytomegalovirus Stunts Naive T Cell Responses via the PD-L1/PD-1 Pathway
Authors: Benedict Chris A. , Loewendorf Andrea , Garcia Zacarias , Blazar Bruce R. , Janssen Edith M.
Published: April 2014

Early during infection, CMV targets dendritic cells (DC) and alters their functions. Herein we show that CMV-infected DC maintain the ability to present both virus-derived and exogenous Ags, but that they actively induce tolerance or anergy in Ag-specific T cells. CMV accomplishes this by selectively maintaining high-level expression of the negative costimulatory molecule programmed death ligand-1 (PD-L1), while commensurately down-regulating positive costimulatory molecules and MHC on the DC surface. Consequently, CD4 and CD8 T cells activated by these infected DC have a stunted phenotype, characterized by poor proliferation, effector function. and recall responses. Blocking PD-L1, but not PD-L2, during direct priming of naive T cells by infected DC significantly restores Ag-specific T cell functions. Using systems where direct and cross-priming of T cells can be distinguished revealed that PD-L1/PD-1 signaling contributes only when naive T cells are primed directly by infected DC, and not upon cross-presentation of viral Ags by uninfected DC. These data suggest that murine CMV programs infected DC during acute infection to inhibit early host adaptive antiviral responses by tipping the balance between negative and positive cosignals.
Age-Related Dysregulation of CD8+ T Cell Memory Specific for a Persistent Virus Is Independent of Viral Replication
Authors: Lang Anna , Brien James D. , Messaoudi Ilhem , Nikolich-Žugich Janko
Published: April 2014

The immune system devotes substantial resources to the lifelong control of persistent pathogens, which were hypothesized to play an important role in immune aging. Specifically, the presence of latent herpesviruses has been correlated with immune exhaustion and shorter lifespan in octogenarians. But neither the causality nor the mechanistic link(s) were established, and the relative roles of persistent antigenic stimulation and of virus-independent homeostatic disturbances in T cell aging remain unresolved. We longitudinally analyzed expansion, contraction, and long-term maintenance of CD8+ T cells responding to localized infection with a latent virus, HSV-1. Young mice exhibited the expected expansion and contraction of HSV-1-specific cells and the stable maintenance of memory T cells into advanced adulthood. However, upon entry into senescence, many (>40%) animals exhibited an accumulation in Ag-specific cells (memory inflation) which in some animals was comparable to that observed in acute infection. Inflation occurred to the same extent in control mice and mice continuously treated with the anti-HSV drug famciclovir, which inhibits viral replication and was able to reduce expression of the glycoprotein B. Age-related inflation was also found long after infection with an acute virus. The inflating cells largely maintained Ag-specific function, and exhibited typical central memory phenotype, with no signs of Ag-specific activation. They exhibited increased expression of CD122 and CD127, akin to the Ag-independent T cell clonal expansions found in old specific pathogen-free laboratory mice. This collectively suggests that, in this model, the inflating cells may be selected for high responsiveness to environmental cytokines largely in an Ag-independent manner.
Complement Factor H-Binding Protein, a Putative Virulence Determinant of Borrelia hermsii, Is an Antigenic Target for Protective B1b Lymphocytes
Authors: Colombo Matthew J. , Alugupalli Kishore R.
Published: April 2014

Vaccination is the most effective way to control infectious diseases. A variety of microbial pathogens use antigenic variation, an immune evasion strategy that poses a challenge for vaccine development. To understand protective immune responses against such pathogens, we have been studying Borrelia hermsii, a bacterium that causes recurrent bacteremia due to antigenic variation. An IgM response is necessary and sufficient to control B. hermsii infection. We have recently found a selective expansion of B1b cells concurrent with the resolution of B. hermsii bacteremia. B1b cells from convalescent but not naive mice confer long-lasting immunity, but the Ag(s) driving the protective IgM responses is unknown. Herein we demonstrate that convalescent B1b cell-derived IgM recognizes complement factor H-binding protein (FhbA), a B. hermsii outer-surface protein and putative virulence factor that does not undergo antigenic variation and is expressed by all clinical isolates. A progressive increase in the IgM response to FhbA correlated with the kinetics of B1b cell expansion, diminished the severity of bacteremic episodes, and led to the eventual resolution of the infection. These data indicate that FhbA is a specific target for protective B1b cell responses. Ags recognized by B1b cells may be considered as an important component in vaccination strategies.
Mouse Mast Cell Tryptase mMCP-6 Is a Critical Link between Adaptive and Innate Immunity in the Chronic Phase ofTrichinella spiralisInfection
Authors: Shin Kichul , Watts Gerald F. M. , Oettgen Hans C. , Friend Daniel S. , Pemberton Alan D. , Gurish Michael F. , Lee David M.
Published: April 2014

Although the innate immune function of mast cells in the acute phase of parasitic and bacterial infections is well established, their participation in chronic immune responses to indolent infection remains incompletely understood. In parasitic infection with Trichinella spiralis, the immune response incorporates both lymphocyte and mast cell-dependent effector functions for pathogen eradication. Among the mechanistic insights still unresolved in the reaction to T. spiralis are the means by which mast cells respond to parasites and the mast cell effector functions that contribute to the immunologic response to this pathogen. We hypothesized that mast cell elaboration of tryptase may comprise an important effector component in this response. Indeed, we find that mice deficient in the tryptase mouse mast cell protease-6 (mMCP-6) display a significant difference in their response to T. spiralis larvae in chronically infected skeletal muscle tissue. Mechanistically, this is associated with a profound inability to recruit eosinophils to larvae in mMCP-6-deficient mice. Analysis of IgE-deficient mice demonstrates an identical defect in eosinophil recruitment. These findings establish that mast cell secretion of the tryptase mMCP-6, a function directed by the activity of the adaptive immune system, contributes to eosinophil recruitment to the site of larval infection, thereby comprising an integral link in the chronic immune response to parasitic infection.
Relative Levels of M-CSF and GM-CSF Influence the Specific Generation of Macrophage Populations during Infection withMycobacterium tuberculosis
Authors: Higgins David M. , Sanchez-Campillo Joaquin , Rosas-Taraco Adrian G. , Higgins Jonathan R. , Lee Eric J. , Orme Ian M. , Gonzalez-Juarrero Mercedes
Published: April 2014

Members of the CSF cytokine family play important roles in macrophage recruitment and activation. However, the role of M-CSF in pulmonary infection with Mycobacterium tuberculosis is not clear. In this study, we show the lungs of mice infected with M. tuberculosis displayed a progressive decrease in M-CSF in contrast to increasing levels of GM-CSF. Restoring pulmonary M-CSF levels during infection resulted in a significant decrease in the presence of foamy macrophages and increased expression of CCR7 and MHC class II, specifically on alveolar macrophages. In response to M-CSF, alveolar macrophages also increased their T cell-stimulating capacity and expression of DEC-205. These studies show that the levels of expression of M-CSF and GM-CSF participate in the progression of macrophages into foamy cells and that these cytokines are important factors in the differentiation and regulation of expression of dendritic cell-associated markers on alveolar macrophages. In addition, these studies demonstrate that M-CSF may have a role in the adaptive immune response to infection with M. tuberculosis.
MDA5 Participates in the Detection of Paramyxovirus Infection and Is Essential for the Early Activation of Dendritic Cells in Response to Sendai Virus Defective Interfering Particles
Authors: Yount Jacob S. , Gitlin Leonid , Moran Thomas M. , López Carolina B.
Published: April 2014

Defective interfering (DI) particles are byproducts of virus replication that potently enhance dendritic cell (DC) maturation by virus infection. DI particles have been reported for many different viruses and are strong inducers of type I IFNs. The cellular mechanisms involved in the response to DI particles are not known. In this study, we show that 1) DI particles are recognized by more than one viral sensor independently of TLRs and type I IFN signaling; 2) The helicase MDA5 participates in the detection of DI genomes as MDA5-deficient DCs respond inefficiently to Sendai virus stocks containing DI particles; 3) DI particles stimulate the expression of IRF3-responsive genes by a uniquely potent mechanism when compared with other prototypic viral stimulus; and 4) the efficient detection of DI particles overcomes virus immune antagonism. These data highlight the outstanding adjuvant capacity of DI particles in stimulating mouse and human DCs. They also offer biological relevance to the previously reported inhibition of MDA5 by different paramyxovirus V proteins. The unique mechanism by which DI particles trigger the maturation of DCs represents a novel strategy that could be further exploited for the development of potent adjuvant molecules.
Adenoviral Expression of Suppressor of Cytokine Signaling-1 Reduces Adenovirus Vector-Induced Innate Immune Responses
Authors: Sakurai Haruna , Tashiro Katsuhisa , Kawabata Kenji , Yamaguchi Tomoko , Sakurai Fuminori , Nakagawa Shinsaku , Mizuguchi Hiroyuki
Published: April 2014

Adenovirus (Ad) vectors are among the most commonly used viral vectors in gene therapy clinical trials. However, the application of Ad vectors has been limited to local injection in many cases, because the systemic administration of Ad vectors triggers innate immune responses such as inflammatory cytokine production and tissue damage. To overcome this limitation, it will be necessary to develop safer Ad vectors less likely to induce the innate immune response. In the present study, we demonstrated that a suppressor of cytokine signaling-1 (SOCS1)-expressing Ad vector, Ad-SOCS1, reduces the innate immune response induced by Ad vectors. RAW264.7-SOCS1, a macrophage-like cell line that stably expresses SOCS1, was shown to produce lower levels of inflammatory cytokines after the transduction of Ad vectors. The systemic administration of Ad-SOCS1 into mice elicited the reduced production of inflammatory cytokines, as compared with that elicited by control Ad vectors, i.e., luciferase-expressing Ad vector, Ad-L2. Furthermore, the coadministration of Ad-L2 with Ad-SOCS1 attenuated inflammatory cytokine production and liver toxicity as compared with injection with Ad-L2 alone, and this was achieved without the suppression of luciferase production in various organs. The JAK/STAT pathway was involved in Ad vector-mediated cytokine production, which was impaired by the overexpression of SOCS1. These findings indicate that Ad-SOCS1 could be useful for reducing Ad vector-mediated innate immunity.
Protection against Influenza A Virus by Memory CD8 T Cells Requires Reactivation by Bone Marrow-Derived Dendritic Cells
Authors: Castiglioni Paola , Hall De Shon , Jacovetty Erica L. , Ingulli Elizabeth , Zanetti Maurizio
Published: April 2014

Influenza A virus is the causative agent of an acute inflammatory disease of the airway. Although Abs can prevent infection, disease and death can be prevented by T cell-mediated immunity. Recently, we showed that protection against lethal influenza A (PR8/34) virus infection is mediated by central memory CD8 T cells (TCM). In this study, using relB−/− mice we began to investigate the role of bone marrow (BM)-derived dendritic cells (DCs) in the mechanism of protection. We found that in the absence of functional DCs, memory CD8 T cells specific for the nucleoprotein epitope (NP366–374) fail to protect even after adoptive transfer into naive recipients. Through an analysis of Ag uptake, activation of memory CD8 T cells, and display of peptide/MHC complex by DCs in draining LNs and spleen early after virus infection, we established that lack of protection is associated with defective Ag presentation by BM-derived DCs and defective homing of memory T cells in the lymph nodes draining the airway tract. Collectively, the data suggest that protection against the influenza A virus requires that memory CD8 T cells be reactivated by Ag presented by BM-derived DCs in the lymph nodes draining the site of infection. They also imply that protection depends both on the characteristics of systemic adaptive immunity and on the coordinated interplay between systemic and local immunity.