bluuebunny/crossref_metadata_2025 huggingface dataset:
Indexation as a Novel Mechanism of Lymphocyte Homeostasis: The Number of CD4+CD25+ Regulatory T Cells Is Indexed to the Number of IL-2-Producing Cells
Authors: Almeida Afonso R. M. , Zaragoza Bruno , Freitas Antonio A.
Published: April 2014

To fulfill its mission, the immune system must maintain a complete set of different cellular subpopulations that play specific roles in immune responses. We have investigated the mechanisms regulating CD4+CD25+ regulatory T (Treg) cell homeostasis. We show that the expression of the high-affinity IL-2Rα endows these cells with the capacity to explore the IL-2 resource, ensuring their presence while keeping their number tied to the number of CD4+ T cells that produce IL-2. We show that such a homeostatic mechanism allows the increased expansion of T cells without causing disease. The indexing of Treg cells to the number of activated IL-2-producing cells may constitute a feedback mechanism that controls T cell expansion during immune responses, thus preventing autoimmune or lymphoproliferative diseases. The present study highlights that maintenance of proportions between different lymphocyte subsets may also be critical for the immune system and are under strict homeostatic control.
Galectin-1-Matured Human Monocyte-Derived Dendritic Cells Have Enhanced Migration through Extracellular Matrix
Authors: Fulcher Jennifer A. , Hashimi Sara T. , Levroney Ernest L. , Pang Mabel , Gurney Kevin B. , Baum Linda G. , Lee Benhur
Published: April 2014

Dendritic cells (DCs) are potent mediators of the immune response, and can be activated by exogenous pathogen components. Galectin-1 is a member of the conserved β-galactoside-binding lectin family that binds galactoside residues on cell surface glycoconjugates. Galectin-1 is known to play a role in immune regulation via action on multiple immune cells. However, its effects on human DCs are unknown. In this study, we show that galectin-1 induces a phenotypic and functional maturation in human monocyte-derived DCs (MDDCs) similar to but distinct from the activity of the exogenous pathogen stimuli, LPS. Immature human MDDCs exposed to galectin-1 up-regulated cell surface markers characteristic of DC maturation (CD40, CD83, CD86, and HLA-DR), secreted high levels of IL-6 and TNF-α, stimulated T cell proliferation, and showed reduced endocytic capacity, similar to LPS-matured MDDCs. However, unlike LPS-matured DCs, galectin-1-treated MDDCs did not produce the Th1-polarizing cytokine IL-12. Microarray analysis revealed that in addition to modulating many of the same DC maturation genes as LPS, galectin-1 also uniquely up-regulated a significant subset of genes related to cell migration through the extracellular matrix (ECM). Indeed, compared with LPS, galectin-1-treated human MDDCs exhibited significantly better chemotactic migration through Matrigel, an in vitro ECM model. Our findings show that galectin-1 is a novel endogenous activator of human MDDCs that up-regulates a significant subset of genes distinct from those regulated by a model exogenous stimulus (LPS). One unique effect of galectin-1 is to increase DC migration through the ECM, suggesting that galectin-1 may be an important component in initiating an immune response.
FOXP3+CD4+CD25+ Adaptive Regulatory T Cells Express Cyclooxygenase-2 and Suppress Effector T Cells by a Prostaglandin E2-Dependent Mechanism
Authors: Mahic Milada , Yaqub Sheraz , Johansson C. Christian , Taskén Kjetil , Aandahl Einar M.
Published: April 2014

CD4+CD25+ regulatory T (TR) cells suppress effector T cells by partly unknown mechanisms. In this study, we describe a population of human suppressive CD4+CD25+ adaptive TR (TRadapt) cells induced in vitro that express cyclooxygenase 2 (COX-2) and the transcription factor FOXP3. TRadapt cells produce PGE2 and suppress effector T cell responses in a manner that is reversed by COX inhibitors and PGE2 receptor-specific antagonists. In resting CD4+CD25− T cells, treatment with PGE2 induced FOXP3 expression. Thus, autocrine and paracrine effects of PGE2 produced by COX-2-positive TRadapt cells may be responsible for both the FOXP3+ phenotype and the mechanism used by these cells to suppress effector T cells.
Lymphotoxin Pathway-Directed, Autoimmune Regulator-Independent Central Tolerance to Arthritogenic Collagen
Authors: Chin Robert K. , Zhu Mingzhao , Christiansen Peter A. , Liu Wenhua , Ware Carl , Peltonen Leena , Zhang Xuejun , Guo Linjie , Han Shuhua , Zheng Biao , Fu Yang-Xin
Published: April 2014

Ectopic expression of peripherally restricted Ags by medullary thymic epithelial cells (mTECs) is associated with negative selection. Autoimmune regulator (AIRE) is considered to be the master regulator of these Ags. We show in this study that the ectopic expression of type II collagen (CII) in mTECs and the corresponding central tolerance to CII are AIRE independent but lymphotoxin dependent. The failure to properly express CII in mTECs of Lta−/− and Ltbr−/− mice leads to overt autoimmunity to CII and exquisite susceptibility to arthritis. These findings define the existence of additional pathways of ectopic peripheral Ag expression, parallel to and independent of AIRE, which may cover an extended spectrum of peripheral Ags in the thymus.
Cutting Edge: Interleukin 17 Signals through a Heteromeric Receptor Complex
Authors: Toy Dean , Kugler David , Wolfson Martin , Bos Tim Vanden , Gurgel Jesse , Derry Jonathan , Tocker Joel , Peschon Jacques
Published: April 2014

IL-17 is an inflammatory cytokine produced primarily by a unique lineage of CD4 T cells that plays critical roles in the pathogenesis of multiple autoimmune diseases. IL-17RA is a ubiquitously expressed receptor that is essential for IL-17 biologic activity. Despite widespread receptor expression, the activity of IL-17 is most classically defined by its ability to induce the expression of inflammatory cytokines, chemokines, and other mediators by stromal cells. The lack of IL-17 responsiveness in mouse stromal cells genetically deficient in IL-17RA is poorly complemented by human IL-17RA, suggesting the presence of an obligate ancillary component whose activity is species specific. This component is IL-17RC, a distinct member of the IL-17R family. Thus, the biologic activity of IL-17 is dependent on a complex composed of IL-17RA and IL-17RC, suggesting a new paradigm for understanding the interactions between the expanded family of IL-17 ligands and their receptors.
Signal Regulatory Protein Molecules Are Differentially Expressed by CD8− Dendritic Cells
Authors: Lahoud Mireille H. , Proietto Anna I. , Gartlan Kate H. , Kitsoulis Susie , Curtis Joan , Wettenhall James , Sofi Mariam , Daunt Carmel , O’Keeffe Meredith , Caminschi Irina , Satterley Keith , Rizzitelli Alexandra , Schnorrer Petra , Hinohara Atsushi , Yamaguchi Yasunori , Wu Li , Smyth Gordon , Handman Emanuela , Shortman Ken , Wright Mark D.
Published: April 2014

A normalized subtracted gene expression library was generated from freshly isolated mouse dendritic cells (DC) of all subtypes, then used to construct cDNA microarrays. The gene expression profiles of the three splenic conventional DC (cDC) subsets were compared by microarray hybridization and two genes encoding signal regulatory protein β (Sirpβ1 and Sirpβ4) molecules were identified as differentially expressed in CD8− cDC. Genomic sequence analysis revealed a third Sirpβ member localized in the same gene cluster. These Sirpβ genes encode cell surface molecules containing extracellular Ig domains and short intracytoplasmic domains that have a charged amino acid in the transmembrane region which can potentially interact with ITAM-bearing molecules to mediate signaling. Indeed, we demonstrated interactions between Sirpβ1 and β2 with the ITAM-bearing signaling molecule Dap12. Real-time PCR analysis showed that all three Sirpβ genes were expressed by CD8− cDC, but not by CD8+ cDC or plasmacytoid pre-DC. The related Sirpα gene showed a similar expression profile on cDC subtypes but was also expressed by plasmacytoid pre-DC. The differential expression of Sirpα and Sirpβ1 molecules on DC was confirmed by staining with mAbs, including a new mAb recognizing Sirpβ1. Cross-linking of Sirpβ1 on DC resulted in a reduction in phagocytosis of Leishmania major parasites, but did not affect phagocytosis of latex beads, perhaps indicating that the regulation of phagocytosis by Sirpβ1 is a ligand-dependent interaction. Thus, we postulate that the differential expression of these molecules may confer the ability to regulate the phagocytosis of particular ligands to CD8− cDC.
A Role for DNA Hypomethylation and Histone Acetylation in Maintaining Allele-Specific Expression of Mouse NKG2A in Developing and Mature NK Cells
Authors: Rogers Sally L. , Rouhi Arefeh , Takei Fumio , Mager Dixie L.
Published: April 2014

The repertoire of receptors that is expressed by NK cells is critical for their ability to kill virally infected or transformed cells. However, the molecular mechanisms that determine whether and when NK receptor genes are transcribed during hemopoiesis remain unclear. In this study, we show that hypomethylation of a CpG-rich region in the mouse NKG2A gene is associated with transcription of NKG2A in ex vivo NK cells and NK cell lines. This observation was extended to various developmental stages of NK cells sorted from bone marrow, in which we demonstrate that the CpGs are methylated in the NKG2A-negative stages (hemopoietic stem cells, NK progenitors, and NKG2A-negative NK cells), and hypomethylated specifically in the NKG2A-positive NK cells. Furthermore, we provide evidence that DNA methylation is important in maintaining the allele-specific expression of NKG2A. Finally, we show that acetylated histones are associated with the CpG-rich region in NKG2A positive, but not negative, cell lines, and that treatment with the histone deacetylase inhibitor trichostatin A alone is sufficient to induce NKG2A expression. Treatment with the methyltransferase inhibitor 5-azacytidine only is insufficient to induce transcription, but cotreatment with both drugs resulted in a significantly greater induction, suggesting a cooperative role for DNA methylation and histone acetylation status in regulating gene expression. These results enhance our understanding of the formation and maintenance of NK receptor repertoires in developing and mature NK cells.
Haemophilus influenzae Surface Fibrils Contribute to Serum Resistance by Interacting with Vitronectin
Authors: Hallström Teresia , Trajkovska Elena , Forsgren Arne , Riesbeck Kristian
Published: April 2014

Vitronectin inhibits the membrane attack complex of the complement system and is found both in plasma and the extracellular matrix. In this study, we have identified the outer membrane protein Haemophilus surface fibrils (Hsf) as the major vitronectin-binding protein in encapsulated H. influenzae type b. A H. influenzae mutant devoid of Hsf showed a significantly decreased binding to both soluble and immobilized vitronectin as compared with the wild-type counterpart. Moreover, Escherichia coli-expressing Hsf at the surface strongly adhered to immobilized vitronectin. Importantly, the H. influenzae Hsf mutant had a markedly reduced survival as compared with the wild-type bacterium when incubated with normal human serum. A series of truncated Hsf fragments were recombinantly manufactured in E. coli. The vitronectin binding regions were located within two separate binding domains. In conclusion, Hsf interacts with vitronectin and thereby inhibits the complement-mediated bactericidal activity, and thus is a major H. influenzae virulence factor.
Altered CD4+ T Cell Phenotype and Function Determine the Susceptibility to Mucosal Candidiasis in Transgenic Mice Expressing HIV-1
Authors: Lewandowski Daniel , Marquis Miriam , Aumont Francine , Lussier-Morin Annie-Claude , Raymond Marianne , Sénéchal Serge , Hanna Zaher , Jolicoeur Paul , de Repentigny Louis
Published: April 2014

The impairments of protective mucosal immunity which cause susceptibility to oropharyngeal candidiasis (OPC) in HIV infection remain undefined. This study used a model of OPC in CD4C/HIV MutA transgenic (Tg) mice expressing Rev, Env, and Nef of HIV-1 to investigate the role of transgene expressing dendritic cells (DCs) and CD4+ T cells in maintenance of chronic oral carriage of Candida albicans. DCs were depleted in the Tg mice and had an immature phenotype, with low expression of MHC class II and IL-12. CD4+ T cells were quantitatively reduced in the oral mucosa, cervical lymph nodes (CLNs) and peripheral blood of the Tg mice, and displayed a polarization toward a nonprotective Th2 response. Proliferation of CLN CD4+ T cells from infected Tg mice in response to C. albicans Ag in vitro was abrogated and the cells failed to acquire an effector phenotype. Coculture of C. albicans-pulsed DCs with CD4+ T cells in vitro showed that Tg expression in either or both of these cell populations sharply reduced the proliferation of CD4+ T cells and their production of IL-2. Finally, transfer of naive non-Tg CD4+ T cells into these Tg mice restored proliferation to C. albicans Ag and sharply reduced oral burdens of C. albicans. Overall, these results indicate that defective CD4+ T cells primarily determine the susceptibility to chronic carriage of C. albicans in these Tg mice.
Evaluation of Immunological Paradigms in a Virus Model: Are Dendritic Cells Critical for Antiviral Immunity and Viral Clearance?
Authors: Ciavarra Richard P. , Stephens Amber , Nagy Sandra , Sekellick Margaret , Steel Christina
Published: April 2014

We have examined the role of dendritic cells (DCs) in the antiviral immune response and viral clearance using a transgenic mouse model (CD11c-diphtheria toxin (DT) receptor GFP) that allows for their conditional ablation in vivo. DT administration systemically ablated conventional and IFN-producing plasmacytoid DCs (pDCs) in transgenic, but not nontransgenic littermates, without elimination of splenic macrophages. Unexpectedly, early (12 and 48 h postinfection) viral clearance of vesicular stomatitis virus was normal in DC-depleted mice despite markedly reduced serum titers of type I IFN. DC-depleted mice remained virus-free with the exception of a subset (∼30%) that developed overwhelming and fatal brain infections 6 days postinfection. However, DT treatment profoundly inhibited clonal expansion of naive CD8+ vesicular stomatitis virus-specific T cells without altering the primary Th1 and Th2 cytokine response. Optimal clonal expansion required pDCs because selective elimination of these cells in vivo with a depleting Ab also suppressed expansion of tetramer+ cells, although Th1/Th2 cytokine production remained unaltered. Collectively, these data indicate that conventional DCs and to a lesser extent pDCs are critical for proliferation of naive antiviral T cells. However, other components of the primary adaptive immune response (Th1/Th2 cytokines) are essentially normal in the absence of DCs, which may account for the efficient viral clearance seen in DC-depleted mice. Thus, sufficient redundancy exists in the immune system to sustain efficient viral clearance despite loss of an APC considered essential for induction of a primary antiviral immune response.
Toll/IL-1R Domain-Containing Adaptor Protein (TIRAP) Is a Critical Mediator of Antibacterial Defense in the Lung against Klebsiella pneumoniae but Not Pseudomonas aeruginosa
Authors: Jeyaseelan Samithamby , Young Scott K. , Yamamoto Masahiro , Arndt Patrick G. , Akira Shizuo , Kolls Jay K. , Worthen G. Scott
Published: April 2014

Bacterial pneumonia is a leading cause of mortality and is associated with extensive neutrophil accumulation. Major pathogens associated with this disease include nonflagellated Klebsiella pneumoniae (Kp) and flagellated Pseudomonas aeruginosa (Pa). TLRs are essential for innate immune defense. TIRAP (Toll/IL-1R domain-containing adaptor protein) is an adaptor in TLR1, TLR2, TLR4, and TLR6 signaling, whereas MyD88 is an adaptor for all TLRs. However, the importance of TIRAP in pulmonary defense against Kp or Pa has not been examined. To demonstrate the role of TIRAP, TIRAP-deficient and wild-type littermates were intratracheally inoculated with Kp or Pa. We found that TIRAP−/− mice had substantial mortality, higher bacterial burden in the lungs, and enhanced dissemination following Kp challenge. Furthermore, Kp-induced neutrophil sequestration, histopathology, and MIP-2, TNF-α, IL-6, and LIX (lipopolysaccharide-induced CXC chemokine) production were attenuated in the lungs of TIRAP−/− mice. In contrast, TIRAP is not required for Pa-induced mortality, pulmonary bacterial burden, bacterial dissemination, neutrophil accumulation, or histopathology, yet it is necessary for MIP-2, TNF-α, and IL-6 production, but not LIX production. However, both Kp- and Pa-induced neutrophil influxes are MyD88 dependent. To determine the mechanisms associated with Pa-induced neutrophil accumulation, we inoculated mice with a flagellin C mutant of Pa (PaΔfliC) or purified flagellin, a TLR5 agonist. PaΔfliC-induced neutrophil sequestration and LIX expression are dependent on TIRAP, whereas flagellin-induced neutrophil influx and LIX expression are independent of TIRAP. These novel findings illustrate a pathogen-specific role for TIRAP in pulmonary defense and suggest that TLR5 plays an essential role for Pa-induced neutrophil influx via LIX production.
C/EBPβ Blocks p65 Phosphorylation and Thereby NF-κB-Mediated Transcription in TNF-Tolerant Cells
Authors: Zwergal Andreas , Quirling Martina , Saugel Bernd , Huth Karin C. , Sydlik Carmen , Poli Valeria , Neumeier Dieter , Ziegler-Heitbrock H. W. Löms , Brand Korbinian
Published: April 2014

TNF is a major mediator of inflammation, immunity, and apoptosis. Pre-exposure to TNF reduces sensitivity to restimulation, a phenomenon known as tolerance, considered as protective in sepsis, but also as a paradigm for immunoparalysis. Earlier experiments in TNF-tolerant cells display inhibition of NF-κB-dependent IL-8 gene expression at the transcriptional level with potential involvement of C/EBPβ. In this study, we have shown that a κB motive was sufficient to mediate transcriptional inhibition under TNF tolerance conditions in monocytic cells. Furthermore, in tolerant cells, TNF-induced NF-κB p65 phosphorylation was markedly decreased, which was accompanied by the formation of C/EBPβ-p65 complexes. Remarkably, in C/EBPβ−/− cells incubated under the conditions of TNF tolerance, neither impairment of transcription nor inhibition of p65 phosphorylation was observed. Finally, we showed that C/EBPβ overexpression reduced p65-mediated transactivation and that association of C/EBPβ with p65 specifically prevented p65 phosphorylation. Our data demonstrate that C/EBPβ is an essential signaling component for inhibition of NF-κB-mediated transcription in TNF-tolerant cells and suggest that this is caused by blockade of p65 phosphorylation. These results define a new molecular mechanism responsible for TNF tolerance in monocytic cells that may contribute to the unresponsiveness seen in patients with sepsis.
Coreceptor Signal Strength Regulates Positive Selection but Does Not Determine CD4/CD8 Lineage Choice in a Physiologic In Vivo Model
Authors: Erman Batu , Alag Amala S. , Dahle Oyvind , van Laethem François , Sarafova Sophia D. , Guinter Terry I. , Sharrow Susan O. , Grinberg Alexander , Love Paul E. , Singer Alfred
Published: April 2014

TCR signals drive thymocyte development, but it remains controversial what impact, if any, the intensity of those signals have on T cell differentiation in the thymus. In this study, we assess the impact of CD8 coreceptor signal strength on positive selection and CD4/CD8 lineage choice using novel gene knockin mice in which the endogenous CD8α gene has been re-engineered to encode the stronger signaling cytoplasmic tail of CD4, with the re-engineered CD8α gene referred to as CD8.4. We found that stronger signaling CD8.4 coreceptors specifically improved the efficiency of CD8-dependent positive selection and quantitatively increased the number of MHC class I (MHC-I)-specific thymocytes signaled to differentiate into CD8+ T cells, even for thymocytes expressing a single, transgenic TCR. Importantly, however, stronger signaling CD8.4 coreceptors did not alter the CD8 lineage choice of any MHC-I-specific thymocytes, even MHC-I-specific thymocytes expressing the high-affinity F5 transgenic TCR. This study documents in a physiologic in vivo model that coreceptor signal strength alters TCR-signaling thresholds for positive selection and so is a major determinant of the CD4:CD8 ratio, but it does not influence CD4/CD8 lineage choice.
Death of CD4+ T Cells from Lymph Nodes during Primary SIVmac251 Infection Predicts the Rate of AIDS Progression
Authors: Viollet Laurence , Monceaux Valérie , Petit Frédéric , Fang Raphaël Ho Tsong , Cumont Marie-Christine , Hurtrel Bruno , Estaquier Jérôme
Published: April 2014

Immunological and virological events that occur during the earliest stages of SIV infection are now considered to have a major impact on subsequent disease progression. In the present study, we demonstrate a clear correlation between progression to AIDS and the rate of in vitro CD4+ (but not CD8+) T cell death in lymph nodes. The dying CD4+ T cells were effector memory T cells, which are critical for the immune response to pathogens. However, there was no correlation between the rate of the viral replication within lymph nodes and the extent of Fas ligand-mediated death, despite the increased sensitivity of CD4+ T cells to death in response to recombinant human Fas ligand. CD4+ T cell death was caspase and apoptosis-inducing factor independent but was clearly associated with mitochondrion damage. Interestingly, higher expression levels of the active form of Bak, a proapoptotic molecule involved in mitochondrial membrane permeabilization, were observed in SIV-infected macaques progressing more rapidly to AIDS. Finally, we demonstrated that the strain of SIV we used requires CCR5 and BOB/GRP15 molecules as coreceptors and caused death of unstimulated noncycling primary CD4+ T cells. Altogether, these results demonstrate that CD4+ T cell death occurring early after SIV infection is a crucial determinant of progression to AIDS and that it is mediated by the intrinsic death pathway.
CXC Chemokine Ligand 13 and CC Chemokine Ligand 19 Cooperatively Render Resistance to Apoptosis in B Cell Lineage Acute and Chronic Lymphocytic Leukemia CD23+CD5+ B Cells
Authors: Chunsong Hu , Yuling He , Li Wang , Jie Xiong , Gang Zhou , Qiuping Zhang , Qingping Gao , Kejian Zhang , Li Qiao , Chang Alfred E. , Youxin Jin , Jinquan Tan
Published: April 2014

CXCL13/CXCR5 and CCL19/CCR7 play a quite important role in normal physiological conditions, but the functions of both chemokine/receptor pairs in pathophysiological events are not well-investigated. We have investigated expression and functions of CXCL13/CXCR5 and CCL19/CCR7 in CD23+CD5+ and CD23+CD5− B cells from cord blood (CB) and patients with B cell lineage acute or chronic lymphocytic leukemia (B-ALL or B-CLL). CXCR5 and CCR7 are selectively expressed on B-ALL, B-CLL, and CB CD23+CD5+ B cells at high frequency, but not on CD23+CD5− B cells. Although no significant chemotactic responsiveness was observed, CXCL13 and CCL19 cooperatively induce significant resistance to TNF-α-mediated apoptosis in B-ALL and B-CLL CD23+CD5+ B cells, but not in the cells from CB. B-ALL and B-CLL CD23+CD5+ B cells express elevated levels of paternally expressed gene 10 (PEG10). CXCL13 and CCL19 together significantly up-regulate PEG10 expression in the same cells. We have found that CXCL13 and CCL19 together by means of activation of CXCR5 and CCR7 up-regulate PEG10 expression and function, subsequently stabilize caspase-3 and caspase-8 in B-ALL and B-CLL CD23+CD5+ B cells, and further rescue the cells from TNF-α-mediated apoptosis. Therefore, we suggest that normal lymphocytes, especially naive B and T cells, use CXCL13/CXCR5 and CCL19/CCR7 for migration, homing, maturation, and cell homeostasis as well as secondary lymphoid tissues organogenesis. In addition, certain malignant cells take advantages of CXCL13/CXCR5 and CCL19/CCR7 for infiltration, resistance to apoptosis, and inappropriate proliferation.
CD8 T Cell Recall Responses Are Regulated by the Tissue Tropism of the Memory Cell and Pathogen
Authors: Klonowski Kimberly D. , Marzo Amanda L. , Williams Kristina J. , Lee Seung-Joo , Pham Quynh-Mai , Lefrançois Leo
Published: April 2014

Whether memory CD8 T cells can be reactivated in nonlymphoid tissues is unclear. Using mice lacking the spleen, lymph nodes, or both, we show that the secondary T cell response, but not homeostatic maintenance of memory cells, required lymphoid tissue. Whereas primary and secondary CD8 T cell responses to vesicular stomatitis virus infection were lymph node dependent, responses to Listeria monocytogenes infection were driven primarily in the spleen. Memory cell subset reactivation was also regulated by location of the responding population and the pathogen. Thus, CD62Llow effector memory T cells (TEM) cells responded nearly as well as CD62Lhigh central memory T cells (TCM) and TCM cells after L. monocytogenes infection, and both subsets generated equivalent populations of secondary memory cells. In contrast, TCM cells, but not TEM cells, mounted a robust response to vesicular stomatitis virus infection. TCM and TEM cells also required lymphoid tissue to mount recall responses, and the bone marrow did not contribute significantly to the response of either subset. Our findings indicated that characteristics of the infectious agent and the migratory preferences of memory cells dictated the secondary lymphoid tissue requirement for the recall response to infection.
T Regulatory and Primed Uncommitted CD4 T Cells Express CD73, Which Suppresses Effector CD4 T Cells by Converting 5′-Adenosine Monophosphate to Adenosine
Authors: Kobie James J. , Shah Pranav R. , Yang Li , Rebhahn Jonathan A. , Fowell Deborah J. , Mosmann Tim R.
Published: April 2014

CD73 (5′-ectonucleotidase) is expressed by two distinct mouse CD4 T cell populations: CD25+ (FoxP3+) T regulatory (Treg) cells that suppress T cell proliferation but do not secrete IL-2, and CD25− uncommitted primed precursor Th (Thpp) cells that secrete IL-2 but do not suppress in standard Treg suppressor assays. CD73 on both Treg and Thpp cells converted extracellular 5′-AMP to adenosine. Adenosine suppressed proliferation and cytokine secretion of Th1 and Th2 effector cells, even when target cells were activated by anti-CD3 and anti-CD28. This represents an additional suppressive mechanism of Treg cells and a previously unrecognized suppressive activity of Thpp cells. Infiltration of either Treg or Thpp cells at inflammatory sites could potentially convert 5′-AMP generated by neutrophils or dying cells into the anti-inflammatory mediator adenosine, thus dampening excessive immune reactions.
Pertussis Toxin Is Superior to TLR Ligands in Enhancing Pathogenic Autoimmunity, Targeted at a Neo-Self Antigen, by Triggering Robust Expansion of Th1 Cells and Their Cytokine Production
Authors: Fujimoto Chiaki , Yu Cheng-Rong , Shi Guangpu , Vistica Barbara P. , Wawrousek Eric F. , Klinman Dennis M. , Chan Chi-Chao , Egwuagu Charles E. , Gery Igal
Published: April 2014

Microbial products are assumed to play a major role in triggering pathogenic autoimmunity. Recently accumulated data have shown that these products stimulate the immune system by interacting with TLRs, expressed on APCs. To examine the capacity of various TLR ligands to trigger pathogenic autoimmunity, we used a system in which naive CD4 cells, specific against hen egg lysozyme (HEL), are injected into recipient mice expressing HEL in their eyes. Only when stimulated, the naive cells acquire pathogenic capacity and induce ocular inflammation. Seven TLR ligands were tested in this system: lipoteichoic acid/peptidoglycan, zymosan, poly (I:C), LPS, pertussis toxin (PTX), flagellin, and CpG oligodeoxynucleotide. Treatment of recipient mice with HEL alone stimulated proliferation of the transferred cells, but no disease, whereas ocular inflammation did develop in recipient mice coinjected with HEL and any one of the seven TLR ligands. Inflammation induced by PTX surpassed by its severity those induced by all other tested TLR ligands and was accompanied by a dramatic increase in number of the transferred cells that acquired features of effector Th1 lymphocytes. Ocular inflammation and number of transferred cells in recipients injected with PTX and HEL were substantially reduced by treatment with Abs against IFN-γ or IL-12, thus indicating the role of these cytokines in the PTX effect. Overall, our observations demonstrate that various TLR ligands are capable of triggering pathogenic autoimmunity and that PTX surpasses other microbial products in this activity, by stimulating excessive proliferation and polarization toward Th1 of naive T cells.
HLA-B*2704, an Allotype Associated with Ankylosing Spondylitis, Is Critically Dependent on Transporter Associated with Antigen Processing and Relatively Independent of Tapasin and Immunoproteasome for Maturation, Surface Expression, and T Cell Recognition: Relationship to B*2705 and B*2706
Authors: Montserrat Verónica , Galocha Begoña , Marcilla Miguel , Vázquez Miriam , López de Castro José A.
Published: April 2014

B*2704 is strongly associated to ankylosing spondylitis in Asian populations. It differs from the main HLA-B27 allotype, B*2705, in three amino acid changes. We analyzed the influence of tapasin, TAP, and immunoproteasome induction on maturation, surface expression, and T cell allorecognition of B*2704 and compared some of these features with B*2705 and B*2706, allotypes not associated to disease. In the tapasin-deficient .220 cell line, this chaperone significantly influenced the extent of folding of B*2704 and B*2705, but not their egress from the endoplasmic reticulum. In contrast, B*2706 showed faster folding and no accumulation in the endoplasmic reticulum in the absence of tapasin. Surface expression of B*2704 was more tapasin dependent than B*2705. However, expression of free H chain decreased in the presence of this chaperone for B*2705 but not B*2704, suggesting that more suboptimal ligands were loaded on B*2705 in the absence of tapasin. Despite its influence on surface expression, tapasin had little effect on allorecognition of B*2704. Both surface expression and T cell recognition of B*2704 were critically dependent on TAP, as established with TAP-deficient and TAP-proficient T2 cells. Both immunoproteasome and surface levels of B*2704 were induced by IFN-γ, but this had little effect on allorecognition. Thus, except for the differential effects of tapasin on surface expression, the tapasin, TAP, and immunoproteasome dependency of B*2704 for maturation, surface expression, and T cell recognition are similar to B*2705, indicating that basic immunological features are shared by the two major HLA-B27 allotypes associated to ankylosing spondylitis in human populations.
Chronic Hyperglycemia Predisposes to Exaggerated Inflammatory Response and Leukocyte Dysfunction in Akita Mice
Authors: Gyurko Robert , Siqueira Camille C. , Caldon Nathaniel , Gao Li , Kantarci Alpdogan , Van Dyke Thomas E.
Published: April 2014

The role of polymorphonuclear neutrophils (PMN) in mediating diabetic tissue damage to the periodontium was investigated in a novel model of chronic hyperglycemia, the Akita mouse. Induction of acute peritoneal inflammation in wild-type (WT) and Akita mice resulted in exaggerated IL-6 response in Akita mice (2.9-fold increase over WT values) and a markedly increased chemokine response (KC, 2.6-fold; MCP-1, 2.6-fold; and MIP-1α, 4.4-fold increase over WT values). Chemotaxis to both fMLP and WKYMVm was significantly reduced in isolated Akita PMN compared with WT PMN as measured in a Boyden chamber. Superoxide release in contrast was significantly increased in Akita PMN as measured with cytochrome c reduction. Bone marrow-derived Akita PMN showed partial translocation of p47phox to the cell membrane without external stimulation, suggesting premature assembly of the superoxide-producing NADPH oxidase in hyperglycemia. In vivo studies revealed that ligature-induced periodontal bone loss is significantly greater in Akita mice compared with WT. Moreover, intravital microscopy of gingival vessels showed that leukocyte rolling and attachment to the vascular endothelium is enhanced in periodontal vessels of Akita mice. These results indicate that chronic hyperglycemia predisposes to exaggerated inflammatory response and primes leukocytes for marginalization and superoxide production but not for transmigration. Thus, leukocyte defects in hyperglycemia may contribute to periodontal tissue damage by impairing the innate immune response to periodontal pathogens as well as by increasing free radical load in the gingival microvasculature.
Inhibition of Helicobacter hepaticus-Induced Colitis by IL-10 Requires the p50/p105 Subunit of NF-κB
Authors: Tomczak Michal F. , Erdman Susan E. , Davidson Anne , Wang Yan Yan , Nambiar Prashant R. , Rogers Arlin B. , Rickman Barry , Luchetti David , Fox James G. , Horwitz Bruce H.
Published: April 2014

Defects within the innate immune system sensitize NF-κB-deficient (p50−/−; p65+/−) mice to Helicobacter hepaticus (Hh)-induced colitis. Because IL-10 plays a central role in the inhibition of Hh-induced colitis, we hypothesized that the ability of IL-10 to inhibit the innate inflammatory response to Hh may be compromised in NF-κB-deficient mice. To test this hypothesis, we evaluated the ability of an IL-10-Ig fusion protein with IL-10-like properties to inhibit Hh-induced colitis in RAG-2−/− (RAG) and p50−/−; p65+/−; RAG-2−/− (3X/RAG) mice. As expected, IL-10-Ig efficiently inhibited the development of colitis in RAG mice. In contrast, the ability of IL-10-Ig to inhibit colitis was compromised in 3X/RAG mice. The defect in response to IL-10-Ig appeared to be primarily the result of the absence of the p50/p105 subunit, because the ability of IL-10-Ig to inhibit colitis was also compromised in p50−/−; RAG-2−/− (p50/RAG) mice. Radiation chimeras demonstrated that the presence of p50/p105 within hemopoietic cells of the innate immune system was necessary for efficient inhibition of colitis by IL-10-Ig. Consistent with a defect in the suppressive effects of IL-10 in the absence of p50/p105, we found that the ability of IL-10 to control LPS-induced expression of IL-12 p40 was significantly compromised in macrophages lacking p50/p105. These results suggest that the absence of the p50/p105 subunit of NF-κB within hemopoietic cells of the innate immune system interferes with the ability of IL-10 to suppress inflammatory gene expression and Hh-induced colitis.
Major Basic Protein Homolog (MBP2): A Specific Human Eosinophil Marker
Authors: Plager Douglas A. , Loegering David A. , Checkel James L. , Tang Junger , Kephart Gail M. , Caffes Patricia L. , Adolphson Cheryl R. , Ohnuki Lyo E. , Gleich Gerald J.
Published: April 2014

Human eosinophil granule major basic protein (MBP1) is an exceedingly basic (isoelectric point >11) 14-kDa protein, comprising the core of the secondary eosinophil granule. Recently, a less cationic homolog of MBP, termed MBPH or simply, MBP2, has been discovered. We prepared a panel of mAbs to MBP2 and used these Abs to localize and quantitate this molecule in leukocytes and biological fluids. Specific mAbs for MBP2 were selected using slot-blot analyses and used in a two-site immunoassay, Western blotting, and immunofluorescence microscopy. The sensitivity of the immunoassay was markedly improved by reduction and alkylation of MBP2. MBP1 is more abundant than MBP2 in lysates of eosinophils and their granules, as judged by immunoassay and Western blotting. By immunofluorescence, MBP1 is present in eosinophils, basophils, and a human mast cell line (HMC1), whereas MBP2 is only detected in eosinophils. Neither MBP1 nor MBP2 could be detected in any other peripheral blood leukocyte. MBP2 levels measured in plasma and serum were essentially identical. In contrast to past measurements for MBP1, MBP2 was not detected above normal levels in sera from pregnant donors. However, measurement of serum MBP2 discriminated patients with elevated eosinophils from normal subjects, and MBP2 was also detectable in other biological specimens, such as bronchoalveolar lavage, sputum, and stool. These results indicate that MBP2 is present only in eosinophils and that it may be a useful biomarker for eosinophil-associated diseases.
Cutting Edge: IL-12 Inversely Regulates T-bet and Eomesodermin Expression during Pathogen-Induced CD8+ T Cell Differentiation
Authors: Takemoto Naofumi , Intlekofer Andrew M. , Northrup John T. , Wherry E. John , Reiner Steven L.
Published: April 2014

Cytokines are critical determinants for specification of lineage-defining transcription factors of CD4+ T cell subsets. Little is known, however, about how cytokines regulate expression of T-bet and eomesodermin (Eomes) in effector and memory CD8+ T cells. We now report that IL-12, a signature of cell-mediated immunity, represses Eomes while positively regulating T-bet in effector CD8+ T cells during infection with Listeria monocytogenes. After resolution of infection and abatement of IL-12 signaling, Eomes expression rises whereas T-bet expression declines in memory CD8+ T cells. Eomes becomes derepressed in effector cells by ablation of IL-12 signaling. In the absence of IL-12, the dynamics of clonal expansion and contraction are also perturbed. Together, these results reveal how a pathogen-associated signal, such as IL-12, could act as a switch, regulating appropriate clonal growth and decline while, in parallel, shaping a unique pattern of fate-determining transcription factors.
CCR4 Is a Key Modulator of Innate Immune Responses
Authors: Ness Traci L. , Ewing Jillian L. , Hogaboam Cory M. , Kunkel Steven L.
Published: April 2014

CCR4 is recognized as a key receptor in Th2-associated immune processes, although very little is known about its role in innate immunity. Previous studies reported increased resistance to LPS-induced lethality in CCR4−/− mice compared with wild-type mice. This study demonstrates that CCR4−/− mice are similarly resistant to challenge with other TLR agonists, as well as bacterial peritonitis. Resistance was associated with enhanced early leukocyte recruitment, increased TLR expression, a skewed type 2 cytokine/chemokine profile, and improved bacterial clearance. Macrophages from CCR4−/− mice exhibited many features consistent with alternative activation, including elevated secretion of type 2 cytokines/chemokines and the found in inflammatory zone 1 (FIZZ1) protein. MyD88-dependent NF-κB signaling was significantly down-regulated in CCR4−/− macrophages, whereas p38 MAPK and JNK activation were conversely increased. These data stress the importance of CCR4 in macrophage differentiation and innate immune responses to pathogens, as well as the involvement of chemokine receptor expression in TLR signaling regulation.
The Chemokine CX3CL1 Reduces Migration and Increases Adhesion of Neurons with Mechanisms Dependent on the β1 Integrin Subunit
Authors: Lauro Clotilde , Catalano Myriam , Trettel Flavia , Mainiero Fabrizio , Ciotti Maria Teresa , Eusebi Fabrizio , Limatola Cristina
Published: April 2014

Fractalkine/CX3CL1 and its specific receptor CX3CR1 are constitutively expressed in several regions of the CNS and are reported to mediate neuron-microglial interaction, synaptic transmission, and neuronal protection from toxic insults. CX3CL1 is released both by neuronal and astrocytic cells, whereas CX3CR1 is mainly expressed by microglial cells and neurons. Microglial cells efficiently migrate in response to CX3CL1, whereas no evidence is reported to date on CX3CL1-induced neuronal migration. For this reason, we have investigated in vitro the effects of CX3CL1 on basal migration of neurons and of the microglial and astrocytic populations, all these cells being obtained from the hippocampus and the cerebellum of newborn rats. We report that CX3CL1 stimulates microglial cell migration but efficiently reduces basal neuronal movement, regardless of the brain source. The effect of CX3CL1 is pertussis toxin (PTX) sensitive and PI3K dependent on hippocampal neurons, while it is PTX sensitive, PI3K dependent, and ERK dependent on cerebellar granules. Interestingly, CX3CL1 also increases neuron adhesion to the extracellular matrix component laminin, with mechanisms dependent on PTX-sensitive G proteins, and on the ERK and PI3K pathways. Both the reduction of migration and the increase of neuron adhesion require the activation of the β1 and α6 integrin subunits with the exception of cerebellar neuron migration, which is only dependent on the β1 subunit. More importantly, in neurons, CX3CL1/CXCL12 cotreatment abolished the effect mediated by a single chemokine on chemotaxis and adhesion. In conclusion, our findings indicate that CX3CL1 reduces neuronal migration by increasing cell adhesion through integrin-dependent mechanisms in hippocampal and cerebellar neurons.
Cyclosporin A Abolishes CD28-Mediated Resistance to CD95-Induced Apoptosis via Superinduction of Caspase-3
Authors: Kerstan Andreas , Armbruster Nicole , Leverkus Martin , Hünig Thomas
Published: April 2014

Costimulation of T cells via CD28 promotes both proliferation and resistance to apoptosis. In this study, we show that the immunosuppressive drug cyclosporin A (CsA) fully reverses resistance to CD95-mediated cell death after TCR/CD28 costimulation or superagonistic anti-CD28 mAb stimulation of primary rat lymph node T cells. This effect correlated with a pronounced superinduction of caspase-3 on both mRNA and protein levels, whereas its main antagonist, X chromosome-linked inhibitor of apoptosis, was unaffected by inclusion of CsA. Apoptosis triggered by CD95 cross-linking was characterized by robust caspase-3 activation. Furthermore, CsA sensitization to CD95-mediated apoptosis of CD28-activated T cells did not alter mRNA stability of superinduced caspase-3 mRNA, suggesting a transcriptional regulation of the caspase-3 gene. Addition of Ca2+ ionophores to TCR/CD28 or superagonistic CD28-stimulated cells reduced caspase-3 levels, further supporting a role for Ca2+-dependent signaling pathways in negatively regulating caspase-3. Taken together, these findings suggest that CsA promotes sensitivity to CD95-mediated apoptosis in CD28-stimulated T cells by superinduction of the caspase-3 gene via a mechanism involving suppression of the calcineurin pathway.
Characterization of Myeloid and Plasmacytoid Dendritic Cells in Human Lung
Authors: Masten Barbara J. , Olson Gwyneth K. , Tarleton Christy A. , Rund Chad , Schuyler Mark , Mehran Reza , Archibeque Tereassa , Lipscomb Mary F.
Published: April 2014

Dendritic cells (DCs) are bone marrow-derived mononuclear cells that play a central role in the initiation of immune responses. Because human lung DCs have been incompletely characterized, we enumerated and phenotyped mononuclear cell populations from excess lung tissue obtained at surgery. Myeloid DCs (MDCs) were identified as CD1c+CD11c+CD14−HLA-DR+ cells and comprised ∼2% of low autofluorescent (LAF) mononuclear cells. Plasmacytoid DCs (PDCs) were characterized as CD123+CD11c−CD14−HLA-DR+ cells and comprised ∼1.0% of the LAF mononuclear cells. Cells enriched in MDCs expressed CD86, moderate CD80, and little CD40, but cells enriched in PDCs had little to no expression of these three costimulatory molecules. CD11c+CD14− lineage-negative (MDC-enriched) LAF cells were isolated and shown to be much more potent in stimulating an alloreaction than CD11c+CD14+ lineage-negative (monocyte-enriched) LAF cells. PDC-enriched cells were more capable of responding to a TLR-7 agonist by secreting IFN-α than MDC-enriched cells. MDC-enriched cells were either CD123+ or CD123−, but both subsets secreted cytokines and chemokines typical of MDC upon stimulation with a TLR-4 agonist and both subsets failed to secrete IFN-α upon stimulation with a TLR-7 agonist. By immunohistochemistry, we identified MDCs throughout different anatomical locations of the lung. However, our method did not allow the localization of PDCs with certainty. In conclusion, in the human lung MDCs were twice as numerous and expressed higher levels of costimulatory molecules than PDCs. Our data suggest that both lung DC subsets exert distinct immune modulatory functions.
Regulation of Dendritic Cell Function and T Cell Priming by the Fatty Acid-Binding Protein aP2
Authors: Rolph Michael S. , Young Timothy R. , Shum Bennett O. V. , Gorgun Cem Z. , Schmitz-Peiffer Carsten , Ramshaw Ian A. , Hotamisligil Gökhan S. , Mackay Charles R.
Published: April 2014

The fatty acid-binding protein (FABP) family consists of a number of conserved cytoplasmic proteins with roles in intracellular lipid transport, storage, and metabolism. Examination of a comprehensive leukocyte gene expression database revealed strong expression of the adipocyte FABP aP2 in human monocyte-derived dendritic cells (DCs). We isolated bone marrow-derived DC from aP2-deficient mice, and showed that expression of DC cytokines including IL-12 and TNF was significantly impaired in these cells. Degradation of IκBα was also impaired in aP2-deficient DCs, indicative of reduced signaling through the IκB kinase-NF-κB pathway. The cytokine defect was selective because there was no effect on Ag uptake or expression of MHC class II, CD40, CD80, or CD86. In an MLR, aP2-deficient DCs stimulated markedly lower T cell proliferation and cytokine production than did wild-type DCs. Moreover, aP2-deficient mice immunized with keyhole limpet hemocyanin/CFA showed reduced production of IFN-γ by restimulated draining lymph node cells, suggesting a similar defect in DC function in vivo. Similarly, infection of aP2-deficient mice with the natural mouse pathogen ectromelia virus resulted in substantially lower production of IFN-γ by CD8+ T cells. Thus, FABP aP2 plays an important role in DC function and T cell priming, and provides an additional link between metabolic processes and the regulation of immune responses.
A Regulatory CD4+ T Cell Subset in the BB Rat Model of Autoimmune Diabetes Expresses Neither CD25 Nor Foxp3
Authors: Hillebrands Jan-Luuk , Whalen Barbara , Visser Jeroen T. J. , Koning Jasper , Bishop Kenneth D. , Leif Jean , Rozing Jan , Mordes John P. , Greiner Dale L. , Rossini Aldo A.
Published: April 2014

Biobreeding (BB) rats model type 1 autoimmune diabetes (T1D). BB diabetes-prone (BBDP) rats develop T1D spontaneously. BB diabetes-resistant (BBDR) rats develop T1D after immunological perturbations that include regulatory T cell (Treg) depletion plus administration of low doses of a TLR ligand, polyinosinic-polycytidylic acid. Using both models, we analyzed CD4+CD25+ and CD4+CD45RC− candidate rat Treg populations. In BBDR and control Wistar Furth rats, CD25+ T cells comprised 5–8% of CD4+ T cells. In vitro, rat CD4+CD25+ T cells were hyporesponsive and suppressed T cell proliferation in the absence of TGF-β and IL-10, suggesting that they are natural Tregs. In contrast, CD4+CD45RC− T cells proliferated in vitro in response to mitogen and were not suppressive. Adoptive transfer of purified CD4+CD25+ BBDR T cells to prediabetic BBDP rats prevented diabetes in 80% of recipients. Surprisingly, CD4+CD45RC−CD25− T cells were equally protective. Quantitative studies in an adoptive cotransfer model confirmed the protective capability of both cell populations, but the latter was less potent on a per cell basis. The disease-suppressing CD4+CD45RC−CD25− population expressed PD-1 but not Foxp3, which was confined to CD4+CD25+ cells. We conclude that CD4+CD25+ cells in the BBDR rat act in vitro and in vivo as natural Tregs. In addition, another population that is CD4+CD45RC−CD25− also participates in the regulation of autoimmune diabetes.