bluuebunny/crossref_metadata_2025 huggingface dataset:
Impaired Recall of CD8 Memory T Cells in Immunologically Privileged Tissue
Authors: Dai Zhenhua , Nasr Isam W. , Reel Michael , Deng Songyan , Diggs Lonnette , Larsen Christian P. , Rothstein David M. , Lakkis Fadi G.
Published: April 2014

Foreign Ags that enter immunologically privileged sites such as the eye, brain, and testis persist for an extended period of time, whereas the same Ags are rapidly eliminated at conventional sites. Immune privilege, therefore, provides unwanted refuge for pathogens and tumor cells but is beneficial for the survival of allogeneic grafts. In this study, we asked whether memory T cells can eliminate foreign Ags deposited at an immunologically privileged site by studying CD8 memory T cell-mediated rejection of pancreatic islet allografts placed either in the testis (a privileged organ) or under the kidney capsule (a nonprivileged site) of diabetic mice. We found that CD8 memory T cells reject intratesticular grafts at a significantly slower rate than the rejection of intrarenal grafts. Delayed graft rejection in the testis was not due to reduced homing or proliferation of memory T cells but due to their increased apoptosis at that site. Apoptosis was mediated by the combined actions of two TNFR family members that are up-regulated on activated memory T cells, Fas, and CD30. Therefore, memory T cells survey immunologically privileged tissues but are subject to the immunosuppressive mechanisms present at these sites.
Different Competitive Capacities of Stat4- and Stat6-Deficient CD4+ T Cells during Lymphophenia-Driven Proliferation
Authors: Sanchez-Guajardo Vanesa , Borghans José A. M. , Marquez Maria-Elena , Garcia Sylvie , Freitas Antonio A.
Published: April 2014

The outcome of an immune response relies on the competitive capacities acquired through differentiation of CD4+ T cells into Th1 or Th2 effector cells. Because Stat4 and Stat6 proteins are implicated in the Th1 vs Th2 generation and maintenance, respectively, we compare in this study the kinetics of Stat4−/− and Stat6−/− CD4+ T cells during competitive bone marrow reconstitution and lymphopenia-driven proliferation. After bone marrow transplantation, both populations reconstitute the peripheral T cell pools equally well. After transfer into lymphopenic hosts, wild-type and Stat6−/− CD4+ T cells show a proliferation advantage, which is early associated with the expression of an active phospho-Stat4 and the down-regulation of Stat6. Despite these differences, Stat4- and Stat6-deficient T cells reach similar steady state numbers. However, when both Stat4−/− and Stat6−/− CD4+ T cells are coinjected into the same hosts, the Stat6−/− cells become dominant and out-compete Stat4−/− cells. These findings suggest that cell activation, through the Stat4 pathway and the down-regulation of Stat6, confers to pro-Th1 T cells a slight proliferation advantage that in a competitive situation has major late repercussions, because it modifies the final homeostatic equilibrium of the populations and favors the establishment of Th1 CD4+ T cell dominance.
Polarized Development of Memory Cell-Like IFN-γ-Producing Cells in the Absence of TCR ζ-Chain
Authors: Krymskaya Ludmila , Lee Wen-Hui , Zhong Lingwen , Liu Chih-Pin
Published: April 2014

TCR/CD3 complex-mediated signals play critical roles in regulating CD4+ Th cell differentiation. In this report, we have examined the in vivo role of a key TCR/CD3 complex molecule ζ-chain in regulating the differentiation of Th cells. We have studied T cells from ζ-chain-deficient mice (ζKO mice), ζ-chain-bearing mice (ζ+ mice), and from ζKO mice expressing a FcRγ chain transgene (FcRγTG, ζKO mice). Our results demonstrated that, compared with those of control mice, CD4+ T cells and not CD8+ T cells from ζKO mice were polarized into IFN-γ-producing cells. Some of these IFN-γ-producing cells could also secrete IL-10. Interestingly, ζKO mouse T cells produced IFN-γ even after they were cultured in a Th2 condition. Our studies to determine the molecular mechanisms underlying the polarized IFN-γ production revealed that the expression level of STAT4 and T-bet were up-regulated in freshly isolated T cells from ζKO mice. Further studies showed that noncultured ζKO mice CD4+ T cells and thymocytes bore a unique memory cell-like CD44high, CD62Llow/neg phenotype. Altogether, these results suggest that, in the absence of the ζ-chain, CD4+ T cells develop as polarized IFN-γ-producing cells that bear a memory cell-like phenotype. The ζ-chain-bearing T cells may produce a large amount of IFN-γ only after they are cultured in a condition favoring Th1 cell differentiation. This study may provide important implications for the down-regulation of ζ-chain in T cells of patients bearing a variety of tumors, chronic inflammatory and infectious diseases.
Induction of Impaired Antitumor Immunity by Fusion of MHC Class II-Deficient Dendritic Cells with Tumor Cells
Authors: Tanaka Yasuhiro , Koido Shigeo , Ohana Masaya , Liu Chunlei , Gong Jianlin
Published: April 2014

To dissect the role of Ag presentation through MHC class I and/or II pathways by dendritic cell (DC)-tumor fusion cells, we have created various types of DC-tumor fusion cells by alternating fusion cell partners. Fusions of MC38/MUC1 carcinoma cells with DC from wild-type (WT-DC), MHC class I knockout (IKO-DC), class II knockout (IIKO-DC), or class I and II knockout (I/IIKO-DC) mice created WTDC-fusion cells (FC), IKO-FC, IIKO-FC, and I/IIKO-FC, respectively. MHC class II- and MUC1-positive fusion cells were constructed by fusion of B16/MUC1 melanoma cells with IKO-DC (IKO/B16-FC). Immunization of MUC1 transgenic mice with 5 × 105 WTDC-FC, IKO-FC, IIKO-FC, or I/IIKO-FC provided 100, 91.7, 61.5, and 15.4% protection, respectively, against tumor challenge with MC38/MUC1 cells. In contrast, all mice immunized with irradiated MC38/MUC1 tumor cells or WT-DC developed tumors. One group of mice was immunized with 5 × 105 IKO/B16-FC and then challenged with B16/Ia+/MUC1 on one flank and MC38/MUC1 on the other flank. Immunization of these mice with IKO/B16-FC resulted in 100 and 78.6% protection against B16/Ia+/MUC1 and MC38/MUC1 tumor challenge, respectively. The antitumor immunity induced by immunization with IKO/B16-FC was able to inhibit the growth of MHC class II-negative tumor. In addition, in vivo results correlated with the induction of Ag-specific CTL. Collectively, the data indicate that MHC class II Ag presentation targeting activation of CD4 T cells is indispensable for antitumor immunity.
IFN-γ Suppresses STAT6 Phosphorylation by Inhibiting Its Recruitment to the IL-4 Receptor
Authors: Huang Zan , Xin Junping , Coleman John , Huang Hua
Published: April 2014

Polarized Th1 cells show a stable phenotype: they become insensitive to IL-4 stimulation and lose the potential to produce IL-4. Previously, we reported that IFN-γ played a critical role in stabilizing Th1 phenotype. However, the mechanism by which IFN-γ stabilizes Th1 phenotype is not clear. In this study, we compared STAT6 phosphorylation in wild-type (WT) and IFN-γ receptor knockout (IFNGR−/−) Th1 cells. We found a striking diminution of STAT6 phosphorylation in differentiated WT Th1 cells, but not in differentiated IFNGR−/− Th1 cells. The impairment of STAT6 phosphorylation in differentiated WT Th1 cells was not due to a lack of IL-4R expression or phosphorylation. Jak1 and Jak3 expression and phosphorylation were comparable in both cell types. No differential expression of suppressor of cytokine signaling 1 (SOCS1), SOCS3, or SOCS5 was observed in the two cell types. In addition, Src homology 2-containing phosphatase mutation did not affect IL-4-induced STAT6 phosphorylation in differentiated Th1 cells derived from viable motheaten (mev/mev) mice. These results led us to focus on a novel mechanism. By using a pulldown assay, we observed that STAT6 in WT Th1 cells bound less effectively to the phosphorylated IL-4R/GST fusion protein than that in IFNGR−/− Th1 cells. Our results suggest that IFN-γ may suppress phosphorylation of STAT6 by inhibiting its recruitment to the IL-4R.
CD70 Signaling Is Critical for CD28-Independent CD8+ T Cell-Mediated Alloimmune Responses In Vivo
Authors: Yamada Akira , Salama Alan D. , Sho Masayuki , Najafian Nader , Ito Toshiro , Forman John P. , Kewalramani Reshma , Sandner Sigrid , Harada Hiroshi , Clarkson Michael R. , Mandelbrot Didier A. , Sharpe Arlene H. , Oshima Hideo , Yagita Hideo , Chalasani Geetha , Lakkis Fadi G. , Auchincloss Hugh , Sayegh Mohamed H.
Published: April 2014

The inability to reproducibly induce robust and durable transplant tolerance using CD28-B7 pathway blockade is in part related to the persistence of alloreactive effector/memory CD8+ T cells that are less dependent on this pathway for their cellular activation. We studied the role of the novel T cell costimulatory pathway, CD27-CD70, in alloimmunity in the presence and absence of CD28-B7 signaling. CD70 blockade prolonged survival of fully mismatched vascularized cardiac allografts in wild-type murine recipients, and in CD28-deficient mice induced long-term survival while significantly preventing the development of chronic allograft vasculopathy. CD70 blockade had little effect on CD4+ T cell function but prevented CD8+ T cell-mediated rejection, inhibited the proliferation and activation of effector CD8+ T cells, and diminished the expansion of effector and memory CD8+ T cells in vivo. Thus, the CD27-CD70 pathway is critical for CD28-independent effector/memory CD8+ alloreactive T cell activation in vivo. These novel findings have important implications for the development of transplantation tolerance-inducing strategies in primates and humans, in which CD8+ T cell depletion is currently mandatory.
Intestinal Dendritic Cell Subsets: Differential Effects of Systemic TLR4 Stimulation on Migratory Fate and Activation In Vivo
Authors: Turnbull Emma L. , Yrlid Ulf , Jenkins Christopher D. , MacPherson G. Gordon
Published: April 2014

Dendritic cells (DC) present peripheral Ags to T cells in lymph nodes, but also influence their differentiation (tolerance/immunity, Th1/Th2). To investigate how peripheral conditions affect DC properties and might subsequently regulate T cell differentiation, we examined the effects of a potent DC-activating, TLR-4-mediated stimulus, LPS, on rat intestinal and hepatic DC in vivo. Steady-state rat intestinal and hepatic lymph DC are αE2 integrinhigh (CD103) and include two subsets, signal regulatory protein α (SIRPα)hi/low, probably representing murine CD8αα−/+ DC. Steady-state lamina propria DC are immature; surface MHC class IIlow, but steady-state lymph DC are semimature, MHC class IIhigh, but CD80/86low. Intravenous LPS induced rapid lamina propria DC emigration and increased lymph DC traffic without altering SIRPαhigh/SIRPαlow proportions. CD80/86 expression on lymph or mesenteric node DC was not up-regulated after i.v. LPS. In contrast, i.v. LPS stimulated marked CD80/86 up-regulation on splenic DC. CD80/86 expression on intestinal lymph DC, however, was increased after in vitro culture with TNF-α or GM-CSF, but not with up to 5 μg/ml LPS. Steady-state SIRPαlow DC localized to T cell areas of mesenteric nodes, spleen, and Peyer’s patch, whereas SIRPαhigh DC were excluded from these areas. Intravenous LPS stimulated rapid and abundant SIRPαhigh DC accumulation in T cell areas of mesenteric nodes and spleen. In striking contrast, i.v. LPS had no effect on DC numbers or distribution in Peyer’s patches. Our results suggest that any explanation of switching between tolerance and immunity as well as involving changes in DC activation status must also take into account differential migration of DC subsets.
Analysis of Marginal Zone B Cell Development in the Mouse with Limited B Cell Diversity: Role of the Antigen Receptor Signals in the Recruitment of B Cells to the Marginal Zone
Authors: Kanayama Naoki , Cascalho Marilia , Ohmori Hitoshi
Published: April 2014

The quasimonoclonal (QM) mouse provides an intelligible model to analyze the B cell selection as the competition between two major 4-hydroxy-3-nitrophenylacetyl-specific B cell populations whose BCR are comprised of the knockin VH17.2.25 (VHT)-encoded H chain and the λ1 or λ2 L chain. In this study, we show the QM system is useful to examine how BCR signals guide a subset of B cells to the marginal zone (MZ). Compared with the control C57BL/6 mice, the QM mice had ∼2.7-fold increased number of B cells exhibiting the MZ B cell phenotype and a larger MZ area in the spleen. Interestingly, VHT/λ2 B cells significantly predominated over VHT/λ1 B cells in MZ-(VHT/λ1:VHT/λ2 ≈ 3:7) and transitional 2-B cell subsets, while these two populations were comparable in immature, transitional 1, and mature counterparts. Thus, the biased use of λ2 in the MZ B cells may be the result of selection in the periphery. The enlargement of MZ B cell compartment and the preferred recruitment of the VHT/λ2 B cells were further augmented by doubling the VHT gene, but dampened by the dysfunction of Bruton’s tyrosine kinase, suggesting a positive role of BCR signaling in this selection. Comparison of Ag specificity between VHT/λ1 and VHT/λ2 IgM mAbs revealed a polyreactive nature of the VHT/λ2 BCR, including the reactivity with ssDNA. Taken together, it is suggested that polyreactivity (including self-reactivity) of BCR is crucial in driving B cells to differentiate into the MZ phenotype.
Differentiation and Expansion of T Cells with Regulatory Function from Human Peripheral Lymphocytes by Stimulation in the Presence of TGF-β
Authors: Rao Patricia E. , Petrone Andria L. , Ponath Paul D.
Published: April 2014

T cells with immunoregulatory function have been described in human and mouse systems. In both systems these cells can be differentiated either in the thymus or from peripheral T cells. To date, more progress has been made in the study of murine regulatory T cells, because it has been very difficult to isolate human regulatory T cells of sufficient purity and in sufficient numbers to permit detailed examinations of their biochemistry. We report in this study that human T cells with regulatory function can be differentiated in vitro from naive (CD4+CD45RA+) cord blood or peripheral T cells by stimulation with anti-CD3 and anti-CD28 in the presence of TGF-β. Cells derived in this manner express a surface phenotype (CD25+, CD122+, HLA-DR+, glucocorticoid-induced TNF receptor-related gene+, CD103+, CTLA-4+) described for human and mouse regulatory T cells and express protein and message for the transcription factor forkhead/winged helix transcription factor (FOXP3). They produce primarily TGF-β and IL-10, with lesser amounts of IFN-γ and IL-13, when stimulated through their TCRs and are capable of inhibiting cytokine production and proliferation by stimulated naive T cells. Unlike Th1 and Th2 cells, these TGF-β-derived regulatory T cells do not appear to be dependent on the protein kinase Cθ pathway of NF-κB activation for Ag-induced responses.
Differential Control of CD28-Regulated In Vivo Immunity by the E3 Ligase Cbl-b
Authors: Krawczyk Connie M. , Jones Russell G. , Atfield Alexandre , Bachmaier Kurt , Arya Sudha , Odermatt Bernhard , Ohashi Pamela S. , Penninger Josef M.
Published: April 2014

The E3 ubiquitin ligase Casitas B cell lymphoma-b (Cbl-b) plays a critical role in the development of autoimmunity and sets the threshold for T cell activation. In the absence of Cbl-b, T cells stimulated via the TCR respond similarly to those that have received a CD28-mediated costimulatory signal, suggesting that the absence of Cbl-b substitutes for CD28-mediated costimulation. In this study, we show that loss of Cbl-b restores Ig class switching and germinal center formation in Vav1 mutant mice in response to an in vivo viral challenge. Genetic inactivation of Cbl-b also rescues impaired antiviral IgG production in CD28-mutant mice. Moreover, loss of CD28 results in disorganization of follicular dendritic cell clusters, which is also rescued by the Cbl-b mutation. Intriguingly, despite restored antiviral in vivo immunity and follicular dendritic cell clusters, loss of Cbl-b did not rescue germinal center formation in CD28-deficient mice. Mechanistically, in vivo vesicular stomatitis virus-induced IL-4 and IFN-γ production and up-regulation of the inducible costimulatory molecule ICOS were dependent on CD28, and could not be rescued by the loss of Cbl-b. These data provide genetic evidence that CD28-dependent in vivo immune responses and Ig class switching can be genetically uncoupled from germinal center formation and ICOS induction by Cbl-b-Vav1-regulated signaling pathways.
Elemental Analysis ofMycobacterium avium-,Mycobacterium tuberculosis-, andMycobacterium smegmatis-Containing Phagosomes Indicates Pathogen-Induced Microenvironments within the Host Cell’s Endosomal System
Authors: Wagner Dirk , Maser Jörg , Lai Barry , Cai Zhonghou , Barry Clifton E. , Höner zu Bentrup Kerstin , Russell David G. , Bermudez Luiz E.
Published: April 2014

Mycobacterium avium and Mycobacterium tuberculosis are human pathogens that infect and replicate within macrophages. Both organisms live in phagosomes that fail to fuse with lysosomes and have adapted their lifestyle to accommodate the changing environment within the endosomal system. Among the many environmental factors that could influence expression of bacterial genes are the concentrations of single elements within the phagosomes. We used a novel hard x-ray microprobe with suboptical spatial resolution to analyze characteristic x-ray fluorescence of 10 single elements inside phagosomes of macrophages infected with M. tuberculosis and M. avium or with avirulent M. smegmatis. The iron concentration decreased over time in phagosomes of macrophages infected with Mycobacterium smegmatis but increased in those infected with pathogenic mycobacteria. Autoradiography of infected macrophages incubated with 59Fe-loaded transferrin demonstrated that the bacteria could acquire iron delivered via the endocytic route, confirming the results obtained in the x-ray microscopy. In addition, the concentrations of chlorine, calcium, potassium, manganese, copper, and zinc were shown to differ between the vacuole of pathogenic mycobacteria and M. smegmatis. Differences in the concentration of several elements between M. avium and M. tuberculosis vacuoles were also observed. Activation of macrophages with recombinant IFN-γ or TNF-α before infection altered the concentrations of elements in the phagosome, which was not observed in cells activated following infection. Siderophore knockout M. tuberculosis vacuoles exhibited retarded acquisition of iron compared with phagosomes with wild-type M. tuberculosis. This is a unique approach to define the environmental conditions within the pathogen-containing compartment.
VCAM-1 Expression on CD8+ Cells Correlates with Enhanced Anti-HIV Suppressing Activity
Authors: Diaz Leyla S. , Foster Hillary , Stone Mars R. , Fujimura Sue , Relman David A. , Levy Jay A.
Published: April 2014

CD8+ cells from HIV-infected individuals showing the CD8+ cell noncytotoxic antiviral response unexpectedly revealed mRNA for VCAM-1, a cell surface molecule found on endothelial cells. Uninfected subjects had undetectable levels of VCAM-1 mRNA in their CD8+ cells. Flow cytometry analysis showed that up to 12% of the CD8+ cells from HIV-positive individuals expressed VCAM-1 compared with 0.8% of the CD8+ cells of HIV-negative individuals. Enrichment of the CD8+VCAM-1+ cell population and subsequent coculture with CD4+ cells acutely infected with HIV-1 showed that the VCAM-1+CD8+ cells were able to suppress viral replication with 50% less input cells than the unseparated CD8+ cell population. This study demonstrates, for the first time, the expression of VCAM-1 on CD8+ cells. Moreover, the CD8+VCAM-1+ cells show enhanced CD8+ cell noncytotoxic antiviral response activity that could have clinical importance in HIV infection.
Profiling Blood Lymphocyte Interactions with Cancer Cells Uncovers the Innate Reactivity of Human γδ T Cells to Anaplastic Large Cell Lymphoma
Authors: Poupot Mary , Pont Frédéric , Fournié Jean-Jacques
Published: April 2014

Quantifying the contacts that circulating lymphocytes have with cancer cells is useful, because their deficit favors malignancy progression. All normal lymphocytes contact, scan, and acquire membrane fragments (trogocytosis) from foreign cells for their immunosurveillance. So in this study, we used the in vitro trogocytosis of PKH67-stained cancer cell lines as a measure of their interactions with bulks of PBMC freshly isolated from healthy donors. Allogeneic PBMC mixed and coincubated in vitro for 1 h did not trogocytosis, whereas in the same conditions CD20+, CD4+, CD8+, γδ T, and CD16+ PBMC interacted strongly with the cancer cells. Although most unprimed lymphoid effectors of innate (NK) and adaptive (B and T) immunity from healthy donors spontaneously trogocytosed different tumoral cell lines, some carcinoma cell lines could escape them in the coculture. This also uncovered the strong interactions of circulating Vγ9/Vδ2+ central memory γδ T cells with anaplastic large cell lymphoma. These interaction profiles were stable upon time for healthy blood donors but were different with other tumors and blood donors. This profiling provides interaction signatures for the immunomonitoring of cancer.
One NY-ESO-1-Derived Epitope That Promiscuously Binds to Multiple HLA-DR and HLA-DP4 Molecules and Stimulates Autologous CD4+ T Cells from Patients with NY-ESO-1-Expressing Melanoma
Authors: Mandic Maja , Castelli Florence , Janjic Bratislav , Almunia Christine , Andrade Pedro , Gillet Daniel , Brusic Vladimir , Kirkwood John M. , Maillere Bernard , Zarour Hassane M.
Published: April 2014

NY-ESO-1 is expressed by a broad range of human tumors and is often recognized by Abs in the sera of cancer patients with NY-ESO-1-expressing tumors. The NY-ESO-1 gene also encodes several MHC class I- and class II-restricted tumor epitopes recognized by T lymphocytes. In this study we report one novel pan-MHC class II-restricted peptide sequence, NY-ESO-1 87–111, that is capable of binding to multiple HLA-DR and HLA-DP4 molecules, including HLA-DRB1*0101, 0401, 0701, and 1101 and HLA-DPB1*0401 and 0402 molecules. We also demonstrate that peptide NY-ESO-1 87–111 stimulates Th1-type and Th-2/Th0-type CD4+ T cells and clones when presented in the context of these HLA-DR and HLA-DP4 molecules. Both bulk CD4+ T cells and CD4+ T cell clones were capable of recognizing not only peptide-pulsed APCs, but also autologous dendritic cells, either loaded with the NY-ESO-1 protein or transfected with NY-ESO-1 cDNAs. Using IFN-γ and IL-5 ELISPOT assays and PBL from patients with NY-ESO-1-expressing tumors, we observed the existence of Th1-type circulating CD4+ T cells recognizing peptide NY-ESO-1 87–111 in the context of HLA-DP4 molecules. Taken together, these data represent the first report of an HLA-DR- and HLA-DP-restricted epitope from a tumor Ag. They also support the relevance of cancer vaccine trials with peptides NY-ESO-1 87–111 in the large number of cancer patients with NY-ESO-1-expressing tumors.
The Expression and Function of Cathepsin E in Dendritic Cells
Authors: Chain Benjamin M. , Free Paul , Medd Patrick , Swetman Claire , Tabor Alethea B. , Terrazzini Nadia
Published: April 2014

Cathepsin E is an aspartic proteinase that has been implicated in Ag processing within the class II MHC pathway. In this study, we document the presence of cathepsin E message and protein in human myeloid dendritic cells, the preeminent APCs of the immune system. Cathepsin E is found in a perinuclear compartment, which is likely to form part of the endoplasmic reticulum, and also a peripheral compartment just beneath the cell membrane, with a similar distribution to that of Texas Red-dextran within 2 min of endocytosis. To investigate the function of cathepsin E in processing, a new soluble targeted inhibitor was synthesized by linking the microbial aspartic proteinase inhibitor pepstatin to mannosylated BSA via a cleavable disulfide linker. This inhibitor was shown to block cathepsin D/E activity in cell-free assays and within dendritic cells. The inhibitor blocked the ability of dendritic cells from wild-type as well as cathepsin D-deficient mice to present intact OVA, but not an OVA-derived peptide, to cognate T cells. The data therefore support the hypothesis that cathepsin E has an important nonredundant role in the class II MHC Ag processing pathway within dendritic cells.
Intestinal γδ T Cells Develop in Mice Lacking Thymus, All Lymph Nodes, Peyer’s Patches, and Isolated Lymphoid Follicles
Authors: Nonaka Satoshi , Naito Tomoaki , Chen Hao , Yamamoto Masafumi , Moro Kazuyo , Kiyono Hiroshi , Hamada Hiromasa , Ishikawa Hiromichi
Published: April 2014

Through analysis of athymic (nu/nu) mice carrying a transgenic gene encoding GFP instead of RAG-2 product, it has recently been reported that, in the absence of thymopoiesis, mesenteric lymph nodes and Peyer’s patches (PP) but not gut cryptopatches are pivotal birthplace of mature T cells such as the thymus-independent intestinal intraepithelial T cells (IEL). To explore and evaluate this important issue, we generated nu/nu mice lacking all lymph nodes (LN) and PP by administration of lymphotoxin-β receptor-Ig and TNF receptor 55-Ig fusion proteins into the timed pregnant nu/+ mice that had been mated with male nu/nu mice (nu/nu LNP− mice). We also generated nu/nu aly/aly (aly, alymphoplasia) double-mutant mice that inherently lacked all LN, PP, and isolated lymphoid follicles. Although γδ-IEL were slightly smaller in number than those in nu/nu mice, substantial colonization of γδ-IEL was found to take place in the intestinal epithelia of nu/nu LNP− and nu/nu aly/aly mice. Notably, the population size of a major CD8αα+ γδ-IEL subset was maintained, the use of TCR-γ-chain variable gene segments by these γδ-IEL was unaltered, and the development of cryptopatches remained intact in these nu/nu LNP− and nu/nu aly/aly mice. These findings indicate that all LN, including mesenteric LN, PP, and isolated lymphoid follicles, are not an absolute requirement for the development of γδ-IEL in athymic nu/nu mice.
IFN-α Regulates TLR-Dependent Gene Expression of IFN-α, IFN-β, IL-28, and IL-29
Authors: Sirén Jukka , Pirhonen Jaana , Julkunen Ilkka , Matikainen Sampsa
Published: April 2014

Toll-like receptors (TLRs) mediate host cell activation by various microbial components. TLR2, TLR3, TLR4, TLR7, TLR8, and TLR9 are the receptors that have been associated with virus-induced immune response. We have previously reported that all these TLRs, except TLR9, are expressed at mRNA levels in human monocyte-derived macrophages. Here we have studied TLR2, TLR3, TLR4, and TLR7/8 ligand-induced IFN-α, IFN-β, IL-28, and IL-29 expression in human macrophages. IFN-α pretreatment of macrophages was required for efficient TLR3 and TLR4 agonist-induced activation of IFN-α, IFN-β, IL-28, and IL-29 genes. TLR7/8 agonist weakly activated IFN-α, IFN-β, IL-28, and IL-29 genes, whereas TLR2 agonist was not able to activate these genes. IFN-α enhanced TLR responsiveness in macrophages by up-regulating the expression of TLR3, TLR4, and TLR7. IFN-α also enhanced the expression of TLR signaling molecules MyD88, TIR domain-containing adaptor inducing IFN-β, IκB kinase-ε, receptor interacting protein 1, and IFN regulatory factor 7. Furthermore, the activation of transcription factor IFN regulatory factor 3 by TLR3 and TLR4 agonists was dependent on IFN-α pretreatment. In conclusion, our results suggest that IFN-α sensitizes cells to microbial recognition by up-regulating the expression of several TLRs as well as adapter molecules and kinases involved in TLR signaling.
High Incidence of Spontaneous Disease in an HLA-DR15 and TCR Transgenic Multiple Sclerosis Model
Authors: Ellmerich Stephan , Mycko Marcin , Takacs Katalin , Waldner Hanspeter , Wahid Faisal N. , Boyton Rosemary J. , King Rosalind H. M. , Smith Paul A. , Amor Sandra , Herlihy Amy H. , Hewitt Rachel E. , Jutton Mark , Price David A. , Hafler David A. , Kuchroo Vijay K. , Altmann Daniel M.
Published: April 2014

Multiple sclerosis (MS) is thought to involve CD4 T cell recognition of self myelin, many studies focusing on a pathogenic role for anti-myelin, HLA-DR15-restricted T cells. In experimental allergic encephalomyelitis, it is known which epitopes trigger disease and that disease is associated with determinant spread of T cell reactivity. Characterization of these events in human MS is critical for the development of peptide immunotherapies, but it has been difficult to define the role of determinant spread or define which epitopes might be involved. In this study, we report humanized transgenic mice, strongly expressing HLA-DR15 with an MS-derived TCR; even on a RAG-2 wild-type background, mice spontaneously develop paralysis. Disease, involving demyelination and axonal degeneration, correlates with inter- and intramolecular spread of the T cell response to HLA-DR15-restricted epitopes of myelin basic protein, myelin oligodendrocyte glycoprotein, and αB-crystallin. Spread is reproducible and progressive, with two of the epitopes commonly described in responses of HLA-DR15 patients. The fact that this pattern is reiterated as a consequence of CNS tissue damage in mice demonstrates the value of the transgenic model in supplying an in vivo disease context for the human responses. This model, encompassing pathologically relevant, spontaneous disease with the presentation of myelin epitopes in the context of HLA-DR15, should offer new insights and predictions about T cell responses during MS as well as a more stringent test bed for immunotherapies.
Extrathymic Hemopoietic Progenitors Committed to T Cell Differentiation in the Adult Mouse
Authors: Arcangeli Marie-Laure , Lancrin Christophe , Lambolez Florence , Cordier Corinne , Schneider Elke , Rocha Benedita , Ezine Sophie
Published: April 2014

The role of the thymus in T cell commitment of hemopoietic precursor is yet controversial. We previously identified a major T cell progenitor activity in precursor cells isolated from bone marrow-derived spleen colonies. In this study, we characterize the properties of these pre-T cells. We demonstrate that they have unique phenotype and can be generated in a total absence of any thymic influence. Indeed, even when studied at the single-cell level, extrathymic T cell-committed precursors express T cell-specific genes. Moreover, these cells are not committed to a particular T cell differentiation pathway because they can generate both extrathymic CD8αα+ intraepithelial lymphocytes and thymus-derived conventional thymocytes. We also compared these pre-T cells with fully T cell-committed thymic progenitors. When tested in vitro or by direct intrathymic transfer, these cells have a low clonogenic activity. However, after i.v. transfer, thymus repopulation is efficient and these precursors generate very high numbers of peripheral T cells. These results suggest the existence of extra steps of pre-T cell maturation that improve thymus reconstitution capacity and that can be delivered even after full T cell commitment. Consequently, our studies identify a source of extrathymic progenitors that will be helpful in defining the role of the thymus in the earliest steps of T cell differentiation.
Immunological Effects of Transgenic Constitutive Expression of the Type 1 Sphingosine 1-Phosphate Receptor by Mouse Lymphocytes
Authors: Gräler Markus H. , Huang Mei-Chuan , Watson Susan , Goetzl Edward J.
Published: April 2014

The type 1 sphingosine 1-phosphate (S1P) G protein-coupled receptor (S1P1) normally transduces S1P effects on lymph node (LN) egress and tissue migration of naive lymphocytes. We now show that persistent expression of S1P1 by lymphocytes of S1P1-transgenic (Tg) mice suppresses delayed-type hypersensitivity and results in production of significantly more IgE Ab and less IgG2 Ab than in wild-type (wt) mice. wt host LN homing of 51Cr-labeled T cells from S1P1-Tg mice was only 30–40% of that for wt T cells. Adoptive-transfer of dye-labeled activated T cells from S1P1-Tg mice into wt mice resulted in 2.2-fold more in blood and 60% less in LNs than for activated wt T cells after 1 day. Proliferative responses of stimulated T cells from S1P1-Tg mice were only 10–34% of those for wt T cells. Disordered cellular and humoral immunity of S1P1-Tg mice thus may be attributable to both altered T cell traffic and depressed T cell functions, suggesting that S1P1-specific agonists may represent a novel therapeutic approach to autoimmunity and transplant rejection.
Direct Regulatory Role of NKT Cells in Allogeneic Graft Survival Is Dependent on the Quantitative Strength of Antigenicity
Authors: Oh Keunhee , Kim Sanghee , Park Se-Ho , Gu Hua , Roopenian Derry , Chung Doo Hyun , Kim Yon Su , Lee Dong-Sup
Published: April 2014

The role of NKT cells during immune responses is diverse, ranging from antiviral and antitumor activity to the regulation of autoimmune diseases; however, the regulatory function of CD1d-dependent NKT cells in rejection responses against allogeneic graft is uncertain. In this study, we demonstrated the direct regulatory effects of CD1d-dependent NKT cells using an allogeneic skin transplantation model. H-Y-mismatched skin graft survival was shortened in CD1d−/− recipients compared with wild-type recipients. Adoptive transfer of syngeneic NKT cells via splenocytes or hepatic mononuclear cells into CD1d−/− recipients restored graft survival times to those of wild-type recipients. α-Galactosylceramide, a specific activator of NKT cells, further prolonged graft survival. Although CD1d-dependent NKT cells did not extend skin graft survival in either major or complete minor histocompatibility-mismatched models, these cells affected graft survival in minor Ag mismatch models according to the magnitude of the antigenic difference. The afferent arm of NKT cell activation during transplantation required CD1d molecules expressed on host APCs and the migration of CD1d-dependent NKT cells into grafts. Moreover, the regulatory effects of CD1d-dependent NKT cells against alloantigen were primarily IL-10 dependent. Taken together, we concluded that CD1d-dependent NKT cells may directly affect the outcome of allogeneic skin graft through an IL-10-dependent regulatory mechanism.
Distinct Requirements for Deletion versus Anergy during CD8 T Cell Peripheral Tolerance In Vivo
Authors: Redmond William L. , Marincek Boris C. , Sherman Linda A.
Published: April 2014

Activation of naive T cells by quiescent APCs results in tolerance through deletion and anergy. The underlying basis for these distinct fates is unclear. Using clone 4 TCR transgenic animals as a source of naive CD8 T cells, we examined the requirements for peripheral deletion in vivo. Our results demonstrate that independent of the amount of Ag used for stimulation, a single dose was insufficient to achieve complete clonal deletion. Instead, further antigenic exposure was required to completely eliminate all of the activated T cells. Additionally, consecutive stimulations with low doses of Ag were highly effective in promoting deletion. In contrast, although stimulation with high doses of Ag initially led to the apoptosis of many of the activated T cells, it induced hyporesponsiveness in a portion of the responding cells, thereby sparing them from further activation and deletion. These data explain why some conditions promote tolerance through clonal deletion whereas others promote anergy. Furthermore, these data provide a framework to devise protocols for effective deletion of potentially autoreactive T cells.
Attenuation of Allergen-Induced Responses in CCR6−/− Mice Is Dependent upon Altered Pulmonary T Lymphocyte Activation
Authors: Lundy Steven K. , Lira Sergio A. , Smit Jetse J. , Cook Donald N. , Berlin Aaron A. , Lukacs Nicholas W.
Published: April 2014

We have established a defect in CCR6−/− mice in response to a cockroach allergen airway challenge characterized by decreased IL-5 production, reduced CD4+ T and B cells as well as decreased eosinophil accumulation. To determine the nature of the defect in CCR6−/− mice T lymphocyte populations from allergen-sensitized wild-type mice were transferred into sensitized CCR6−/− mice. The reconstituted response was characterized by an increase in IL-5 levels, eosinophil accumulation, and serum IgE levels in recipient CCR6−/− mice. Analysis of lymphocytes from draining lymph nodes of CCR6+/+ and CCR6−/− sensitized or challenged mice demonstrated a significant decrease in IL-5 and IL-13 production in CCR6−/− mice. In contrast, the systemic response in allergen-rechallenged spleen cells demonstrated no significant alteration in allergen-induced cytokine production. Transfer of isolated splenic T lymphocytes from sensitized CCR6+/+ mice induced airway hyperresponsiveness in wild-type but not CCR6−/− naive mice, suggesting that T cells alone were not sufficient to induce airway hyperresponsiveness in CCR6−/− mice. Additional analysis demonstrated decreased CD11c+, CD11b+ and CD11c, and B220 subsets of dendritic cells in the lungs of CCR6−/− mice after allergen challenge. Using in vitro cell mixing studies with isolated pulmonary CD4+ T cells and CD11c+ cells from CCR6+/+ or CCR6−/− mice, we demonstrate alterations in both CCR6−/− T cells and CCR6−/− pulmonary APCs to elicit IL-5 responses. Altogether, the defect in CCR6−/− mice appears to be primarily due to an alteration in T cell activation, but also appears to include local pulmonary APC defects.
Complement Inhibitor of C5 Activation from the Soft Tick Ornithodoros moubata
Authors: Nunn Miles A. , Sharma Ankit , Paesen Guido C. , Adamson Sharon , Lissina Olga , Willis Anthony C. , Nuttall Patricia A.
Published: April 2014

Blood-feeding ticks must control C activation or be damaged by the host inflammatory response. We report the characterization and expression of a novel, relatively small, broad-acting C inhibitory protein (termed OmCI) from the soft tick Ornithodoros moubata. The native 17-kDa nonglycosylated protein inhibits both human and guinea pig classical and alternative C activation pathways. The IC50 values for each pathway were 12 and 27 nM, respectively, in hemolytic assays using human serum diluted 40-fold. The cDNA encodes a protein of 168 aa, including an 18-aa secretion signal sequence that is absent in the mature form. The inhibitor has 46% amino acid identity with moubatin, a platelet aggregation inhibitor also from O. moubata that is an outlying member of the lipocalin family. Native OmCI had no inhibitory effect on the addition of C8 and C9 to preformed C5b-C7 and C5b-C8 to form the membrane attack complex and no effect on the rate of C3a production by the C3 convertase enzymes C4bC2a, C3(H2O)Bb, or C3bBb. Both recombinant and native OmCI abolish production of C5a by human classical (C4bC3bC2a) and alternative (C3bC3bBb) C5 convertases. Addition of excess C5 but not C3 competes away the inhibitory activity of OmCI, indicating that OmCI targets C5 itself rather than inhibiting the C5 convertase C4bC3bC2a itself. Direct binding of OmCI to C5 was demonstrated by Western blotting and gel filtration chromatography using 125I-labeled proteins. OmCI is the first lipocalin family member shown to inhibit C and also the first natural inhibitor that specifically targets the C5 activation step.
Two Autoimmune Diabetes Loci Influencing T Cell Apoptosis Control Susceptibility to Experimental Autoimmune Myocarditis
Authors: Guler Mehmet L. , Ligons Davinna L. , Wang Yan , Bianco Michael , Broman Karl W. , Rose Noel R.
Published: April 2014

The pathogenesis of immune-mediated myocarditis depends on genetic and environmental factors. To study the genetic mechanisms, we have developed a model of experimental autoimmune myocarditis in the A.SW mouse. Here we provide evidence that loci on murine chromosome 6, and possibly chromosome 1, are involved in regulating susceptibility. Moreover, these loci overlap with loci implicated in other autoimmune diseases including diabetes in the NOD mouse. These two loci also regulate apoptosis in thymocytes as well as peripheral T cells in the NOD mouse, and we report further that A.SW mice demonstrate the same characteristics in apoptosis. These results suggest that common pathogenetic mechanisms involving apoptosis of both thymic and peripheral T cells are shared by multiple autoimmune diseases.
Preferential Apoptosis of HIV-1-Specific CD4+ T Cells
Authors: Yue Feng Yun , Kovacs Colin M. , Dimayuga Rowena C. , Gu Xiao Xiao Jenny , Parks Paul , Kaul Rupert , Ostrowski Mario A.
Published: April 2014

In contrast to other viral infections such as CMV, circulating frequencies of HIV-1-specific CD4+ T cells in peripheral blood are quantitatively diminished in the majority of HIV-1-infected individuals. One mechanism for this quantitative defect is preferential infection of HIV-1-specific CD4+ T cells, although <10% of HIV-1-specific CD4+ T cells are infected. Apoptosis has been proposed as an important contributor to the pathogenesis of CD4+ T cell depletion in HIV/AIDS. We show here that, within HIV-1-infected individuals, a greater proportion of ex vivo HIV-1-specific CD4+ T cells undergo apoptosis compared with CMV-specific CD4+ T cells (45 vs 7.4%, respectively, p < 0.05, in chronic progressors). The degree of apoptosis within HIV-1-specific CD4+ T cells correlates with viral load and disease progression, and highly active antiretroviral therapy abrogates these differences. The data support a mechanism for apoptosis in these cells similar to that found in activation-induced apoptosis through the TCR, resulting in oxygen-free radical production, mitochondrial damage, and caspase-9 activation. That HIV-1 proteins can also directly enhance activation-induced apoptosis supports a mechanism for a preferential induction of apoptosis of HIV-1-specific CD4+ T cells, which contributes to a loss of immunological control of HIV-1 replication.
IL-2 and Autocrine IL-4 Drive the In Vivo Development of Antigen-Specific Th2 T Cells Elicited by Nematode Parasites
Authors: Liu Zhugong , Liu Qian , Hamed Hossein , Anthony Robert M. , Foster Anthony , Finkelman Fred D. , Urban Joseph F. , Gause William C.
Published: April 2014

The intestinal nematode parasite, Nippostrongylus brasiliensis, triggers potent type 2 immunity. Using OVA peptide as a model Ag, we have examined the adjuvant effects of this parasite on the in vivo development of Ag-specific Th2 cells from naive DO11.10 T cells. Our findings show that Th2 cells can develop from transferred naive OVA-specific DO11.10 T cells in recipient IL-4−/− mice inoculated with N. brasiliensis plus OVA. However, autocrine IL-4 is required for in situ Th2 cell differentiation since transferred IL-4Rα-deficient DO11.10 T cells showed greatly reduced Th2 cell development in inoculated IL-4−/− recipient mice. Surprisingly, we also found that IL-2 blockade promoted B7-dependent T cell cycling, but inhibited the development of OVA-specific Th2 cells. Furthermore, the effects of IL-2 occurred independently of CD25+ T regulatory cells. These studies establish a previously unrecognized requirement for autocrine IL-4 and IL-2 in Th2 responses elicited by nematode parasites.
Potentiation of Glucocorticoid Activity in Hypoxia through Induction of the Glucocorticoid Receptor
Authors: Leonard Martin O. , Godson Catherine , Brady Hugh R. , Taylor Cormac T.
Published: April 2014

Tissue hypoxia is intimately associated with chronic inflammatory disease and may signal to the resolution of inflammatory processes. Glucocorticoid signaling through the glucocorticoid receptor (GR) represents a clinically important endogenous anti-inflammatory pathway. Microarray analysis reveals that the GR is transcriptionally up-regulated by hypoxia in human renal proximal tubular epithelial cells. Hypoxic up-regulation of the GR was confirmed at the level of promoter activity, mRNA, and protein expression. Furthermore, functional potentiation of glucocorticoid activity in hypoxia was observed as an enhancement of dexamethasone-induced glucocorticoid response element promoter activity and enhanced dexamethasone-mediated inhibition of IL-1β-stimulated IL-8 expression and hypoxia-induced vascular endothelial growth factor expression. Knockdown of enhanced GR gene expression in hypoxia using specific GR small inhibitory RNA (siRNA) resulted in an attenuation of the enhanced glucocorticoid sensitivity. A role for the hypoxia-inducible transcription factor, HIF-1α, in the regulation of GR expression and the associated potentiation of glucocorticoid activity in hypoxia was also demonstrated. These results reveal a novel signaling aspect responsible for the incorporation of hypoxic and glucocorticoid stimuli, which we hypothesize to be an important co-operative pathway for the control of gene expression observed in complex tissue microenvironments in inflamed states.
Multiple β1 Integrins Mediate Enhancement of Human Airway Smooth Muscle Cytokine Secretion by Fibronectin and Type I Collagen
Authors: Peng Qi , Lai Dilys , Nguyen Trang T.-B. , Chan Vivien , Matsuda Takeshi , Hirst Stuart J.
Published: April 2014

Altered airway smooth muscle (ASM) function and enrichment of the extracellular matrix (ECM) with interstitial collagen and fibronectin are major pathological features of airway remodeling in asthma. We have previously shown that these ECM components confer enhanced ASM proliferation in vitro, but their action on its newly characterized secretory function is unknown. Here, we examined the effects of fibronectin and collagen types I, III, and V on IL-1β-dependent secretory responses of human ASM cells, and characterized the involvement of specific integrins. Cytokine production (eotaxin, RANTES, and GM-CSF) was evaluated by ELISA, RT-PCR, and flow cytometry. Function-blocking integrin mAbs and RGD (Arg-Gly-Asp)-blocking peptides were used to identify integrin involvement. IL-1β-dependent release of eotaxin, RANTES, and GM-CSF was enhanced by fibronectin and by fibrillar and monomeric type I collagen, with similar changes in mRNA abundance. Collagen types III and V had no effect on eotaxin or RANTES release but did modulate GM-CSF. Analogous changes in intracellular cytokine accumulation were found, but in <25% of the total ASM cell population. Function-blocking Ab and RGD peptide studies revealed that α2β1, α5β1, αvβ1, and αvβ3 integrins were required for up-regulation of IL-1β-dependent ASM secretory responses by fibronectin, while α2β1 was an important transducer for type I collagen. Thus, fibronectin and type I collagen enhance IL-1β-dependent ASM secretory responses through a β1 integrin-dependent mechanism. Enhancement of cytokine release from ASM by these ECM components may contribute to airway wall inflammation and remodeling in asthma.
Heparin Binds to Lipopolysaccharide (LPS)-Binding Protein, Facilitates the Transfer of LPS to CD14, and Enhances LPS-Induced Activation of Peripheral Blood Monocytes
Authors: Heinzelmann Michael , Bosshart Herbert
Published: April 2014

Heparin is one of the most effective drugs for preventing and treating thromboembolic complications in surgical patients. Recent evidence suggests that heparin enhances the proinflammatory responses of human peripheral blood monocytes to Gram-negative endotoxin (LPS). We have identified LPS-binding protein (LBP) as a novel heparin-binding plasma protein. The affinity of LPB to heparin was KD = 55 ± 8 nM, as measured by surface plasmon resonance. Using a fluorescence-based assay, we showed that clinically used heparin preparations significantly enhance the ability of LBP to catalytically disaggregate and transfer LPS to CD14, the LPS receptor. The presence of clinically relevant heparin concentrations in human whole blood increased LPS-induced production of the proinflammatory cytokine IL-8. Fondaparinux, which is identical with the antithrombin III-binding pentasaccharide in heparin, did not bind to LBP or alter LBP function. Thus, this novel anticoagulant drug is a potential candidate for safe administration to patients who have endotoxemia and require anticoagulation.