bluuebunny/crossref_metadata_2025 huggingface dataset:
Notch2 Haploinsufficiency Results in Diminished B1 B Cells and a Severe Reduction in Marginal Zone B Cells
Authors: Witt Colleen M. , Won Woong-Jai , Hurez Vincent , Klug Christopher A.
Published: April 2014

Recent studies have implicated a role for Notch in the generation of marginal zone (MZ) B cells. To further investigate the role of Notch in the B cell lineage, we have analyzed the effects of reduced Notch2 signaling in mice expressing one functional allele of Notch2 (Notch2+/−). Notch2+/− mice have reduced B1 B cells of the peritoneal cavity and show a severe reduction in MZ B cells of the spleen. The reduction in MZ B cells was not due to the disruption of splenic architecture, disregulated terminal differentiation, nor to increased apoptosis within the MZ B cell compartment. Rather, our data suggest that Notch2 haploinsufficiency leads to impaired development of MZ B cells, possibly by impacting the formation of immediate MZ B precursors. These results provide evidence that Notch2 plays a determining role in the development and/or the maintenance of B1 B and MZ B cells.
Role of TRAIL and IFN-γ in CD4+ T Cell-Dependent Tumor Rejection in the Anterior Chamber of the Eye
Authors: Wang Shixuan , Boonman Zita F. H. M. , Li Hao-Chuan , He YuGuang , Jager Martine J. , Toes Rene E. M. , Niederkorn Jerry Y.
Published: April 2014

Although the anterior chamber of the eye expresses immune privilege, some ocular tumors succumb to immune rejection. Previous studies demonstrated that adenovirus-induced tumors, adenovirus type 5 early region 1 (Ad5E1), underwent immune rejection following transplantation into the anterior chamber of syngeneic mice. Intraocular tumor rejection required CD4+ T cells, but did not require the following: 1) CD8+ T cells, 2) B cells, 3) TNF, 4) perforin, 5) Fas ligand, or 6) NK cells. This study demonstrates that CD4+ T cell-dependent tumor rejection does not occur in IFN-γ-deficient mice. Ad5E1 tumor cells expressed DR5 receptor for TRAIL and were susceptible to TRAIL-induced apoptosis. Although IFN-γ did not directly induce apoptosis of the tumor cells, it rendered them 3-fold more susceptible to TRAIL-induced apoptosis. Both CD4+ T cells and corneal endothelial cells expressed TRAIL and induced apoptosis of Ad5E1 tumor cells. The results suggest that Ad5E1 tumor rejection occurs via TRAIL-induced apoptosis as follows: 1) tumor cells express TRAIL-R2 and are susceptible to TRAIL-induced apoptosis, 2) IFN-γ enhances TRAIL expression on CD4+ T cells and ocular cells, 3) IFN-γ enhances tumor cell susceptibility to TRAIL-induced apoptosis, 4) apoptotic tumor cells are found in the eyes of rejector mice, but not in the eyes of IFN-γ knockout mice that fail to reject intraocular tumors, 5) CD4+ T cells and corneal endothelial cells express TRAIL and induce apoptosis of tumor cells, and 6) apoptosis induced by either CD4+ T cells or corneal cells can be blocked with anti-TRAIL Ab.
CCL9 Is Secreted by the Follicle-Associated Epithelium and Recruits Dome Region Peyer’s Patch CD11b+ Dendritic Cells
Authors: Zhao Xinyan , Sato Ayuko , Dela Cruz Charles S. , Linehan Melissa , Luegering Andreas , Kucharzik Torsten , Shirakawa Aiko-Konno , Marquez Gabriel , Farber Joshua M. , Williams Ifor , Iwasaki Akiko
Published: April 2014

The follicle-associated epithelium (FAE) secretes chemokines important in the recruitment of various cell types including CCL20 (MIP-3α). CCL20 is chemotactic to the CD11b+ dendritic cells (DCs) distributed in the subepithelial dome regions of the Peyer’s patches, and mice deficient in the receptor for CCL20, CCR6, have been reported to be devoid of the CD11b+ DCs in the dome regions. Here, we describe another chemokine specifically secreted from the FAE of mouse Peyer’s patches, CCL9 (MIP-1γ, CCF18, MRP-2). By in situ hybridization, we demonstrated that CCL9 mRNA was expressed by the FAE but not by the villus epithelium. At the protein level, CCL9 was detected on the FAE and on extracellular matrix structures within the dome regions of the Peyer’s patches. By RT-PCR, we demonstrated that one of the putative receptors for CCL9, CCR1, was expressed by the Peyer’s patch CD11b+ DCs and in a chemotaxis assay, CD11b+ DCs migrated toward CCL9. To compare the abilities of the chemokines CCL20 and CCL9 to recruit CD11b+ DCs to the dome regions, we examined the in vivo distribution of these cells in CCR6-deficient, CCL9-blocked wild type, or CCL9-blocked CCR6-deficient mice. To our surprise, using a sensitive immunofluorescence analysis, we observed that CD11b+ DCs were present in the dome regions of the CCR6-deficient mice. In contrast, Ab neutralization of CCL9 in vivo resulted in significant reduction of the CD11b+ DC number in the subepithelial dome regions of Peyer’s patches of both wild type and CCR6 −/− mice. Taken together, these results demonstrate an important role of CCL9 in CD11b+ DC recruitment to the dome regions of mouse Peyer’s patches.
Expression and Functional Importance of Collagen-Binding Integrins, α1β1 and α2β1, on Virus-Activated T Cells
Authors: Andreasen Susanne Ø. , Thomsen Allan R. , Koteliansky Victor E. , Novobrantseva Tatiana I. , Sprague Andrew G. , de Fougerolles Antonin R. , Christensen Jan P.
Published: April 2014

Adhesive interactions are crucial to cell migration into inflammatory sites. Using murine lymphocytic choriomeningitis virus as an Ag model system, we have investigated expression and function of collagen-binding integrins, α1β1 and α2β1, on activated and memory T cells. Using this system and MHC tetramers to define Ag-specific T cells, we demonstrate that contrary to being VLAs, expression of α1β1 and α2β1 can be rapidly induced on acutely activated T cells, that expression of α1β1 remains elevated on memory T cells, and that expression of α1β1 parallels that of viral-specific effector CD8+ T cells (defined by tetramer and IFN-γ staining). In an adoptive transfer model, mAb-mediated blockade of these integrins on activated effector and memory T cells inhibited Ag-specific delayed-type hypersensitivity responses; similar decreased responses were seen upon transfer of α1-deficient activated/memory T cells. Thus, expression of α1β1 and α2β1 integrins on activated T cells is directly functionally important for generation of inflammatory responses within tissues. Finally, the inhibitory effect of α1β1 blockade on the delayed-type hypersensitivity response could be bypassed by direct injection of Ag-specific T cells to inflammatory sites, demonstrating for the first time in vivo that collagen-binding integrins are involved in leukocyte migration into tissues.
CXCR3 Is Induced Early on the Pathway of CD4+ T Cell Differentiation and Bridges Central and Peripheral Functions
Authors: Rabin Ronald L. , Alston Marc A. , Sircus Janna C. , Knollmann-Ritschel Barbara , Moratz Chantal , Ngo Diana , Farber Joshua M.
Published: April 2014

Chemokine receptors on T cells are frequently categorized as functioning either in immune system homeostasis within lymphoid organs, or in peripheral inflammation. CXCR3 is in the latter category and is reported to be expressed selectively on Th1 cells. We found that CXCR3 was expressed in vivo on newly activated tonsillar CD4+ T cells. Using CD4+ T cells from cord blood, we found that CXCR3 was induced by cellular activation in vitro independently of the cytokine milieu, although on resting cells, expression was maintained preferentially on those that had been activated in type 1 conditions. In inflamed tonsils, CXCR3+CD4+ T cells were localized around and within germinal centers. The inference that CXCR3 has a role in germinal center reactions was supported by the finding that the CXCR3 ligand CXC chemokine ligand 9 was expressed in a pattern demarcating a subset of germinal centers both in tonsil and in lymph nodes from an HIV-infected individual. We next investigated the role of CXCR3 on peripheral effector/memory CD4+ T cells by comparing its pattern of expression with that of CCR5, another Th1-cell associated chemokine receptor. Analysis of cells directly from peripheral blood and after activation in vitro suggested that CXCR3 expression preceded that of CCR5, supporting a model of sequential induction of chemokine receptors during CD4+ T cell differentiation. Taken together, our data show that CXCR3 can be expressed at all stages of CD4+ T cell activation and differentiation, bridging central function in lymphoid organs and effector function in peripheral tissues.
Modeling TCR Signaling Complex Formation in Positive Selection
Authors: Hare Katherine J. , Pongracz Judit , Jenkinson Eric J. , Anderson Graham
Published: April 2014

T cell receptor signaling in the thymus can result in positive selection, and hence progressive maturation to the CD4+8− or CD4−8+ stage, or induction of apoptosis by negative selection. Although it is poorly understood how TCR ligation at the CD4+8+ stage can lead to such different cell fates, it is thought that the strength of signal may play a role in determining the outcome of TCR signaling. In this study, we have characterized the formation of an active signaling complex in thymocytes undergoing positive selection as a result of interaction with thymic epithelial cells. Although this signaling complex involves redistribution of cell surface and intracellular molecules, reminiscent of that observed in T cell activation, accumulation of GM1-containing lipid rafts was not observed. However, enforced expression of the costimulatory molecule CD80 on thymic epithelium induced GM1 polarization in thymocytes, and was accompanied by reduced positive selection and increased apoptosis. We suggest that the presence or absence of CD80 costimulation influences the outcome of TCR signaling in CD4+8+ thymocytes through differential lipid raft recruitment, thus determining overall signal strength and influencing developmental cell fate.
Sustained Response Initiation Is Required for T Cell Clonal Expansion But Not for Effector or Memory Development In Vivo
Authors: Lefrançois Leo , Marzo Amanda , Williams Kristina
Published: April 2014

The factors determining whether an immune response is productive are poorly understood. To understand the circumstances affecting the early stage of the immune response which determine whether memory is generated, the CD8 T cell response was mapped in detail following immunization with live or heat-killed bacteria. Our results demonstrate that even in response to a weak immunogen, functional memory cell development is linked to effector cell induction in lymphoid and nonlymphoid tissues. The main defect in the response to killed microorganisms is inefficient induction of clonal expansion. This failure is due to a contracted, but costimulation-dependent activation phase in the lymphoid tissues, resulting in rapid but abortive growth. Conversely, the response to live bacteria is characterized by protracted early T cell sequestration in lymphoid tissues. Thus, memory development requires effector induction, while optimal clonal expansion is regulated by the duration of response initiation.
A Failure to Repair Self-Proteins Leads to T Cell Hyperproliferation and Autoantibody Production
Authors: Doyle Hester A. , Gee Renelle J. , Mamula Mark J.
Published: April 2014

It is clear that many factors can perturb T cell homeostasis that is critical in the maintenance of immune tolerance. Defects in the molecules that regulate homeostasis can lead to autoimmune pathology. This simple immunologic concept is complicated by the fact that many self-proteins undergo spontaneous posttranslational modifications that affect their biological functions. This is the case in the spontaneous conversion of aspartyl residues to isoaspartyl residues, a modification occurring at physiological pH and under conditions of cell stress and aging. We have examined the effect of isoaspartyl modifications on the effector functions of T lymphocytes in vivo using mice lacking the isoaspartyl repair enzyme protein carboxyl methyltransferase (PCMT). PCMT−/− CD4+ T cells exhibit increased proliferation in response to mitogen and Ag receptor stimulation as compared with wild-type CD4+ T cells. Hyperproliferation is marked by increased phosphorylation of members of both the TCR and CD28 signaling pathways. Wild-type mice reconstituted with PCMT−/− bone marrow develop high titers of anti-DNA autoantibodies and kidney pathology typical of that found in systemic lupus erythematosus. These observations, coupled with the fact that humans have polymorphisms in the pcmt gene, suggest that isoaspartyl self-proteins may alter the maintenance of peripheral immune tolerance.
Involvement of Inducible Costimulator-B7 Homologous Protein Costimulatory Pathway in Murine Lupus Nephritis
Authors: Iwai Hideyuki , Abe Masaaki , Hirose Sachiko , Tsushima Fumihiko , Tezuka Katsunari , Akiba Hisaya , Yagita Hideo , Okumura Ko , Kohsaka Hitoshi , Miyasaka Nobuyuki , Azuma Miyuki
Published: April 2014

Inducible costimulator (ICOS)-B7 homologous protein (B7h) is a new member of the CD28-B7 family of costimulatory molecules that regulates T cell-dependent humoral immune responses. In this study, we examined the involvement of this costimulatory pathway in the development and progression of lupus in NZB/W F1 mice. Expression of ICOS on T cells was enhanced with disease progression, whereas B7h expression on B cells was down-regulated. Administration of anti-B7h mAb before the onset of renal disease significantly delayed the onset of proteinuria and prolonged survival. Blockade of B7h effectively inhibited all subclasses of IgG autoantibody production and accumulation of both Th1 and Th2 cells. Hypercellularity and deposition of IgG and C3 in glomeruli were significantly reduced. B7h blockade after the onset of proteinuria prevented the disease progression and improved the renal pathology. Our results demonstrated the involvement of the ICOS-B7h costimulatory pathway in the pathogenesis of lupus nephritis, and the blockade of this pathway may be beneficial for the treatment of human systemic lupus erythematosus.
Ultraviolet Light Exposure Suppresses Contact Hypersensitivity by Abrogating Endothelial Intercellular Adhesion Molecule-1 Up-Regulation at the Elicitation Site
Authors: Komura Kazuhiro , Hasegawa Minoru , Hamaguchi Yasuhito , Saito Eriko , Kaburagi Yuko , Yanaba Koichi , Kawara Shigeru , Takehara Kazuhiko , Seki Makoto , Steeber Douglas A. , Tedder Thomas F. , Sato Shinichi
Published: April 2014

Hapten sensitization through UV-exposed skin induces systemic immune suppression, which is experimentally demonstrated by inhibition of contact hypersensitivity (CHS). Although this UV-induced effect has been shown to be mediated by inhibition of the afferent phase of the CHS, the UV effects on the efferent (elicitation) phase remain unknown. In this study, UV effects on endothelial ICAM-1 expression at elicitation sites were first examined. Mice were sensitized by hapten application onto UV-exposed back skin, and ears were challenged 5 days later. ICAM-1 up-regulation at nonirradiated elicitation sites following hapten challenge was eliminated by UV exposure on sensitization sites distant from elicitation sites. To assess whether loss of the ICAM-1 up-regulation at elicitation sites contributed to UV-induced immunosuppression, we examined CHS responses in UV-exposed ICAM-1-deficient (ICAM-1−/−) mice that genetically lacked the ICAM-1 up-regulation. ICAM-1−/− mice exhibited reduced CHS responses without UV exposure, but UV exposure did not further reduce CHS responses in ICAM-1−/− mice. Furthermore, ICAM-1 deficiency did not affect the afferent limb, because ICAM-1−/− mice had normal generation of hapten-specific suppressor and effector T cells. This UV-induced immunosuppression was associated with a lack of TNF-α production after Ag challenge at elicitation sites. Local TNF-α injection before elicitation abrogated the UV-induced CHS inhibition with increased endothelial ICAM-1 expression. TNF-α production at elicitation sites was down-regulated by IL-10, a possible mediator produced by hapten-specific suppressor T cells that are generated by UV exposure. These results indicate that UV exposure inhibits CHS by abrogating up-regulation of endothelial ICAM-1 expression after Ag challenge at elicitation sites.
Infectious Nickel Tolerance: A Reciprocal Interplay of Tolerogenic APCs and T Suppressor Cells That Is Driven by Immunization
Authors: Roelofs-Haarhuis Karin , Wu Xianzhu , Nowak Michael , Fang Min , Artik Suzan , Gleichmann Ernst
Published: April 2014

Previously, we reported that tolerance to nickel, induced by oral administration of Ni2+ ions, can be adoptively transferred to naive mice with only 102 splenic T cells. Here we show that 102 T cell-depleted spleen cells (i.e., APCs) from orally tolerized donors can also transfer nickel tolerance. This cannot be explained by simple passive transfer of the tolerogen. The APCs from orally tolerized donors displayed a reduced allostimulatory capacity, a tolerogenic phenotype, and an increased expression of CD38 on B cells. In fact, it was B cells among the APCs that carried the thrust of tolerogenicity. Through serial adoptive transfers with Ly5.1+ donors and two successive sets of Ly5.2+ recipients, we demonstrated that nickel tolerance was infectiously spread from donor to host cells. After the transfer of either T cells or APCs from orally tolerized donors, the spread of tolerance to the opposite cell type of the recipients (i.e., APCs and T cells, respectively) required recipient immunization with NiCl2/H2O2. For the spread of tolerance from a given donor cell type, T cell or APC, to the homologous host cell type, the respective opposite cell type in the host was required as intermediate. We conclude that T suppressor cells and tolerogenic APCs induced by oral administration of nickel are part of a positive feedback loop that can enhance and maintain tolerance when activated by Ag associated with a danger signal. Under these conditions, APCs and T suppressor effector cells infectiously spread the tolerance to naive T cells and APCs, respectively.
Genetic Control of NKT Cell Numbers Maps to Major Diabetes and Lupus Loci
Authors: Esteban Luis M. , Tsoutsman Tatiana , Jordan Margaret A. , Roach Daniel , Poulton Lynn D. , Brooks Andrew , Naidenko Olga V. , Sidobre Stephane , Godfrey Dale I. , Baxter Alan G.
Published: April 2014

Natural killer T cells are an immunoregulatory population of lymphocytes that plays a critical role in controlling the adaptive immune system and contributes to the regulation of autoimmune responses. We have previously reported deficiencies in the numbers and function of NKT cells in the nonobese diabetic (NOD) mouse strain, a well-validated model of type 1 diabetes and systemic lupus erythematosus. In this study, we report the results of a genetic linkage analysis of the genes controlling NKT cell numbers in a first backcross (BC1) from C57BL/6 to NOD.Nkrp1b mice. The numbers of thymic NKT cells of 320 BC1 mice were determined by fluorescence-activated cell analysis using anti-TCR Ab and CD1/α-galactosylceramide tetramer. Tail DNA of 138 female BC1 mice was analyzed for PCR product length polymorphisms at 181 simple sequence repeats, providing greater than 90% coverage of the autosomal genome with an average marker separation of 8 cM. Two loci exhibiting significant linkage to NKT cell numbers were identified; the most significant (Nkt1) was on distal chromosome 1, in the same region as the NOD mouse lupus susceptibility gene Babs2/Bana3. The second most significant locus (Nkt2) mapped to the same region as Idd13, a NOD-derived diabetes susceptibility gene on chromosome 2.
Inducible Expression of Macrophage Receptor Marco by Dendritic Cells Following Phagocytic Uptake of Dead Cells Uncovered by Oligonucleotide Arrays
Authors: Grolleau Annabelle , Misek David E. , Kuick Rork , Hanash Samir , Mulé James J.
Published: April 2014

The efficient Ag presenting and immunostimulatory capacity of dendritic cells (DCs) has led to the use of tumor Ag-pulsed DCs in treatment regimens for cancer. Although vaccine studies involving tumor lysate-pulsed DCs have been performed, little, if any, information is available on the effects of phagocytic uptake of tumor lysate on DC biology and function. We have investigated gene expression pattern differences between unpulsed DCs and tumor lysate-pulsed-DCs, using Affymetrix MG-U74Av2 oligonucleotide arrays, which contain ∼12,000 genes and expressed sequence tags. Upon 24 h tumor lysate pulsing, the levels of 87 transcripts increased at least 3-fold while the levels of 121 transcripts were reduced by one-third or more, with accompanying p values <0.01. Most of these genes encoded proteins important for DC effector functions including cytokines, chemokines, and receptors, such as IL-12p40, macrophage inflammatory protein-2, and IL-6; Ag presentation, such as carboxypeptidase D and H2-DM; cell adhesion (e.g., EGF-like module containing, mucin-like, hormone receptor-like sequence 1, rhoB); and T cell activation. Interestingly, we observed a high level of expression of a novel member of the class A scavenger receptor family, macrophage receptor with collagenous structure (Marco). Marco is thought to play an important role in the immune response by mediating binding and phagocytosis, but also in the formation of lamellipodia-like structures and of dendritic processes. Therefore, we have identified a repertoire of genes that likely play a role in DC function.
Activating and Inhibitory Ly49 Receptors Modulate NK Cell Chemotaxis to CXC Chemokine Ligand (CXCL) 10 and CXCL12
Authors: Inngjerdingen Marit , Rolstad Bent , Ryan James C.
Published: April 2014

NK cells can migrate into sites of inflammatory responses or malignancies in response to chemokines. Target killing by rodent NK cells is restricted by opposing signals from inhibitory and activating Ly49 receptors. The rat NK leukemic cell line RNK16 constitutively expresses functional receptors for the inflammatory chemokine CXC chemokine ligand (CXCL)10 (CXCR3) and the homeostatic chemokine CXCL12 (CXCR4). RNK-16 cells transfected with either the activating Ly49D receptor or the inhibitory Ly49A receptor were used to examine the effects of NK receptor ligation on CXCL10- and CXCL12-mediated chemotaxis. Ligation of Ly49A, either with Abs or its MHC class I ligand H2-Dd, led to a decrease in chemotactic responses to either CXCL10 or CXCL12. In contrast, Ly49D ligation with Abs or H2-Dd led to an increase in migration toward CXCL10, but a decrease in chemotaxis toward CXCL12. Ly49-dependent effects on RNK-16 chemotaxis were not the result of surface modulation of CXCR3 or CXCR4 as demonstrated by flow cytometry. A mutation of the Src homology phosphatase-1 binding motif in Ly49A completely abrogated Ly49-dependent effects on both CXCL10 and CXCL12 chemotaxis, suggesting a role for Src homology phosphatase-1 in Ly49A/chemokine receptor cross-talk. Ly49D-transfected cells were pretreated with the Syk kinase inhibitor Piceatannol before ligation, which abrogated the previously observed changes in migration toward CXCL10 and CXCL12. Piceatannol also abrogated Ly49A-dependent inhibition of chemotaxis toward CXCL10, but not CXCL12. Collectively, these data suggest that Ly49 receptors can influence NK cell chemotaxis within sites of inflammation or tumor growth upon interaction with target cells.
Notch Signaling Augments T Cell Responsiveness by Enhancing CD25 Expression
Authors: Adler Scott H. , Chiffoleau Elise , Xu Lanwei , Dalton Nicole M. , Burg Jennifer M. , Wells Andrew D. , Wolfe Michael S. , Turka Laurence A. , Pear Warren S.
Published: April 2014

Notch receptors signal through a highly conserved pathway to influence cell fate decisions. Notch1 is required for T lineage commitment; however, a role for Notch signaling has not been clearly defined for the peripheral T cell response. Notch gene expression is induced, and Notch1 is activated in primary CD4+ T cells following specific peptide-Ag stimulation. Notch activity contributes to the peripheral T cell response, as inhibition of endogenous Notch activation decreases the proliferation of activated T cells in a manner associated with the diminished production of IL-2 and the expression of the high affinity IL-2R (CD25). Conversely, forced expression of a constitutively active Notch1 in primary T cells results in increased surface expression of CD25, and renders these cells more sensitive to both cognate Ag and IL-2, as measured by cell division. These data suggest an important role for Notch signaling during CD4+ T cell responses, which operates through augmenting a positive feedback loop involving IL-2 and its high affinity receptor.
Phenotypic and Functional Characterization of Long-Term Cultured Rhesus Macaque Spleen-Derived NKT Cells
Authors: Gansuvd Balgansuren , Hubbard William J. , Hutchings Anne , Thomas Francis T. , Goodwin Jeanine , Wilson S. Brian , Exley Mark A. , Thomas Judith M.
Published: April 2014

Natural killer T cells are immunoregulatory cells, which have important roles in tolerance and autoimmunity, as demonstrated primarily in mice and humans. In this study, we define the phenotype and function of Vα24+ T cells derived from the spleens of rhesus macaques, a species increasingly used in models of immune tolerance. Vα24+ cells were isolated and expanded with monocyte-derived immature dendritic cells in the presence of α-galactosylceramide, IL-2, and IL-15. Rhesus NKT cells were stained with mAbs against both Vα24 and the invariant complementarity-determining region 3 epitope of the human Vα24/JαQ TCR. The cells were CD4, CD8 double negative and expressed CD56. Rhesus NKT cells also exhibited moderate to high expression of CD95, CD45RO, CD11a, and β7 integrin, but did not express CD45 RA, CD62L, CCR7, CD28, and other activation, costimulatory molecules (CD69 and CD40L). By intracellular staining, >90% of unstimulated rhesus NKT cells expressed IL-10, but not IFN-γ. However, the latter was strongly expressed after stimulation. Rhesus NKT secreted large amounts of TGF-β, IL-13, and IL-6, and modest levels of IFN-γ, whereas IL-10 secretion was negligible and no detectable IL-4 was observed either intracellularly or in culture supernatants. Functionally, the NKT cells and their supernatants suppressed T cell proliferation in allogeneic MLR. We conclude that long-term cultured rhesus macaque spleen-derived Vα24+ T cells are semi-invariant double-negative cells with effector memory phenotype. These cells are semianergic, polarized to a uniquely Th3 > T regulatory-1 regulatory cell phenotype, and have regulatory/suppressive function in vitro.
In Vitro Generation and Life Span Extension of Human Papillomavirus Type 16-Specific, Healthy Donor-Derived CTL Clones
Authors: Schreurs Marco W. J. , Scholten Kirsten B. J. , Kueter Esther W. M. , Ruizendaal Janneke J. , Meijer Chris J. L. M. , Hooijberg Erik
Published: April 2014

Human papillomavirus (HPV) type 16 infection is strongly associated with the development of cervical carcinoma (CxCa) in women. The HPV16-derived oncoproteins E6 and E7, responsible for both onset and maintenance of malignant transformation, are expressed constitutively in CxCa cells and represent tumor-associated Ags. As a result, E6 and E7 constitute potential targets for adoptive CTL-mediated immunotherapy of CxCa. However, the availability to date of well-characterized HPV16-specific, CxCa-reactive human CTLs is extremely limited. The current study describes the in vitro generation and isolation of HPV16 E7-specific, CxCa-reactive human CTL clones from low-frequency healthy donor-derived CD8β-positive precursors. For this purpose, an in vitro CTL induction protocol was used involving mature monocyte-derived dendritic cells as stimulator cells loaded with an HLA-A2.1-restricted, E711–20-derived high-affinity altered peptide ligand. A double tetramer-guided isolation procedure and subsequent limiting-dilution cloning resulted in Ag-specific CTL clones. Stringent CTL characterization clearly indicated Ag-specific, HLA-A2.1-restricted reactivity against different HPV16-transformed CxCa cell lines. To allow expansion of E711–20-specific CTL clones to numbers required for prolonged in vitro as well as in vivo application, their life span was significantly extended by ectopic expression of human telomerase reverse transcriptase. Collectively, our results show that optimized CTL induction and stringent CTL selection procedures, followed by human telomerase reverse transcriptase-mediated life span extension will allow continued availability of low-frequency HPV16-specific, CxCa-reactive human CTL clones. This may enhance the prospects of HPV16-specific adoptive CTL immunotherapy in CxCa patients.
Recombinant CD63/ME491/Neuroglandular/NKI/C-3 Antigen Inhibits Growth of Established Tumors in Transgenic Mice
Authors: Li Jian , Li Weiping , Liang Shaohong , Cai Dewei , Kieny Marie Paule , Jacob Lutz , Linnenbach Alban , Abramczuk Jan W. , Bender Hans , Sproesser Katrin , Swoboda Rolf , Somasundaram Rajasekharan , Guerry DuPont , Herlyn Dorothee
Published: April 2014

Attempts to vaccinate against tumors can be hindered by the induction of immunological tolerance to the target Ag as a result of Ag expression on normal tissues. In this study, we find that transgenic mice expressing the melanoma-associated Ag CD63/ME491/neuroglandular/NKI/C-3 on their normal tissues do, in fact, exhibit immunological tolerance to the Ag, recapitulating the conditions in cancer patients. In these mice, growth of murine melanoma cells expressing the Ag after gene transfer was inhibited by immunization with Ag-expressing recombinant vaccinia virus combined with IL-2, but not by immunization with the protein alone, anti-idiotypic Abs, or irradiated tumor cells. The effect of the recombinant virus was demonstrated both for nonestablished and established tumors. Infiltration with both CD4+ and CD8+ T lymphocytes was significantly more extensive in tumors from experimental mice than in tumors from control mice. MHC class I-positive, but not class I-negative, tumors were inhibited by the vaccine, suggesting that MHC class I-restricted T lymphocytes play a role in the antitumor effects. Abs did not appear to be involved in the vaccine effects. CD63 was immunogenic in 2 of 13 melanoma patients, pointing to the potential of this Ag, combined with IL-2, as a vaccine for melanoma patients.
An IL-2-Dependent Switch Between CD95 Signaling Pathways Sensitizes Primary Human T Cells Toward CD95-Mediated Activation-Induced Cell Death
Authors: Schmitz Ingo , Krueger Andreas , Baumann Sven , Schulze-Bergkamen Henning , Krammer Peter H. , Kirchhoff Sabine
Published: April 2014

The CD95 (APO-1/Fas) system plays a critical role in activation-induced cell death (AICD) of T cells. We previously described two distinct CD95 (APO-1/Fas) signaling pathways: 1) type I cells show strong death-inducing signaling complex (DISC) formation and mitochondria-independent apoptosis and 2) DISC formation is reduced in type II cells, leading to mitochondria-dependent apoptosis. To investigate the relevance of these pathways, we set up an in vitro model that mimics the initiation and the down phase of an immune response, respectively. Freshly activated human T cells (initiation) are resistant toward CD95-mediated AICD despite high expression of CD95. We previously reported that these T cells show reduced DISC formation. In this study, we show that freshly activated T cells are CD95-type II cells that show high expression levels of Bcl-xL and display a block in the mitochondrial apoptosis pathway. Furthermore, we show that, upon prolonged culture (down phase), human T cells undergo a switch from type II to type I cells that renders T cells sensitive to CD95-mediated AICD. Finally, we demonstrate that this switch is dependent on the presence of IL-2. Our observations reveal for the first time that the existence of coexisting CD95 signaling pathways is of physiological relevance.
Assessment of Requirements for IL-15 and IFN Regulatory Factors in Uterine NK Cell Differentiation and Function During Pregnancy
Authors: Ashkar Ali A. , Black Gordon P. , Wei Qingxia , He Hong , Liang Luchuan , Head Judith R. , Croy B. Anne
Published: April 2014

In mouse and human, precursors of NK cell lineage home to decidualizing uteri. To assess the requirement for IL-15, an essential cytokine for NK differentiation in lymphoid tissue, on uterine NK (uNK) cell differentiation, implantation sites from IL-15−/− mice were analyzed histologically. IL-15−/− implantation sites had no uNK cells, no spiral-artery modification, and lacked the decidual integrity found in normal mice. IL-15−/− recipients of C57BL/6 marrow displayed similar pathology. However, implantation sites from recombination-activating gene-2−/−γc−/− (alymphoid) recipients of IL-15−/− marrow showed normal uNK cells, modified spiral arteries, and well-developed decidua basalis. Deletion of the IFN-regulatory factor (IRF)-1, but not IRF-2 (factors important in peripheral NK cell differentiation) limited but did not prevent uNK cell development. In situ hybridization localized IRF-1 largely to placental trophoblast cells. IRF-1−/− marrow transplanted into recombination-activating gene-2−/−γc−/− displayed competence for full uNK cell differentiation. IL-15 mRNA expression at implantation sites of IRF-1−/− and C57BL/6 was similar, suggesting that, unlike in bone marrow and spleen, IRF-1 does not regulate IL-15 in the pregnant uterus. Terminal differentiation of uNK cells was not promoted in pregnant IRF-1−/− mice by 5-day infusion of murine rIL-15, suggesting that IRF-1 deficiency rather than IL-15 deficiency limits uNK cell differentiation in these mice. Further, IRF-1 regulates placental growth, birth weight, and postnatal growth of offspring. These studies indicate that uNK cell development and maturation share some aspects with NK cell development in other tissues, but also display distinctive tissue-specific regulation.
Alterations in the Expression of MHC Class I Glycoproteins by B16BL6 Melanoma Cells Modulate Insulin Receptor-Regulated Signal Transduction and Augments Resistance to Apoptosis
Authors: Assa-Kunik Efrat , Fishman Daniel , Kellman-Pressman Sigal , Tsory Sylvia , Elhyany Shira , Baharir Ofer , Segal Shraga
Published: April 2014

In a variety of malignancies, the immune-escape phenotype is associated, in part, with the inability of tumor cells to properly present their Ags to CTLs due to a deranged expression of MHC class I glycoproteins. However, these molecules were found to possess broader nonimmune functions, including participation in signal transduction and regulation of proliferation, differentiation, and sensitivity to apoptosis-inducing factors; processes, which are characteristically impaired during malignant transformation. We investigated whether the deranged expression of MHC class I expression by tumor cells could affect proper receptor-mediated signal transduction and accentuate their malignant phenotype. The malignant and H-2K murine MHC class I-deficient B16BL6 melanoma cells were characterized by an attenuated capacity to bind insulin due to the retention of corresponding receptor in intracellular stores. The restoration of H-2K expression in these cells, which abrogated their capacity to form tumors in mice, enhanced membrane translocation of the receptor, presumably, by modulating its glycosylation. The addition of insulin to H-2K-expressing melanoma cells cultured in serum-free conditions precluded apoptotic death by up-regulating the activity of protein kinase B (PKB)/Akt. In contrast, the deficiency for H-2K characteristic to the malignant clones was associated with a constitutive high activity of PKB/Akt, which rendered them resistant to apoptosis, induced by deprivation of serum-derived growth factors. The possibility to correct the regulation of PKB/Akt activity by restoration of H-2K expression in B16BL6 melanoma cells may be considered as an attractive approach for cancer therapy, since an aberrant activation of this enzyme is characteristic to resistant malignancies.
In Vivo Antitumor Activity of NKT Cells Activated by the Combination of IL-12 and IL-18
Authors: Baxevanis Constantin N. , Gritzapis Angelos D. , Papamichail Michael
Published: April 2014

Interleukin-12 and IL-18 have been demonstrated to potentiate innate immunity in a variety of experimental tumor models, but the functional roles of NK and/or NKT cells and their mechanism of action in these models have not been fully addressed. Through adoptive transfer of NKT cells activated in vitro with a combination of IL-12 plus IL-18 (IL-12/IL-18 NKT) into syngeneic animals, we demonstrate in this study that IL-12/IL-18 NKT cells are essential and collaborate with the host’s own NK cells in natural host immunity against the growth of ALC and MC57X syngeneic tumors. The relative roles of the adoptively transferred IL-12/IL-18 NKT cells and endogenous NK cells in host protection were first shown in normal C57BL/6 (B6) mice treated with anti-asialo GM1 Ab that selectively depletes NK cells; second, in B6.TCRJα281−/− mice specifically deficient for NKT cells; and third, in B6.scid mice that also lack NKT cells. Furthermore, by injecting normal B6 mice with anti-IL-2 and/or anti-IFN-γ mAb, we could demonstrate that effective innate immunity against both types of syngeneic tumors was dependent on the production of IL-2 and IFN-γ by the adoptively transferred NKT cells. In vitro studies confirmed both the secretion of IL-2 and IFN-γ by the IL-12/IL-18-activated NKT cells and their collaborative role with NK cells for lysis of ALC and MC57X syngeneic tumor targets. This is the first description of an antitumor function of IL-12/IL-18 NKT cells adoptively transferred into syngeneic hosts that provides the basis for a new modality in the cellular immunotherapy of cancer.
Differential Chemokine Responses and Homing Patterns of Murine TCRαβ NKT Cell Subsets
Authors: Johnston Brent , Kim Chang H. , Soler Dulce , Emoto Masashi , Butcher Eugene C.
Published: April 2014

NKT cells play important roles in the regulation of diverse immune responses. Therefore, chemokine receptor expression and chemotactic responses of murine TCRαβ NKT cells were examined to define their homing potential. Most NKT cells stained for the chemokine receptor CXCR3, while >90% of Vα14i-positive and ∼50% of Vα14i-negative NKT cells expressed CXCR6 via an enhanced green fluorescent protein reporter construct. CXCR4 expression was higher on Vα14i-negative than Vα14i-positive NKT cells. In spleen only, subsets of Vα14i-positive and -negative NKT cells also expressed CXCR5. NKT cell subsets migrated in response to ligands for the inflammatory chemokine receptors CXCR3 (monokine induced by IFN-γ/CXC ligand (CXCL)9) and CXCR6 (CXCL16), and regulatory chemokine receptors CCR7 (secondary lymphoid-tissue chemokine (SLC)/CC ligand (CCL)21), CXCR4 (stromal cell-derived factor-1/CXCL12), and CXCR5 (B cell-attracting chemokine-1/CXCL13); but not to ligands for other chemokine receptors. Two NKT cell subsets migrated in response to the lymphoid homing chemokine SLC/CCL21: CD4− Vα14i-negative NKT cells that were L-selectinhigh and enriched for expression of Ly49G2 (consistent with the phenotype of most NKT cells found in peripheral lymph nodes); and immature Vα14i-positive cells lacking NK1.1 and L-selectin. Mature NK1.1+ Vα14i-positive NKT cells did not migrate to SLC/CCL21. BCA-1/CXCL13, which mediates homing to B cell zones, elicited migration of Vα14i-positive and -negative NKT cells in the spleen. These cells were primarily CD4+ or CD4−CD8− and were enriched for Ly49C/I, but not Ly49G2. Low levels of chemotaxis to CXCL16 were only detected in Vα14i-positive NKT cell subsets. Our results identify subsets of NKT cells with distinct homing and localization patterns, suggesting that these populations play specialized roles in immunological processes in vivo.
Enhancing DNA Vaccine Potency by Combining a Strategy to Prolong Dendritic Cell Life with Intracellular Targeting Strategies
Authors: Kim Tae Woo , Hung Chien-Fu , Boyd David , Juang Jeremy , He Liangmei , Kim Jeong Won , Hardwick J. Marie , Wu T.-C.
Published: April 2014

We have recently shown that intradermal coadministration of DNA encoding Ag with DNA encoding inhibitors of apoptosis, including Bcl-xL, prolongs dendritic cell (DC) life and thereby enhances the potency of DNA vaccines in vivo. We have also demonstrated that DNA vaccines targeting Ag to subcellular compartments, using proteins such as Mycobacterium tuberculosis heat shock protein 70, calreticulin, or the sorting signal of the lysosome-associated membrane protein type 1 (LAMP-1), enhanced DNA vaccine potency. In this study, we reasoned that the combination of a strategy to prolong DC life with intracellular targeting strategies might produce a more effective DNA vaccine against human papillomavirus E7. We showed that coadministration of DNA encoding Bcl-xL with DNA encoding E7/heat shock protein 70, calreticulin/E7, or Sig/E7/LAMP-1 resulted in further enhancement of the E7-specific CD8+ T cell response for all three constructs. Of these strategies, mice vaccinated with Sig/E7/LAMP-1 DNA mixed with Bcl-xL DNA showed the greatest increase in E7-specific CD8+ T cells (∼13-fold increase). This combination of strategies resulted in increased CD8+ T cell functional avidity, an increased E7-specific CD4+ Th1 cell response, enhanced tumor treatment ability, and stronger long-term tumor protection when compared with mice vaccinated without Bcl-xL DNA. Therefore, DNA vaccines that combine strategies to enhance intracellular Ag processing and prolong DC life have potential clinical implications for control of viral infection and neoplasia.
Identification of a Novel Subpopulation of Human Cord Blood CD34−CD133−CD7−CD45+Lineage− Cells Capable of Lymphoid/NK Cell Differentiation After In Vitro Exposure to IL-15
Authors: Rutella Sergio , Bonanno Giuseppina , Marone Maria , de Ritis Daniela , Mariotti Andrea , Voso Maria Teresa , Scambia Giovanni , Mancuso Salvatore , Leone Giuseppe , Pierelli Luca
Published: April 2014

The hemopoietic stem cell (HSC) compartment encompasses cell subsets with heterogeneous proliferative and developmental potential. Numerous CD34− cell subsets that might reside at an earlier stage of differentiation than CD34+ HSCs have been described and characterized within human umbilical cord blood (UCB). We identified a novel subpopulation of CD34−CD133−CD7−CD45dimlineage (lin)− HSCs contained within human UCB that were endowed with low but measurable extended long-term culture-initiating cell activity. Exposure of CD34−CD133−CD7−CD45dimlin− HSCs to stem cell factor preserved cell viability and was associated with the following: 1) concordant expression of the stem cell-associated Ags CD34 and CD133, 2) generation of CFU-granulocyte-macrophage, burst-forming unit erythroid, and megakaryocytic aggregates, 3) significant extended long-term culture-initiating cell activity, and 4) up-regulation of mRNA signals for myeloperoxidase. At variance with CD34+lin− cells, CD34−CD133−CD7−CD45dimlin− HSCs maintained with IL-15, but not with IL-2 or IL-7, proliferated vigorously and differentiated into a homogeneous population of CD7+CD45brightCD25+CD44+ lymphoid progenitors with high expression of the T cell-associated transcription factor GATA-3. Although they harbored nonclonally rearranged TCRγ genes, IL-15-primed CD34−CD133−CD7−CD45dimlin− HSCs failed to achieve full maturation, as manifested in their CD3−TCRαβ−γδ− phenotype. Conversely, culture on stromal cells supplemented with IL-15 was associated with the acquisition of phenotypic and functional features of NK cells. Collectively, CD34−CD133−CD7−CD45dimlin− HSCs from human UCB displayed an exquisite sensitivity to IL-15 and differentiated into lymphoid/NK cells. Whether the transplantation of CD34−lin− HSCs possessing T/NK cell differentiation potential may impact on immunological reconstitution and control of minimal residual disease after HSC transplantation for autoimmune or malignant diseases remains to be determined.
The WD Motif-Containing Protein RACK-1 Functions as a Scaffold Protein Within the Type I IFN Receptor-Signaling Complex
Authors: Usacheva Anna , Tian Xinyong , Sandoval Raudel , Salvi Debra , Levy David , Colamonici Oscar R.
Published: April 2014

The WD repeat-containing protein receptor for activated protein kinase C (RACK)-1 has been linked to a variety of signaling systems including protein kinase C, growth factors, and IFNs. In the IFN system, RACK-1 functions as an adaptor recruiting the transcription factor STAT1 to the receptor complex. However, RACK-1 should play a broader role in type I IFN signaling because mutation of the RACK-1 binding site in the IFN-α receptor 2/β subunit of the type I IFN receptor abrogates not only STAT1, but also STAT2, activation. In this study, we demonstrate that RACK-1 serves as a scaffold protein for a multiprotein complex that includes the IFN-α receptor 2/β-chain of the receptor, STAT1, Janus kinase 1, and tyrosine kinase 2. In vitro data further suggest that within this complex tyrosine kinase 2 is the tyrosine kinase responsible for the phosphorylation of STAT1. Finally, we provide evidence that RACK-1 may also serve as a scaffold protein in other cytokine systems such as IL-2, IL-4, and erythropoietin.
In Vivo Impact of CpG1826 Oligodeoxynucleotide on CD8 T Cell Primary Responses and Survival
Authors: Beloeil Laurent , Tomkowiak Martine , Angelov Georgi , Walzer Thierry , Dubois Patrice , Marvel Jacqueline
Published: April 2014

CpG oligodeoxynucleotide (ODN) promotes maturation of APCs in vivo and induces strong type 1 T cell responses in mice. In this study, we have investigated the ability of CpG1826 to modulate peptide-specific CD8 T cell responses in a context where CD4 T cells are likely to play a minor role. The effects of CpG1826 were evaluated in a system where a population of NP68-specific F5 TCR transgenic CD8 T cells is diluted into a polyclonal host following adoptive transfer into C57BL/10 syngeneic recipients. Using this approach, we found that CpG1826 enhanced the ability of F5 CD8 T cells to undergo multiple divisions in vivo, to express IFN-γ ex vivo, and to up-regulate memory-associated cell surface markers such as CD122 (IL-2Rβ) and Ly-6C. Moreover, CpG1826 greatly increased in vivo cytotoxic activity. Using tetramer detection, we found that CpG1826 promoted long-term survival of Ag-specific CD8 T cells after immunization while no NP68-specific cells were detected when the cognate peptide was injected alone. These results indicate that CpG1826 acts as an adjuvant which increases CD8 T cell effector responses and promotes long-term survival of NP68 peptide-specific cells in vivo. They also suggest that this adjuvant can modulate CD8 T cell responses in a system which is likely to be independent of CD4 T cell help.
TCR Comodulation of Nonengaged TCR Takes Place by a Protein Kinase C and CD3γ Di-Leucine-Based Motif-Dependent Mechanism
Authors: Bonefeld Charlotte Menné , Rasmussen Anette B. , Lauritsen Jens Peter H. , von Essen Marina , Ødum Niels , Andersen Peter S. , Geisler Carsten
Published: April 2014

One of the earliest events following TCR triggering is TCR down-regulation. However, the mechanisms behind TCR down-regulation are still not fully known. Some studies have suggested that only directly triggered TCR are internalized, whereas others studies have indicated that, in addition to triggered receptors, nonengaged TCR are also internalized (comodulated). In this study, we used transfected T cells expressing two different TCR to analyze whether comodulation took place. We show that TCR triggering by anti-TCR mAb and peptide-MHC complexes clearly induced internalization of nonengaged TCR. By using a panel of mAb against the Tiβ chain, we demonstrate that the comodulation kinetics depended on the affinity of the ligand. Thus, high-affinity mAb (KD = 2.3 nM) induced a rapid but reversible comodulation, whereas low-affinity mAb (KD = 6200 nM) induced a slower but more permanent type of comodulation. Like internalization of engaged TCR, comodulation was dependent on protein tyrosine kinase activity. Finally, we found that in contrast to internalization of engaged TCR, comodulation was highly dependent on protein kinase C activity and the CD3γ di-leucine-based motif. Based on these observations, a physiological role of comodulation is proposed and the plausibility of the TCR serial triggering model is discussed.