bluuebunny/crossref_metadata_2025 huggingface dataset:
Cross-Presentation of Tumor Antigens to Effector T Cells Is Sufficient to Mediate Effective Immunotherapy of Established Intracranial Tumors
Authors: Plautz Gregory E. , Mukai Shigehiko , Cohen Peter A. , Shu Suyu
Published: April 2014

The systemic adoptive transfer of tumor-sensitized T cells, activated ex vivo, can eliminate established intracranial tumors. Regression of MHC class II negative MCA 205 fibrosarcomas occurs optimally following adoptive transfer of both CD4 and CD8 tumor-sensitized T cells, indicating an important function for tumor-infiltrating APC. Here, we demonstrate that during an effector response, indirect presentation of tumor Ags to transferred T cells is sufficient to mediate intracranial tumor regression. BALB/c → CB6F1 (H-2bxd) bone marrow chimeras were challenged with the MCA 205 fibrosarcoma (H-2b). The tumor grew progressively in the H-2b-tolerant chimeras and stimulated an immune response in tumor-draining lymph nodes. Tumor-sensitized lymph node T cells were activated ex vivo with anti-CD3 and IL-2, then adoptively transferred to sublethally irradiated BALB/c or C57BL/6 recipients bearing established intracranial MCA 205 tumors. The transferred T cells eradicated MCA 205 tumors in BALB/c recipients and demonstrated tumor specificity, but had no therapeutic efficacy in the C57BL/6 recipients. These data establish that tumor-associated host cell constituents provide sufficient Ag presentation to drive effector T cell function in the complete absence of direct tumor recognition. This effector mechanism has an evident capacity to remain operative in circumstances of immune escape, where the tumor does not express the relevant MHC molecules, and may have importance even at times when direct CTL recognition also remains operative.
Effects of a Constitutively Active Form of Calcineurin on T Cell Activation and Thymic Selection
Authors: Hayden-Martinez Kristine , Kane Lawrence P. , Hedrick Stephen M.
Published: April 2014

Calcineurin is a calcium/calmodulin-dependent phosphatase whose activity is required for the induction of T cell lymphokine production and proliferation. Although its specific role in T cell development is less well defined, studies with the immunosuppressive drugs cyclosporin A and FK-506 suggest that it is involved in both positive and negative selection of immature thymocytes. To more completely characterize a role for calcineurin in T cell development in vivo, we have generated transgenic mice that express an activated form of this enzyme in thymocytes and peripheral T cells. We find that the transgene causes a block in early thymic development, resulting in a reduction in the steady-state number of CD4 and CD8 double positives, but not on the number of mature T cells. We also find that thymocytes and mature T cells expressing this transgene are more sensitive to signals through their TCR. In thymocytes this sensitivity difference is manifested as an increase in positive selection, although negative selection seems to remain unaffected. Therefore, these studies confirm and extend past reports that suggested a role for calcineurin in thymic development and selection.
Galectin-1 Induces Partial TCR ζ-Chain Phosphorylation and Antagonizes Processive TCR Signal Transduction
Authors: Chung Chan D. , Patel Viresh P. , Moran Miriana , Lewis Linda A. , Miceli M. Carrie
Published: April 2014

Galectin-1 is an endogenous lectin with known T cell immunoregulatory activity, though the molecular basis by which galectin-1 influences Ag specific T cell responses has not been elucidated. Here, we characterize the ability of galectin-1 to modulate TCR signals and responses by T cells with well defined hierarchies of threshold requirements for signaling distinct functional responses. We demonstrate that galectin-1 antagonizes TCR responses known to require costimulation and processive protein tyrosine phosphorylation, such as IL-2 production, but is permissive for TCR responses that only require partial TCR signals, such as IFN-γ production, CD69 up-regulation, and apoptosis. Galectin-1 binding alone or together with Ag stimulation induces partial phosphorylation of TCR-ζ and the generation of inhibitory pp21ζ. Galectin-1 antagonizes Ag induced signals and TCR/costimulator dependent lipid raft clustering at the TCR contact site. We propose that galectin-1 functions as a T cell “counterstimulator” to limit required protein segregation and lipid raft reorganization at the TCR contact site and, thus, processive and sustained TCR signal transduction. These findings support the concept that TCR antagonism can arise from the generation of an inhibitory pp21ζ-based TCR signaling complex. Moreover, they demonstrate that TCR antagonism can result from T cell interactions with a ligand other than peptide/MHC.
The Mechanism of Unresponsiveness to Circulating Tumor Antigen MUC1 Is a Block in Intracellular Sorting and Processing by Dendritic Cells
Authors: Hiltbold Elizabeth M. , Vlad Anda M. , Ciborowski Pawel , Watkins Simon C. , Finn Olivera J.
Published: April 2014

Immunity to tumor Ags in patients is typically weak and not therapeutic. We have identified a new mechanism by which potentially immunogenic glycoprotein tumor Ags, such as MUC1, fail to stimulate strong immune responses. MUC1 is a heavily glycosylated membrane protein that is also present in soluble form in sera and ascites of cancer patients. We show that this soluble protein is readily taken up by dendritic cells (DC), but is not transported to late endosomes or MHC class II compartments for processing and binding to class II MHC. MUC1 uptake is mediated by the mannose receptor, and the protein is then retained long term in early endosomes without degradation. Long-term retention of MUC1 does not interfere with the ability of DC to process and present other Ags. We also demonstrate inhibited processing of another important glycoprotein tumor Ag, HER-2/neu. This may, therefore, be a frequent obstacle to presentation of tumor Ags and an important consideration in the design of cancer vaccines. It should be possible to overcome this obstacle by providing DC with a form of tumor Ag that can be better processed. For MUC1 we show that a 140-aa-long synthetic peptide is very efficiently processed by DC.
The CD85/LIR-1/ILT2 Inhibitory Receptor Is Expressed by All Human T Lymphocytes and Down-Regulates Their Functions
Authors: Saverino Daniele , Fabbi Marina , Ghiotto Fabio , Merlo Andrea , Bruno Silvia , Zarcone Daniela , Tenca Claudya , Tiso Micaela , Santoro Giuseppe , Anastasi Giuseppe , Cosman David , Grossi Carlo E. , Ciccone Ermanno
Published: April 2014

The inhibitory molecule CD85/LIR-1/ILT2 has been detected previously on the surface of a small proportion of T lymphocytes. In this study, evidence is provided that, although only a fraction of CD3+ cells are stained by mAb specific for CD85/LIR-1/ILT2 on their surface, this inhibitory receptor is present in the cytoplasm of all T lymphocytes, and that it is detectable on the surface of all T cell clones by the M402 mAb. Biochemical analyses further demonstrate that CD85/LIR-1/ILT2 is present in all T clones analyzed, and that the protein is tyrosine-phosphorylated. Expression of mRNA coding for CD85/LIR-1/ILT2 has been assessed by RT-PCR. Notably, in the NKL cell line and in one T cell clone, amplification of the messenger required 30 cycles only, whereas, in other T cell clones, an amplification product was detected by increasing the number of cycles. CD85/LIR-1/ILT2 inhibits CD3/TCR-mediated activation in both CD4+ and CD8+ clones, and it down-regulates Ag recognition by CD8+ cells in a clonally distributed fashion. Addition of anti-ILT2 HP-F1 mAb in the cytolytic assay enhances target cell lysis mediated by Ag-specific CTL. This could be due to interference of the mAb with receptor/ligand interactions. In contrast, HP-F1 mAb cross-linking triggers inhibitory signals that reduce cytotoxicity. CD85/LIR-1/ILT2 also controls responses to recall Ags and, in low responders, its engagement sharply increases T cell proliferation. The inhibitory function of the molecule is also confirmed by its ability to reduce CD3/TCR-induced intracellular Ca2+ mobilization.
A Monoclonal Antibody Against the 66-kDa Protein Expressed in Mouse Spleen and Thymus Inhibits Ly-6A.2-Dependent Cell-Cell Adhesion
Authors: English Andrea , Kosoy Roman , Pawlinski Rafel , Bamezai Anil
Published: April 2014

The Ly-6 locus encodes several cell surface proteins of 10–12 kDa. Some members of this multigene family may function in cell signaling and/or cell adhesion processes. T lymphocytes overexpressing Ly-6A.2 (one member of the Ly-6 gene family) protein homotypically aggregate when cultured in vitro. Further analysis of this homotypic aggregation suggests that Ly-6A.2 participates in cell-cell adhesion. These observations indicated the presence of a Ly-6 ligand(s) on the surface of lymphoid cells. In this study we report generation of a hamster mAb, 9AB2, that blocks Ly-6A.2-dependent cell-cell adhesion. The 9AB2 Ab recognizes a 66-kDa glycoprotein with unique tissue expression. The 9AB2 mAb does not bind Ly-6A.2, but coimmunoprecipitates Ly-6A.2 molecule. Moreover, 9AB2 Ag-expressing thymocytes specifically bind to Chinese hamster ovary cells overexpressing Ly-6A.2 protein, and this binding is specifically blocked by 9AB2 and anti-Ly-6A.2 Abs. These results suggest that the 66-kDa protein recognized by 9AB2 mAb is the putative ligand for Ly-6A.2.
Plasmid Vaccine Expressing Granulocyte-Macrophage Colony-Stimulating Factor Attracts Infiltrates Including Immature Dendritic Cells into Injected Muscles
Authors: Haddad Diana , Ramprakash Jayanthi , Sedegah Martha , Charoenvit Yupin , Baumgartner Roxanne , Kumar Sanjai , Hoffman Stephen L. , Weiss Walter R.
Published: April 2014

Plasmid-encoded GM-CSF (pGM-CSF) is an adjuvant for genetic vaccines; however, little is known about how pGM-CSF enhances immunogenicity. We now report that pGM-CSF injected into mouse muscle leads to a local infiltration of potential APCs. Infiltrates reached maximal size on days 3 to 5 after injection and appeared in several large discrete clusters within the muscle. Immunohistological studies in muscle sections from mice injected with pGM-CSF showed staining of cells with the macrophage markers CD11b, Mac-3, IAd/Ed and to the granulocyte marker GR-1 from day 1 through day 14. Cells staining with the dendritic cell marker CD11c were detected only on days 3 to 5. Muscles injected with control plasmids did not stain for CD11c but did stain for CD11b, Mac-3, IAd/Ed, and GR-1. No staining was observed with the APC activation markers, B7.1 or CD40, or with markers for T or B cells. These findings are consistent with the infiltrating cells in the pGM-CSF-injected muscles being a mixture of neutrophils, macrophages, and immature dendritic cells and suggest that the i.m. APCs may be enhancing immune responses to coinjected plasmid Ags. This hypothesis is supported by data showing that 1) separation of injections with pGM-CSF and Ag-expressing plasmid into different sites did not enhance immune responses and 2) immune enhancement was associated with the presence of CD11c+ cells in the infiltrates. Thus, pGM-CSF enhancement may depend on APC recruitment to the i.m. site of injection.
Type 1 IFN Maintains the Survival of Anergic CD4+ T Cells
Authors: Lombardi Giovanna , Dunne Pádraic J. , Scheel-Toellner Dagmar , Sanyal Tina , Pilling Darrell , Taams Leonie S. , Life Paul , Lord Janet M. , Salmon Mike , Akbar Arne N.
Published: April 2014

Anergic T cells have immunoregulatory activity and can survive for extended periods in vivo. It is unclear how anergic T cells escape from deletion, because both anergy and apoptosis can occur after TCR ligation. Stimulation of human CD4+ T cell clones reactive to influenza hemagglutinin peptides can occur in the absence of APCs when MHC class II-expressing, activated T cells present peptide to each other. This T:T peptide presentation can induce CD95-mediated apoptosis, while the cells that do not die are anergic. We found that the death after peptide or anti-CD3 treatment of a panel of CD4+ T cell clones is blocked by IFN-β secreted by fibroblasts and also by IFN-α. This increases cell recovery after stimulation, which is not due to T cell proliferation. This mechanism for apoptosis inhibition rapidly stops protein kinase C-δ translocation from the cytoplasm to the nucleus, which is an early event in the death process. A central observation was that CD4+ T cells that are rescued from apoptosis after T:T presentation of peptide by IFN-αβ remain profoundly anergic to rechallenge with Ag-pulsed APCs. However, anergized cells retain the ability to respond to IL-2, showing that they are nonresponsive but functional. The prevention of peptide-induced apoptosis in activated T cells by IFN-αβ is a novel mechanism that may enable the survival and maintenance of anergic T cell populations after TCR engagement. This has important implications for the persistence of anergic T cells with the potential for immunoregulatory function in vivo.
The Role of the DAP12 Signal in Mouse Myeloid Differentiation
Authors: Aoki Naoko , Kimura Shoji , Takiyama Yumi , Atsuta Yoshiaki , Abe Asami , Sato Keisuke , Katagiri Makoto
Published: April 2014

DAP12 is a recently cloned, immunoreceptor tyrosine-based activation motif-bearing transmembrane adapter molecule that is associated with the NK-activating receptors. Previous reports showed that the DAP12 message could be detected not only in NK cells but also in granulocytes, monocytes, dendritic cells, and macrophages. In this study we found a significant level of DAP12 protein expression in macrophage-related cell lines and organs. Additionally, we observed increased expression of DAP12 after LPS-induced differentiation of M1 cells into macrophages. To examine the role of DAP12 in the myeloid cell lineage, we established M1 FLAG-DAP12 transfectants (FDAP-M1) and demonstrated the marked morphological changes in FDAP-M1 cells caused by signaling through DAP12. Cell surface phenotypic analysis showed up-regulation of macrophage markers CD11b, 2.4G2, and adhesion molecule B7-2. Additionally, after stimulation through DAP12, phosphorylated FLAG -DAP12 could be immunoprecipitated using anti-phosphotyrosine mAbs. Collectively, these findings indicate that direct DAP12 signaling has an important role in macrophage differentiation.
Dendritic Cells Capture Killed Tumor Cells and Present Their Antigens to Elicit Tumor-Specific Immune Responses
Authors: Nouri-Shirazi Mahyar , Banchereau Jacques , Bell Diana , Burkeholder Susan , Kraus Elizabeth T. , Davoust Jean , Palucka Karolina A.
Published: April 2014

Due to their capacity to induce primary immune responses, dendritic cells (DC) are attractive vectors for immunotherapy of cancer. Yet the targeting of tumor Ags to DC remains a challenge. Here we show that immature human monocyte-derived DC capture various killed tumor cells, including Jurkat T cell lymphoma, malignant melanoma, and prostate carcinoma. DC loaded with killed tumor cells induce MHC class I- and class II-restricted proliferation of autologous CD8+ and CD4+ T cells, demonstrating cross-presentation of tumor cell-derived Ags. Furthermore, tumor-loaded DC elicit expansion of CTL with cytotoxic activity against the tumor cells used for immunization. CTL elicited by DC loaded with the PC3 prostate carcinoma cell bodies kill another prostate carcinoma cell line, DU145, suggesting recognition of shared Ags. Finally, CTL elicited by DC loaded with killed LNCap prostate carcinoma cells, which express prostate specific Ag (PSA), are able to kill PSA peptide-pulsed T2 cells. This demonstrates that induced CTL activity is not only due to alloantigens, and that alloantigens do not prevent the activation of T cells specific for tumor-associated Ags. This approach opens the possibility of using allogeneic tumor cells as a source of tumor Ag for antitumor therapies.
Direct Tumor Lysis by NK Cells Uses a Ras-Independent Mitogen-Activated Protein Kinase Signal Pathway
Authors: Wei Sheng , Gilvary Danielle L. , Corliss Brian C. , Sebti Said , Sun Jiazhi , Straus David B. , Leibson Paul J. , Trapani Joseph A. , Hamilton Andrew D. , Weber Michael J. , Djeu Julie Y.
Published: April 2014

Destruction of tumor cells is a key function of lymphocytes, but the molecular processes driving it are unclear. Analysis of signal molecules indicated that mitogen-activated protein kinase (MAPK)/extracellular regulated kinase 2 critically controlled lytic function in human NK cells. We now have evidence to indicate that target ligation triggers a Ras-independent MAPK pathway that is required for lysis of the ligated tumor cell. Target engagement caused NK cells to rapidly activate MAPK within 5 min, and PD098059 effectively blocked both MAPK activation and tumoricidal function in NK cells. Target engagement also rapidly activated Ras, detected as active Ras-GTP bound to GST-Raf-RBD, a GST fusion protein linked to the Raf protein fragment containing the Ras-GTP binding domain. However, Ras inactivation by pharmacological disruption with the farnesyl transferase inhibitor, FTI-277, had no adverse effect on the ability of NK cells to lyse tumor cells or to express MAPK activation upon target conjugation. Notably, MAPK inactivation with PD098059, but not Ras inactivation with FTI-277, could interfere with perforin and granzyme B polarization within NK cells toward the contacted target cell. Using vaccinia delivery of N17 Ras into NK cells, we demonstrated that IL-2 activated a Ras-dependent MAPK pathway, while target ligation used a Ras-independent MAPK pathway to trigger lysis in NK cells.
Phosphatidylinositol 3-Kinase and NF-κB/Rel Are at the Divergence of CD40-Mediated Proliferation and Survival Pathways
Authors: Andjelic Sofija , Hsia Constance , Suzuki Harumi , Kadowaki Takashi , Koyasu Shigeo , Liou Hsiou-Chi
Published: April 2014

CD40 receptor ligation evokes several crucial outcomes for the fate of an activated B cell, including proliferation and survival. Although multiple signaling molecules in the CD40 pathways have been identified, their specific roles in regulating proliferation and maintaining cell viability are still obscure. In this report, we demonstrate that the activation of both phosphatidylinositol 3-kinase (PI-3K) and NF-κB/Rel transcription factors is crucial for CD40-mediated proliferation. Furthermore, our data indicate that PI-3K is indispensable for CD40-mediated NF-κB/Rel activation. This is achieved via activation of AKT and the degradation of IκBα. Furthermore, we show that PI-3K activity is necessary for the degradation of cyclin-dependent kinase inhibitor p27kip. Therefore, both of these events comprise the mechanism by which PI-3K controls cell proliferation. In contrast to the absolute requirement of PI-3K and NF-κB/Rel for proliferation, these signaling molecules are only partially responsible for CD40-mediated survival, as blocking of PI-3K activity did not lead to apoptosis of anti-CD40-treated cells. However, the PI-3K/NF-κB pathway is still required for CD40-induced Bcl-X gene expression. Taken together, our data indicate that multiple survival pathways are triggered via this receptor, whereas NF-κB/Rel and PI-3K are crucial for CD40-induced proliferation.
Functional Analysis of Ligand-Binding and Signal Transduction Domains of CD69 and CD23 C-Type Lectin Leukocyte Receptors
Authors: Sancho David , Santis Ana G. , Alonso-Lebrero José L. , Viedma Fernando , Tejedor Reyes , Sánchez-Madrid Francisco
Published: April 2014

CD69 and CD23 are leukocyte receptors with distinctive pattern of cell expression and functional features that belong to different C-type lectin receptor subfamilies. To assess the functional equivalence of different domains of these structurally related proteins, a series of CD69/CD23 chimeras exchanging the carbohydrate recognition domain, the neck region, and the transmembrane and cytoplasmic domains were generated. Biochemical analysis revealed the importance of the neck region (Cys68) in the dimerization of CD69. Functional analysis of these chimeras in RBL-2H3 mast cells and Jurkat T cell lines showed the interchangeability of structural domains of both proteins regarding Ca2+ fluxes, serotonin release, and TNF-α synthesis. The type of the signal transduced mainly relied on the cytoplasmic domain and was independent of receptor oligomerization. The cytoplasmic domain of CD69 transduced a Ca2+-mediated signaling that was dependent on the extracellular uptake of Ca2+. Furthermore, a significant production of TNF-α was induced through the cytoplasmic domain of CD69 in RBL-2H3 cells, which was additive to that promoted via FcεRI, thus suggesting a role for CD69 in the late phase of reactions mediated by mast cells. Our results provide new important data on the functional equivalence of homologous domains of these two leukocyte receptors.
Restriction in Vκ Gene Use and Antigen Selection in Anti-Myeloperoxidase Response in Mice
Authors: Jethwa Hitendra S. , Clarke Stephen H. , Itoh-Lindstrom Yoshie , Falk Ronald J. , Jennette J. Charles , Nachman Patrick H.
Published: April 2014

Anti-neutrophil cytoplasmic Abs, directed primarily toward myeloperoxidase (MPO) and proteinase 3, are detected in the majority of patients with distinct forms of small vessel vasculitides and pauci-immune necrotizing glomerulonephritis. However, the origin of these autoantibodies remains unknown. We studied the V region gene use in murine anti-MPO Abs derived from Spontaneous Crescentic Glomerulonephritis/Kinjoh mice. A total of 13 anti-MPO-producing hybridomas were generated from four unimmunized mice. Ten of the 13 hybridomas (corresponding to 3 of 4 clones) expressed Vκ1C but differed in their use of VH genes. The remaining three hybridomas expressed a Vκ5 gene. Anti-MPO hybridomas from individual mice were derived from single clones as deduced by sequence similarity and splice-site identity. We found a statistically significant bias of amino acid replacement mutations to the complementarity-determining regions (CDR) in the Vκ1C-expressing hybridomas. Intriguingly, all 10 Vκ1C hybridomas share a lysine to glutamate mutation in the CDR1. To determine the effects of somatic V gene mutations on binding to MPO, we generated an anti-MPO Ab with an unmutated Vκ1C L chain and compared its ability to bind MPO with its mutated counterpart. The mutated hybridoma-derived Ab has a 4.75-fold higher avidity for MPO than the unmutated Ab. These results suggest that: 1) the L chain plays a dominant role in determining Ab specificity to MPO, 2) the anti-MPO Ab response is oligoclonal, consistent with Ag selection, and 3) MPO is a driving Ag in the murine anti-MPO Ab response.
Identification of a Novel Cytokine Response Element in the Human IFN Regulatory Factor-1 Gene Promoter
Authors: Imanishi Daisuke , Yamamoto Kazuo , Tsushima Hideki , Miyazaki Yasushi , Kuriyama Kazutaka , Tomonaga Masao , Matsuyama Toshifumi
Published: April 2014

The present study investigates the regulatory mechanisms involved in the cooperation between IFN-γ and TNF-α to promote transcription from IFN regulatory factor-1 (IRF-1). A transient transfection analysis revealed that the region between −218 and −144, where +1 is the transcription start site, as well as previously reported downstream elements, ppκB and IFN-γ activation site/κB, were required for the optimal response to the two cytokines. A subsequent DNase I footprint analysis showed that the region between −171 and −144 was inducibly protected with stimulation by TNF-α, and this protection was significantly enhanced with the combination of IFN-γ and TNF-α. In an EMSA with the protected region as a probe, a TNF-α-inducible complex (C1) and an IFN-γ-inducible complex (C2), but no synergy-specific DNA-protein complexes, were recognized. The C1 complex consisted of a pre-existing factor (p65/p50), whereas the C2 complex consisted of a newly synthesized IRF-1-related factor. A methylation interference assay revealed the critical G residues (from −167 to −151) for the DNA-protein complex formation specific to the cytokine response, and within this region the novel κB sequence, the promoter distal κB (pdκB) element (5′-GGGGAAGTAC-3′), was identified. Because the base substitutions over the pdκB region (from −171 to −144) affected not only the TNF-α-response but also that of IFN-γ, this region might contribute to the cooperative action of the NF-κB subunits with the IRF-1-related factor. Finally, we demonstrated that none of the cis-acting elements, ppκB, pdκB, or IFN-γ activation site/κB, is dispensable for the optimal synergism in response to IFN-γ and TNF-α.
Distinct Effects of Surfactant Protein A or D Deficiency During Bacterial Infection on the Lung
Authors: LeVine Ann Marie , Whitsett Jeffrey A. , Gwozdz Jodie A. , Richardson Theresa R. , Fisher James H. , Burhans Michael S. , Korfhagen Thomas R.
Published: April 2014

Mice lacking surfactant protein (SP)-A (SP-A−/−) or SP-D (SP-D−/−) and wild-type mice were infected with group B streptococcus or Haemophilus influenzae by intratracheal instillation. Although decreased killing of group B streptococcus and H. influenzae was observed in SP-A−/− mice but not in SP-D−/− mice, deficiency of either SP-A or SP-D was associated with increased inflammation and inflammatory cell recruitment in the lung after infection. Deficient uptake of bacteria by alveolar macrophages was observed in both SP-A- and SP-D-deficient mice. Isolated alveolar macrophages from SP-A−/− mice generated significantly less, whereas those from SP-D−/− mice generated significantly greater superoxide and hydrogen peroxide compared with wild-type alveolar macrophages. In SP-D−/− mice, bacterial killing was associated with increased lung inflammation, increased oxidant production, and decreased macrophage phagocytosis. In contrast, in the absence of SP-A, bacterial killing was decreased and associated with increased lung inflammation, decreased oxidant production, and decreased macrophage phagocytosis. Increased oxidant production likely contributes to effective bacterial killing in the lungs of SP-D−/− mice. The collectins, SP-A and SP-D, play distinct roles during bacterial infection of the lung.
Role of Cystic Fibrosis Transmembrane Conductance Regulator in Pulmonary Clearance of Pseudomonas aeruginosa In Vivo
Authors: Chroneos Zissis C. , Wert Susan E. , Livingston Jennifer L. , Hassett Daniel J. , Whitsett Jeffrey A.
Published: April 2014

Cystic fibrosis (CF)2 is a fatal genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) that is commonly associated with chronic pulmonary infections with mucoid Pseudomonas aeruginosa (PA). To test the hypothesis that CFTR plays a direct role in PA adhesion and clearance, we have used mouse lines expressing varying levels of human (h) or mouse (m) CFTR. A subacute intratracheal dose of 3 × 106 bacteria was cleared with similar kinetics in control wild-type (WT) and transgenic mice overexpressing hCFTR in the lung from the surfactant protein C (SP-C) promoter (SP-C-hCFTR+/−). In a second series of experiments, the clearance of an acute intratracheal dose of 1.5 × 107 PA bacteria was also similar in WT, hemizygous SP-C-hCFTR+/−, and bitransgenic gut-corrected FABP-hCFTR+/+-mCFTR−/−, the latter lacking expression of mCFTR in the lung. However, a small but significant decrease in bacterial killing was observed in lungs of homozygote SP-C-hCFTR+/+ mice. Lung pathology in both WT and SP-C-hCFTR+/+ mice was marked by neutrophilic inflammation and bacterial invasion of perivascular and subepithelial compartments. Bacteria were associated primarily with leukocytes and were not associated with alveolar type II or bronchiolar epithelial cells, the cellular sites of SP-C-hCFTR+/+ transgene expression. The results indicate that there is no direct correlation between levels of CFTR expression and bacterial clearance or association of bacteria with epithelial cells in vivo.
Granzyme Activity in the Inflamed Lung Is Not Controlled by Endogenous Serine Proteinase Inhibitors
Authors: Tremblay Guy M. , Wolbink Angela M. , Cormier Yvon , Hack C. Erik
Published: April 2014

Numerous lung diseases, such as hypersensitivity pneumonitis (HP), are characterized by the presence of activated alveolar CTL and NK cells. Since these cells produce granzymes, granzyme A and B levels in bronchoalveolar lavage (BAL) fluids from 14 normal subjects and 12 patients with HP were measured by ELISA. Median (range) BAL granzyme A and B levels were 4 (0–37) and 0 (0–6) pg/ml in normal subjects. BAL granzyme levels were significantly higher in HP patients, being at 74 (0–1889) and 10 (0–78) pg/ml for granzymes A and B, respectively. In vitro, neither of the three main serine protease inhibitors of the lung, namely α1-antitrypsin, secretory leukocyte protease inhibitor, and elafin, showed any effect on granzyme A or B activity. In addition, granzyme A was shown to be fully active in BAL fluids. Hence, these data show that granzyme activity may be poorly controlled by protease inhibitors in inflamed tissues. Thus, granzymes could contribute to tissue remodeling and inflammation characterizing HP.
Nitric Oxide-Dependent Ribosomal RNA Cleavage Is Associated with Inhibition of Ribosomal Peptidyl Transferase Activity in ANA-1 Murine Macrophages
Authors: Cai Charles Q. , Guo Hongtao , Schroeder Rebecca A. , Punzalan Cecile , Kuo Paul C.
Published: April 2014

NO can regulate specific cellular functions by altering transcriptional programs and protein reactivity. With respect to global cellular processes, NO has also been demonstrated to inhibit total protein synthesis and cell proliferation. The underlying mechanisms are unknown. In a system of ANA-1 murine macrophages, iNOS expression and NO production were induced by exposure to endotoxin (LPS). In selected instances, cells were exposed to an exogenous NO donor, S-nitroso-N-acetylpenicillamine or a substrate inhibitor of NO synthesis. Cellular exposure to NO, from both endogenous and exogenous sources, was associated with a significant time-dependent decrease in total protein synthesis and cell proliferation. Gene transcription was unaltered. In parallel with decreased protein synthesis, cells exhibited a distinctive cleavage pattern of 28S and 18S rRNA that were the result of two distinct cuts in both 28S and 18S rRNA. Total levels of intact 28S rRNA, 18S rRNA, and the composite 60S ribosome were significantly decreased in the setting of cell exposure to NO. Finally, 60S ribosome-associated peptidyl transferase activity, a key enzyme for peptide chain elongation, was also significantly decreased. Our data suggest that NO-mediated cleavage of 28S and 18S rRNA results in decreased 60S ribosome associated peptidyl transferase activity and inhibition of total protein synthesis.
GM-CSF Regulates Bleomycin-Induced Pulmonary Fibrosis Via a Prostaglandin-Dependent Mechanism
Authors: Moore Bethany B. , Coffey Michael J. , Christensen Paul , Sitterding Stephanie , Ngan Rose , Wilke Carol A. , McDonald Rod , Phare Susan M. , Peters-Golden Marc , Paine Robert , Toews Galen B.
Published: April 2014

To characterize the role of GM-CSF in pulmonary fibrosis, we have studied bleomycin-induced fibrosis in wild-type mice vs mice with a targeted deletion of the GM-CSF gene (GM-CSF−/− mice). Without GM-CSF, pulmonary fibrosis was worse both histologically and quantitatively. These changes were not related to enhanced recruitment of inflammatory cells because wild-type and GM-CSF−/− mice recruited equivalent numbers of cells to the lung following bleomycin. Interestingly, recruitment of eosinophils was absent in GM-CSF−/− mice. We investigated whether the enhanced fibrotic response in GM-CSF−/− animals was due to a deficiency in an endogenous down-regulator of fibrogenesis. Analysis of whole lung homogenates from saline- or bleomycin-treated mice revealed that GM-CSF−/− animals had reduced levels of PGE2. Additionally, alveolar macrophages were harvested from wild-type and GM-CSF−/− mice that had been exposed to bleomycin. Although bleomycin treatment impaired the ability of alveolar macrophages from wild-type mice to synthesize PGE2, alveolar macrophages from GM-CSF−/− mice exhibited a significantly greater defect in PGE2 synthesis than did wild-type cells. Exogenous addition of GM-CSF to alveolar macrophages reversed the PGE2 synthesis defect in vitro. Administration of the PG synthesis inhibitor, indomethacin, to wild-type mice during the fibrogenic phase postbleomycin worsened the severity of fibrosis, implying a causal role for PGE2 deficiency in the evolution of the fibrotic lesion. These data demonstrate that GM-CSF deficiency results in enhanced fibrogenesis in bleomycin-induced pulmonary fibrosis and indicate that one mechanism for this effect is impaired production of the potent antifibrotic eicosanoid, PGE2.
CD4+ T Lymphocytes with Constitutive CD40 Ligand in Preautoimmune (NZB × NZW)F1 Lupus-Prone Mice: Phenotype and Possible Role in Autoreactivity
Authors: Lettesjö Helene , Burd Gary P. , Mageed Rizgar A.
Published: April 2014

Lupus disease is marked by B lymphocyte hyperactivity and the production of Abs to dsDNA. The production of these anti-dsDNA Abs is T lymphocyte dependent. However, it is not clear how CD4+ T lymphocytes provide help for B lymphocytes to produce IgG anti-dsDNA Abs. One possible mechanism is suggested by studies showing that human patients with systemic lupus erythematosus and lupus mice have increased numbers of CD40 ligand (CD40L)+ T and B lymphocytes. The results described in this study reveal that young, clinically healthy lupus-prone New Zealand Black × New Zealand White F1 (BWF1) mice have naive CD4+ T cells with preformed CD40L. These cells contribute to a brisk response to immunization and to the production of anti-dsDNA Abs. In vitro experiments revealed that CD4+ T cells with preformed CD40L could, upon stimulation, provide antiapoptotic signals for B cells but could not induce proliferation or reduce activation threshold. These results suggest that the direct target cells for the effect of T cells with preformed CD40L in lupus may not be B lymphocytes.
Expression of a Human Coxsackie/Adenovirus Receptor Transgene Permits Adenovirus Infection of Primary Lymphocytes
Authors: Schmidt Madelyn R. , Piekos Brian , Cabatingan Mark S. , Woodland Robert T.
Published: April 2014

Replication-defective adenoviruses are effective vehicles for gene transfer, both for the repair of defective genes and for studies of gene function in primary cells. Many cell types, including lymphocytes, are refractory to adenovirus infection because they lack the Coxsackie/adenovirus receptor (CAR) needed for virus attachment. To extend the advantages of adenovirus-mediated gene transfer to primary lymphoid populations and other cell types lacking endogenous CAR, we produced a mouse that expresses human (h) CAR as a transgene under control of a murine MHC class I promoter. hCAR protein is expressed on T and B lymphocytes from a variety of organs (spleen, lymph node, bone marrow, thymus, and peritoneum). These lymphocytes are susceptible to adenovirus infection, as demonstrated by reporter green fluorescent protein gene expression, with the fraction of expressing cells as high as 70%. Some lymphocyte subpopulations required stimulation subsequent to adenovirus infection for reporter expression. This activation requirement is a restriction imposed by the promoter used in the adenovirus construct. In subpopulations requiring activation, the elongation factor 1 promoter was far superior to a hCMV promoter for directing green fluorescent protein production. We also find that hCAR mRNA is produced in nonlymphoid tissues from all founder lines, including tissues that do not express endogenous murine CAR, suggesting the opportunity for effecting gene delivery to and testing gene function in a wide variety of primary cell types previously resistant to gene transfer.
Cutting Edge: Gab2 Mediates an Inhibitory Phosphatidylinositol 3′-Kinase Pathway in T Cell Antigen Receptor Signaling
Authors: Pratt Joanne C. , Igras Vivien E. , Maeda Hiroyuki , Baksh Shairaz , Gelfand Erwin W. , Burakoff Steven J. , Neel Benjamin G. , Gu Haihua
Published: April 2014

Phosphatidylinositol 3′-kinase (PI3K) is a key component of multiple signaling pathways, where it typically promotes survival, proliferation, and/or adhesion. Here, we show that in TCR signaling, the scaffolding adapter Gab2 delivers an inhibitory signal via PI3K. Overexpression of Gab2 in T cell lines inhibits TCR-evoked activation of the IL-2 promoter, blocking NF-AT- and NF-κB-directed transcription. Inhibition is abrogated by mutating the Gab2 p85-binding sites, by treatment with PI3K inhibitors or by cotransfection of phosphatase homolog of tensin. Our findings provide the first evidence of a negative function for a scaffolding adapter in T cells and identify Gab2/PI3K-containing complexes as novel regulators of TCR signaling.
Phosphorothioate Backbone Modification Modulates Macrophage Activation by CpG DNA
Authors: Sester David P. , Naik Shalin , Beasley Shannon J. , Hume David A. , Stacey Katryn J.
Published: April 2014

Macrophages respond to unmethylated CpG motifs present in nonmammalian DNA. Stabilized phosphorothioate-modified oligodeoxynucleotides (PS-ODN) containing CpG motifs form the basis of immunotherapeutic agents. In this study, we show that PS-ODN do not perfectly mimic native DNA in activation of macrophages. CpG-containing PS-ODN were active at 10- to 100-fold lower concentrations than corresponding phosphodiester ODN in maintenance of cell viability in the absence of CSF-1, in induction of NO production, and in activation of the IL-12 promoter. These enhancing effects are attributable to both increased stability and rate of uptake of the PS-ODN. By contrast, PS-ODN were almost inactive in down-modulation of the CSF-1R from primary macrophages and activation of the HIV-1 LTR. Delayed or poor activation of signaling components may contribute to this, as PS-ODN were slower and less effective at inducing phosphorylation of the extracellular signal-related kinases 1 and 2. In addition, at high concentrations, non-CpG PS-ODN specifically inhibited responses to CpG DNA, whereas nonstimulatory phosphodiester ODN had no such effect. Although nonstimulatory PS-ODN caused some inhibition of ODN uptake, this did not adequately explain the levels of inhibition of activity. The results demonstrate that the phosphorothioate backbone has both enhancing and inhibitory effects on macrophage responses to CpG DNA.
Vγ1+ T Cells Suppress and Vγ4+ T Cells Promote Susceptibility to Coxsackievirus B3-Induced Myocarditis in Mice
Authors: Huber Sally A. , Graveline Danielle , Newell M. Karen , Born Willi K. , O’Brien Rebecca L.
Published: April 2014

Coxsackievirus B3 infections of C57BL/6 mice, which express the MHC class II IA but not IE Ag, results in virus replication in the heart but minimal myocarditis. In contrast, Bl.Tg.Eα mice, which are C57BL/6 mice transgenically induced to express IE Ag, develop significant myocarditis upon Coxsackievirus B3 infection. Despite this difference in inflammatory damage, cardiac virus titers are similar between C57BL/6 and Bl.Tg.Eα mice. Removing γδ T cells from either strain by genetic manipulation (γδ knockout(ko)) changes the disease phenotype. C57BL/6 γδ ko mice show increased myocarditis. In contrast, Bl.Tg.Eα γδ ko mice show decreased cardiac inflammation. Flow cytometry revealed a difference in the γδ cell subsets in the two strains, with Vγ1 dominating in C57BL/6 mice, and Vγ4 predominating Bl.Tg.Eα mice. This suggests that these two Vγ-defined subsets might have different functions. To test this possibility, we used mAb injection to deplete each subset. Mice depleted of Vγ1 cells showed enhanced myocarditis, whereas those depleted of Vγ4 cells suppressed myocarditis. Adoptively transfusing enriched Vγ4+ cells to the C57BL/6 and Bl.Tg.Eα γδ ko strains confirmed that the Vγ4 subset promoted myocarditis. Th subset analysis suggests that Vγ1+ cells biased the CD4+ T cells to a dominant Th2 cell response, whereas Vγ4+ cells biased CD4+ T cells toward a dominant Th1 cell response.
Accumulation of RXRα During Activation of Cycling Human T Lymphocytes: Modulation of RXRE Transactivation Function by Mitogen-Activated Protein Kinase Pathways
Authors: Ishaq Mohammad , Fan Ming , Natarajan Ven
Published: April 2014

We have previously reported that the activation of resting human immature peripheral blood T (PBT) lymphocytes is associated with the loss of retinoid X receptor α (RXRα) expression. In the present study, we have demonstrated that, unlike resting cells, activation of cycling human mature PBT lymphocytes, and T lymphocyte leukemia cell lines is accompanied by the accumulation of RXRα mRNA and protein. Interestingly, cyclosporin A further augmented RXRα expression, indicating the involvement of calcineurin pathways in the process. 9-cis retinoic acid inhibited the accumulation, suggesting that retinoids can regulate the synthesis of their own receptors during T cell activation. Transfection analysis in Jurkat cells, using RXRE-dependent reporter assays, showed that RXRα accumulated during T cell activation was transcriptionally inactive. To investigate the mechanism of such inhibition, the role of two mitogen-activated protein kinase pathways, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), in modulating RXRE-dependent transcription, was explored. The expression of constitutively active MAP/ERK kinase kinase 1 (MEKK1) inhibited RXRE-dependent transcription, whereas dominant negative MEKK1 increased the transcription, indicating the involvement of JNK signaling pathways in the process. In contrast, expression of constitutively active MEK1, which activates ERK pathway, enhanced RXRE-dependent activation. When both were activated simultaneously, JNK pathway was dominant over ERK pathway and resulted in inhibition of RXRE-mediated transcription. These data demonstrate a dual regulatory control of RXRα expression during the activation of resting and cycling T lymphocytes and indicate a dynamic balance between JNK and ERK pathways in modulating RXRE-mediated transactivation.
Divergent Roles for CD4+ T Cells in the Priming and Effector/Memory Phases of Adoptive Immunotherapy
Authors: Hu Hong-Ming , Winter Hauke , Urba Walter J. , Fox Bernard A.
Published: April 2014

The requirement for CD4+ Th cells in the cross-priming of antitumor CTL is well accepted in tumor immunology. Here we report that the requirement for T cell help can be replaced by local production of GM-CSF at the vaccine site. Experiments using mice in which CD4+ T cells were eliminated, either by Ab depletion or by gene knockout of the MHC class II β-chain (MHC II KO), revealed that priming of therapeutic CD8+ effector T cells following vaccination with a GM-CSF-transduced B16BL6-D5 tumor cell line occurred independently of CD4+ T cell help. The adoptive transfer of CD8+ effector T cells, but not CD4+ effector T cells, led to complete regression of pulmonary metastases. Regression of pulmonary metastases did not require either host T cells or NK cells. Transfer of CD8+ effector T cells alone could cure wild-type animals of systemic tumor; the majority of tumor-bearing mice survived long term after treatment (>100 days). In contrast, adoptive transfer of CD8+ T cells to tumor-bearing MHC II KO mice improved survival, but eventually all MHC II KO mice succumbed to metastatic disease. WT mice cured by adoptive transfer of CD8+ T cells were resistant to tumor challenge. Resistance was mediated by CD8+ T cells in mice at 50 days, while both CD4+ and CD8+ T cells were important for protection in mice challenged 150 days following adoptive transfer. Thus, in this tumor model CD4+ Th cells are not required for the priming phase of CD8+ effector T cells; however, they are critical for both the complete elimination of tumor and the maintenance of a long term protective antitumor memory response in vivo.
Autoregulation of Human Monocyte-Derived Dendritic Cell Maturation and IL-12 Production by Cyclooxygenase- 2-Mediated Prostanoid Production
Authors: Whittaker Donna S. , Bahjat Keith S. , Moldawer Lyle L. , Clare-Salzler Michael J.
Published: April 2014

PG added to cell culture profoundly affect the in vitro maturation and function of monocyte-derived dendritic cells (MDC). Because unstimulated monocytes express cyclooxygenase (COX)-1, and COX-2 when activated, we examined whether MDC express these enzymes and produce prostanoids that autoregulate maturation and IL-12 production. Immature MDC (I-MDC) and mature MDC express COX-1, but, unlike monocytes, both MDC populations constitutively express COX-2. However, COX-2 regulation in both MDC populations differs from monocytes, as IL-4 does not suppress enzyme expression. COX-2 is functional in MDC as a specific inhibitor, NS-398, significantly reduces PGE2 production. I-MDC undergoing maturation with soluble CD40 ligand (sCD40L) increase PGE2 synthesis, but prostanoid synthesis is switched to COX-1. However, with IFN-γ present, sCD40L-stimulated PG metabolism is redirected to COX-2, and PGE2 synthesis increases severalfold. Endogenous PG production by MDC does not regulate CD40, CD80, CD86, or HLA DR expression; however, it does promote MDC maturation, as NS-398 significantly reduces CD83 expression in I-MDC matured with sCD40L/IFN-γ. PG produced through COX-2 also autoregulate IL-12, but the effects are dependent on the MDC maturation state. Blocking COX-2 reduces I-MDC secretion of IL-12p40, whereas it increases IL-12p40 and p70 production by maturing MDC. COX-2-mediated PG production impacts MDC function as maturing these cells in the presence of NS-398 yields MDC that stimulate significantly more IFN-γ in an allogeneic mixed lymphocyte response than MDC matured without this inhibitor. These studies demonstrate that MDC express both COX isoforms constitutively and produce prostanoids, which autoregulate their maturation and function.
Enhancement of T Cell Receptor Signaling by a Mild Oxidative Shift in the Intracellular Thiol Pool
Authors: Hehner Steffen P. , Breitkreutz Raoul , Shubinsky George , Unsoeld Heike , Schulze-Osthoff Klaus , Schmitz M. Lienhard , Dröge Wulf
Published: April 2014

Exposure of T cells to the macrophage products hydrogen peroxide (HP) or l-lactate (LAC) was previously shown to enhance IL-2 production and to modulate glutathione (GSH) status. We now found that 50 μM HP and 30 mM LAC enhanced strongly the transcription from the IL-2 promoter in Jurkat T cells after stimulation with anti-CD28 together with or without anti-CD3 but not with anti-CD3 Abs alone. Therefore, we used anti-CD3 plus anti-CD28-stimulated cells to investigate the effect of the GSH reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on the signal cascade. BCNU enhanced the transcription to a similar extent as HP or LAC. Lowering the intracellular GSH/GSH disulfide ratio by BCNU, HP, or NO resulted in all cases in the fulminant enhancement of Jun-N-terminal kinase and p38 mitogen-activated protein kinase but not extracellular signal-regulated kinase 1/2. Jun-N-terminal kinase and NF-κB activation was enhanced through pathways involving Rac, Vav1, PKCΘ, p56lck, p59fyn, and IκB kinases. In a cell-free system, the autophosphorylation of rFyn was stimulated by GSH disulfide but not by HP. These findings suggest that the oxidation of the cellular thiol pool may play a role as an amplifying mechanism for TCR/CD3 signals in immune responses.
Unexpected Autoantibody Production in Membrane Ig-μ-Deficient/lprMice
Authors: Melamed Doron , Miri Engelmayer , Leider Nira , Nemazee David
Published: April 2014

In the B lymphocyte lineage, Fas-mediated cell death is important in controlling activated mature cells, but little is known about possible functions at earlier developmental stages. In this study we found that in mice lacking the IgM transmembrane tail exons (μMT mice), in which B cell development is blocked at the pro-B stage, the absence of Fas or Fas ligand allows significant B cell development and maturation, resulting in high serum Ig levels. These B cells demonstrate Ig heavy chain isotype switching and autoimmune reactivity, suggesting that lack of functional Fas allows maturation of defective and/or self-reactive B cells in μMT/lpr mice. Possible mechanisms that may allow maturation of these B cells are discussed.