bluuebunny/crossref_metadata_2025 huggingface dataset:
Immature CD4+CD8+ Thymocytes Do Not Polarize Lipid Rafts in Response to TCR-Mediated Signals
Authors: Ebert Peter J. R. , Baker Josh F. , Punt Jennifer A.
Published: April 2014

TCR-mediated stimulation induces activation and proliferation of mature T cells. When accompanied by signals through the costimulatory receptor CD28, TCR signals also result in the recruitment of cholesterol- and glycosphingolipid-rich membrane microdomains (lipid rafts), which are known to contain several molecules important for T cell signaling. Interestingly, immature CD4+CD8+ thymocytes respond to TCR/CD28 costimulation not by proliferating, but by dying. In this study, we report that, although CD4+CD8+ thymocytes polarize their actin cytoskeleton, they fail to recruit lipid rafts to the site of TCR/CD28 costimulation. We show that coupling of lipid raft mobilization to cytoskeletal reorganization can be mediated by phosphoinositide 3-kinase, and discuss the relevance of these findings to the interpretation of TCR signals by immature vs mature T cells.
Thymocytes Express thegolliProducts of the Myelin Basic Protein Gene and Levels of Expression Are Stage Dependent
Authors: Feng Ji-Ming , Givogri Irene M. , Bongarzone Ernesto R. , Campagnoni Celia , Jacobs Erin , Handley Vance W. , Schonmann Vilma , Campagnoni Anthony T.
Published: April 2014

The golli products of the myelin basic protein gene have been shown to be expressed in mouse thymus and brain. The full repertoire of thymic cell types expressing golli products has not yet been determined, although immunoreactivity has been found in some macrophages. We have analyzed the cellular expression of golli mRNAs and proteins in the thymus. The results showed that MTS5+ cortical/MTS10+ medullary epithelial cells and NLDC145+ dendritic cells did not express golli, while some macrophages did exhibit strong immunoreactivity. Golli mRNAs were not detected in macrophages by in situ hybridization. Thymocytes expressed significant levels of golli mRNAs and proteins by in situ hybridization and immunohistochemistry. Interestingly, golli immunoreactivity varied with thymocyte stage of differentiation. For example, CD4−CD8− (double-negative) thymocytes expressed relatively high levels of golli. Upon further differentiation into CD4−CD8− (double-positive) thymocytes, golli protein expression declined dramatically. When thymocytes developed into CD8− or CD4+ (single-positive) thymocytes, golli protein expression increased again, but it never achieved the levels found in double-negative thymocytes. Thus, the altered levels of expression of golli proteins in developing thymocytes correlated with the transitions from double-negative to double-positive and double-positive to single-positive stages. The lack of significant golli expression in thymic stromal cells may offer an alternative explanation for the mechanism of inefficient negative selection of those autoreactive thymocytes with specificity for myelin basic proteins.
Commitment of B Lymphocytes to a Plasma Cell Fate Is Associated with Blimp-1 Expression In Vivo
Authors: Angelin-Duclos Cristina , Cattoretti Giorgio , Lin Kuo-I , Calame Kathryn
Published: April 2014

B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcriptional repressor that is sufficient to trigger terminal differentiation in the B cell lymphoma BCL-1. In this study, we have determined the expression pattern of Blimp-1 in vivo in primary and secondary lymphoid organs of humans and immunized mice. Blimp-1 is expressed in plasma cells derived from either a T-independent or T-dependent response in plasma cells that have undergone isotype switching and those resulting from secondary immunization. Blimp-1 is also present in long-lived plasma cells residing in the bone marrow. However, Blimp-1 was not detected in memory B cells. This expression pattern provides further evidence of a critical role for Blimp-1 in plasma cell development, supporting earlier studies in cultured lines. Significantly, Blimp-1 was also found in a fraction (4–15%) of germinal center B cells in murine spleen and human tonsils. Blimp-1 expression in the germinal center is associated with an interesting subset of cells with a phenotype intermediate between germinal center B cells and plasma cells. In the mouse, Blimp-1+ germinal center B cells peak at day 12 postimmunization and disappear soon thereafter. They are not apoptotic, some are proliferating, they express germinal center markers peanut agglutinin or CD10 but not Bcl-6, and most express CD138 (syndecan-1), IRF4, and cytoplasmic Ig. Together, these data support a model in which B cell fate decisions occur within the germinal center and Blimp-1 expression is critical for commitment to a plasma cell, rather than a memory cell, fate.
IFN-γ and IFN-α Posttranscriptionally Down-Regulate the IL-4-Induced IL-4 Receptor Gene Expression
Authors: So Eui-Young , Park Hyun-Hee , Lee Choong-Eun
Published: April 2014

As Th1 and Th2 cytokines, IFN-γ/α and IL-4 counterregulate diverse immune functions. In particular, IFN-γ and IFN-α have been reported to markedly suppress the IL-4-induced IgE production and type II IgE receptor (FcεRII/CD23) expression. Because modulation of IL-4R may be an important mechanism in the regulation of IL-4 response, we have investigated the effect of IFN-γ/α on IL-4R expression and signal transduction mechanisms involved in this process. In human mononuclear cells and B cells isolated from tonsil or peripheral blood, IL-4 up-regulates IL-4R(α) expression at surface protein and mRNA levels, and the IL-4-induced IL-4R(α) is significantly down-regulated by both IFN-γ and IFN-α to a similar extent. The inhibitory effects of IFN-γ/α on the IL-4R mRNA expression require a lag period of about 8 h, and are sensitive to cycloheximide treatment, which suggests that the suppressive effect of IFNs on IL-4R gene expression is a secondary response requiring de novo synthesis of IFN-induced factors. Under such conditions that the inhibitory effects of IFNs are observed, IFNs do not affect the IL-4-induced STAT6 activation and IL-4R transcription, as analyzed by EMSA and nuclear run-on assays, respectively. Subsequently, mRNA stability studies have indicated that the action of IFN-γ/α is primarily mediated by an accelerated decay of IL-4-induced IL-4R mRNA. Thus, it appears that, as already shown in the case of the IL-4-induced FcεRII regulation, posttranscriptional inhibition of IL-4-inducible genes by mRNA destabilization is a common mechanism by which type I and II IFNs antagonize the IL-4 response in human immune cells.
CD95 (Fas) Ligand-Expressing Vesicles Display Antibody-Mediated, FcR-Dependent Enhancement of Cytotoxicity
Authors: Jodo Satoshi , Hohlbaum Andreas M. , Xiao Sheng , Chan Derek , Strehlow David , Sherr David H. , Marshak-Rothstein Ann , Ju Shyr-Te
Published: April 2014

Bioactive Fas ligand (FasL)-expressing vesicles were generated (vesicle preparation, VP) from two cell lines overexpressing FasL. The effect of NOK-1 anti-FasL mAb (mouse IgG1) on the cytotoxicity of FasL VP against various targets was determined. At high concentrations (1–10 μg/ml), NOK-1 inhibited the cytotoxicity. By contrast, NOK-1 in the dose range of 1–100 ng/ml significantly enhanced cytotoxicity against the FcR+ LB27.4, M59, and LF+ targets, but not the FcR− Jurkat and K31H28 hybridoma T cell targets. The ability to enhance FasL VP-mediated cytotoxicity could be blocked by the FcR-specific mAb 2.4G2. Enhancement was also observed with FcR+ A20 B lymphoma but not with the FcR− A20 variant. Enhancement of FasL VP cytotoxicity was observed with five IgG anti-FasL mAbs, but not with an IgM anti-FasL mAb. Inhibition was observed with high doses of all mAb except the IgG anti-FasL mAb G247-4, which is specific to a segment outside the FasL binding site. Interestingly, under identical conditions but in the presence of 2.4G2, G247-4 inhibited the cytotoxicity of FasL VP. In addition, G247-4 inhibited the FasL VP-mediated killing of FcR− Jurkat. The data demonstrate that FasL-expressing bioactive vesicles display a property heretofore unknown in bioactive agents that express FasL-mediated cytotoxicity. The mechanism of the Ab-mediated, FcR-dependent enhancement of cytotoxicity of bioactive vesicles and its physiological significance are discussed.
Th1 and Th2 Cell Clones to a Poorly Immunogenic Tumor Antigen Initiate CD8+ T Cell-Dependent Tumor Eradication In Vivo
Authors: Fallarino Francesca , Grohmann Ursula , Bianchi Roberta , Vacca Carmine , Fioretti Maria C. , Puccetti Paolo
Published: April 2014

Although CD8+ T cells play a central role as immune effectors, CD4+ T cells act to control the activation and persistence of the CD8+ T cell response in autoimmune disease, antiviral immunity, and experimental systems with immunogenic model tumor Ag. However, little information is available on the effects of CD4+ T cells on the function of endogenous CD8+ T lymphocytes recognizing authentic tumor rejection Ag with limited immunogenicity. We report here that the prophylactic or postchallenge administration of T helper Th1-type and Th2-type CD4+ clones specific for an unmutated rejection Ag (murine P815AB, resembling tumor-specific shared Ag in humans) leads to the induction of P815AB-specific reactivity in vivo and concomitant tumor destruction, with quantitative rather than qualitative differences characterizing the antitumor activity of Th1 vs Th2 cells. Because the transferred CD4+ cells lacked direct antitumor activity in vitro and required the de novo generation of P815AB-specific CD8+ T cells in vivo, these findings suggest that CD4+ lymphocytes can enhance the ability of host APC to initiate an endogenous CD8+ T cell response to authentic, poorly immunogenic tumor rejection Ag.
IFN-γ Can Promote Tumor Evasion of the Immune System In Vivo by Down-Regulating Cellular Levels of an Endogenous Tumor Antigen
Authors: Beatty Gregory L. , Paterson Yvonne
Published: April 2014

Although IFN-γ has been generally thought to enhance antitumor immune responses, we found that IFN-γ can promote tumor escape in the CT26 colon carcinoma by down-regulating the protein expression of an endogenous tumor Ag. gp70, the env product of the endogenous ecotropic murine leukemia virus, has been reported to be the immunodominant Ag of CT26. We show that IFN-γ down-regulates intracellular and surface levels of gp70 protein resulting in a reduced lysis by CTL, which is restored by pulsing IFN-γ-treated CT26 with the Ld-restricted immunodominant AH1 epitope derived from gp70. To investigate the role of CT26 sensitivity to IFN-γ in vivo, we constructed two variants of CT26, CT26.mugR and CT26.IFN, that are unresponsive to IFN-γ or express IFN-γ, respectively. We demonstrate using these variants that tumor responsiveness to IFN-γ promotes a reduction in tumor immunogenicity in vivo that is correlated with an increased tumor incidence in immune mice. Analysis of the tumors from mice challenged with CT26 or CT26.mugR revealed infiltration of CD8 T cells secreting IFN-γ. We conclude that IFN-γ secreted by tumor-infiltrating T cells promotes tumor escape through the down-regulation of the endogenous tumor Ag gp70. These findings have impact on the design of effective antitumor vaccine strategies.
Eosinophil Major Basic Protein-1 Does Not Contribute to Allergen-Induced Airway Pathologies in Mouse Models of Asthma
Authors: Denzler Karen L. , Farmer Steven C. , Crosby Jeffrey R. , Borchers Michael , Cieslewicz Grzegorz , Larson Kirsten A. , Cormier-Regard Stephania , Lee Nancy A. , Lee James J.
Published: April 2014

The relationship between eosinophils and the development of Ag-induced pulmonary pathologies, including airway hyper-responsiveness, was investigated using mice deficient for the secondary granule component, major basic protein-1 (mMBP-1). The loss of mMBP-1 had no effect on OVA-induced airway histopathologies or inflammatory cell recruitment. Lung function measurements of knockout mice demonstrated a generalized hyporeactivity to methacholine-induced airflow changes (relative to wild type); however, this baseline phenotype was observable only with methacholine; no relative airflow changes were observed in response to another nonspecific stimulus (serotonin). Moreover, OVA sensitization/aerosol challenge of wild-type and mMBP-1−/− mice resulted in identical dose-response changes to either methacholine or serotonin. Thus, the airway hyper-responsiveness in murine models of asthma occurs in the absence of mMBP-1.
Inhibition of IgG1 and IgE Production by Stimulation of the B Cell CTLA-4 Receptor
Authors: Pioli Claudio , Gatta Lucia , Ubaldi Vanessa , Doria Gino
Published: April 2014

Although a large amount of information is available on the activity of CTLA-4 in T cells, the role of this receptor in B cells has not been previously characterized. Our results show that CD40 or LPS stimulation in the presence of IL-4 induces CTLA-4 expression in purified B cells; the maximum level is reached in both membrane and intracellular compartments after 48–72 h. Engagement of the B cell CTLA-4 by immobilized mAb inhibits IgG1 and IgE production and reduces the frequency of IgG1- and IgE-expressing B cells. Cε and Cγ1 germline mRNA expression as well as NF-κB and STAT6 activation, events required for isotype switching, are also inhibited by CTLA-4 engagement. Together these findings show the critical role of CTLA-4 in the control of IL-4-driven isotype switching and suggest new approaches for modulating immediate-type hypersensitivity responses.
Fas Ligand Costimulates the In Vivo Proliferation of CD8+ T Cells
Authors: Suzuki Ivy , Martin Stefan , Boursalian Tamar E. , Beers Courtney , Fink Pamela J.
Published: April 2014

Fas ligand (FasL/CD95L/APO-1L) is one of a growing number of TNF family members whose triggering costimulates maximal proliferation of activated T cells. In this study we show that maximal Ag-dependent accumulation of transferred TCR-transgenic CD8+ T cells requires Fas (CD95/APO-1) expression by the adoptive hosts. Additionally, adoptively transferred FasL+ CD8+ T cells demonstrate a 2-fold advantage in Ag-driven expansion over their FasL−counterparts. This study illustrates the in vivo role of TCR-dependent FasL costimulation in the Ag-specific proliferation of both heterogeneous and homogeneous populations of primary CD8+ T cells and long-term CTL lines. Thus, cross-linking FasL on naive and Ag-experienced CD8+ T cells whose Ag-specific TCRs are engaged is required to drive maximal cellular proliferation in vivo.
Molecular Mechanisms of B Lymphocyte Activation by the Immune Response Modifier R-848
Authors: Bishop Gail A. , Hsing Yina , Hostager Bruce S. , Jalukar Sangita V. , Ramirez Luis M. , Tomai Mark A.
Published: April 2014

The imidazoquinoline R-848, originally identified as a highly effective antiviral agent, has recently been shown to be capable of potent B lymphocyte activation. The B cell-activating properties of R-848 are strikingly similar to the effects of the CD40 ligand CD154. The present study demonstrates that this similarity extends to the intracellular signaling pathways triggered by the compound, although both overlapping and distinct mechanisms of signaling were seen. Like CD40 ligation, R-848 stimulated activation of the stress-activated protein kinases c-Jun kinase and p38 and activated the NF-κB family of transcription factors. Both R-848- and CD40-mediated B cell differentiation were dependent upon NF-κB activation, although the relative importance of individual NF-κB family members appeared to differ between R-848- and CD40-mediated signals. Both signals were partially dependent upon induction of TNF-α and IL-6, and the cytoplasmic adaptor molecule TNF receptor-associated factor 2 is involved in both R-848- and CD40-mediated differentiation.
A Critical Role for B Cells in the Development of Memory CD4 Cells
Authors: Linton Phyllis-Jean , Harbertson Judith , Bradley Linda M.
Published: April 2014

Activated B cells express high levels of class II MHC and costimulatory molecules and are nearly as effective as dendritic cells in their APC ability. Yet, their importance as APC in vivo is controversial and their role, if any, in the development of CD4 memory is unknown. We compared responses of CD4 cells from normal and B cell-deficient mice to keyhole limpet hemocyanin over 6 mo and observed diminished IL-2 production by cells primed in the absence of B cells. This was due to lower frequencies of Ag-responsive cells and not to decreased levels of IL-2 secretion per cell. The absence of B cells did not affect the survival of memory CD4 cells since frequencies remained stable. Despite normal dendritic cell function, multiple immunizations of B cell-deficient mice did not restore frequencies of memory cells. However, the transfer of B cells restored memory cell development. Ag presentation was not essential since B cells activated in vitro with irrelevant Ag also restored frequencies of memory cells. The results provide unequivocal evidence that B cells play a critical role in regulating clonal expansion of CD4 cells and, as such, are requisite for the optimal priming of memory in the CD4 population.
Role of STAT4 and STAT6 Signaling in Allograft Rejection and CTLA4-Ig-Mediated Tolerance
Authors: Zhou Ping , Szot Greg L. , Guo Zhong , Kim Oliver , He Gang , Wang Jun , Grusby Michael J. , Newell Kenneth A. , Thistlethwaite J. Richard , Bluestone Jeffrey A. , Alegre Maria-Luisa
Published: April 2014

STAT4−/− mice have impaired type 1 T cell differentiation, whereas STAT6−/− mice fail to generate type 2 responses. The role of type 1 and type 2 T cell differentiation in acute cardiac allograft rejection and in the induction of tolerance was examined in wild-type, STAT4−/−, and STAT6−/− recipients. All recipients rejected the grafts promptly. Analysis of in situ cytokine gene expression in the allografts confirmed decreased levels of IFN-γ in STAT4−/− recipients and undetectable levels of IL-4 and IL-5 in STAT6−/− mice. Blockade of the CD28/B7 costimulatory pathway prolonged cardiac graft survival for >100 days in 100% of wild-type and STAT4−/− mice. However, 14% of CTLA4-Ig-treated STAT6−/− mice rejected their grafts between 20 and 100 days. Moreover, of those animals followed past 100 days, 60% of the STAT6−/− mice rejected their grafts. Splenocytes harvested on day 145 posttransplant from CTLA4-Ig-treated rejecting STAT6−/− recipients were transfused into syngeneic SCID mice transplanted with donor or third party cardiac allografts. Both donor and third party grafts were rejected, indicating that the initial graft loss may be due to an immunological rejection. In contrast, when splenocytes from CTLA4-Ig-treated wild-type or nonrejecting STAT6−/− mice were transferred into SCID recipients, donor allografts were accepted, but third party hearts were rejected. Thus, long-term prolongation of cardiac allograft survival by CTLA4-Ig is STAT4-independent but, at least in part, STAT6-dependent. These data suggest that the balance of type 1 and type 2 T lymphocyte differentiation is not critical for acute rejection but influences the robust tolerance induced by CD28/B7 blockade in this model.
The Neuropeptide Substance P Activates p38 Mitogen-Activated Protein Kinase Resulting in IL-6 Expression Independently from NF-κB
Authors: Fiebich Bernd L. , Schleicher Sandra , Butcher Russel D. , Craig Alexander , Lieb Klaus
Published: April 2014

Substance P (SP), a member of the tachykinin peptide family, is a major mediator of neuroimmunomodulatory activities and neurogenic inflammation within the central and peripheral nervous system. SP has been shown to induce the expression of proinflammatory cytokines such as IL-6, which might be implicated in the etiopathology of several human brain disorders. We showed in a previous study that nanomolar concentrations of SP triggered activation of NF-κB, a transcription factor involved in the control of cytokine expression. However, activation of NF-κB was not involved in SP-induced expression of IL-6. Here, we describe p38 mitogen-activated protein kinase (p38 MAPK) as a signal transduction component that operates independently from NF-κB activation and that mediates SP-induced IL-6 expression in the human astrocytoma cell line U373 MG. SP induced the phosphorylation of p38 MAPK within 10 min, and this activation persisted up to 30 min and was independent from p42/44 MAPKs and protein kinase C activation, which all are induced after stimulation with SP. As shown by EMSA, p38 MAPK was not involved in SP-induced activation of NF-κB. p38 MAPK, however, mediated SP-induced IL-6 expression as shown by the use of specific inhibitors of this kinase. Our results suggest that activation of p38 MAPK is an important component controlling neurogenic inflammation within the CNS independently from NF-κB. Drugs targeting this MAPK may therefore interfere with SP-correlated neuropsychiatric disorders and may represent a therapeutic approach in these disorders.
TNF Regulates Thymocyte Production by Apoptosis and Proliferation of the Triple Negative (CD3−CD4−CD8−) Subset
Authors: Baseta Juana Gonzalez , Stutman Osias
Published: April 2014

TNF is a proinflammatory cytokine with opposing death/no-death effects in vivo and in vitro. Our studies showed that TNF regulates mouse thymocyte production, inducing both apoptosis and proliferation of the most immature CD3−CD4−CD8− triple negative (TN) subset within a broad range of dosages (101–105 pg/ml) in the presence of IL-7. TNF apoptosis affected only the TN3 (CD44−CD25+) and TN4 (CD44−CD25−) subsets that expressed both TNFR-p55 and -p75. Although each TNFR alone could mediate TNF apoptosis, maximal apoptosis was seen in C57BL/6J wild type, which expressed both TNFRs. TNF also induced proliferation of TN3 cells at higher doses (104–105 pg/ml) mediated only by TNFR-p75. Both anti-TNFR-p55 and -TNFR-p75 mAb inhibited apoptosis but only anti-p75 inhibited proliferation. TNF also regulated TN proliferation to IL-7 because TNFR knockout (KO), TNF KO, and TNF/lymphotoxin α and β triple KO mice showed 2- to 3-fold increased responses not seen in C57BL/6J wild type. In vivo, TNFR KO mice showed thymic hypertrophy with a 60% increase in total thymocytes, with no effect on the CD4/CD8 subsets. We conclude that TNF maintains homeostatic control of total thymocyte production by negative selection of TN3 and TN4 prothymocytes and down-regulation of their proliferation to endogenous IL-7.
Mechanisms of Tolerance Induction by a Gene-Transferred Peptide-IgG Fusion Protein Expressed in B Lineage Cells
Authors: El-Amine Moustapha , Melo Marco , Kang Yubin , Nguyen Hao , Qian Jiahua , Scott David W.
Published: April 2014

A gene therapy model has been designed to induce tolerance to multiple epitopes expressed in-frame on a soluble IgG fusion protein scaffold. Tolerance to the λ repressor cI sequence p1-102 or its immunodominant epitopes (p12-26, p73-88) can be elicited when bone marrow (BM) or LPS blasts are transduced and injected into naive or even primed recipients. To explore the mechanism of tolerance, class II−/− (knockout, KO) BM cells were transduced with p1-102-IgG and transferred to irradiated recipients. These cells failed to induce tolerance to challenge with p1-102 epitopes, whereas transduced +/+ BM cells did. This supports the importance of class II MHC on the tolerogenic APC rather than secretion and representation in tolerogenesis. When BM cells from μMT KO mice were transfected with p12-26-IgG and injected into irradiated mice, these transduced BM cells also failed to induce tolerance to an immunodominant epitope. These results suggest the direct involvement of B cells in tolerance to p1-102 epitopes. IL-10 KO BM cells infected with a p12-26-IgG construct were still tolerogenic. Importantly, anti-CTLA-4 injections reversed tolerance in primed, but not in naive, recipients of transduced LPS blasts. These data emphasize the importance of MHC class II presentation, B cell involvement, and CTLA-4 engagement in induction and/or maintenance of tolerance.
Kb, Kd, and Ld Molecules Share Common Tapasin Dependencies as Determined Using a Novel Epitope Tag
Authors: Myers Nancy B. , Harris Michael R. , Connolly Janet M. , Lybarger Lonnie , Yu Yik Y. L. , Hansen Ted H.
Published: April 2014

The endoplasmic reticulum protein tapasin is considered to be a class I-dedicated chaperone because it facilitates peptide loading by proposed mechanisms such as peptide editing, endoplasmic reticulum retention of nonpeptide-bound molecules, and/or localizing class I near the peptide source. Nonetheless, the primary functions of tapasin remain controversial as do the relative dependencies of different class I molecules on tapasin for optimal peptide loading and surface expression. Tapasin dependencies have been addressed in previous studies by transfecting different class I alleles into tapasin-deficient LCL721.220 cells and then monitoring surface expression and Ag presentation to T cells. Indeed, by these criteria, class I alleles have disparate tapasin-dependencies. In this study, we report a novel and more direct method of comparing tapasin dependency by monitoring the ratio of folded vs open forms of the different mouse class I heavy chains, Ld, Kd, and Kb. Furthermore, we determine the amount of de novo heavy chain synthesis required to attain comparable expression in the presence vs absence of tapasin. Our findings show that tapasin dramatically improves peptide loading of all three of these mouse molecules.
Export of the High Affinity IgE Receptor From the Endoplasmic Reticulum Depends on a Glycosylation-Mediated Quality Control Mechanism
Authors: Albrecht Bettina , Woisetschläger Maximilian , Robertson Michael W.
Published: April 2014

The high affinity IgE receptor (FcεRI) is a multisubunit complex comprised of either αγ2 or αβγ2 chains. The cotranslational assembly of the IgE-binding α-chain with a dimer of γ-chains occurs in a highly controlled manner and is proposed to involve masking of a dilysine motif present at the cytoplasmic C terminus of the FcεRI α-chain that targets localization of this subunit to the endoplasmic reticulum (ER). Here, we show that ER quality control modulates export from the ER of newly synthesized αγ2 and αβγ2 receptors. We demonstrate that the presence of untrimmed N-linked core glycans (Glc3Man9GlcNAc2) on the FcεRI α-chain activates the ER quality control mechanism to retain this subunit in the ER, despite the presence of γ-chains. At the same time, the untrimmed, ER-localized α-chain exhibits IgE-binding activity, suggesting that FcεRI α-chain folding occurs before constitutive glucose trimming. In additional experiments, we demonstrate that cell surface expression of an α-chain C-terminal truncation mutant is also dependent on glucose trimming, but not on γ-chain coexpression. We suggest that glucosidase trimming of terminal glucose residues is a critical control step in the export of FcεRIα from the ER. Finally, we show that the constitutive ER FcεRI α-chain, expressed in the absence of the other FcεRI subunits, associates with the ER lectin-like chaperone calnexin, but not the structurally similar ER chaperone calreticulin, presumably through interaction with monoglucosylated α-chain ER glycoforms.
Helper-Independent, L-Selectinlow CD8+ T Cells with Broad Anti-Tumor Efficacy Are Naturally Sensitized During Tumor Progression
Authors: Peng Liaomin , Kjaergaard Jørgen , Plautz Gregory E. , Weng David E. , Shu Suyu , Cohen Peter A.
Published: April 2014

We recently reported that the CD4+ T cell subset with low L-selectin expression (CD62Llow) in tumor-draining lymph nodes (TDLN) can be culture activated and adoptively transferred to eradicate established pulmonary and intracranial tumors in syngeneic mice, even without coadministration of IL-2. We have extended these studies to characterize the small subset of L-selectinlow CD8+ T cells naturally present in TDLN of mice bearing weakly immunogenic tumors. Isolated L-selectinlow CD8+ T cells displayed the functional phenotype of helper-independent T cells, and when adoptively transferred could consistently eradicate, like L-selectinlow CD4+ T cells, both established pulmonary and intracranial tumors without coadministration of exogenous IL-2. Whereas adoptively transferred L-selectinlow CD4+ T cells were more potent on a cell number basis for eradicating 3-day intracranial and s.c. tumors, L-selectinlow CD8+ T cells were more potent against advanced (10-day) pulmonary metastases. Although the presence of CD4+ T cells enhanced generation of L-selectinlow CD8+ effector T cells, the latter could also be obtained from CD4 knockout mice or normal mice in vivo depleted of CD4+ T cells before tumor sensitization. Culture-activated L-selectinlow CD8+ T cells did not lyse relevant tumor targets in vitro, but secreted IFN-γ and GM-CSF when specifically stimulated with relevant tumor preparations. These data indicate that even without specific vaccine maneuvers, progressive tumor growth leads to independent sensitization of both CD4+ and CD8+ anti-tumor T cells in TDLN, phenotypically L-selectinlow at the time of harvest, each of which requires only culture activation to unmask highly potent stand-alone effector function.
Modulation of Innate and Acquired Immune Responses by Escherichia coli Heat-Labile Toxin: Distinct Pro- and Anti-Inflammatory Effects of the Nontoxic AB Complex and the Enzyme Activity
Authors: Ryan Elizabeth J. , McNeela Edel , Pizza Mariagrazia , Rappuoli Rino , O’Neill Luke , Mills Kingston H. G.
Published: April 2014

We have examined the roles of enzyme activity and the nontoxic AB complex of heat-labile toxin (LT) from Escherichia coli on its adjuvant and immunomodulatory properties. LTK63, an LT mutant that is completely devoid of enzyme activity, enhanced Th1 responses to coinjected Ags at low adjuvant dose. In contrast, LTR72, a partially detoxified mutant, enhanced Th2 responses and when administered intranasally to mice before infection with Bordetella pertussis suppressed Th1 responses and delayed bacterial clearance from the lungs. LTR72 or wild-type LT inhibited Ag-induced IFN-γ production by Th1 cells, and LT enhanced IL-5 production by Th2 cells in vitro. Each of the toxins enhanced B7-1 expression on macrophages, but enhancement of B7-2 expression was dependent on enzyme activity. We also observed distinct effects of the nontoxic AB complex and enzyme activity on inflammatory cytokine production. LT and LTR72 suppressed LPS and IFN-γ induced TNF-α and IL-12 production, but enhanced IL-10 secretion by macrophages in vitro and suppressed IL-12 production in vivo in a murine model of LPS-induced shock. In contrast, LTK63 augmented the production of IL-12 and TNF-α. Furthermore, LTK63 enhanced NF-κB translocation, whereas low doses of LTR72 or LT failed to activate NF-κB, but stimulated cAMP production. Thus, E. coli LT appears to be capable of suppressing Th1 responses and enhancing Th2 responses through the modulatory effects of enzyme activity on NF-κB activation and IL-12 production. In contrast, the nontoxic AB complex can stimulate acquired immune responses by activating components of the innate immune system.
Monocytic Cells Synthesize, Adhere to, and Migrate on Laminin-8 (α4β1γ1)
Authors: Pedraza Claudio , Geberhiwot Tarekegn , Ingerpuu Sulev , Assefa Daniel , Wondimu Zenebech , Kortesmaa Jarkko , Tryggvason Karl , Virtanen Ismo , Patarroyo Manuel
Published: April 2014

Laminins, a growing family of large heterotrimeric proteins with cell adhesive and signaling properties, are major components of vascular and other basement membranes. Expression, recognition, and use of laminin isoforms by leukocytes are poorly understood. In monoblastic THP-1 cells, transcripts for laminin γ1-, β1-, and α4-chains were detected by RT-PCR. Following immunoaffinity purification on a laminin β1 Ab-Sepharose column, laminin β1- (220 kDa), γ1- (200 kDa), and α4- (180/200 kDa) chains were detected by Western blotting in THP-1 cells and in two other monoblastic cell lines, U-937 and Mono Mac 6. After cell permeabilization, a mAb to laminin γ1-chain reacted with practically all blood monocytes by immunofluorescence flow cytometry, and laminin-8 (α4β1γ1) could be isolated also from these cells. Monoblastic JOSK-I cells adhered constitutively to immobilized recombinant laminin-8, less than to laminin-10/11 (α5β1γ1/α5β2γ1) but to a higher level than to laminin-1 (α1β1γ1). Compared with the other laminin isoforms, adhesion to laminin-8 was preferentially mediated by α6β1 and β2 integrins. Laminin-8 and, to a lower extent, laminin-1 promoted spontaneous and chemokine-induced migration of blood monocytes, whereas laminin-10/11 was inhibitory. Altogether, the results indicate that leukocytes, as other cell types, are able to synthesize complete laminin molecules. Expression, recognition, and use of laminin-8 by leukocytes suggest a major role of this laminin isoform in leukocyte physiology.
Deficiency of Src Homology 2-Containing Phosphatase 1 Results in Abnormalities in Murine Neutrophil Function: Studies inMotheatenMice
Authors: Kruger Joshua , Butler Jeffrey R. , Cherapanov Vera , Dong Qin , Ginzberg Hedy , Govindarajan Anand , Grinstein Sergio , Siminovitch Katherine A. , Downey Gregory P.
Published: April 2014

Neutrophils, an essential component of the innate immune system, are regulated in part by signaling pathways involving protein tyrosine phosphorylation. While protein tyrosine kinase functions in regulating neutrophil behavior have been extensively investigated, little is known about the role for specific protein tyrosine phosphatases (PTP) in modulating neutrophil signaling cascades. A key role for Src homology 2 domain-containing phosphatase 1 (SHP-1), a PTP, in neutrophil physiology is, however, implied by the overexpansion and inappropriate activation of granulocyte populations in SHP-1-deficient motheaten (me/me) and motheaten viable (mev/mev) mice. To directly investigate the importance of SHP-1 to phagocytic cell function, bone marrow neutrophils were isolated from both me/me and mev/mev mice and examined with respect to their responses to various stimuli. The results of these studies revealed that both quiescent and activated neutrophils from motheaten mice manifested enhanced tyrosine phosphorylation of cellular proteins in the 60- to 80-kDa range relative to that detected in wild-type congenic control neutrophils. Motheaten neutrophils also demonstrated increased oxidant production, surface expression of CD18, and adhesion to protein-coated plastic. Chemotaxis, however, was severely diminished in the SHP-deficient neutrophils relative to control neutrophils, which was possibly attributable to a combination of defective deadhesion and altered actin assembly. Taken together, these results indicate a significant role for SHP-1 in modulating the tyrosine phosphorylation-dependent signaling pathways that regulate neutrophil microbicidal functions.
A Targeted DNA Vaccine Augments the Natural Immune Response to Self TNF-α and Suppresses Ongoing Adjuvant Arthritis
Authors: Wildbaum Gizi , Youssef Sawsan , Karin Nathan
Published: April 2014

Depending on the mode of immunization, a single administration of CFA may result in the development of a local inflammatory process or chronic poly adjuvant-induced arthritis (AA). Administration of naked DNA encoding TNF-α results in the generation of immunological memory to its gene product. Upon induction of AA, this memory effectively inhibited the development of disease. Self-specific Abs developed in DNA-vaccinated animals were neutralizing in vitro and could adoptively transfer the beneficial effect of the vaccine. Administration of CFA to induce a local delayed-type hypersensitivity response rather than AA did not lead to an elicited production of Abs to the gene product of the above vaccine. Thus, elicitation of protective immunity is dependent on the development of an autoimmune condition. Most importantly, the administration of the TNF-α DNA construct after the onset of disease led to a rapid, long-lasting remission. This suggests a highly effective way by which a DNA vaccine encoding an autologous proinflammatory cytokine can be used to reprogram the immune system to generate protective immunity to its own potentially harmful activities.
Chemically Modified Ribozyme to V Gene Inhibits Anti-DNA Production and the Formation of Immune Deposits Caused by Lupus Lymphocytes
Authors: Suzuki Yoko , Funato Tadao , Munakata Yasuhiko , Sato Kanako , Hirabayashi Yasuhiko , Ishii Tomonori , Takasawa Naruhiko , Ootaka Tetsuya , Saito Takao , Sasaki Takeshi
Published: April 2014

A variety of autoantibodies is responsible for the tissue injury in autoimmune diseases. We have demonstrated that the human anti-DNA Ab O-81, of which Ids are commonly detected in renal glomeruli of active lupus nephritis, uses the V3-7 gene. We tried to develop a new therapy for lupus nephritis by using chemically modified ribozymes to specifically inhibit the expression of the mRNA of Ig V gene. The transfection of hammerhead ribozyme or the addition of chemically modified ribozyme against the flanking region of V3-7 caused a potent and selective inhibition of anti-DNA production in V3-7-using B cell clones, but not in irrelevant V gene-using clones in vitro. Chemically modified ribozyme was long-acting and resistant to RNase, and nonspecific cytotoxicity of the ribozyme was negligible. To know the efficacy of the ribozyme in vivo, we used a model of immune complex nephritis in SCID mice in which 5 × 106 PBLs from patients with active lupus nephritis (lupus PBL) were transferred twice. The injection of lupus PBL in combination with chemically modified ribozyme to increase resistance to RNase significantly reduced anti-DNA Ab levels in blood and decreased levels of urinary protein in the immune deposit models. Immunofluorescence study also revealed a marked decrease in IgG deposits at renal glomeruli in the ribozyme-treated group. These results indicate an efficacy of chemically modified ribozyme therapy for autoantibody-mediated immune diseases.
Comparative Analysis of T Lymphocytes Recovered from the Lungs of Mice Genetically Susceptible, Resistant, and Hyperresistant toMycobacterium tuberculosis-Triggered Disease
Authors: Lyadova Irina V. , Eruslanov Evgenyi B. , Khaidukov Sergei V. , Yeremeev Vladimir V. , Majorov Konstantin B. , Pichugin Alexander V. , Nikonenko Boris V. , Kondratieva Tatiana K. , Apt Alexander S.
Published: April 2014

Genetic control of susceptibility to tuberculosis (TB) is being intensively studied, and immune responses to mycobacteria are considerably well characterized. However, it remains largely unknown which parameters of response distinguish resistant and susceptible TB phenotypes. Mice of I/St and A/Sn inbred strains and (A/Sn × I/St)F1 hybrids were previously categorized as, respectively, susceptible, resistant, and hyperresistant to Mycobacterium tuberculosis-triggered disease. In the present work we compared parameters of lung T cell activation and response following M. tuberculosis challenge. In all mice, the disease progression was accompanied by a marked accumulation in the lungs of activated CD4+ (CD44high/CD45RBlow) and CD8+ (CD44high/CD45RB+) T cells capable of secreting IFN-γ and of activating macrophages for NO production and mycobacterial growth inhibition. However, significantly more CD8+ T cells were accumulated in the lungs of resistant A/Sn and F1 compared with I/St mice. About 80% A/Sn and F1 CD8+ cells expressed CD44high/CD45RB+ phenotype, while about 40% I/St CD8+ cells did not express CD45RB marker at week 5 of infection. In contrast, in susceptible I/St mice lung CD4+ cells proliferated much more strongly in response to mycobacterial sonicate, and a higher proportion of these cells expressed CD95 and underwent apoptosis compared with A/Sn cells. Unseparated lung cells and T cells of I/St origin produced more IL-5 and IL-10, respectively, whereas their A/Sn and F1 counterparts produced more IFN-γ following infection. F1 cells overall expressed an intermediate phenotype between the two parental strains. Such a more balanced type of immune reactivity could be linked to a better TB defense.
Generation of T Cells Specific for the Wild-Type Sequence p53264–272 Peptide in Cancer Patients: Implications for Immunoselection of Epitope Loss Variants
Authors: Hoffmann Thomas K. , Nakano Koji , Elder Elaine M. , Dworacki Grzegorz , Finkelstein Sydney D. , Appella Ettore , Whiteside Theresa L. , DeLeo Albert B.
Published: April 2014

Alterations in the p53 gene occur frequently and can lead to accumulation of p53 protein in squamous cell carcinomas of the head and neck (SCCHN). Since accumulation of p53 is associated with enhanced presentation of wild-type sequence (wt) p53 peptides to immune cells, the development of pan vaccines against SCCHN has focused on wt p53 epitopes. We used the HLA-A2.1-restricted wt p53264–272 epitope to generate CTL from circulating precursor T cells of HLA-A2.1+ healthy donors and patients with SCCHN. Autologous peptide-pulsed dendritic cells were used for in vitro sensitization. CTL specific for the wt p53264–272 peptide were generated from PBMC obtained from two of seven normal donors and three of seven patients with SCCHN. These CTL were HLA class I restricted and responded to T2 cells pulsed with p53264–272 peptide as well as HLA-A2-matched SCCHN cell lines naturally presenting the epitope. Paradoxically, none of the tumors in the three patients who generated CTL could adequately present the epitope; two had a wt p53 genotype and no p53 protein accumulation, while the third tumor expressed a point mutation (R to H) in codon 273 that prevents presentation of the p53264–272 epitope. In contrast, patients who did not generate CTL had tumors that accumulated altered p53 and potentially could present the p53264–272 epitope. These findings suggest that in vivo, CTL specific for the wt p53264–272 peptide might play a role in the elimination of tumor cells expressing this epitope and in immunoselection of epitope-loss tumor cells. Immunoselection of tumors that become resistant to anti-p53 immune responses has important implications for future p53-based vaccination strategies.
Cutting Edge: B Cell Antigen Receptor Signaling Occurs Outside Lipid Rafts in Immature B Cells
Authors: Sproul Tim W. , Malapati Sunil , Kim Julie , Pierce Susan K.
Published: April 2014

B cell Ag receptor (BCR) signaling changes dramatically during B cell development, resulting in activation in mature B cells and apoptosis, receptor editing, or anergy in immature B cells. BCR signaling in mature B cells was shown to be initiated by the translocation of the BCR into cholesterol- and sphingolipid-enriched membrane microdomains that include the Src family kinase Lyn and exclude the phosphatase CD45. Subsequently the BCR is rapidly internalized into the cell. Here we show that the BCR in the immature B cell line, WEHI-231, does not translocate into lipid rafts following cross-linking nor is the BCR rapidly internalized. The immature BCR initiates signaling from outside lipid rafts as evidenced by the immediate induction of an array of phosphoproteins and subsequent apoptosis. The failure of the BCR in immature B cells to enter lipid rafts may contribute to the dramatic difference in the outcome of signaling in mature and immature B cells.
BDCA-2, BDCA-3, and BDCA-4: Three Markers for Distinct Subsets of Dendritic Cells in Human Peripheral Blood
Authors: Dzionek Andrzej , Fuchs Anja , Schmidt Petra , Cremer Sabine , Zysk Monika , Miltenyi Stefan , Buck David W. , Schmitz Jürgen
Published: April 2014

We have generated a panel of mAbs that identify three presumably novel human dendritic cell Ags: BDCA-2, BDCA-3, and BDCA-4. In blood, BDCA-2 and BDCA-4 are expressed on CD11c− CD123bright plasmacytoid dendritic cells, whereas BDCA-3 is expressed on small population of CD11c+ CD123− dendritic cells. All three Ags are not detectable on a third blood dendritic cell population, which is CD1c+ CD11cbright CD123dim, or on any other cells in blood. BDCA-4 is also expressed on monocyte-derived and CD34+ cell-derived dendritic cells. Expression of all three Ags dramatically changes once blood dendritic cells undergo in vitro maturation. BDCA-2 is completely down-regulated on plasmacytoid CD11c− CD123bright dendritic cells, expression of BDCA-3 is up-regulated on both plasmacytoid CD11c− CD123bright dendritic cells and CD1c+ CD11cbright CD123dim dendritic cells, and expression of BDCA-4 is up-regulated on CD1c+ CD11cbright CD123dim dendritic cells. BDCA-2 is rapidly internalized at 37°C after mAb labeling. The three presumably novel Ags serve as specific markers for the respective subpopulations of blood dendritic cells in fresh blood and will be of great value for their further analysis and to evaluate their therapeutic potential.
Tumor-Specific CD4+ T Cells Have a Major “Post-Licensing” Role in CTL Mediated Anti-Tumor Immunity
Authors: Marzo Amanda L. , Kinnear Beverley F. , Lake Richard A. , Frelinger Jeffrey J. , Collins Edward J. , Robinson Bruce W. S. , Scott Bernadette
Published: April 2014

A number of tumor studies have indicated a link between CD4 help and the magnitude and persistence of CTL activity; however, the mechanisms underlying this have been largely unclear. To evaluate and determine the mechanisms by which CD4+ T cells synergize with CD8+ T cells to prevent tumor growth, we used the novel technique of monitoring in vivo CTL by labeling target cells with CFSE. This approach was supported by the direct visualization of CTL using peptide-MHC tetramers to follow tumor-specific T cells. The data presented demonstrate that while cotransfer of Ag-specific CD4+ T cells was not required for the generation of CTLs, because adoptive transfer of CD8+ T cells alone was sufficient, CD4+ T cells were required for the maintenance of CD8+ T cell numbers. Our data suggest that there is a correlation among the number of CD8+ T cells, in vivo CTL function, and IFN-γ production, with no evidence of a partial or nonresponsive phenotype among tetramer-positive cells. We also show that CD4+ T cells are required for CD8+ T cell infiltration of the tumor.
Interaction ofMycobacterium avium-Containing Phagosomes with the Antigen Presentation Pathway
Authors: Ullrich Heinz-Joachim , Beatty Wandy L. , Russell David G.
Published: April 2014

Pathogenic mycobacteria infect macrophages where they replicate in phagosomes that minimize contact with late endosomal/lysosomal compartments. Loading of Ags to MHC class II molecules occurs in specialized compartments with late endosomal characteristics. This points to a sequestration of mycobacteria-containing phagosomes from the sites where Ags meet MHC class II molecules. Indeed, in resting macrophages MHC class II levels decreased strongly in phagosomes containing M. avium during a 4-day infection. Phagosomal MHC class II of early (4 h) infections was partly surface-derived and associated with peptide. Activation of host macrophages led to the appearance of H2-M, a chaperon of Ag loading, and to a strong increase in MHC class II molecules in phagosomes of acute (1 day) infections. Comparison with the kinetics of MHC class II acquisition by IgG-coated bead-containing phagosomes suggests that the arrest in phagosome maturation by mycobacteria limits the intersection of mycobacteria-containing phagosomes with the intracellular trafficking pathways of Ag-presenting molecules.