bluuebunny/crossref_metadata_2025 huggingface dataset:
Redundancy of Direct Priming and Cross-Priming in Tumor-Specific CD8+ T Cell Responses
Authors: Wolkers Monika C. , Stoetter Gerd , Vyth-Dreese Florry A. , Schumacher Ton N. M.
Published: April 2014

Against a subset of human cancers, vigorous tumor-specific CD8+ T cell responses can develop either spontaneously or upon allogeneic transplantation. However, the parameters that determine the induction of such pronounced anti-tumor immunity remain ill defined. To dissect the conditions required for the induction of high magnitude T cell responses, we have developed a murine model system in which tumor-specific T cell responses can be monitored directly ex vivo by MHC tetramer technology. In this model, tumor challenge of naive mice with Ag-bearing tumor cells results in a massive Ag-specific T cell response, followed by CD8+ T cell-dependent tumor rejection. We have subsequently used this model to assess the contribution of direct priming and cross-priming in the induction of tumor immunity in a well-defined system. Our results indicate that direct priming of T cells and Ag cross-priming are redundant mechanisms for the induction of tumor-specific T cell immunity. Moreover, T cell responses that arise as a consequence of Ag cross-presentation can occur in the absence of CD4+ T cell help and are remarkably robust.
L-Selectin Shedding Is Independent of Its Subsurface Structures and Topographic Distribution
Authors: Fors Brian P. , Goodarzi Katayoon , von Andrian Ulrich H.
Published: April 2014

L-selectin (CD62L), a lectin-like adhesion molecule, mediates lymphocyte homing and leukocyte accumulation at sites of inflammation. Its transmembrane (TM) and intracellular (IC) domains confer clustering of L-selectin on microvilli of resting leukocytes, which is important for L-selectin function. Following activation of protein kinase C (PKC) or calmodulin inhibition, the wild-type (WT) protein is rapidly cleaved in its membrane-proximal ectodomain. To examine whether L-selectin topography or TM/IC domains are involved in this shedding process, we used stable transfectants expressing WT L-selectin (on microvilli) or chimeric molecules consisting of the L-selectin ectodomain linked to the TM/IC domains of CD44 (excluded from microvilli) or CD31 (randomly distributed). PKC activation by PMA altered the cells’ surface morphology, but did not induce a redistribution of L-selectin ectodomains. All cell lines shed ectodomains upon PMA activation in a dose-dependent fashion and with similar kinetics. Calmodulin inhibition by trifluoperazine induced shedding in both WT and chimera transfectants. At high trifluoperazine concentrations, shedding of WT L-selectin was significantly more pronounced than that of chimeric molecules. Regardless of the activating stimulus, shedding was blocked by a hydroxamate-based metalloprotease inhibitor, suggesting that ectodomain down-regulation occurred through proteolytic cleavage by identical protease(s). These results show that the recognition site(s) for PKC-induced L-selectin shedding is exclusively contained within the ectodomain; the nature of subsurface structures and surface topography are irrelevant. Shedding induced by calmodulin inhibition has two components: one requires the L-selectin TM/IC domain, and the other is independent of it.
CD84 Functions as a Homophilic Adhesion Molecule and Enhances IFN-γ Secretion: Adhesion Is Mediated by Ig-Like Domain 1
Authors: Martin Margarita , Romero Xavier , de la Fuente Miguel Angel , Tovar Victoria , Zapater Núria , Esplugues Enric , Pizcueta Pilar , Bosch Jaime , Engel Pablo
Published: April 2014

CD84 is a member of the CD2 subset of the Ig superfamily of cell surface molecules. Its cytoplasmic tail binds to Src homology 2 domain-containing protein 1A (signaling lymphocytic activation molecule-associated protein), a protein encoded by the X-linked lymphoproliferative disease gene. It is preferentially expressed on B lymphocytes, monocytes, and platelets. We show that it is also expressed on thymocytes and T cells. CD84 was positive on CD4−CD8− thymocytes, and its expression decreased with cell maturation. It is expressed on mature T cells preferentially on CD45RO+. To identify the CD84 ligand, we generated a soluble Ig fusion protein containing the human CD84 extracellular domains (CD84-Ig). Because receptor-ligand interactions occur between several members of this subfamily, we assayed CD84-Ig binding with all members of the CD2 family. CD84-Ig bound to CD84-transfected cells, whereas no binding was detected with cells expressing other CD2 subfamily receptors, showing that CD84 binds to itself. Anti-CD84 mAbs recognizing epitopes wholly within domain 1 of CD84 blocked the binding of the CD84-Ig fusion protein to CD84-transfected cells and platelets. Data from CD84 domain human/mouse chimeras further revealed that only the first extracellular domain of the molecule is involved in the ligand receptor recognition. The CD84-CD84 interaction was independent of its cytoplasmic tail. Finally, concurrent ligation of human CD84 with mAbs or CD84-Ig and CD3 enhanced IFN-γ secretion in human lymphocytes. Thus, CD84 is its own ligand and acts as a costimulatory molecule.
Histamine Polarizes Human Dendritic Cells into Th2 Cell-Promoting Effector Dendritic Cells
Authors: Caron Gersende , Delneste Yves , Roelandts Edith , Duez Catherine , Bonnefoy Jean-Yves , Pestel Joel , Jeannin Pascale
Published: April 2014

Allergic disorders are characterized by allergen-specific Th2-biased responses. Signals controlling Th2 cell polarization, especially those acting by polarizing dendritic cells (DC) into Th2-promoting DC (DC2), are not well known. Histamine, a mediator released by allergen-stimulated mast cells from allergic subjects, has been reported to activate human immature DC. We have therefore tested whether histamine affects DC polarization. We report here that histamine inhibits LPS-induced IL-12 production and polarizes uncommitted maturing DC into effector DC2. DC matured in the presence of histamine fail to produce IL-12 upon subsequent stimulation and prime Th2 responses, even in presence of IFN-γ, a potent DC1-driving factor. All these effects are mediated through both H1 and H2 receptors. These data show that histamine is a potent DC2-polarizing factor and provide evidence for a novel mechanism that explains the initiation and maintenance of a predominant Th2 response in allergic disorders.
Infiltration of Canonical Vγ4/Vδ1 γδ T Cells in an Adriamycin-Induced Progressive Renal Failure Model
Authors: Ando Takashi , Wu Huiling , Watson Debbie , Hirano Tadashi , Hirakata Hideki , Fujishima Masatoshi , Knight John F.
Published: April 2014

We have previously reported an infiltration of renal interstitial γδ T cells in Adriamycin-induced progressive glomerulosclerosis in the rat kidney. The TCR repertoire and sequences used by these γδ T cells have now been studied. Two injections of Adriamycin 14 days apart caused segmental glomerulosclerosis, massive interstitial infiltration of mononuclear cells, and end-stage renal failure. Flow cytometry of lymphocyte subpopulations with Abs to CD3, the γδ TCR, and the αβ TCR showed that γδ T cells as a proportion of CD3+ cells were increased in Adriamycin-treated kidneys (8.5 ± 5.4%), but not in lymph nodes (1.3 ± 0.4%). A semiquantitative score of glomerular damage (r = 0.65; p < 0.01) and creatinine (r = 0.62; p < 0.01) correlated significantly with the presence of γδ T cells. TCR Vγ repertoire analysis by RT-PCR and Southern blotting showed that Vγ2 was the dominant subfamily in lymph nodes, whereas Vγ4 became the predominant subfamily in advanced stages of the rat Adriamycin-treated kidney. Sequencing of the Vγ4-Jγ junctional region showed an invariant sequence. The amino acid sequence of the junctional region of the Vγ4 TCR was the same as the reported mouse canonical Vγ4 TCR sequence. Analysis of the kidney Vδ repertoire showed dominant expression of Vδ1, and sequencing again revealed the selective expression of a canonical Vδ1 gene. Semiquantitative RT-PCR for cytokine gene expression showed that γδ T cells from the kidneys expressed TGF-β, but not IL-4, IL-10, or IFN-γ. These results suggest that the predominant γδ T cells in the Adriamycin kidney use an invariant Vγ4/Vδ1 receptor.
Active Vaccination Against IL-5 Bypasses Immunological Tolerance and Ameliorates Experimental Asthma
Authors: Hertz Marc , Mahalingam Surendran , Dalum Iben , Klysner Steen , Mattes Joerg , Neisig Anne , Mouritsen Søren , Foster Paul S. , Gautam Anand
Published: April 2014

Current therapeutic approaches to asthma have had limited impact on the clinical management and resolution of this disorder. By using a novel vaccine strategy targeting the inflammatory cytokine IL-5, we have ameliorated hallmark features of asthma in mouse models. Delivery of a DNA vaccine encoding murine IL-5 modified to contain a promiscuous foreign Th epitope bypasses B cell tolerance to IL-5 and induces neutralizing polyclonal anti-IL-5 Abs. Active vaccination against IL-5 reduces airways inflammation and prevents the development of eosinophilia, both hallmark features of asthma in animal models and humans. The reduced numbers of inflammatory T cells and eosinophils in the lung also result in a marked reduction of Th2 cytokine levels. Th-modified IL-5 DNA vaccination reduces the expression of IL-5 and IL-4 by ∼50% in the airways of allergen-challenged mice. Most importantly, Th-modified IL-5 DNA vaccination restores normal bronchial hyperresponsiveness to β-methacholine. Active vaccination against IL-5 reduces key pathological events associated with asthma, such as Th2 cytokine production, airways inflammation, and hyperresponsiveness, and thus represents a novel therapeutic approach for the treatment of asthma and other allergic conditions.
The Murine Cytomegalovirus Immune Evasion Protein m4/gp34 Forms Biochemically Distinct Complexes with Class I MHC at the Cell Surface and in a Pre-Golgi Compartment
Authors: Kavanagh Daniel G. , Koszinowski Ulrich H. , Hill Ann B.
Published: April 2014

We have recently demonstrated that the murine CMV (MCMV) gene m4 is an immune evasion gene that protects MCMV-infected targets from some virus-specific CTL clones. m4 encodes m4/gp34, a 34-kDa glycoprotein that binds to major histocompatibility complex class I in the endoplasmic reticulum and forms a detergent-stable complex that is exported to the surface of the cell. To investigate how m4/gp34 promotes CTL evasion, we analyzed the assembly and export of m4/gp34-Kb complexes. We found that 50–70% of Kb exported over the course of MCMV infection was m4/gp34 associated. Because these complexes are present at the cell surface, it is possible that m4 mediates CTL evasion by interfering with contact between class I and receptors on the T cell. In addition, we found that Kb retained by the MCMV immune evasion gene m152 formed a novel type of complex with Endo H-sensitive m4/gp34; these complexes are distinguished from the exported complexes by being stable in 1% digitonin and unstable in 1% Nonidet P-40. Because this association occurs in a pre-Golgi compartment, m4/gp34 might also interfere with Ag presentation by affecting some aspect of class I assembly, such as peptide loading. Although m4/gp34 requires β2-microglobulin to bind class I, there was no significant binding of m4/gp34 to β2-microglobulin in the absence of class I H chain, demonstrating that m4/gp34 forms Nonidet P-40-stable complexes specifically with folded conformations of class I. We conclude that m4/gp34 promotes immune evasion by a novel mechanism involving altered assembly and/or T cell recognition of class I molecules.
Apoptotic Deletion of Th Cells Specific for the 19-kDa Carboxyl-Terminal Fragment of Merozoite Surface Protein 1 During Malaria Infection
Authors: Wipasa Jiraprapa , Xu Huji , Stowers Anthony , Good Michael F.
Published: April 2014

Immunity induced by the 19-kDa fragment of merozoite surface protein 1 is dependent on CD4+ Th cells. However, we found that adoptively transferred CFSE-labeled Th cells specific for an epitope on Plasmodium yoelii 19-kDa fragment of merozoite surface protein 1 (peptide (p)24), but not OVA-specific T cells, were deleted as a result of P. yoelii infection. As a result of infection, spleen cells recovered from infected p24-specific T cell-transfused mice demonstrated reduced response to specific Ag. A higher percentage of CFSE-labeled p24-specific T cells stained positive with annexin and anti-active caspase-3 in infected compared with uninfected mice, suggesting that apoptosis contributed to deletion of p24-specific T cells during infection. Apoptosis correlated with increased percentages of p24-specific T cells that stained positive for Fas from infected mice, suggesting that P. yoelii-induced apoptosis is, at least in part, mediated by Fas. However, bystander cells of other specificities also showed increased Fas expression during infection, suggesting that Fas expression alone is not sufficient for apoptosis. These data have implications for the development of immunity in the face of endemic parasite exposure.
Local Costimulation Reinvigorates Tumor-Specific Cytolytic T Lymphocytes for Experimental Therapy in Mice with Large Tumor Burdens
Authors: Bai Xue-Feng , Bender Jonathan , Liu Jinqing , Zhang Huiming , Wang Yin , Li Ou , Du Peishuang , Zheng Pan , Liu Yang
Published: April 2014

Cytotoxic T cells recognize tumor Ags and destroy cancer cells in vitro. Adoptive transfer studies with transgenic T cells specific for tumor Ags have demonstrated that CTL are effective only in mice with small tumor burdens and thus appear to have limited potential in cancer immunotherapy. Here we used transgenic mice that express the TCR specific for an unmutated tumor Ag P1A and multiple lineages of P1A-expressing tumors to address this critical issue. We found that local costimulation, either by expression of B7-1 on the tumor cells or by local administration of anti-CD28 mAb 37N, reinvigorated the function of CTL specific for the tumor Ag, as it substantially increased the efficacy of CTL therapy for mice with large tumor burdens. Our study suggests that CTL-based immunotherapy can be manipulated to deal with large tumors.
Cutting Edge: IL-17F, a Novel Cytokine Selectively Expressed in Activated T Cells and Monocytes, Regulates Angiogenesis and Endothelial Cell Cytokine Production
Authors: Starnes Trevor , Robertson Michael J. , Sledge George , Kelich Stephanie , Nakshatri Harikrishna , Broxmeyer Hal E. , Hromas Robert
Published: April 2014

A novel secreted cytokine, termed IL-17F, was cloned using nested RACE PCR. This cytokine bears homology to IL-17. IL-17F was expressed only in activated CD4+ T cells and activated monocytes. Recombinant human IL-17F did not stimulate the proliferation of hematopoietic progenitors or the migration of mature leukocytes. However, it markedly inhibited the angiogenesis of human endothelial cells and induced endothelial cells to produce IL-2, TGF-β, and monocyte chemoattractant protein-1.
Cutting Edge: The Absence of C3 Demonstrates a Role for Complement in Th2 Effector Functions in a Murine Model of Pulmonary Allergy
Authors: Drouin Scott M. , Corry David B. , Kildsgaard Jens , Wetsel Rick A.
Published: April 2014

Asthma is a chronic disease of the lung resulting from airway obstruction. Although the initiating causes are not entirely clear, the airway inflammation in asthma is associated with Th2 lymphocytes and their cytokines, particularly IL-4, which play a prominent role in this disease by regulating airway hyperresponsiveness, eosinophil activation, and IgE synthesis. Historically, complement was not thought to contribute to the pathogenesis of asthma. However, using C3-deficient mice in an allergen-induced model of pulmonary allergy, we demonstrate that complement may impact key features of this disease. When challenged with allergen, mice deficient in C3 exhibit diminished airway hyperresponsiveness and lung eosinophilia. Furthermore, these mice also have dramatically reduced numbers of IL-4-producing cells and attenuated Ag-specific IgE and IgG1 responses. Collectively, these results demonstrate that C3-deficient mice have significantly altered allergic lung responses and indicate a role for the complement system in promoting Th2 effector functions in asthma.
Induction of IL-10 and Inhibition of Experimental Arthritis Are Specific Features of Microbial Heat Shock Proteins That Are Absent for Other Evolutionarily Conserved Immunodominant Proteins
Authors: Prakken Berent J. , Wendling Uwe , van der Zee Ruurd , Rutten Victor P. M. G. , Kuis Wietse , van Eden Willem
Published: April 2014

Bacterial heat shock proteins (hsp) are evolutionary conserved immunodominant proteins that manifest amino acid homologies with hsp present in mammalian cells. Preimmunization with mycobacterial hsp65 has been found to protect against various forms of experimental arthritis. As these protective effects have previously been attributed to induction of self homologue cross-reactive T cell responses, the question was raised as to whether this protective effect could be extended to other highly conserved and immunodominant microbial Ags with mammalian homologues. Therefore, we immunized Lewis rats with conserved bacterial Ags (superoxide dismutase, aldolase, GAPDH, and hsp70). Although all Ags appeared highly immunogenic, we only found a protective effect in experimental arthritis after immunization with bacterial hsp70. The protective effect of hsp70 was accompanied with a switch in the subclasses of hsp70-specific Abs, suggesting the induction of Th2-like response. The most striking difference between immunization with hsp70 and all other immunodominant Ags was the expression of IL-10 found after immunization with hsp70. Even more, while immunization with hsp70 led to Ag-induced production of IL-10 and IL-4, immunization with aldolase led to increased production of IFN-γ and TNF-α. Thus, the protective effect of conserved immunodominant proteins in experimental arthritis seems to be a specific feature of hsp. Therefore, hsp may offer unique possibilities for immunological intervention in inflammatory diseases.
Persistence of Dominant T Cell Clones in Accepted Solid Organ Transplants
Authors: Baron Christophe , McMorrow Isabel , Sachs David H , LeGuern Christian
Published: April 2014

Donor/recipient MHC class II matching is beneficial to the survival of allogeneic kidneys in humans and swine. In the latter, tolerance to class I-disparate grafts can be induced by a short course of immunosuppression, a peripheral mechanism that implicates regulatory T cells. Absence of treatment will lead to prompt rejection. Rejected grafts are infiltrated by dominant alloaggressive T cells, whereas there is still speculation on the specificity and function of T cells invading accepted tissues. To characterize the TCR repertoire of graft-infiltrating T cells (GITC) in accepted kidneys, we have used the RT-PCR-based spectratyping technique to assess the length polymorphism of the porcine TCRβ chain complementary-determining region 3 (CDR3). Results show that T cells infiltrating accepted kidneys (n = 5) express a restricted polymorphism of the CDR3 length, whereas PBL from the same animal have the polymorphic distribution of CDR3 lengths found in naive animals; that the skewed Vβ repertoire in accepted grafts involved distinct Vβ subfamilies in otherwise MHC-identical recipient animals; that GITC clonal dominance is not caused by immunosuppression because a second kidney, accepted without drug treatment, exhibits the same TCR Vβ CDR3 profiles than those detected in the first graft; and that intragraft clonal dominance intensifies with time, indicating progressive preeminence of nonaggressive GITC clones. Collectively, these data represent the first example, in a preclinical model, of the emergence of nonaggressive intragraft clones, which may be involved in the induction/maintenance of local tolerance to allogeneic tissues.
Enhancement of Mast Cell Survival: A Novel Function of Some Secretory Phospholipase A2 Isotypes
Authors: Fonteh Alfred N. , Marion Chad R. , Barham Brooke J. , Edens Michelle B. , Atsumi Gen-ichi , Samet James M. , High Kevin P. , Chilton Floyd H.
Published: April 2014

This study tested the hypothesis that certain secretory phospholipase A2 (sPLA2) isotypes act in a cytokine-like fashion through cell surface receptors to influence mast cell survival. Initial experiments revealed that sPLA2 activity and sPLA2 receptor expression are increased, and mast cells lost their capacity to maintain membrane asymmetry upon cytokine depletion. Groups IB and III, but not group IIA PLA2, prevented the loss of membrane asymmetry. Similarly, group IB prevented nucleosomal DNA fragmentation in mast cells. Providing putative products of sPLA2 hydrolysis to cytokine-depleted mast cells did not influence survival. Furthermore, catalytic inactivation of sPLA2 did not alter its capacity to prevent apoptosis. Inhibition of protein synthesis using cycloheximide or actinomycin reversed the antiapoptotic effect of sPLA2. Additionally, both wild-type and catalytically inactive group IB PLA2 induced IL-3 synthesis in mast cells. However, adding IL-3-neutralizing Ab did not change Annexin VFITC binding and only partially inhibited thymidine incorporation in sPLA2-supplemented mast cells. In contrast, IL-3-neutralizing Ab inhibited both Annexin VFITC binding and thymidine incorporation in mast cells maintained with IL-3. sPLA2 enhanced phosphoinositide 3′-kinase activity, and a specific inhibitor of phosphoinositide 3′-kinase reversed the antiapoptotic effects of sPLA2. Likewise, sPLA2 increased the degradation of I-κBα, and specific inhibitors of nuclear factor κ activation (NF-κB) reversed the antiapoptotic effects of sPLA2. Together, these experiments reveal that certain isotypes of sPLA2 enhance the survival of mast cells in a cytokine-like fashion by activating antiapoptotic signaling pathways independent of IL-3 and probably via sPLA2 receptors rather than sPLA2 catalytic products.
Unique Signaling Properties of B Cell Antigen Receptor in Mature and Immature B Cells: Implications for Tolerance and Activation
Authors: Benschop Robert J. , Brandl Erin , Chan Andrew C. , Cambier John C.
Published: April 2014

Immature B cells display increased sensitivity to tolerance induction compared with their mature counterparts. The molecular mechanisms underlying these differences are poorly defined. In this study, we demonstrate unique maturation stage-dependent differences in B cell Ag receptor (BCR) signaling, including BCR-mediated calcium mobilization responses. Immature B cells display greater increases in intracellular calcium concentrations following Ag stimulation. This has consequences for the induction of biologically relevant responses: immature B cells require lower Ag concentrations for activation than mature B cells, as measured by induction of receptor editing and CD86 expression, respectively. BCR-induced tyrosine phosphorylation of CD79a, Lyn, B cell linker protein, and phospholipase Cγ2 is enhanced in immature B cells and they exhibit greater capacitative calcium entry in response to Ag. Moreover, B cell linker protein, Bruton’s tyrosine kinase, and phospholipase Cγ2, which are crucial for the induction of calcium mobilization responses, are present at ∼3-fold higher levels in immature B cells, potentially contributing to increased mobilization of calcium. Consistent with this possibility, we found that the previously reported lack of inositol-1,4,5-triphosphate production in immature B cells may be explained by enhanced inositol-1,4,5-triphosphate breakdown. These data demonstrate that multiple mechanisms guarantee increased Ag-induced mobilization of calcium in immature B cells and presumably ensure elimination of autoreactive B cells from the repertoire.
Regulation of NKT Cells by Ly49: Analysis of Primary NKT Cells and Generation of NKT Cell Line
Authors: Maeda Motoi , Lohwasser Stefan , Yamamura Takashi , Takei Fumio
Published: April 2014

TCRαβ+NK1.1+ (NKT) cells are known to express various NK cell-associated molecules including the Ly49 family of receptors for MHC class I, but its functional significance has been unclear. Here, we examined the expression of Ly49A, C/I and G2 on various NKT cell populations from normal and MHC class I-deficient C57BL/6 mice as well as their responsiveness to α-galactosylceramide (α-GalCer), a potent stimulator of CD1d-restricted NKT cells. The frequency and the level of Ly49 expression varied among NKT cells from different tissues, and were regulated by the expression of MHC class I and CD1d in the host. Stimulation of various NKT cells with α-GalCer suggested that Ly49 expression inversely correlates with the responsiveness of NKT cells to α-GalCer. Moreover, α-GalCer presented by normal dendritic cells stimulated purified Ly49−, but not Ly49+, splenic NKT cells, whereas MHC class I-deficient dendritic cells presented α-GalCer to both Ly49+ and Ly49− NKT cells equally well. Therefore, MHC class I on APCs seems to inhibit activation of NKT cells expressing Ly49. To further characterize CD1d-restricted NKT cells, we generated an α-GalCer-responsive NKT cell line from thymocytes. The line could only be generated from Ly49−NK1.1+CD4+ thymocytes but not from other NKT cell subsets, and it lost expression of NK1.1 and CD4 during culture. Together, these results indicate the functional significance of Ly49 expression on NKT cells.
Neonatal Exposure to Antigen Primes the Immune System to Develop Responses in Various Lymphoid Organs and Promotes Bystander Regulation of Diverse T Cell Specificities
Authors: Pack Christopher D. , Cestra Aimee E. , Min Booki , Legge Kevin L. , Li Lequn , Caprio-Young Jacque C. , Bell J. Jeremiah , Gregg Randal K. , Zaghouani Habib
Published: April 2014

Neonatal exposure to Ag has always been considered suppressive for immunity. Recent investigations, however, indicated that the neonatal immune system could be guided to develop immunity. For instance, delivery of a proteolipid protein (PLP) peptide on Ig boosts the neonatal immune system to develop responses upon challenge with the PLP peptide later. Accordingly, mice given Ig-PLP at birth and challenged with the PLP peptide as adults developed proliferative T cells in the lymph node that produced IL-4 instead of the usual Th1 cytokines. However, the spleen was unresponsive unless IL-12 was provided. Herein, we wished to determine whether such a neonatal response is intrinsic to the PLP peptide or could develop with an unrelated myelin peptide as well as whether the T cell deviation is able to confer resistance to autoimmunity involving diverse T cell specificities. Accordingly, the amino acid sequence 87–99 of myelin basic protein was expressed on the same Ig backbone, and the resulting Ig-myelin basic protein chimera was tested for induction of neonatal immunity and protection against experimental allergic encephalomyelitis. Surprisingly, the results indicated that immunity developed in the lymph node and spleen, with deviation of T cells occurring in both organs. More striking, the splenic T cells produced IL-10 in addition to IL-4, providing an environment that facilitated bystander deviation of responses to unrelated epitopes and promoted protection against experimental allergic encephalomyelitis involving diverse T cell specificities. Thus, neonatal exposure to Ag can prime responses in various organs and sustain regulatory functions effective against diverse autoreactive T cells.
Purification of Ag-Specific T Lymphocytes After Direct Peripheral Blood Mononuclear Cell Stimulation Followed by CD25 Selection. I. Application to CD4+ or CD8+ Cytomegalovirus Phosphoprotein pp65 Epitope Determination
Authors: Gallot Géraldine , Vivien Régine , Ibisch Catherine , Lulé Jaqueline , Davrinche Christian , Gaschet Joëlle , Vié Henri
Published: April 2014

The two main constraints that currently limit a broader usage of T cell therapy against viruses are the delay required to obtain specific T cells and the safety of the selection procedure. In the present work we developed a generally applicable strategy that eliminates the need for APC for timing reasons, and the need for infectious viral strains for safety concerns. As a model, we used the selection of T lymphocytes specific for the immunodominant CMV phosphoprotein pp65. PBMC from healthy seropositive donors were first depleted of IL-2R α-chain CD25+ cells and were then stimulated for 24–96 h with previously defined peptide Ags or with autologous PBMC infected with a canarypox viral vector encoding the total pp65 protein (ALVAC-pp65). Subsequent immunomagnetic purification of newly CD25-expressing cells allowed efficient recovery of T lymphocytes specific for the initial stimuli, i.e., for the already known immunodominant epitope corresponding to the peptides used as a model or for newly defined epitopes corresponding to peptides encoded by the transfected pp65 protein. Importantly, we demonstrated that direct PBMC stimulation allowed recovery not only of CD8+ memory T lymphocytes, but also of the CD4+ memory T cells, which are known to be crucial to ensure persistence of adoptively transferred immune memory. Finally, our analysis of pp65-specific T cells led to the identification of several new helper and cytotoxic epitopes. This work thus demonstrates the feasibility of isolating memory T lymphocytes specific for a clinically relevant protein without the need to prepare APC, to use infectious viral strains, or to identify immunodominant epitopes.
Peptide Length Variants p2Ca and QL9 Present Distinct Conformations to Ld-Specific T Cells
Authors: Hornell Tara M. C. , Martin Shiloh M. , Myers Nancy B. , Connolly Janet M.
Published: April 2014

Recent advances have provided insights into how the TCR interacts with MHC/peptide complexes and a rationale to predict optimal epitopes for MHC binding and T cell recognition. For example, peptides of nine residues are predicted to be optimal for binding to H2-Ld, although 8 mer epitopes have also been identified. It has been predicted that 8 mer and 9 mer length variant peptides bound to Ld present identical epitopes to T cells. However, in contrast to this prediction, we demonstrate here that the 8 mer peptide p2Ca and its 9 mer length variant QL9, extended by an N-terminal glutamine, assume distinct conformations when bound to Ld. We generated self-Ld-restricted CTL clones specific for p2Ca that recognize Ld/QL9 poorly if at all. This result is in sharp contrast to what has been observed with Ld-alloreactive T cells that possess a much higher affinity for Ld/QL9 than for Ld/p2Ca. Alanine substitutions of the N-terminal residues of the QL9 peptide rescue detection by these self-Ld/p2Ca-specific T cells, but decrease recognition by the Ld-alloreactive 2C T cell clone. In addition, 2C T cell recognition of the p2Ca peptide is affected by different alanine substitutions compared with 2C T cell recognition of the QL9 peptide. These data clearly demonstrate that the p2Ca and QL9 peptides assume distinct conformations when bound to Ld and, furthermore, demonstrate that there is flexibility in peptide binding within the MHC class I cleft.
Induction of CTL and Nonpolarized Th Cell Responses by CD8α+ and CD8α− Dendritic Cells
Authors: Schlecht Géraldine , Leclerc Claude , Dadaglio Gilles
Published: April 2014

Two distinct dendritic cell (DC) subpopulations have been evidenced in mice on the basis of their differential CD8α expression and their localization in lymphoid organs. Several reports suggest that CD8α+ and CD8α− DC subsets could be functionally different. In this study, using a panel of MHC class I- and/or class II-restricted peptides, we analyzed CD4+ and CD8+ T cell responses obtained after i.v. injection of freshly purified peptide-pulsed DC subsets. First, we showed that both DC subsets efficiently induce specific CTL responses and Th1 cytokine production in the absence of CD4+ T cell priming. Second, we showed that in vivo activation of CD4+ T cells by CD8α+ or CD8α− DC, injected i.v., leads to a nonpolarized Th response with production of both Th1 and Th2 cytokines. The CD8α− subset induced a higher production of Th2 cytokines such as IL-4 and IL-10 than the CD8α+ subset. However, IL-5 was produced by CD4+ T cells activated by both DC subsets. When both CD4+ and CD8+ T cells were primed by DC injected i.v., a similar pattern of cytokines was observed, but, under these conditions, Th1 cytokines were mainly produced by CD8+ T cells, while Th2 cytokines were produced by CD4+ T cells. Thus, this study clearly shows that CD4+ T cell responses do not influence the development of specific CD8+ T cell cytotoxic responses induced either by CD8α+ or CD8α− DC subsets.
Immunotoxins Containing Recombinant Anti-CTLA-4 Single-Chain Fragment Variable Antibodies and Saporin: In Vitro Results and In Vivo Effects in an Acute Rejection Model
Authors: Tazzari Pier-Luigi , Polito Letizia , Bolognesi Andrea , Pistillo Maria-Pia , Capanni Paolo , Palmisano Giulio Lelio , Lemoli Roberto M. , Curti Antonio , Biancone Luigi , Camussi Giovanni , Conte Roberto , Ferrara Giovanni B. , Stirpe Fiorenzo
Published: April 2014

Immunotoxins containing recombinant human-derived single-chain fragment variable (scFv) reagents (83 and 40) against CTLA-4 (CD152) linked to saporin, a ribosome-inactivating protein, were prepared and tested on CD3/CD28-activated T lymphocytes, MLRs, CTLA-4-positive cell lines, and hemopoietic precursors. Immunotoxins induced apoptosis in activated T lymphocytes and were able to specifically inhibit MLR between T lymphocytes and dendritic cells. The 83-saporin immunotoxin also inhibited the T cell activation in an MLR between T lymphocytes and an EBV-positive lymphoblastoid B cell line. Toxicity tests on hemopoietic precursors showed little or no effects in inhibiting colonies’ growth. As the 83 scFv Ab was reactive also with activated mouse T lymphocytes, 83-saporin was tested in a model of tumor rejection consisting of C57BL/6 mice bearing a murine H.end endothelioma cell line, derived from DBA/2 mice. The lymphoid infiltration due to the presence of the tumor was reduced to a high extent, demonstrating that the immunotoxin was actually available and active in vivo. Thus, taking the results altogether, this study might represent a new breakthrough for immunotherapy, showing the possibility of targeting CTLA-4 to kill activated T cells, using conjugates containing scFv Abs and type 1 ribosome-inactivating protein.
Inhibition of the Death Receptor Pathway by cFLIP Confers Partial Engraftment of MHC Class I-Deficient Stem Cells and Reduces Tumor Clearance in Perforin-Deficient Mice
Authors: Taylor Mesha Austin , Chaudhary Preet M. , Klem Jennifer , Kumar Vinay , Schatzle John D. , Bennett Michael
Published: April 2014

NK cells mediate acute rejection of MHC class I-deficient bone marrow cell (BMC) grafts. However, the exact cytotoxic mechanisms of NK cells during acute BMC graft rejection are not well defined. Although the granule exocytosis pathway plays a major role in NK cell-mediated rejection, alternative perforin-independent mechanisms also exist. By analyzing the anti-apoptotic effects of cellular Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein (cFLIP) overexpression, we investigated the possible role of death receptor-induced apoptosis in NK cell-mediated cytotoxicity. In the absence of perforin, we found that cFLIP overexpression reduces lysis of tumor cells by NK cells in vitro and in vivo. In addition, perforin-deficient NK cells were impaired in their ability to acutely reject cFLIP-overexpressing TAP-1 knockout stem cells. These results emphasize the importance of NK cell death receptor-mediated killing during BMC grafts in the absence of perforin.
HIV Envelope Protein Inhibits MHC Class I Presentation of a Cytomegalovirus Protective Epitope
Authors: López Daniel , Samino Yolanda , Koszinowski Ulrich H. , Del Val Margarita
Published: April 2014

CTL recognize peptides that derive from viral protein Ags by proteolytic processing and are presented by MHC class I molecules. In this study we tested whether coexpression of viral Ags in the same cell leads to competition between them. To this end, two Ld-restricted epitopes derived from HIV-1 envelope gp160 (ENV) and from CMV pp89 phosphoprotein were coexpressed. HIV ENV strain IIIB, but not MN variant, impaired recognition by specific CTL of CMV pp89 epitope 9pp89. Susceptibility to inhibition after ENV coexpression was inversely related to the amount of antigenic 9pp89 peptide processed from different antigenic constructs. In line with it, competition decreased the yield of naturally processed antigenic 9pp89 peptide bound to MHC class I molecules in coinfected cells. Also, point mutants of the presenting MHC class I molecule differed in their competition pattern. Collectively, the data imply that competition operates at the step of MHC-peptide complex assembly or stabilization. We conclude that, although not the rule, in certain combinations there is interference between different Ags expressed in the same cell and presented by the same MHC class I allele. These studies have implications for vaccine development and for understanding immunodominance.
Activation of CD25+CD4+ Regulatory T Cells by Oral Antigen Administration
Authors: Zhang Xingmin , Izikson Leonid , Liu Liming , Weiner Howard L.
Published: April 2014

CD25+CD4+ T cells are naturally occurring regulatory T cells that are anergic and have suppressive properties. Although they can be isolated from the spleens of normal mice, there are limited studies on how they can be activated or expanded in vivo. We found that oral administration of OVA to OVA TCR transgenic mice resulted in a modification of the ratio of CD25+CD4+ to CD25−CD4+ cells with an increase of CD25+CD4+ T cells accompanied by a decrease of CD25−CD4+ T cells. The relative increase in CD25+CD4+ T cells persisted for as long as 4 wk post feeding. We also found that CTLA-4 was dominantly expressed in CD25+CD4+ T cells and there was an increase in the percentage of CD25+CD4+ T cells expressing CTLA-4 in OVA-fed mice. In contrast to CD25−CD4+ cells, CD25+CD4+ cells from fed mice proliferated only minimally to OVA or anti-CD3 and secreted IL-10 and elevated levels of TGF-β1 following anti-CD3 stimulation. CD25+CD4+ cells from fed mice suppressed the proliferation of CD25−CD4+ T cells in vitro more potently than CD25+CD4+ T cells isolated from unfed mice, and this suppression was partially reversible by IL-10 soluble receptor or TGF-β soluble receptor and high concentration of anti-CTLA-4. With anti-CD3 stimulation, CD25+CD4+ cells from unfed mice secreted IFN-γ, whereas CD25+CD4+ cells from fed mice did not. Adoptive transfer of CD25+CD4+ T cells from fed mice suppressed in vivo delayed-type hypersensitivity responses in BALB/c mice. These results demonstrate an Ag-specific in vivo method to activate CD25+CD4+ regulatory T cells and suggest that they may be involved in oral tolerance.
Visualization and Characterization of Respiratory Syncytial Virus F-Specific CD8+ T Cells During Experimental Virus Infection
Authors: Chang Jun , Srikiatkhachorn Anon , Braciale Thomas J.
Published: April 2014

CTL play a major role in the clearance of respiratory syncytial virus (RSV) during experimental pulmonary infection. The fusion (F) glycoprotein of RSV is a protective Ag that elicits CTL and Ab response against RSV infection in BALB/c mice. We used the strategy of screening a panel of overlapping synthetic peptides corresponding to the RSV F protein and identified an immunodominant H-2Kd-restricted epitope (F85–93; KYKNAVTEL) recognized by CD8+ T cells from BALB/c mice. We enumerated the F-specific CD8+ T cell response in the lungs of infected mice by flow cytometry using tetramer staining and intracellular cytokine synthesis. During primary infection, F85–93-specific effector CD8+ T cells constitute ∼4.8% of pulmonary CD8+ T cells at the peak of the primary response (day 8), whereas matrix 2-specific CD8+ T cells constituted ∼50% of the responding CD8+ T cell population in the lungs. When RSV F-immune mice undergo a challenge RSV infection, the F-specific CD8+ T cell response is accelerated and dominates, whereas the primary response to the matrix 2 epitope in the lungs is reduced by ∼20-fold. In addition, we found that activated F-specific effector CD8+ T cells isolated from the lungs of RSV-infected mice exhibited a lower than expected frequency of IFN-γ-producing CD8+ T cells and were significantly impaired in ex vivo cytolytic activity compared with competent F-specific effector CD8+ T cells generated in vitro. The significance of these results for the regulation of the CD8+ T cell response to RSV is discussed.
Resting and Activation-Dependent Ion Channels in Human Mast Cells
Authors: Duffy S. Mark , Lawley Wendy J. , Conley Edward C. , Bradding Peter
Published: April 2014

The mechanism of mediator secretion from mast cells in disease is likely to include modulation of ion channel activity. Several distinct Ca2+, K+, and Cl− conductances have been identified in rodent mast cells, but there are no data on human mast cells. We have used the whole-cell variant of the patch clamp technique to characterize for the first time macroscopic ion currents in purified human lung mast cells and human peripheral blood-derived mast cells at rest and following IgE-dependent activation. The majority of both mast cell types were electrically silent at rest with a resting membrane potential of around 0 mV. Following IgE-dependent activation, >90% of human peripheral blood-derived mast cells responded within 2 min with the development of a Ca2+-activated K+ current exhibiting weak inward rectification, which polarized the cells to around −40 mV and a smaller outwardly rectifying Ca2+-independent Cl− conductance. Human lung mast cells showed more heterogeneity in their response to anti-IgE, with Ca2+-activated K+ currents and Ca2+-independent Cl− currents developing in ∼50% of cells. In both cell types, the K+ current was blocked reversibly by charybdotoxin, which along with its electrophysiological properties suggests it is carried by a channel similar to the intermediate conductance Ca2+-activated K+ channel. Charybdotoxin did not consistently attenuate histamine or leukotriene C4 release, indicating that the Ca2+-activated K+ current may enhance, but is not essential for, the release of these mediators.
CD4+CD25+ T Cells Facilitate the Induction of T Cell Anergy
Authors: Ermann Joerg , Szanya Veronika , Ford Gregory S. , Paragas Violette , Fathman C. Garrison , Lejon Kristina
Published: April 2014

T cell anergy is characterized by the inability of the T cell to produce IL-2 and proliferate. It is reversible by the addition of exogenous IL-2. A similar state of unresponsiveness is observed when the proliferative response of murine CD4+CD25− T cells is suppressed in vitro by coactivated CD4+CD25+ T cells. We have developed a suppression system that uses beads coated with anti-CD3 and anti-CD28 Abs as surrogate APCs to study the interaction of CD4+CD25+ and CD4+CD25− T cells in vitro. CD4+CD25+ T cell-induced suppression, in this model, was not abrogated by blocking the B7-CTLA-4 pathway. When the CD4+CD25− T cells were separated from the CD4+CD25+ suppressor cells after 24 h of coactivation by the Ab-coated beads, the CD4+CD25− T cells were unable to proliferate or to produce IL-2 upon restimulation. The induction of this anergic phenotype in the CD4+CD25− T cells correlated with the up-regulated expression of the gene related to anergy in lymphocytes (GRAIL), a novel anergy-related gene that acts as a negative regulator of IL-2 transcription. This system constitutes a novel mechanism of anergy induction in the presence of costimulation.
Hapten Addition to an MHC Class I-Binding Peptide Causes Substantial Adjustments of the TCR Structure of the Responding CD8+ T Cells
Authors: Honda Shinichiro , Zhang Weijia , Kalergis Alexis M. , DiLorenzo Teresa P. , Wang Fuming , Nathenson Stanley G.
Published: April 2014

T cell responses against hapten-modified peptides play an important role in the pathogenesis of certain diseases, including contact dermatitis and allergy. However, the structural features of TCRs recognizing bulky, potentially mobile hapten groups remain poorly defined. To analyze the structural basis of TCR recognition of defined hapten-modified peptides, the immunodominant octapeptide derived from vesicular stomatitis virus nucleoprotein (VSV8) was modified with a trinitrophenyl (TNP) group at the primary TCR contact residues (position 4 or 6) and used for immunization of mice carrying either the TCR α- or β-chain of a VSV8 (unmodified)/H-2Kb-specific CTL clone as a transgene. Such mice allow independent analysis of one TCR chain by maintaining the other fixed. The TCR V gene usage of the responding T cell population was specifically altered depending upon the presence of the TNP group and its position on the peptide. The CDR3 sequences of the TNP-modified peptide-specific TCRs showed a preferential J region usage in both the CDR3α and β loops, indicating that the J regions of both CDR3s are critical for recognition of TNP-modified peptides. In contrast to our previous observations showing the prime importance of CDR3β residues encoded by D-segment or N-addition nucleotides for recognition of position 6 of unmodified VSV8, our studies of TNP-modified peptides demonstrate the importance of the Jβ region, while the Jα region was crucial for recognizing both TNP-modified and unmodified peptides. These data suggest that different structural strategies are utilized by the CDR3α and β loops to allow interaction with a haptenated peptide.
Regression of Extensive Pulmonary Metastases in Mice by Adoptive Transfer of Antigen-Specific CD8+ CTL Reactive Against Tumor Cells Expressing a Naturally Occurring Rejection Epitope
Authors: Ryan Mary Hilburger , Bristol J. Andrew , McDuffie Elwood , Abrams Scott I.
Published: April 2014

In this study, we developed a mouse model of adoptive immunotherapy reflecting immune recognition of syngeneic tumor cells naturally expressing an endogenous rejection Ag. Specifically, in a pulmonary metastases model, we examined the potency and maintenance of an antitumor CD8+ CTL response in vivo, as well as its effectiveness against an “extensive” tumor burden. The approach taken was to first generate tumor-specific CTL from mice challenged with the CMS4 sarcoma coadministered with anti-CTLA4 mAb, which has been shown to facilitate the induction of Ag-specific T cell responses in vivo. An H-2Ld-restricted nonamer peptide, derived from an endogenous murine leukemia provirus was identified as a CMS4-reactive CTL epitope based upon the following: CTL cross-recognition of another syngeneic tumor cell line (CT26 colon carcinoma) previously characterized to express that gene product; sensitization of Ag-negative lymphoblasts or P815 targets with the peptide; and by cold target inhibition assays. In vivo, the adoptive transfer of CMS4-reactive CTL (≥1 × 106) resulted in nearly the complete regression of 3-day established lung metastases. Furthermore, mice that rejected CMS4 following a single adoptive transfer of CTL displayed antitumor activity to a rechallenge 45 days later, not only in the lung, but also at a s.c. distal site. Lastly, the adoptive transfer of CTL to mice harboring extensive pulmonary metastases (>150 nodules) led to a substantial reduction in tumor burden. Overall, these data suggest that the adoptive transfer of tumor-specific CTL may have therapeutic potential for malignancies that proliferate in or metastasize to the lung.