bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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

Abstract
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

AbstractThe 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

Abstract
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

AbstractTwo 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

Abstract
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

Abstract
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

AbstractCTL 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

AbstractCD25+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

Abstract
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

Abstract
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, &gt;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

Abstract
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

Abstract
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

Abstract
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 (&gt;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.