bluuebunny/crossref_metadata_2025 huggingface dataset:
Visualization of Early APC/T Cell Interactions in the Mouse Lung Following Intranasal Challenge
Authors: Byersdorfer Craig A. , Chaplin David D.
Published: April 2014

We have used fluorescent latex beads, with or without covalently conjugated OVA, to facilitate study of Ag trafficking in the mouse lung and draining peribronchial lymph node (LN). At 6 h, and up to 48 h after intranasal administration, beads were observed as intracellular clusters in the tissue parenchyma. Flow cytometry of bead-positive (bead+) cells from the bronchoalveolar lavage demonstrated that a majority of these cells are CD11c+, F4/80+, and CD11b−. Furthermore, fluorescent microscopy confirmed that a major subset of bead+ cells in the lung tissue was also CD11c+. In the draining peribronchial LNs, small numbers of beads were present in the subcapsular sinus as early as 6 h after inhalation. By 12 h and beyond, bead+ cells had localized exclusively to the LN T zone. OVA-conjugated latex beads, in addition to stimulating brisk proliferation of naive, OVA-specific DO11.10 transgenic T cells in vitro, could also recruit OVA-specific T cells in vivo. In some cases, bead+ APCs and CD4+ Th1 cells were found adjacently localized in the lung tissue 6 h after airway challenge. Thus, interactions of bead+ APCs with Ag-specific CD4+ T cells occurred earlier in the peripheral airways than these same interactions occurred in the draining peribronchial LN. Lastly, after adoptive transfer, in vitro differentiated Th1 cells accumulated at peripheral sites in the lung tissue and airways before Ag challenge and therefore were ideally positioned to influence subsequent immune reactions of the airway.
Pre-Existing Tumor-Sensitized T Cells Are Essential for Eradication of Established Tumors by IL-12 and Cyclophosphamide Plus IL-12
Authors: Le Hop N. , Lee Natalie C. , Tsung Kangla , Norton Jeffrey A.
Published: April 2014

The antitumor immune response activated by IL-12, especially by a combination of cyclophosphamide and IL-12 (Cy+IL-12), is clinically significant in certain experimental tumor models, in that a number of well-established (10–20 mm in diameter) s.c. tumors are completely eradicated. Furthermore, Cy+IL-12 treatment is also able to eradicate well-established grossly detectable experimental lung metastases and advanced ascites tumors. Despite the dramatic antitumor effects seen in some tumor models, Cy+IL-12 fails to induce regression of other established tumors. Characterization of tumor immunogenicity shows that all tumors responding to IL-12 and Cy+IL-12 treatments are immunogenic tumors, in that an antitumor immune response is detectable in tumor-bearing hosts upon tumor establishment. In contrast, none of the nonimmunogenic tumor responds to IL-12 and Cy+IL-12 treatments. Analysis of cellular requirements for successful tumor rejection through an adoptive cell transfer approach reveals that the presence of tumor-sensitized, but not naive, T cells is essential for tumor rejection by IL-12 and Cy+IL-12. Transfer of these tumor-sensitized T cells must be conducted before, but not after, IL-12 treatment in order for tumor rejection to occur. The requirement of sensitized T cells is also tumor specific. In mice bearing immunogenic tumors, the presence of pre-existing tumor-sensitized T cells is demonstrated by adoptive cell transfer experiments using purified spleen T cells from these mice. Results from our study show that Cy+IL-12-based immunotherapy of cancer may be highly effective and that pre-existing tumor-sensitized T cells are essential for the success of the therapy.
Essential Contribution of Germline-Encoded Lysine Residues in Jγ1.2 Segment to the Recognition of Nonpeptide Antigens by Human γδ T Cells
Authors: Miyagawa Fumi , Tanaka Yoshimasa , Yamashita Seiji , Mikami Bunzo , Danno Kiichiro , Uehara Masami , Minato Nagahiro
Published: April 2014

Human γδ T cells display unique repertoires of Ag specificities largely imposed by selective usages of distinct Vγ and Vδ genes. Among them, Vγ2/Vδ2+ T cells predominate in the circulation of healthy adults and respond to various microbial small molecular mass nonpeptide Ags. The present results indicate that the primary Vγ2/Vδ2+ T cells stimulated with the distinct groups of nonpeptide Ags, including monoethyl pyrophosphate, isobutyl amine, and aminobisphosphonate, invariably exhibit Jγ1.2 in the Vγ2+ TCR-γ chains. Gene transfer studies revealed that most of the randomly cloned Vγ2/Jγ1.2+ TCR-γ genes bearing diverse Vγ/Jγ junctional sequences could confer the responsiveness to all these nonpeptide Ags, while none of the Vγ2/Jγ1.1+ or Vγ2/Jγ1.3+ TCR-γ genes could do so. Furthermore, mutation of the lysine residues encoded by the Jγ1.2 gene, which are unique in human Jγ1.2 and absent in other human or mouse Jγ segments, completely abrogated the responsiveness to all the nonpeptide Ags without affecting the response to anti-CD3 mAb. These results strongly suggested that the positively charged lysine residues in the TCR-γ chain CDR3 region encoded by the germline Jγ1.2 gene play a key role in the recognition of diverse small molecular mass nonpeptide Ags.
Resolution of Three Nonproliferative Immature Splenic B Cell Subsets Reveals Multiple Selection Points During Peripheral B Cell Maturation
Authors: Allman David , Lindsley R. Coleman , DeMuth William , Rudd Kristina , Shinton Susan A. , Hardy Richard R.
Published: April 2014

Although immature/transitional peripheral B cells may remain susceptible to selection pressures before full maturation, the nature and timing of these selection events remain unclear. We show that correlated expression of surface (s) IgM (sIgM), CD23, and AA4 defines three nonproliferative subpopulations of immature/transitional peripheral B cells. We designate these populations transitional (T) 1 (AA4+CD23−sIgMhigh), T2 (AA4+CD23+sIgMhigh), and T3 (AA4+CD23+sIgMlow). Cells within all three subsets are functionally immature as judged by their failure to proliferate following sIgM cross-linking in vitro, and their rapid rate of turnover in vivo as assessed by 5-bromo-2′-deoxyuridine labeling. These labeling studies also reveal measurable cell loss at both the T1-T2 and T2-T3 transitions, suggesting the existence of multiple selection points within the peripheral immature B cell pool. Furthermore, we find that Btk-deficient (xid) mice exhibit an incomplete developmental block at the T2-T3 transition within the immature B cell pool. This contrasts markedly with lyn−/− mice, which exhibit depressed numbers but normal ratios of each immature peripheral B cell subset and severely reduced numbers of mature B cells. Together, these data provide evidence for multiple selection points among immature peripheral B cells, suggesting that the B cell repertoire is shaped by multiple unique selection events that occur within the immature/transitional peripheral B cell pool.
CR2/CD21 Proximal Promoter Activity Is Critically Dependent on a Cell Type-Specific Repressor
Authors: Ulgiati Daniela , Holers V. Michael
Published: April 2014

Transcription of the human complement receptor type 2 (CR2/CD21) gene is controlled by both proximal promoter and intronic elements. CR2 is primarily expressed on B cells from the immature through mature cell stages. We have previously described the presence of an intronic element that is required for both cell- and stage-specific expression of CR2. In this study, we report the identification of a cell type-specific repressor element within the proximal promoter. This repressor sequence is shown by linker scanning mutagenesis to comprise an E box motif. By supershift analysis this element binds members of the basic helix-loop-helix family of proteins, in particular E2A gene products. Mutational analysis demonstrates that binding of E2A proteins is critical for functioning of this repressor. Thus, E2A activity is key not only for early B cell development, but also for controlling CR2 expression, a gene expressed only during later stages of ontogeny.
Localizing a Control Region in the Pathway to Leukotriene C4 Secretion Following Stimulation of Human Basophils with Anti-IgE Antibody
Authors: Miura Katsushi , Lavens-Phillips Sandra , MacGlashan Donald W.
Published: April 2014

Mediator release from human basophils is a self-limited process, but down-regulation of the signaling cascades leading to secretion of leukotriene C4 (LTC4) is controlled independently of the pathway leading to IL-4 secretion. In the current studies, we have explored the regulation of upstream signaling events leading to activation of extracellular signal-related kinases (ERKs; previously shown to be required for LTC4 generation) in human basophils. IgE-, but not FMLP-mediated activation, induced sustained tyrosine phosphorylation of syk, of shc, and an association of shc to the Grb2/son of sevenless 2 complex. In contrast, IgE-mediated activation resulted in transient activation of p21ras and mitogen-activated protein/ERK kinase 1, which were kinetically associated with phosphorylation of ERKs. The canonical Shc/Grb2/son of sevenless pathway to activation of p21ras is therefore sustained, while p21ras activity is not. We have previously shown that phosphatidylinositol 3 kinase activity is required for p21ras activity and, in the current studies, we show that of the p85-sensitive forms of p110 possible, basophils express only p110 δ and that there are no changes in association between p21ras and p110 δ in stimulated basophils. We used the generation of phospho-Akt as a marker of the presence of phosphatidylinositol-3,4,5-trisphosphate and found that phospho-Akt is transient on a time scale consistent with p21ras activity. On the basis of information obtained in these and other studies, we localize down-regulation of IgE-mediated LTC4 secretion to a region of the signaling cascade antecedent to p21ras activation, downstream of phosphatidylinositol 3 kinase activity and probably involving regulation of phosphatidylinositol-3,4,5-trisphosphate levels.
Expression and Release of IL-18 Binding Protein in Response to IFN-γ
Authors: Paulukat Jens , Bosmann Markus , Nold Marcel , Garkisch Stefanie , Kämpfer Heiko , Frank Stefan , Raedle Jochen , Zeuzem Stefan , Pfeilschifter Josef , Mühl Heiko
Published: April 2014

IL-18 and IL-18 binding protein (IL-18BP) are two newly described opponents in the cytokine network. Local concentrations of these two players may determine biological functions of IL-18 in the context of inflammation, infection, and cancer. As IL-18 appears to be involved in the pathogenesis of Crohn’s disease and may modulate tumor growth, we investigated the IL-18/IL-18BPa system in the human colon carcinoma/epithelial cell line DLD-1. In this study, we report that IFN-γ induces expression and release of IL-18BPa from DLD-1 cells. mRNA induction and secretion of IL-18BPa immunoreactivity were associated with an activity that significantly impaired release of IFN-γ by IL-12/IL-18-stimulated PBMC. Inducibility of IL-18BPa by IFN-γ was also observed in LoVo, Caco-2, and HCT116 human colon carcinoma cell lines and in the human keratinocyte cell line HaCaT. Induction of IL-18BPa in colon carcinoma/epithelial cell lines was suppressed by coincubation with sodium butyrate. IFN-γ-mediated IL-18BPa and its suppression by sodium butyrate were confirmed in organ cultures of intestinal colonic biopsy specimens. In contrast, sodium butyrate did not modulate expression of IL-18. The present data suggest that IFN-γ may limit biological functions of IL-18 at sites of colonic immune activation by inducing IL-18BPa production. Down-regulation of IL-18BPa by sodium butyrate suggests that reinforcement of local IL-18 activity may contribute to actions of this short-chain fatty acid in the colonic microenvironment.
CXCR3 Internalization Following T Cell-Endothelial Cell Contact: Preferential Role of IFN-Inducible T Cell α Chemoattractant (CXCL11)
Authors: Sauty Alain , Colvin Richard A. , Wagner Ludwig , Rochat Sophie , Spertini Francois , Luster Andrew D.
Published: April 2014

Chemokine receptors are rapidly desensitized and internalized following ligand binding, a process that attenuates receptor-mediated responses. However, the physiological settings in which this process occurs are not clear. Therefore, we examined the fate of CXCR3, a chemokine receptor preferentially expressed on activated T cells following contact with endothelial cells. By immunofluorescence microscopy and flow cytometry, we found that CXCR3 was rapidly internalized when T cells were incubated with IFN-γ-activated human saphenous vein endothelial cells (HSVEC), but not with resting HSVEC. Similar results were obtained using human CXCR3-transfected murine 300-19 B cells. CXCR3 down-regulation was significantly more pronounced when T cells were in contact with HSVEC than with their supernatants, suggesting that CXCR3 ligands were efficiently displayed on the surface of HSVEC. Using neutralizing mAbs to IFN-induced protein-10 (CXCL10), monokine induced by IFN-γ (CXCL9), and IFN-inducible T cell α chemoattractant (I-TAC; CXCL11), we found that even though I-TAC was secreted from IFN-γ-activated HSVEC to lower levels than IFN-induced protein-10 or the monokine induced by IFN-γ, it was the principal chemokine responsible for CXCR3 internalization. This correlated with studies using recombinant chemokines, which revealed that I-TAC was the most potent inducer of CXCR3 down-regulation and of transendothelial migration. Known inhibitors of chemokine-induced chemotaxis, such as pertussis toxin or wortmannin, did not reduce ligand-induced internalization, suggesting that a distinct signal transduction pathway mediates internalization. Our data demonstrate that I-TAC is the physiological inducer of CXCR3 internalization and suggest that chemokine receptor internalization occurs in physiological settings, such as leukocyte contact with an activated endothelium.
T Cell Epitopes of Human Myelin Oligodendrocyte Glycoprotein Identified in HLA-DR4 (DRB1*0401) Transgenic Mice Are Encephalitogenic and Are Presented by Human B Cells
Authors: Forsthuber Thomas G. , Shive Carey L. , Wienhold Wolfgang , de Graaf Katrien , Spack Edward G. , Sublett Robert , Melms Arthur , Kort Jens , Racke Michael K. , Weissert Robert
Published: April 2014

Myelin oligodendrocyte glycoprotein (MOG) is an Ag present in the myelin sheath of the CNS thought to be targeted by the autoimmune T cell response in multiple sclerosis (MS). In this study, we have for the first time characterized the T cell epitopes of human MOG restricted by HLA-DR4 (DRB1*0401), an MHC class II allele associated with MS in a subpopulation of patients. Using MHC binding algorithms, we have predicted MOG peptide binding to HLA-DR4 (DRB1*0401) and subsequently defined the in vivo T cell reactivity to overlapping MOG peptides by testing HLA-DR4 (DRB1*0401) transgenic mice immunized with recombinant human (rh)MOG. The data indicated that MOG peptide 97–108 (core 99–107, FFRDHSYQE) was the immunodominant HLA-DR4-restricted T cell epitope in vivo. This peptide has a high in vitro binding affinity for HLA-DR4 (DRB1*0401) and upon immunization induced severe experimental autoimmune encephalomyelitis in the HLA-DR4 transgenic mice. Interestingly, the same peptide was presented by human B cells expressing HLA-DR4 (DRB1*0401), suggesting a role for the identified MOG epitopes in the pathogenesis of human MS.
Activation of βγ Subunits of Gi2 and Gi3 Proteins by Basic Secretagogues Induces Exocytosis Through Phospholipase Cβ and Arachidonate Release Through Phospholipase Cγ in Mast Cells
Authors: Ferry Xavier , Eichwald Virginie , Daeffler Laurent , Landry Yves
Published: April 2014

Mast cells are activated by Ag-induced clustering of IgE bound to FcεRI receptors or by basic secretagogues that stimulate pertussis toxin-sensitive heterotrimeric G proteins. The cell response includes the secretion of stored molecules, such as histamine, through exocytosis and of de novo synthesized mediators, such as arachidonate metabolites. The respective roles of G proteins α and βγ subunits as well as various types of phospholipase C (PLC) in the signaling pathways elicited by basic secretagogues remain unknown. We show that a specific Ab produced against the C-terminus of Gαi3 and an anti-recombinant Gαi2 Ab inhibited, with additive effects, both exocytosis and arachidonate release from permeabilized rat peritoneal mast cells elicited by the basic secretagogues mastoparan and spermine. A specific Ab directed against Gβγ dimers prevented both secretions. Anti-PLCβ Abs selectively prevented exocytosis. The selective phosphatidylinositol 3-kinase inhibitor LY 294002 prevented arachidonate release without modifying exocytosis. Gβγ coimmunoprecipitated with PLCβ and phosphatidylinositol 3-kinase. The anti-PLCγ1 and anti-phospholipase A2 Abs selectively blocked arachidonate release. Protein tyrosine phosphorylation was inhibited by anti-Gβγ Abs, LY294002, and anti PLCγ1 Abs. These data show that the early step of basic secretagogue transduction is common to both signaling pathways, involving βγ subunits of Gi2 and Gi3 proteins. Activated Gβγ interacts, on one hand, with PLCβ to elicit exocytosis and, on the other hand, with phosphatidylinositol 3-kinase to initiate the sequential activation of PLCγ1, tyrosine kinases, and phospholipase A2, leading to arachidonate release.
Primary Tumor Tissue Lysates Are Enriched in Heat Shock Proteins and Induce the Maturation of Human Dendritic Cells
Authors: Somersan Selin , Larsson Marie , Fonteneau Jean Francois , Basu Sreyashi , Srivastava Pramod , Bhardwaj Nina
Published: April 2014

Upon exposure to lysates or supernatants of necrotic transformed cell lines, human dendritic cells (DCs) undergo maturation. In contrast, DCs exposed to apoptotic transformed cell lines or necrotic lysates of primary cells remain immature. Analysis of supernatants of necrotic transformed cell lines showed them to be enriched in the heat shock proteins (hsp)70 and gp96, in contrast to supernatants of primary cells. Likewise, cells from a variety of primary human tumors contained considerably higher levels of hsp than their normal autologous tissue counterparts. Of the majority of human tumors enriched in hsps (hsp70 and/or gp96), their corresponding lysates matured DCs. The maturation effect of tumor cell lysates was abrogated by treatment with boiling, proteinase K, and geldanamycin, an inhibitor of hsps, suggesting that hsps rather than endotoxin or DNA were the responsible factors. Supporting this idea, highly purified, endotoxin-depleted hsp70, induced DC maturation similar to that seen with standard maturation stimuli LPS and monocyte conditioned medium. These results suggest that the maturation activity inherent within tumor cells and lines is mediated at least in part by hsps. The release of hsps in vivo as a result of cell injury should promote immunity through the maturation of resident DCs.
Staining of Celiac Disease-Relevant T Cells by Peptide-DQ2 Multimers
Authors: Quarsten Hanne , McAdam Stephen N. , Jensen Tore , Arentz-Hansen Helene , Molberg Øyvind , Lundin Knut E. A. , Sollid Ludvig M.
Published: April 2014

Gluten-specific T cells in the small intestinal mucosa are thought to play a central role in the pathogenesis of celiac disease (CD). The vast majority of these T cells recognize gluten peptides when presented by HLA-DQ2 (DQA1*05/DQB1*02), a molecule which immunogenetic studies have identified as conferring susceptibility to CD. We have previously identified and characterized three DQ2-restricted gluten epitopes that are recognized by intestinal T cells isolated from CD patients, two of which are immunodominant. Because almost all of the gluten epitopes are restricted by DQ2, and because we have detailed knowledge of several of these epitopes, we chose to develop peptide-DQ2 tetramers as a reagent to further investigate the role of these T cells in CD. In the present study, stable soluble DQ2 was produced such that it contained leucine zipper dimerization motif and a covalently coupled peptide. We have made four different peptide-DQ2 staining reagents, three containing the gluten epitopes and one containing a DQ2-binding self-peptide that provides a negative control for staining. We show in this study that peptide-DQ2 when adhered to plastic specifically stimulates T cell clones and that multimers comprising these molecules specifically stain peptide-specific T cell clones and lines. Interestingly, T cell activation caused severe reduction in staining intensities obtained with the multimers and an Ab to the TCR. The problem of TCR down-modulation must be taken into consideration when using class II multimers to stain T cells that may have been recently activated in vivo.
Dendritic Cell-Derived IL-12 Is Not Required for the Generation of Cytotoxic, IFN-γ-Secreting, CD8+ CTL In Vivo
Authors: Wan Yonghong , Lu Lingmin , Bramson Jonathan L. , Baral Stefan , Zhu Qing , Pilon Andrew , Dayball Kelley
Published: April 2014

By using adoptive transfer of Ag-loaded bone marrow-derived dendritic cells (BMDC), we have established an in vivo model of CTL priming. Activation of CTL in these experiments required both CD4+ T cells and CD154, demonstrating that this model reflects CD4+ T cell-dependent dendritic cell (DC) licensing. Because IL-12 has been suggested to play an important role in CTL activation by DC, we examined the ability of BMDC to prime CTL in the complete absence of IL-12 using p40-deficient mice. We observed that the absence of IL-12 does not affect the phenotype or allostimulatory function of BMDC after in vitro maturation. Moreover, there was no difference in the ability of Ag-loaded DC to elicit CTL cytotoxicity, whether the Ag was delivered by virus infection or peptide pulsing. Equal frequencies of Ag-specific, IFN-γ-secreting CD8+ T cells developed in both wild-type and IL-12-deficient backgrounds. Finally, CTL generated in the IL-12-deficient environment were capable of protecting immunized mice against tumor challenge, demonstrating that these CTL were fully functional, despite the absence of IL-12 during the maturation process in vivo. These results indicate that IL-12 is not critical for the development of IFN-γ secreting, CD8+ T cells and that another mechanism must be used by licensed DC to prime and activate CTL.
Targeting of Tumor Cells for Human γδ T Cells by Nonpeptide Antigens
Authors: Kato Yu , Tanaka Yoshimasa , Miyagawa Fumi , Yamashita Seiji , Minato Nagahiro
Published: April 2014

Human Vγ2/Vδ2+ γδ T cells respond to low molecular-mass nonpeptide Ags in a γδ TCR-dependent manner. Although requirements of Ag presentation have remained controversial, we have indicated that specific responses of the primary γδ T cells to pamidronate were dependent on monocytic adherent cells for Ag presentation. Here, we show that human tumor cells can efficiently present aminobisphosphonate and pyrophosphomonoester compounds to γδ T cells, inducing specific proliferation and IFN-γ production. γδ TCR dependency of the response to Ag-pulsed tumor cells was confirmed by using a Jurkat line transfected with a Vγ2/Vδ2 γδ TCR. Furthermore, γδ T cells exhibited markedly enhanced cytotoxicity against the Ag-pulsed tumor cells as compared with untreated tumor cells. Survey of a number of human tumor cell lines of different origins revealed that the majority of them became susceptible for γδ T cell-mediated cytotoxicity following the Ag pulsing except for breast cancer lines so far examined, while normal PHA blast cells remained resistant. The results not only imply a unique mode of nonpeptide Ag recognition by human γδ T cells but also may provide a novel strategic clue for immunotherapy of human malignancy.
PGRL Is a Major CD81-Associated Protein on Lymphocytes and Distinguishes a New Family of Cell Surface Proteins
Authors: Clark Krista L. , Zeng Zhihong , Langford Amy L. , Bowen Starla M. , Todd Scott C.
Published: April 2014

CD81 exerts a range of interesting effects on T cells including early thymocyte differentiation, LFA-1 activation, and provision of costimulation. To better understand the mechanisms by which CD81 influences T cell function we evaluated CD81 molecular complexes on T cells. The most prominent CD81-associated cell surface protein on thymocytes as well as a number of T cell and B cell lines has an apparent molecular mass of 75 kDa. The 75-kDa protein was purified and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry followed by postsource-decay profiling. p75 is a novel type I transmembrane protein of the Ig superfamily which is most similar to FPRP. We cloned and sequenced both human and mouse PG regulatory-like protein (PGRL) and characterized mouse PGRL expression in both lymphocytes and nonlymphoid tissues. The discovery of PGRL allows for the clustering of a small family of related proteins including PGRL, FPRP, V7/CD101, and IGSF3. Expression constructs containing various domains of PGRL with an epitope tag were coexpressed with CD81 and used to determine that the interaction of CD81 with PGRL requires the membrane distal Ig3–Ig4 domains of PGRL. Although it remains to be determined whether PGRL possesses PG regulatory functions, transwell chamber experiments show that PGs and CD81 coordinately regulate T cell motility.
The Initiation of B Cell Clonal Expansion Occurs Independently of Pre-B Cell Receptor Formation
Authors: Kline Gregory H. , Hayden Tracy A. , Riegert Patricia
Published: April 2014

Current models of B cell development posit that clonal expansion occurs as a direct result of Ig H chain expression. To test this hypothesis, we isolated a population of early B cells in which H chain recombination is initiated and assessed VHDJH rearrangements in both cycling and noncycling cells. We found that actively dividing cells within this population are enriched for H chain rearrangements that are productive when compared with their counterparts in G0/G1, apparently supporting a role for H chain expression in initiating early B cell division; entrance into the cell cycle was accompanied by VH gene-dependent H chain selection. However, we also identified a phenotypically identical population of actively cycling early B cells in the absence of H chain expression in recombination activating gene knockout mice. In addition, actively cycling early B cells could be detected in pre-B cell receptor (pBCR)-negative λ5 knockout mice, but we found no evidence for VH-dependent H chain selection in this population. Given these results, we suggest that the initiation of clonal expansion, at this early stage in B cell development, occurs independently of H chain expression. Although the cycling cell pool is enriched for pBCR-positive cells in mice expressing surrogate L chain, pBCR formation is not required for the initiation of cell division.
Efficient Chromosomal Mapping of a Methylcholanthrene- Induced Tumor Antigen by CTL Immunoselection
Authors: Akilesh Shreeram , Dudley Mark E. , Eden Peter A. , Roopenian Derry C.
Published: April 2014

It has been difficult to genetically map the genes encoding tumor Ags because they arise as a consequence of somatic mutational events. CTL-mediated immunoselection can impose potent immunoselective pressure against tumor cells, resulting in the survival of rare tumor Ag-loss variants. We subjected a heterozygous 3-methylcholanthrene-induced murine sarcoma cell line to CTL immunoselection, selecting for the loss of a tumor-specific Ag, recognized antigen from MCA-induced tumor 1 (Ram1). Several variants eluded CTL recognition by genetic loss of the hemizygously expressed tumor-specific Ag epitope. A frequently observed genetic escape mechanism was spontaneous mitotic recombination resulting in loss of heterozygosity on chromosome 4. Higher density genetic analyses along with functional confirmation with an independently produced chromosome 4 loss of heterozygosity variant positioned the Ram1 locus to a distal 7.1 cM interval on chromosome 4. This region of the mouse genome is rich in tumor-modifier genes and this positioning of Ram1 may thus provide insight into the genetic basis of 3-methycholanthrene-induced tumor Ags.
Transmembrane Domain-Mediated Colocalization of HLA-DM and HLA-DR Is Required for Optimal HLA-DM Catalytic Activity
Authors: Weber Dominique A. , Dao Chinh T. , Jun Julie , Wigal Jerrod L. , Jensen Peter E.
Published: April 2014

HLA-DM catalyzes peptide loading and exchange reactions by MHC class II molecules. Soluble recombinant DM, lacking transmembrane and cytoplasmic domains, was observed to have 200- to 400-fold less activity compared with the full-length protein in assays measuring DM-catalyzed peptide dissociation from purified HLA-DR1 in detergent solutions. Additional studies with truncated soluble DR1 demonstrated that transmembrane domains in DR1 molecules are also required for optimal activity. The potential requirement for specific interaction between the transmembrane domains of DM and DR was ruled out in experiments with chimeric DR1 molecules containing transmembrane domains from either DM or the unrelated protein CD80. These results suggested that the major role of the transmembrane domains is to facilitate colocalization of DM and DR in detergent micelles. The latter conclusion was further supported by the observation that HLA-DM-catalyzed peptide binding to certain murine class II proteins is increased by reducing the volume of detergent micelles. The importance of membrane colocalization was directly demonstrated in experiments in which DM and DR were reconstituted separately or together into membrane bilayers in unilamellar liposomes. Our findings demonstrate the importance of membrane anchoring in DM activity and underscore the potential importance of membrane localization in regulating peptide exchange by class II molecules.
The Role of the Individual Domains in the Structure and Function of the Catalytic Region of a Modular Serine Protease, C1r
Authors: Kardos József , Gál Péter , Szilágyi László , Thielens Nicole M. , Szilágyi Katalin , Lõrincz Zsolt , Kulcsár Péter , Gráf László , Arlaud Gérard  J. , Závodszky Péter
Published: April 2014

The first enzymatic event in the classical pathway of complement activation is autoactivation of the C1r subcomponent of the C1 complex. Activated C1r then cleaves and activates zymogen C1s. C1r is a multidomain serine protease consisting of N-terminal α region interacting with other subcomponents and C-terminal γB region mediating proteolytic activity. The γB region consists of two complement control protein modules (CCP1, CCP2) and a serine protease domain (SP). To clarify the role of the individual domains in the structural and functional properties of the γB region we produced the CCP1-CCP2-SP (γB), the CCP2-SP, and the SP fragments in recombinant form in Escherichia coli. We successfully renatured the inclusion body proteins. After renaturation all three fragments were obtained in activated form and showed esterolytic activity on synthetic substrates similar to each other. To study the self-activation process in detail zymogen mutant forms of the three fragments were constructed and expressed. Our major statement is that the ability of autoactivation and C1s cleavage is an inherent property of the SP domain. We observed that the CCP2 module significantly increases proteolytic activity of the SP domain on natural substrate, C1s. Therefore, we propose that CCP2 module provides accessory binding sites. Differential scanning calorimetric measurements demonstrated that CCP2 domain greatly stabilizes the structure of SP domain. Deletion of CCP1 domain from the CCP1-CCP2-SP fragment results in the loss of the dimeric structure. Our experiments also provided evidence that dimerization of C1r is not a prerequisite for autoactivation.
Cytokine Requirements for Induction of Systemic and Mucosal CTL After Nasal Immunization
Authors: Staats Herman F. , Bradney Curtis P. , Gwinn William M. , Jackson Shawn S. , Sempowski Gregory D. , Liao Hua-Xin , Letvin Norman L. , Haynes Barton F.
Published: April 2014

Cholera toxin (CT) is frequently used as an experimental adjuvant intranasally for the induction of systemic and mucosal immunity. However, CT is highly reactogenic and not approved for use in humans. To define the cytokine requirements for the nasal activation of the systemic and mucosal immune system, and to design new adjuvants with efficacy similar to CT, we defined the cytokines that were able to replace CT as a nasal adjuvant for the induction of CTL. BALB/c mice were nasally immunized with an HIV immunogen that contains an MHC class I-restricted CTL epitope ± cytokines and tested for HIV-specific immune responses. We found that combinations of IL-1α plus IL-18, IL-1α plus IL-12, and IL-1α plus IL-12 plus GM-CSF each induced optimal splenocyte anti-HIV CTL responses in immunized mice (range 60–71% peptide-specific 51Cr release). Peak H-2Dd-peptide tetramer-binding T cell responses induced by cytokine combinations were up to 5.5% of CD8+ PBMC. Nasal immunization with HIV immunogen and IL-1α, IL-12, and GM-CSF also induced Ag-specific IFN-γ-secreting cells in the draining cervical lymph node and the lung. The use of IL-1α, IL-12, and GM-CSF as nasal adjuvants was associated with an increased expression of MHC class II and B7.1 on nonlymphocytes within the nasal-associated lymphoid tissue/nasal mucosa. Thus, IL-1α, IL-12, IL-18, and GM-CSF are critical cytokines for the induction of systemic and mucosal CTL after nasal immunization. Moreover, these cytokines may serve as effective adjuvants for nasal vaccine delivery.
Donor IFN-γ Receptors Are Critical for Acute CD4+ T Cell-Mediated Cardiac Allograft Rejection
Authors: Wiseman Alexander C. , Pietra Biagio A. , Kelly Brian P. , Rayat Gina R. , Rizeq Mona , Gill Ronald G.
Published: April 2014

Recent studies using mouse models demonstrate that CD4+ T cells are sufficient to mediate acute cardiac allograft rejection in the absence of CD8+ T cells and B cells. However, the mechanistic basis of CD4-mediated rejection is unclear. One potential mechanism of CD4-mediated rejection is via elaboration of proinflammatory cytokines such as IFN-γ. To determine whether IFN-γ is a critical cytokine in CD4-mediated acute cardiac allograft rejection, we studied whether the expression of IFN-γ receptors on the donor heart was required for CD4-mediated rejection. To investigate this possibility, purified CD4+ T cells were transferred into immune-deficient mice bearing heterotopic cardiac allografts from IFN-γ receptor-deficient (GRKO) donors. While CD4+ T cells triggered acute rejection of wild-type heart allografts, they failed to trigger rejection of GRKO heart allografts. The impairment in CD4-mediated rejection of GRKO hearts appeared to primarily involve the efferent phase of the immune response. This conclusion was based on the findings that GRKO stimulator cells provoked normal CD4 proliferation in vitro and that intentional in vivo challenge of CD4 cells with wild-type donor APC or the adoptive transfer of in vitro primed CD4 T cells failed to provoke acute rejection of GRKO allografts. In contrast, unseparated lymph node cells acutely rejected both GRKO and wild-type hearts with similar time courses, illustrating the existence of both IFN-γ-dependent and IFN-γ-independent mechanisms of acute allograft rejection.
Immature and Mature CD8α+ Dendritic Cells Prolong the Survival of Vascularized Heart Allografts
Authors: O’Connell Peta J. , Li Wei , Wang Zhiliang , Specht Susan M. , Logar Alison J. , Thomson Angus W.
Published: April 2014

CD8α+ and CD8α− dendritic cells (DCs) arise from committed bone marrow progenitors and can induce or regulate immune reactivity. Previously, the maturational status of CD8α− (myeloid) DCs has been shown to influence allogeneic T cell responses and allograft survival. Although CD8α+ DCs have been implicated in central tolerance and found to modulate peripheral T cell function, their influence on the outcome of organ transplantation has not been examined. Consistent with their equivalent high surface expression of MHC and costimulatory molecules, sorted mature C57BL/10J (B10; H2b) DCs of either subset primed naive, allogeneic C3H/HeJ (C3H; H2k) recipients for Th1 responses. Paradoxically and in contrast to their CD8α− counterparts, mature CD8α+ B10 DCs given systemically 7 days before transplant markedly prolonged B10 heart graft survival in C3H recipients. This effect was associated with specific impairment of ex vivo antidonor T cell proliferative responses, which was not reversed by exogenous IL-2. Further analyses of possible underlying mechanisms indicated that neither immune deviation nor induction of regulatory cells was a significant contributory factor. In contrast to the differential capacity of the mature DC subsets to affect graft outcome, immature CD8α+ and CD8α− DCs administered under the same experimental conditions significantly prolonged transplant survival. These observations demonstrate for the first time the innate capacity of CD8α+ DCs to regulate alloimmune reactivity and transplant survival, independent of their maturation status. Mobilization of such a donor DC subset with capacity to modulate antidonor immunity may have significant implications for the therapy of allograft rejection.
The CD8 T Cell Compartment Plays a Dominant Role in the Deficiency of Brown-Norway Rats to Mount a Proper Type 1 Immune Response
Authors: Cautain Bastien , Damoiseaux Jan , Bernard Isabelle , Xystrakis Emmanuel , Fournié Emmanuelle , van Breda Vriesman Peter , Druet Philippe , Saoudi Abdelhadi
Published: April 2014

Differential cytokine production by T cells plays an important role in regulating the nature of an immune response. In the rat, Brown-Norway (BN) and Lewis (LEW) strains differ markedly in their susceptibility to develop either type 1 or type 2-mediated autoimmune manifestations. BN rats are susceptible to type 2-dependent systemic autoimmunity, while LEW rats are resistant. Conversely, type 1-mediated, organ-specific autoimmune disease can be easily induced in LEW, but not in BN, rats. The mechanisms involved in the differential development of type 1 and type 2 immune responses by these two strains are still unknown. In the present study we analyzed the contributions of APC, CD4 and CD8 T cells, and MHC molecules in the difference between LEW and BN rats to develop a type 1 immune response. First, we show that the defect of BN T cells to produce type 1 cytokines in vitro does not require the presence of APC and, by using an APC-independent stimulation assay, we have localized the defect within the T cell compartment. Both CD4 and CD8 T cells are involved in the defect of BN rats to develop a type 1 immune response with a major contribution of the CD8 T cell compartment. This defect is associated with an increase in the type 2 cytokine IL-4 in both BN T cell populations, but neutralization of this cytokine does not restore this defect. Finally, by using MHC congenic rats, we show that the MHC haplotype is not involved in the defect of BN T cells to mount a proper type 1 cytokine response.
Conservation and Variation in Human and Common Chimpanzee CD94 and NKG2 Genes
Authors: Shum Benny P. , Flodin Laura R. , Muir David G. , Rajalingam Raja , Khakoo Salim I. , Cleland Sophia , Guethlein Lisbeth A. , Uhrberg Markus , Parham Peter
Published: April 2014

To assess polymorphism and variation in human and chimpanzee NK complex genes, we determined the coding-region sequences for CD94 and NKG2A, C, D, E, and F from several human (Homo sapiens) donors and common chimpanzees (Pan troglodytes). CD94 is highly conserved, while the NKG2 genes exhibit some polymorphism. For all the genes, alternative mRNA splicing variants were frequent among the clones obtained by RT-PCR. Alternative splicing acts similarly in human and chimpanzee to produce the CD94B variant from the CD94 gene and the NKG2B variant from the NKG2A gene. Whereas single chimpanzee orthologs for CD94, NKG2A, NKG2E, and NKG2F were identified, two chimpanzee paralogs of the human NKG2C gene were defined. The chimpanzee Pt-NKG2CI gene encodes a protein similar to human NKG2C, whereas in the chimpanzee Pt-NKG2CII gene the translation frame changes near the beginning of the carbohydrate recognition domain, causing premature termination. Analysis of a panel of chimpanzee NK cell clones showed that Pt-NKG2CI and Pt-NKG2CII are independently and clonally expressed. Pt-NKG2CI and Pt-NKG2CII are equally diverged from human NKG2C, indicating that they arose by gene duplication subsequent to the divergence of chimpanzee and human ancestors. Genomic DNA from 80 individuals representing six primate species were typed for the presence of CD94 and NKG2. Each species gave distinctive typing patterns, with NKG2A and CD94 being most conserved. Seven different NK complex genotypes within the panel of 48 common chimpanzees were due to differences in Pt-NKG2C and Pt-NKG2D genes.
Prostaglandin E2-Mediated Activation of HIV-1 Long Terminal Repeat Transcription in Human T Cells Necessitates CCAAT/Enhancer Binding Protein (C/EBP) Binding Sites in Addition to Cooperative Interactions Between C/EBPβ and Cyclic Adenosine 5′-Monophosphate Response Element Binding Protein
Authors: Dumais Nancy , Bounou Salim , Olivier Martin , Tremblay Michel J.
Published: April 2014

Previous work indicates that treatment of human T cells with PGE2 results in an increase of HIV-1 long terminal repeat (LTR) transcriptional activity. The noticed PGE2-mediated activation of virus gene activity required the participation of specific intracellular second messengers such as calcium and two transcription factors, i.e., NF-κB and CREB. We report in this work that the nuclear transcription factor CCAAT/enhancer binding protein (C/EBP) is also important for PGE2-dependent up-regulation of HIV-1 LTR-driven gene activity. The implication of C/EBP was shown by using a trans-dominant negative inhibitor of C/EBP (i.e., liver-enriched transcriptional inhibitory protein) and several molecular constructs carrying site-directed mutations in the C/EBP binding sites located within the HIV-1 LTR. Mutated HIV-1 LTR constructs also revealed the involvement of the two most proximal C/EBP binding sites. Data from cotransfection experiments with vectors coding for dominant negative mutants and gel mobility shift assays indicated that PGE2-mediated induction of HIV-1 LTR activity results from a cooperative interaction between C/EBPβ and CREB, two members of the basic leucine zipper family of transcription factors. Altogether these findings indicate that treatment of human T cells with PGE2 induces HIV-1 LTR activity through a complex interplay between C/EBPβ and CREB. Such a combinatorial regulation may represent a mechanism that permits a fine regulation of HIV-1 expression by PGE2 in human T cells.
Mast Cell α-Chymase Reduces IgE Recognition of Birch Pollen Profilin by Cleaving Antibody-Binding Epitopes
Authors: Mellon Matthew B. , Frank Brendon T. , Fang Kenneth C.
Published: April 2014

In sensitized individuals birch pollen induces an allergic response characterized by IgE-dependent mast cell degranulation of mediators, such as α-chymase and other serine proteases. In birch and other plant pollens, a major allergen is profilin. In mammals, profilin homologues are found in an intracellular form bound to cytoskeletal or cytosolic proteins or in a secreted form that may initiate signal transduction. IgE specific to birch profilin also binds human profilin I. This cross-reactivity between airborne and endogenous proteins may help to sustain allergy symptoms. The current work demonstrates that cultured mast cells constitutively secrete profilin I, which is susceptible to degranulation-dependent proteolysis. Coincubation of chymase-rich BR mastocytoma cells with Ala-Ala-Pro-Phe-chloromethylketone (a chymase inhibitor) blocks profilin cleavage, which does not occur in degranulated HMC-1 mast cells, which are rich in tryptase, but chymase deficient. These data implicate chymase as the serine protease cleaving secreted mast cell profilin. Sequencing of chymase-cleaved profilins reveals hydrolysis at Tyr6-Val7 and Trp35-Ala36 in birch profilin and at Trp32-Ala33 in human profilin, with all sites lying within IgE-reactive epitopes. IgE immunoblotting studies with sera from birch pollen-allergic individuals demonstrate that cleavage by chymase attenuates binding of birch profilin to IgE. Thus, destruction of IgE-binding epitopes by exocytosed chymase may limit further mast cell activation by this class of common plant allergens, thereby limiting the allergic responses in sensitized individuals.
Vaccine Protection Against Functional CTL Abnormalities in Simian Human Immunodeficiency Virus-Infected Rhesus Monkeys
Authors: McKay Paul F. , Schmitz Jörn E. , Barouch Dan H. , Kuroda Marcelo J. , Lifton Michelle A. , Nickerson Christine E. , Gorgone Darci A. , Letvin Norman L.
Published: April 2014

Accumulating evidence suggests that HIV-specific CD8+ CTL are dysfunctional in HIV-infected individuals with progressive clinical disease. In the present studies, cytokine production by virus-specific CTL was assessed in the rhesus monkey model for AIDS to determine its contribution to the functional impairment of CTL. CTL from monkeys infected with nonpathogenic isolates of simian and simian-human immunodeficiency virus expressed high levels of IFN-γ, TNF-α, and IL-2 after in vitro exposure to a nonspecific mitogen or the optimal peptide representing a dominant virus-specific CTL epitope. However, similarly performed studies assessing these capabilities in CTL from monkeys infected with pathogenic immunodeficiency virus isolates demonstrated a significant dysfunction in the ability of the CTL to produce IL-2 and TNF-α. Importantly, CTL from vaccinated monkeys that effectively controlled the replication of a highly pathogenic simian-human immunodeficiency virus isolate following challenge demonstrated a preserved capacity to produce these cytokines. These experiments suggest that defects in cytokine production may contribute to CTL dysfunction in chronic HIV or SIV infection. Moreover, an AIDS vaccine that confers protection against clinical disease evolution in this experimental model also preserves the functional capacity of these CTL to produce both IL-2 and TNF-α.
Toll-Like Receptor 2 Is Required for Innate, But Not Acquired, Host Defense to Borrelia burgdorferi
Authors: Wooten R. Mark , Ma Ying , Yoder R. Alyson , Brown Jeanette P. , Weis John H. , Zachary James F. , Kirschning Carsten J. , Weis Janis J.
Published: April 2014

Borrelia burgdorferi lipoproteins activate inflammatory cells through Toll-like receptor 2 (TLR2), suggesting that TLR2 could play a pivotal role in the host response to B. burgdorferi. TLR2 does play a critical role in host defense, as infected TLR2−/− mice harbored up to 100-fold more spirochetes in tissues than did TLR2+/+ littermates. Spirochetes persisted at extremely elevated levels in TLR2-deficient mice for at least 8 wk following infection. Infected TLR2−/− mice developed normal Borrelia-specific Ab responses, as measured by quantity of Borrelia-specific Ig isotypes, the kinetics of class switching to IgG, and the complexity of the Ags recognized. These findings indicate that the failure to control spirochete levels in tissues is not due to an impaired acquired immune response. While macrophages from TLR2−/− mice were not responsive to lipoproteins, they did respond to nonlipoprotein components of sonicated spirochetes. These TLR2-independent responses could play a role during the inflammatory response to B. burgdorferi, as infected TLR2−/− mice developed greater ankle swelling than wild-type littermates. Thus, while TLR2-dependent signaling pathways play a major role in the innate host defense to B. burgdorferi, both inflammatory responses and the development of the acquired humoral response can occur in the absence of TLR2.