bluuebunny/crossref_metadata_2025 huggingface dataset:
Type I Collagen Is the Autoantigen in Experimental Autoimmune Anterior Uveitis
Authors: Bora Nalini S. , Sohn Jeong-Hyeon , Kang Shin-Goo , Cruz Jose M. C. , Nishihori Hiroki , Suk Hye-Jung , Wang Yali , Kaplan Henry J. , Bora Puran S.
Published: April 2014

This study was undertaken to identify and characterize the Ag responsible for the induction of experimental autoimmune anterior uveitis (EAAU). Melanin-associated Ag isolated from bovine iris and ciliary body was digested with the proteolytic enzyme V8 protease to solubilize the proteins and the pathogenic protein was purified to homogeneity. Lewis rats were sensitized to various fractions and investigated for the development of anterior uveitis and an immune response to the purified Ag. The uveitogenic Ag had a mass of 22 kDa (SDS-PAGE) and an isoelectric point of 6.75. The N-terminal amino acid sequence of this protein demonstrated 100% homology with the bovine type I collagen α-2 chain starting from amino acid 385 and will be referred to as CI-α2 (22 kDa). Animals immunized with bovine CI-α2 (22 kDa) developed both cellular and humoral immunity to the Ag. They developed anterior uveitis only if the CI-α2 chain underwent proteolysis and if the bound carbohydrates were intact. EAAU induced by CI-α2 (22 kDa) can be adoptively transferred to naive syngenic rats by primed CD4+ T cells. EAAU could not be induced by the adoptive transfer of sera obtained from animals immunized with CI-α2 (22 kDa). The α-1 and α-2 chains (intact or proteolytically cleaved) of type I collagen from calfskin were not pathogenic. Although human anterior uveitis has been historically characterized as a collagen disease, this is first time collagen has been directly identified as the target autoantigen in uveitis.
Phosphatidylinositol 3-Kinase Is Involved in α2(I) Collagen Gene Expression in Normal and Scleroderma Fibroblasts
Authors: Asano Yoshihide , Ihn Hironobu , Yamane Kenichi , Jinnin Masatoshi , Mimura Yoshihiro , Tamaki Kunihiko
Published: April 2014

TGF-β is implicated in the pathogenesis of fibrotic disorders. It has been shown that Smad3 promotes the human α2(I) collagen (COL1A2) gene expression by TGF-β1 in human dermal fibroblasts. Here, we investigated the role of phosphatidylinositol 3-kinase (PI3K) in the COL1A2 gene expression in normal and scleroderma fibroblasts. In normal fibroblasts, the PI3K inhibitor, LY294002, significantly decreased the basal and the TGF-β1-induced increased stability of COL1A2 mRNA. The TGF-β1-induced COL1A2 promoter activity, but not the basal activity, was significantly attenuated by LY294002 or the dominant negative mutant of p85 subunit of PI3K, while the constitutive active mutant of p110 subunit of PI3K did not affect the basal or the TGF-β1-induced COL1A2 promoter activity. LY294002 significantly decreased the phosphorylation of Smad3 induced by TGF-β1. Furthermore, the transient overexpression of 2xFYVE, which induces the mislocalization of FYVE domain proteins, decreased the TGF-β1-induced Smad3 phosphorylation to a similar extent to LY294002. In scleroderma fibroblasts, the blockade of PI3K significantly decreased the mRNA stability and the promoter activity of the COL1A2 gene. Furthermore, LY294002 and the transient overexpression of 2xFYVE completely diminished the constitutive phosphorylation of Smad3. These results indicate that 1) the basal activity of PI3K is necessary for the COL1A2 mRNA stabilization in normal and scleroderma fibroblasts, 2) there is an unidentified FYVE domain protein specifically interacting with Smad3, and 3) the basal activity of PI3K and the FYVE domain protein are indispensable for the efficient TGF-β/Smad3 signaling in normal fibroblasts and for the establishment of the constitutive activation of TGF-β/Smad3 signaling in scleroderma fibroblasts.
Induction of Human Dendritic Cell Maturation Using Transfection with RNA Encoding a Dominant Positive Toll-Like Receptor 4
Authors: Cisco Robin M. , Abdel-Wahab Zeinab , Dannull Jens , Nair Smita , Tyler Douglas S. , Gilboa Eli , Vieweg Johannes , Daaka Yehia , Pruitt Scott K.
Published: April 2014

Maturation of dendritic cells (DC) is critical for the induction of Ag-specific immunity. Ag-loaded DC matured with LPS, which mediates its effects by binding to Toll-like receptor 4 (TLR4), induce Ag-specific CTL in vitro and in vivo in animal models. However, clinical use of LPS is limited due to potential toxicity. Therefore, we sought to mimic the maturation-inducing effects of LPS on DC by stimulating TLR4-mediated signaling in the absence of exogenous LPS. We developed a constitutively active TLR4 (caTLR4) and demonstrated that transfection of human DC with RNA encoding caTLR4 led to IL-12 and TNF-α secretion. Transfection with caTLR4 RNA also induced a mature DC phenotype. Functionally, transfection of DC with caTLR4 RNA enhanced allostimulation of CD4+ T cells. DC transfected with RNA encoding the MART (Melan-A/MART-1) melanoma Ag were then used to stimulate T cells in vitro. Cotransfection of these DC with caTLR4 RNA enhanced the generation of MART-specific CTL. This CTL activity was superior to that seen when DC maturation was induced using either LPS or a standard mixture of cytokines (TNF-α, IL-6, IL-1β, and PGE2). We conclude that transfection of DC with RNA encoding a functional signaling protein, such as caTLR4, may provide a new tool for studying TLR signaling in DC and may be a promising approach for the induction of DC maturation for tumor immunotherapy.
Identification of an SSX-2 Epitope Presented by Dendritic Cells to Circulating Autologous CD4+ T Cells
Authors: Ayyoub Maha , Hesdorffer Charles S. , Metthez Genevieve , Stevanovic Stefan , Ritter Gerd , Chen Yao-Tseng , Old Lloyd J. , Speiser Daniel , Cerottini Jean-Charles , Valmori Danila
Published: April 2014

Accumulating evidence supports the requirement for both tumor-specific CD8+ and CD4+ T cell responses for efficient tumor rejection to occur. Because of its expression in different tumor types, the cancer/testis Ag encoded by the synovial sarcoma X breakpoint 2 (SSX-2) gene is among the most relevant candidates for the development of generic cancer vaccines. The immunogenicity of SSX-2 has been previously corroborated by detection of specific humoral and CD8+ T cell responses in cancer patients. In this study we report identification of the first CD4+ T cell epitope encoded by SSX-2. The identified epitope mapped to the 19–34 region of the protein and was recognized by CD4+ T cells from an Ag-expressing melanoma patient in association with HLA-DPB1*0101. The absence of detectable response in healthy donors and other patients suggests that SSX-2-specific CD4+ T cells in the responder patient had been previously expanded in vivo in response to the autologous tumor. The epitope did not appear to be presented on the surface of tumor cells at levels sufficient to allow direct recognition. In contrast, it was efficiently presented by autologous dendritic cells, supporting the concept that processing by professional APC is the main pathway through which the CD4+ T cell immunoresponse to tumor Ags occurs in vivo.
NF-κB and Oct-2 Synergize to Activate the Human 3′ Igh hs4 Enhancer in B Cells
Authors: Sepulveda Manuel A. , Emelyanov Alexander V. , Birshtein Barbara K.
Published: April 2014

In B cells, the Igh gene locus contains several DNase I-hypersensitive (hs) sites with enhancer activity. These include the 3′ Igh enhancers, which are located downstream of the Cα gene(s) in both mouse and human. In vivo experiments have implicated murine 3′ enhancers, hs3B and/or hs4, in class switching and somatic hypermutation. We previously reported that murine hs4 was regulated by NF-κB, octamer binding proteins, and Pax5 (B cell-specific activator protein). In this study we report that human hs4 is regulated differently. EMSAs and Western analysis of normal B cells before and after stimulation with anti-IgM plus anti-CD40 showed the same complex binding pattern formed by NF-κB, Oct-1, and Oct-2 (but not by Pax5). A similar EMSA pattern was detected in mature human B cell lines (BL-2, Ramos, and HS-Sultan) and in diffuse large B cell lymphoma cell lines, although yin yang 1 protein (YY1) binding was also observed. We have confirmed the in vivo association of these transcription factors with hs4 in B cells by chromatin immunoprecipitation assays. The diffuse large B cell lymphoma cell lines had a distinctive slow-migrating complex containing YY1 associated with Rel-B. We have confirmed by endogenous coimmunoprecipitation an association of YY1 with Rel-B, but not with other NF-κΒ family members. Transient transfection assays showed robust hs4 enhancer activity in the mature B cell lines, which was dependent on synergistic interactions between NF-κB and octamer binding proteins. In addition, human hs4 enhancer activity required Oct-2 and correlated with expression of Oct coactivator from B cells (OCA-B).
Role of Commensal Bacteria in Development of Gut-Associated Lymphoid Tissues and Preimmune Antibody Repertoire
Authors: Rhee Ki-Jong , Sethupathi Periannan , Driks Adam , Lanning Dennis K. , Knight Katherine L.
Published: April 2014

Intestinal bacteria are required for development of gut-associated lymphoid tissues (GALT), which mediate a variety of host immune functions, such as mucosal immunity and oral tolerance. In rabbits, the intestinal microflora are also required for developing the preimmune Ab repertoire by promoting somatic diversification of Ig genes in B cells that have migrated to GALT. We studied the mechanism of bacteria-induced GALT development. Bacteria were introduced into rabbits in which the appendix had been rendered germfree by microsurgery (we refer to these rabbits as germfree-appendix rabbits). We then identified specific members of the intestinal flora that promote GALT development. The combination of Bacteroides fragilis and Bacillus subtilis consistently promoted GALT development and led to development of the preimmune Ab repertoire, as shown by an increase in somatic diversification of VDJ-Cμ genes in appendix B cells. Neither species alone consistently induced GALT development, nor did Clostridium subterminale, Escherichia coli, or Staphylococcus epidermidis. B. fragilis, which by itself is immunogenic, did not promote GALT development; hence, GALT development in rabbits does not appear to be the result of an Ag-specific immune response. To identify bacterial pathways required for GALT development, we introduced B. fragilis along with stress-response mutants of B. subtilis into germfree-appendix rabbits. We identified two Spo0A-controlled stress responses, sporulation and secretion of the protein YqxM, which are required for GALT development. We conclude that specific members of the commensal, intestinal flora drive GALT development through a specific subset of stress responses.
Identification of a Novel FcγRIIIaα-Associated Molecule That Contains Significant Homology to Porcine Cathelin
Authors: Sweeney Susan E. , Kim Yoon B.
Published: April 2014

The following studies are the first to demonstrate the association of porcine FcγRIIIaα with a molecule that contains significant homology to the cathelin family of antimicrobial proteins. We performed immunoprecipitation of the porcine FcγRIIIaα multisubunit complex from Brij 96 lysates of polymorphonuclear leukocytes using the G7 mAb, which binds to FcγRIIIaα on the surface of porcine NK cells and phagocytes. Previous results indicate that the transmembrane α subunit of the FcγRIIIa complex is associated with the γ subunit on the surface of porcine polymorphonuclear leukocytes and with several other unique proteins that surface iodinate and migrate at ∼15, 20, and 25 kDa when analyzed by reducing SDS-PAGE. Through characterization of the porcine FcγRIIIa complex, we identified the 15-kDa molecule as a unique FcγR-associated protein that has not been described in other systems. We now report an association between FcγRIIIaα and a 15-kDa molecule that shares homology to cathelin, a protein of undetermined function initially identified in porcine leukocytes. A domain with a high degree of homology to cathelin is found in the proregions of a family of antibiotic proteins referred to as cathelicidins. The results of our studies indicate the presence of a novel FcγRIIIa complex in the porcine system, and may provide new insights into the function of this antimicrobial protein homologue in relation to the variety of responses mediated through FcγRs.
Bone Marrow-Derived Progenitor Cells Are Important for Lung Repair after Lipopolysaccharide-Induced Lung Injury
Authors: Yamada Mitsuhiro , Kubo Hiroshi , Kobayashi Seiichi , Ishizawa Kota , Numasaki Muneo , Ueda Shinsaku , Suzuki Takashi , Sasaki Hidetada
Published: April 2014

Tissue repair often occurs in organs damaged by an inflammatory response. Inflammatory stimuli induce a rapid and massive release of inflammatory cells including neutrophils from the bone marrow. Recently, many studies suggested that bone marrow cells have the potential to differentiate into a variety of cell types. However, whether inflammatory stimuli induce release of bone marrow-derived progenitor cells (BMPCs), or how much impact the suppression of BMPCs has on the injured organ is not clear. Here we show that LPS, a component of Gram-negative bacterial cell walls, in the lung airways, induces a rapid mobilization of BMPCs into the circulation in mice. BMPCs accumulate within the inflammatory site and differentiate to become endothelial and epithelial cells. Moreover, the suppression of BMPCs by sublethal irradiation before intrapulmonary LPS leads to disruption of tissue structure and emphysema-like changes. Reconstitution of the bone marrow prevents these changes. These data suggest that BMPCs are important and required for lung repair after LPS-induced lung injury.
Potential Targets of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitor for Multiple Sclerosis Therapy
Authors: Nath Narender , Giri Shailendra , Prasad Ratna , Singh Avtar K. , Singh Inderjit
Published: April 2014

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors or statins are newly identified immunomodulators. In vivo treatment of SJL/J mice with lovastatin reduced the duration and clinical severity of active and passive experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Lovastatin induced the expression of GATA3 and the phosphorylation of STAT6, whereas it inhibited tyrosine phosphorylation of Janus kinase 2, tyrosine kinase 2, and STAT4. Inhibition of the Janus kinase-STAT4 pathway by lovastatin modulated T0 to Th1 differentiation and reduced cytokine (IFN-γ and TNF-α) production, thus inducing Th2 cytokines (IL-4, IL-5, and IL-10). It inhibited T-bet (T box transcription factor) and NF-κB in activated T cells and significantly reduced infiltration of CD4- and MHC class II-positive cells to CNS. Further, it stabilized IL-4 production and GATA-3 expression in differentiated Th2 cells, whereas in differentiated Th1 cells it inhibited the expression of T-bet and reduced the production of IFN-γ. Moreover, lovastatin-exposed macrophage and BV2 (microglia) in allogeneic MLRs induced the production of the anti-inflammatory cytokine IL-10. These observations indicate that the anti-inflammatory effects of lovastatin are mediated via T cells as well as APCs, because it modulates the polarization patterns of naive T cell activation in an APC-independent system. Together, these findings reveal that lovastatin may have possible therapeutic value involving new targets (in both APCs and T cells) for the treatment of multiple sclerosis and other inflammatory diseases.
Cutting Edge: NF-κB2 Is a Negative Regulator of Dendritic Cell Function
Authors: Speirs Kendra , Lieberman Linda , Caamano Jorge , Hunter Christopher A. , Scott Phillip
Published: April 2014

RelB, a member of the NF-κB family of transcription factors, is essential for dendritic cell (DC) maturation. Recent findings indicate that RelB is exclusively regulated through its interaction with cytoplasmic NF-κB2/p100. The studies presented in this report show that DCs lacking NF-κB2 have dramatically enhanced RelB activity, associated with increased MHC class II and costimulatory molecule expression and an enhanced ability to induce CD4+ T cell responses. These studies identify a novel role for NF-κB2 in the negative regulation of RelB-induced DC maturation, with critical consequences for the regulation of adaptive immune responses.
Invariant Chain and the MHC Class II Cytoplasmic Domains Regulate Localization of MHC Class II Molecules to Lipid Rafts in Tumor Cell-Based Vaccines
Authors: Dolan Brian P. , Phelan Timothy P. , Ilkovitch Dan , Qi Ling , Wade William F. , Laufer Terri M. , Ostrand-Rosenberg Suzanne
Published: April 2014

Cell-based tumor vaccines, consisting of MHC class I+ tumor cells engineered to express MHC class II molecules, stimulate tumor-specific CD4+ T cells to mediate rejection of established, poorly immunogenic tumors. Previous experiments have demonstrated that these vaccines induce immunity by functioning as APCs for endogenously synthesized, tumor-encoded Ags. However, coexpression of the MHC class II accessory molecule invariant chain (Ii), or deletion of the MHC class II cytoplasmic domain abrogates vaccine immunogenicity. Recent reports have highlighted the role of lipid microdomains in Ag presentation. To determine whether Ii expression and/or truncation of MHC class II molecules impact vaccine efficacy by altering MHC class II localization to lipid microdomains, we examined the lipid raft affinity of MHC class II molecules in mouse M12.C3 B cell lymphomas and SaI/Ak sarcoma vaccine cells. Functional MHC class II heterodimers were detected in lipid rafts of both cell types. Interestingly, expression of Ii in M12.C3 cells or SaI/Ak cells blocked the MHC class II interactions with cell surface lipid rafts. In both cell types, truncation of either the α- or β-chain decreased the affinity of class II molecules for lipid rafts. Simultaneous deletion of both cytoplasmic domains further reduced localization of class II molecules to lipid rafts. Collectively, these data suggest that coexpression of Ii or deletion of the cytoplasmic domains of MHC class II molecules may reduce vaccine efficacy by blocking the constitutive association of MHC class II molecules with plasma membrane lipid rafts.
Regulation of Autoimmune Diabetes by Complete Freund’s Adjuvant Is Mediated by NK Cells
Authors: Lee I-Fang , Qin Huilian , Trudeau Jacqueline , Dutz Jan , Tan Rusung
Published: April 2014

Autoimmune (type 1) diabetes results from a loss of β cells that is mediated by self-reactive T cells. Previous studies have shown that a single injection of CFA prevents diabetes in nonobese diabetic (NOD) mice, but the mechanism(s) of protection remain unknown. We show here that NOD mice immunized with CFA have a markedly reduced incidence of diabetes and that this reduced incidence is associated with a decrease in the number of β cell-specific, autoreactive CTL. In addition, the adoptive transfer of diabetes into syngeneic NOD/SCID recipients was prevented by CFA immunization, and the protective effects of CFA were lost when cells expressing the NK cell marker, asialo GM1, were removed from both donor cells and recipient mice. Returning a population of CD3−DX5+ cells to the adoptive transfer restored the protective effects of CFA. Therefore, NK cells mediate the protective effects of CFA possibly through the down-regulation of autoreactive CTL and stimulation of NK cells represents a novel approach to the prevention of autoimmune diabetes.
IL-12p70-Dependent Th1 Induction by Human B Cells Requires Combined Activation with CD40 Ligand and CpG DNA
Authors: Wagner Moritz , Poeck Hendrik , Jahrsdoerfer Bernd , Rothenfusser Simon , Prell Domenik , Bohle Barbara , Tuma Evelyn , Giese Thomas , Ellwart Joachim W. , Endres Stefan , Hartmann Gunther
Published: April 2014

The detection of microbial molecules via Toll-like receptors (TLR) in B cells is not well characterized. In this study, we found that both naive and memory B cells lack TLR4 (receptor for LPS) but express TLR9 (receptor for CpG motifs) and produce IL-6, TNF-α, and IL-10 upon stimulation with CpG oligonucleotides (ODN), synthetic mimics of microbial DNA. Consistent with the lack of TLR4, purified B cells failed to respond to LPS. Similar to CpG ODN, CD40 ligand (CD40L) alone induced IL-6, TNF-α, and IL-10. Production of these cytokines as well as IgM synthesis was synergistically increased when both CpG ODN and CD40L were combined. Unlike IL-6, TNF-α, and IL-10, the Th1 cytokine IL-12p70 was detected only when both CpG ODN and CD40L were present, and its induction was independent of B cell receptor cross-linking. CpG ODN did not increase the capacity of CD40L-activated B cells to induce proliferation of naive T cells. However, B cells activated with CpG ODN and CD40L strongly enhanced IFN-γ production in developing CD4 T cells via IL-12. Together, these results demonstrate that IL-12p70 production in human B cells is under the dual control of microbial stimulation and T cell help. Our findings provide a molecular basis for the potent adjuvant activity of CpG ODN to support humoral immune responses observed in vivo, and for the limited value of LPS.
Early Growth Response Gene 3 Regulates Thymocyte Proliferation during the Transition from CD4−CD8− to CD4+CD8+1
Authors: Xi Hongkang , Kersh Gilbert J.
Published: April 2014

In thymocytes developing in the αβ lineage, the transition from CD4, CD8 double negative (DN) to CD4, CD8 double positive (DP) is associated with several rounds of cell division and changes in the expression of multiple genes. This transition is induced by the formation of a pre-TCR that includes a rearranged TCR β-chain and the pre-TCR α-chain. The mechanism by which the pre-TCR influences both gene expression and proliferation has not been defined. We have evaluated the role played by early growth response gene 3 (Egr3) in translating pre-TCR signals into differentiation and proliferation. Egr3 is a transcriptional regulator that contains a zinc-finger DNA binding domain. We find that Egr3-deficient mice have a reduced number of thymocytes compared with wild-type mice, and that this is due to poor proliferation during the DN to DP transition. Treatment of both Egr3+/+ and Egr3−/− mice on the Rag1−/− background with anti-CD3ε Ab in vivo results in similar differentiation events, but reduced cell recovery in the Egr3−/− mice. We have also generated transgenic mice that express high levels of Egr3 constitutively, and when these mice are bred onto a Rag1−/− background they exhibit increased proliferation in the absence of stimulation and have pre-TCR α-chain and CD25 down-regulation, as well as increased Cα expression. The results show that Egr3 is an important regulator of proliferation in response to pre-TCR signals, and that it also may regulate some specific aspects of differentiation.
Induction of Tumor Regression by Administration of B7-Ig Fusion Proteins: Mediation by Type 2 CD8+ T Cells and Dependence on IL-4 Production
Authors: Yamaguchi Nobuya , Hiraoka Shin-ichiro , Mukai Takao , Takeuchi Noritami , Zhou Xu-Yu , Ono Shiro , Kogo Mikihiko , Dunussi-Joannopoulos Kyriaki , Ling Vincent , Wolf Stanley , Fujiwara Hiromi
Published: April 2014

CD28 signals contribute to either type 1 or type 2 T cell differentiation. Here, we show that administration of B7.2-Ig fusion proteins to tumor-bearing mice induces tumor regression by promoting the differentiation of antitumor type 2 CD8+ effector T cells along with IL-4 production. B7.2-Ig-mediated regression was not induced in IL-4−/− and STAT6−/− mice. However, it was elicited in IFN-γ−/− and STAT4−/− mice. By contrast, IL-12-induced tumor regression occurred in IL-4−/− and STAT6−/− mice, but not in IFN-γ−/− and STAT4−/− mice. Moreover, B7.2-Ig treatment was effective in a tumor model not responsive to IL-12. B7.2-Ig administration elicited elevated levels of IL-4 production. Tumor regression was predominantly mediated by CD8+ T cells, although the induction of these effector cells required CD4+ T cells. Tumor regression induced by CD8+ T cells alone was inhibited by neutralizing the IL-4 produced during B7.2-Ig treatment. Thus, these results indicate that stimulation in vivo of CD28 with B7.2-Ig in tumor-bearing mice results in enhanced induction of antitumor type 2 CD8+ T cells (Tc2) leading to Tc2-mediated tumor regression.
Characterization of Defective CD4−CD8− T Cells in Murine Tumors Generated Independent of Antigen Specificity
Authors: Prins Robert M. , Incardona Francesca , Lau Regan , Lee Paul , Claus Sarah , Zhang Wenxuan , Black Keith L. , Wheeler Christopher J.
Published: April 2014

Immune-based therapy confers limited benefits to hosts bearing late-stage tumors. Mounting evidence points to local suppression of T cell function as the most substantial barrier to effective antitumor immunity in hosts with large tumor burdens. Despite this, events responsible for locally defective T cells and immune suppression in tumors remain unclear. We describe in this study a predominant T cell population localized within two murine tumors that is characterized by expression of apoptotic markers and TCRαβ/CD3, but not CD4, CD8, or NK-associated markers. These defective cells resembled double negative (DN) T cells in lpr mice, harbored defects in the expression of T cell signaling molecules, and produced the anti-inflammatory cytokine, IL-10. Conditions known to increase or decrease the accumulation of lpr DN T cells had corresponding effects on local DN tumor infiltrating lymphocyte (TIL) levels and inversely impacted host survival. Adoptive transfer into s.c. tumors demonstrated that naive CD8+ T cells were highly susceptible to becoming DN TIL, and local supplementation of tumors with nontumor Ag-bearing MHC class I-expressing fibroblasts decreased both this susceptibility and endogenous DN TIL levels. These findings identify a major defective T cell population with suppressive potential within tumors. The data also suggest that local T cell defectiveness is controlled by the tumor environment independent of cognate Ag specificity per se. Decreasing defective DN TIL levels by increasing noncognate peptide MHC class I availability, or modulating TCR or cytokine signaling may facilitate host survival by bolstering endogenous immunity to late-stage tumors, and may help improve therapeutic tumor vaccines.
Nucleotide Sequencing of Psoriatic Arthritis Tissue before and during Methotrexate Administration Reveals a Complex Inflammatory T Cell Infiltrate with Very Few Clones Exhibiting Features That Suggest They Drive the Inflammatory Process by Recognizing Autoantigens
Authors: Curran Shane A. , FitzGerald Oliver M. , Costello Patrick J. , Selby Jeanette M. , Kane David J. , Bresnihan Barry , Winchester Robert
Published: April 2014

Psoriatic arthritis is an interesting MHC class I allele associated autoimmune disease where injury is likely mediated exclusively by T cells. We used TCR β-chain nucleotide sequencing to gain insight into the adaptive immune events responsible for this injury and determine whether the numerous oligoclonal expansions of this disease represent extreme determinant spreading among driving clones that recognize autoantigen or were non-Ag-driven, inflammation-related expansions. Because methotrexate suppresses but does not eliminate this inflammation, we hypothesized that clones persisting during methotrexate treatment would likely drive the inflammation. Seventy-six percent of the T cell clones in active tissue were polyclonal and unexpanded, accounting for 31% of transcripts. They were decreased greatly by methotrexate. Strikingly, most expanded clones in the inflamed joint did not persist during methotrexate treatment, were found only in inflammatory sites, exhibited no structural homology to one another, and were either CD4 or CD8 in lineage, suggesting they were non-autoantigen-driven, inflammation-related expansions. Only 12% of the expanded clones could be grouped into clonal sets distinguished by structurally homologous CDR3 β-chain amino acid motifs suggesting Ag drive. These were exclusively CD8 in lineage, persisted during methotrexate administration, and were present in both joint fluid and blood implying they were candidate driver clones that recognized an autoantigen. However, a major set of putative driver clones exhibited a previously described EBV-specific β-chain motif, emphasizing that the dominant feature of the disease was activation of multiple clones apparently lacking specificity for an inciting autoantigen.
CD86 and CD80 Differentially Modulate the Suppressive Function of Human Regulatory T Cells
Authors: Zheng Yong , Manzotti Claire N. , Liu Michael , Burke Fiona , Mead Karen I. , Sansom David M.
Published: April 2014

Regulatory T cells (Treg) are important in maintaining tolerance to self tissues. As both CD28 and CTLA-4 molecules are implicated in the function of Treg, we investigated the ability of their two natural ligands, CD80 and CD86, to influence the Treg-suppressive capacity. During T cell responses to alloantigens expressed on dendritic cells, we observed that Abs against CD86 potently enhanced suppression by CD4+CD25+ Treg. In contrast, blocking CD80 enhanced proliferative responses by impairing Treg suppression. Intriguingly, the relative expression levels of CD80 and CD86 on dendritic cells are modulated during progression from an immature to a mature state, and this correlates with the ability of Treg to suppress responses. Our data show that CD80 and CD86 have opposing functions through CD28 and CTLA-4 on Treg, an observation that has significant implications for manipulation of immune responses and tolerance in vivo.
In Vivo VL-Targeted Activation-Induced Apoptotic Supraclonal Deletion by a Microbial B Cell Toxin
Authors: Goodyear Carl S. , Narita Masami , Silverman Gregg J.
Published: April 2014

To interfere with host immune responses, some microbial pathogens produce proteins with the properties of superantigens, which can interact via conserved V region framework subdomains of the Ag receptors of lymphocytes rather than the complementarity-determining region involved in the binding of conventional Ags. In recent studies, we have elucidated how a model B cell superantigen affects the host immune system by targeting a conserved VH site on the Ag receptors of B lymphocytes. To determine whether these findings represent a general paradigm, we investigated the in vivo immunobiologic properties of protein L of Peptostreptococcus magnus (PpL), a microbial Ig-binding protein specific for a V region site on Ig L chains. Our studies confirmed that PpL binding is restricted to a subset of murine Vκ-expressing B cells, and found that B cells with stronger PpL-binding activity are associated with certain B cell subsets: splenic marginal zone (CD21high CD23low), splenic CD1+, peritoneal B-1a (IgDlow CD5+), and CD21high CD24high B cells in peripheral lymph nodes, mesenteric lymph nodes, and Peyer’s patches. Infusion of PpL triggered a sequence of events in B cell receptor (BCR)-targeted B cells, with rapid down-regulation of BCR, the induction of an activation phenotype, and limited rounds of proliferation. Apoptosis followed through a process heralded by the dissipation of mitochondrial membrane potential, the induction of the caspase pathway, DNA fragmentation, and the deposition of B cell apoptotic bodies. These studies define a common pathway by which microbial toxins that target V region-associated BCR sites induce programmed cell death.
Targeting of Secretory IgA to Peyer’s Patch Dendritic and T Cells after Transport by Intestinal M Cells
Authors: Rey Jacques , Garin Nathalie , Spertini François , Corthésy Blaise
Published: April 2014

In addition to being instrumental to the protection of mucosal epithelia, secretory IgA (SIgA) adheres to and is transported by intestinal Peyer’s patch (PP) M cells. The possible functional reason for this transport is unknown. We have thus examined in mice the outcome of SIgA delivered from the intestinal lumen to the cells present in the underlying organized mucosa-associated lymphoreticular tissue. We show selective association of SIgA with dendritic cells and CD4+ T and B lymphocytes recovered from PP in vitro. In vivo, exogenously delivered SIgA is able to enter into multiple PP lining the intestine. In PP, SIgA associates with and is internalized by dendritic cells in the subepithelial dome region, whereas the interaction with CD4+ T cells is limited to surface binding. Interaction between cells and SIgA is mediated by the IgA moiety and occurs for polymeric and monomeric molecular forms. Thus, although immune exclusion represents the main function of SIgA, transport of the Ab by M cells might promote Ag sampling under neutralizing conditions essential to the homeostasis of mucosal surfaces.
Postsecretory Processing Generates Multiple Cathelicidins for Enhanced Topical Antimicrobial Defense
Authors: Murakami Masamoto , Lopez-Garcia Belen , Braff Marissa , Dorschner Robert A. , Gallo Richard L.
Published: April 2014

The production of antimicrobial peptides and proteins is essential for defense against infection. Many of the known human antimicrobial peptides are multifunctional, with stimulatory activities such as chemotaxis while simultaneously acting as natural antibiotics. In humans, eccrine appendages express DCD and CAMP, genes encoding proteins processed into the antimicrobial peptides dermcidin and LL-37. In this study we show that after secretion onto the skin surface, the CAMP gene product is processed by a serine protease-dependent mechanism into multiple novel antimicrobial peptides distinct from the cathelicidin LL-37. These peptides show enhanced antimicrobial action, acquiring the ability to kill skin pathogens such as Staphylococcus aureus and Candida albicans. Furthermore, although LL-37 may influence the host inflammatory response by stimulating IL-8 release from keratinocytes, this activity is lost in subsequently processed peptides. Thus, a single gene product encoding an important defense molecule alters structure and function in the topical environment to shift the balance of activity toward direct inhibition of microbial colonization.
Leishmania infantum Promotes Replication of HIV Type 1 in Human Lymphoid Tissue Cultured Ex Vivo by Inducing Secretion of the Proinflammatory Cytokines TNF-α and IL-1α
Authors: Zhao Chenqi , Papadopoulou Barbara , Tremblay Michel J.
Published: April 2014

Parasitic infections such as leishmaniasis can modulate the life cycle of HIV-1 and disease progression. Coinfection with HIV-1 and Leishmania has emerged as a serious threat in countries where both pathogenic agents are widespread. Although there are numerous clinical reports illustrating the cofactor role played by Leishmania in HIV-1-infected patients, there is still no information on the contribution of Leishmania to the biology of HIV-1 in human lymphoid tissue that is considered a major in vivo site of virus production. In this study we explored the modulatory effect of Leishmania on the process of HIV-1 infection using ex vivo cultured human tonsillar tissue. We found that the protozoan parasite Leishmania enhances both HIV-1 transcription and virus production after infection of human tonsillar tissue infected ex vivo with viral strains bearing various coreceptor usage profiles. Studies conducted with pentoxifylline and neutralizing Abs revealed that the Leishmania-mediated increase in HIV-1 production was linked to a higher production of TNF-α and IL-1α. Our findings help to unravel the molecular mechanism(s) through which the two microorganisms interact and provide information that may be useful for the design of more effective therapeutic strategies aimed at controlling disease progression in persons dually infected with HIV-1 and Leishmania. This work also indicates that histocultures of human lymphoid tissue infected by both pathogens represent an ideal experimental cell system to dissect interactions occurring between HIV-1 and an opportunist pathogen in a human microenvironment that approximates conditions prevailing under physiological situations.
Coordinate Expression of Cytokines and Chemokines by NK Cells during Murine Cytomegalovirus Infection
Authors: Dorner Brigitte G. , Smith Hamish R.C. , French Anthony R. , Kim Sungjin , Poursine-Laurent Jennifer , Beckman Diana L. , Pingel Jeanette T. , Kroczek Richard A. , Yokoyama Wayne M.
Published: April 2014

Cytokines and chemokines activate and direct effector cells during infection. We previously identified a functional group of five cytokines and chemokines, namely, IFN-γ, activation-induced T cell-derived and chemokine-related cytokine/lymphotactin, macrophage-inflammatory protein 1α, macrophage-inflammatory protein 1β, and RANTES, coexpressed in individual activated NK cells, CD8+ T cells, and CD4+ Th1 cells in vitro and during in vivo infections. However, the stimuli during infection were not known. In murine CMV (MCMV) infection, the DAP12/KARAP-associated Ly49H NK cell activation receptor is crucial for resistance through recognition of MCMV-encoded m157 but NK cells also undergo in vivo nonspecific responses to uncharacterized stimuli. In this study, we show that Ly49H ligation by m157 resulted in a coordinated release of all five cytokines/chemokines from Ly49H+ NK cells. Whereas other cytokines also triggered the release of these cytokines/chemokines, stimulation was not confined to the Ly49H+ population. At the single-cell level, the production of the five mediators showed strong positive correlation with each other. Interestingly, NK cells were a major source of these five cytokines/chemokines in vitro and in vivo, whereas infected macrophages produced only limited amounts of macrophage-inflammatory protein 1α, macrophage-inflammatory protein1β, and RANTES. These findings suggest that both virus-specific and nonspecific NK cells play crucial roles in activating and directing other inflammatory cells during MCMV infection.
Toll-Like Receptor 2 Plays a Role in the Early Inflammatory Response to Murine Pneumococcal Pneumonia but Does Not Contribute to Antibacterial Defense
Authors: Knapp Sylvia , Wieland Catharina W. , van ’t Veer Cornelis , Takeuchi Osamu , Akira Shizuo , Florquin Sandrine , van der Poll Tom
Published: April 2014

Toll-like receptors (TLR) are crucial pattern recognition receptors in innate immunity. The importance of TLR2 in host defense against Gram-positive bacteria has been suggested by the fact that this receptor recognizes major Gram-positive cell wall components, such as peptidoglycan and lipoteichoic acid. To determine the role of TLR2 in pulmonary Gram-positive infection, we first established that TLR2 is indispensable for alveolar macrophage responsiveness toward Streptococcus pneumoniae. Nonetheless, TLR2 gene-deficient mice intranasally inoculated with S. pneumoniae at doses varying from nonlethal (with complete clearance of the infection) to lethal displayed only a modestly reduced inflammatory response in their lungs and an unaltered antibacterial defense when compared with normal wild-type mice. These data suggest that TLR2 plays a limited role in the innate immune response to pneumococcal pneumonia, and that additional pattern recognition receptors likely are involved in host defense against this common respiratory pathogen.
Comprehensive Early and Lasting Loss of Memory CD8 T Cells and Functional Memory during Acute and Persistent Viral Infections
Authors: Kim Sung-Kwon , Welsh Raymond M.
Published: April 2014

Viral infections have been shown to induce lymphopenias that lower memory CD8 T cell frequencies, and they also have been shown to cause a permanent loss of memory cells specific to previously encountered pathogens. In this study, the patterns and significance of virus-induced memory CD8 T cell depletion were examined in mice immune to heterologous (Pichinde, vesicular stomatitis, vaccinia) viruses and subsequently challenged with acute or persistent lymphocytic choriomeningitis virus infections. Memory CD8 T cell loss was comprehensive and occurred in both lymphoid and peripheral tissues of the immune host. The impact of the loss of memory T cells was reflected by in vivo cytotoxicity assays, which showed decreased clearance of epitope-expressing targets. Memory CD8 T cell loss occurred very early (day 2) after infection, and was thereafter sustained, consistent more with an active deletion model than with a competition model. Cross-reactive T cells, in contrast, increased in number, but memory cells were reduced whether or not there was competition from cross-reactive T cells. Memory T cell loss was more profound during persistent infection than after acute infection. Adoptive transfer studies showed that, unlike the resolved acute infection, in which the reduced memory frequencies became stable, memory T cell loss was a continuously ongoing process during persistent infection. This study therefore links an early virus-induced lymphopenia to a subsequent long-term loss of CD8 T cell memory and offers a new mechanism for immune deficiency during persistent viral infections.
Depletion of CD4+ T Cells during Immunization with Nonviable Listeria monocytogenes Causes Enhanced CD8+ T Cell-Mediated Protection against Listeriosis
Authors: Kursar Mischo , Köhler Anne , Kaufmann Stefan H. E. , Mittrücker Hans-Willi
Published: April 2014

Immunization of mice with nonviable Listeria monocytogenes generates an insufficient CD8+ T cell response and consequently only limited protection against subsequent L. monocytogenes infection. We have recently demonstrated that depletion of regulatory CD4+ T cells during immunization significantly enhances CD8+ T cell responses. In the present study, we determined the impact of CD4+ T cell depletion on the CD8+ T cell response against heat-killed Listeria. Treatment of mice with anti-CD4 mAb during boost immunization with heat-killed Listeria significantly increased numbers of Listeria-specific CD8+ T cells and improved protection against subsequent infection with L. monocytogenes. During challenge infection, numbers of Listeria-specific CD8+ T cells were enhanced, and these cells expressed effector functions in terms of IFN-γ production. In summary, we demonstrate that combining nonviable L. monocytogenes vaccination and CD4+ T cell depletion improves generation of long-lasting and functional Listeria-specific CD8+ memory T cells.
Cryptic Epitope Identified in Rat and Human Cardiac Myosin S2 Region Induces Myocarditis in the Lewis Rat
Authors: Li Ya , Heuser Janet S. , Kosanke Stanley D. , Hemric Mark , Cunningham Madeleine W.
Published: April 2014

Myocarditis is a common cause of dilated cardiomyopathy leading to heart failure. Chronic stages of myocarditis may be initiated by autoimmune responses to exposed cardiac Ags after myocyte damage. Cardiac myosin, a heart autoantigen, induced experimental autoimmune myocarditis (EAM) in susceptible animals. Although cardiac myosin-induced myocarditis has been reported in Lewis rats, the main pathogenic epitope has not been identified. Using overlapping synthetic peptides of the S2 region of human cardiac myosin, we identified an amino acid sequence, S2–16 (residues 1052–1076), that induced severe myocarditis in Lewis rats. The myocarditic epitope was localized to a truncated S2–16 peptide (residues 1052–1073), which contained a sequence identical in human and rat cardiac myosin. The S2–16 peptide was not myocarditic for three other strains of rats, in which the lack of myocarditis was accompanied by the absence of strong S2–16-specific lymphocyte responses in vitro. For Lewis rats, S2–16 was characterized as a cryptic epitope of cardiac myosin because it did not recall lymphocyte and Ab responses after immunization with cardiac myosin. Lymphocytes from S2–16 immunized rats recognized not only S2–16, but also peptides in the S2–28 region. Furthermore, peptide S2–28 was the dominant epitope recognized by T cells from cardiac myosin immunized rats. S2–16 was presented by Lewis rat MHC class II molecules, and myocarditis induction was associated with an up-regulation of inflammatory cytokine production. S2–16-induced EAM provides a defined animal model to investigate mechanisms of EAM and modulation of immune responses to prevent autoimmune myocarditis.
Long-Term Suppression of Tumor Growth by TNF Requires a Stat1- and IFN Regulatory Factor 1-Dependent IFN-γ Pathway but Not IL-12 or IL-18
Authors: Wu Terry H. , Pabin Christine N. , Qin Zhihai , Blankenstein Thomas , Philip Mary , Dignam James , Schreiber Karin , Schreiber Hans
Published: April 2014

Tumor cells engineered to secrete TNF were used as a model to examine how persistently high local concentrations of TNF suppress tumor growth. TNF secretion had no effect on tumor cell proliferation in vitro but caused a very impressive growth arrest in vivo that was dependent on both bone marrow- and non-bone marrow-derived host cells expressing TNFR. Suppression also required an endogenous IFN-γ pathway consisting minimally of IFN-γ, IFN-γ receptor, Stat1, and IFN regulatory factor 1 since mice with targeted disruption of any of the four genes failed to arrest tumor growth. The ability of these mice to suppress tumor growth was restored after they were reconstituted with bone marrow cells from Wt mice. Interestingly, mice lacking the major IFN-γ-inducing cytokines IL-12 and IL-18 or T cells, B cells, and the majority of NK cells that are potential sources of IFN-γ nevertheless inhibited tumor development. Moreover, multiple lines of evidence indicated that local release of IFN-γ was not required to inhibit tumor formation. These results strongly suggest a novel function for the endogenous IFN-γ pathway that without measurable IFN-γ production or activity affects the ability of TNF to suppress tumor development.
Role of Specific CD8+ T Cells in the Severity of a Fulminant Zoonotic Viral Hemorrhagic Fever, Hantavirus Pulmonary Syndrome
Authors: Kilpatrick Elizabeth D. , Terajima Masanori , Koster Frederick T. , Catalina Michelle D. , Cruz John , Ennis Francis A.
Published: April 2014

We report on the role of specific CD8+ T cells in the pathogenesis of a highly lethal human viral disease, hantavirus pulmonary syndrome (HPS). HPS is a zoonotic disease caused by transmission of Sin Nombre virus (SNV) from chronically infected deer mice. In humans, this fulminant infection is characterized by lung capillary leakage, respiratory failure, and cardiogenic shock. Individuals with HLA-B*3501 have an increased risk of developing severe HPS, suggesting that CD8+ T cell responses to SNV contribute to pathogenesis. We identified three CD8+ T cell epitopes in SNV presented by HLA-B*3501 and quantitated circulating SNV-specific CD8+ T cells in 11 acute HPS patients using HLA/peptide tetramers. We found significantly higher frequencies of SNV-specific T cells in patients with severe HPS requiring mechanical ventilation (up to 44.2% of CD8+ T cells) than in moderately ill HPS patients hospitalized but not requiring mechanical ventilation (up to 9.8% of CD8+ T cells). These results imply that virus-specific CD8+ T cells contribute to HPS disease outcome. Intense CD8+ T cell responses to SNV may be induced by the encounter of the unnatural human host to this zoonotic virus without coevolution. This may also be the immunopathologic basis of other life-threatening human virus infections.