bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytokine Polymorphisms and Histologic Expression in Autopsy Studies: Contribution of TNF-α and TGF-β1 to the Pathogenesis of Autoimmune-Associated Congenital Heart Block
Authors: Clancy Robert M. , Backer Chelsea B. , Yin Xiaoming , Kapur Raj P. , Molad Yair , Buyon Jill P.
Published: April 2014

Although Abs to SSA/Ro-SSB/La are necessary for the development of congenital heart block (CHB), the low frequency suggests that fetal factors are contributory. Because CHB involves a cascade from inflammation to scarring, polymorphisms of the TNF-α promoter region and codons 10 and 25 of the TGF-β gene were evaluated in 88 children (40 CHB, 17 rash, 31 unaffected siblings) and 74 mothers from the Research Registry for Neonatal Lupus (NL). Cytokine expression was assessed in autopsy material from two fetuses with CHB. Significantly increased frequency of the −308A (high-producer) allele of TNF-α was observed in all NL groups compared with controls. In contrast, the TGF-β polymorphism Leu10 (associated with increased fibrosis) was significantly higher in CHB children (genotypic frequency 60%, allelic frequency 78%) than unaffected offspring (genotypic frequency 29%, p = 0.016; allelic frequency 56%, p = 0.011) and controls, while there were no significant differences between controls and other NL groups. For the TGF-β polymorphism, Arg25, there were no significant differences between NL groups and controls. In fetal CHB hearts, protein expression of TGF-β, but not TNF-α, was demonstrated in septal regions, extracellularly in the fibrous matrix, and intracellularly in macrophage infiltrates. Age-matched fetal hearts from voluntary terminations expressed neither cytokine. TNF-α may be one of several factors that amplify susceptibility; however, the genetic studies, backed by the histological data, more convincingly link TGF-β to the pathogenesis of CHB. This profibrosing cytokine and its secretion/activation circuitry may provide a novel direction for evaluating fetal factors in the development of a robust animal model of CHB as well as therapeutic strategies in humans.
Cytokine Milieu of Atopic Dermatitis, as Compared to Psoriasis, Skin Prevents Induction of Innate Immune Response Genes
Authors: Nomura Ichiro , Goleva Elena , Howell Michael D. , Hamid Quatyba A. , Ong Peck Y. , Hall Clifton F. , Darst Marc A. , Gao Bifeng , Boguniewicz Mark , Travers Jeffrey B. , Leung Donald Y. M.
Published: April 2014

Atopic dermatitis (AD) and psoriasis are the two most common chronic skin diseases. However patients with AD, but not psoriasis, suffer from frequent skin infections. To understand the molecular basis for this phenomenon, skin biopsies from AD and psoriasis patients were analyzed using GeneChip microarrays. The expression of innate immune response genes, human β defensin (HBD)-2, IL-8, and inducible NO synthetase (iNOS) was found to be decreased in AD, as compared with psoriasis, skin (HBD-2, p = 0.00021; IL-8, p = 0.044; iNOS, p = 0.016). Decreased expression of the novel antimicrobial peptide, HBD-3, was demonstrated at the mRNA level by real-time PCR (p = 0.0002) and at the protein level by immunohistochemistry (p = 0.0005). By real-time PCR, our data confirmed that AD, as compared with psoriasis, is associated with elevated skin production of Th2 cytokines and low levels of proinflammatory cytokines such as TNF-α, IFN-γ, and IL-1β. Because HBD-2, IL-8, and iNOS are known to be inhibited by Th2 cytokines, we examined the effects of IL-4 and IL-13 on HBD-3 expression in keratinocyte culture in vitro. We found that IL-13 and IL-4 inhibited TNF-α- and IFN-γ-induced HBD-3 production. These studies indicate that decreased expression of a constellation of antimicrobial genes occurs as the result of local up-regulation of Th2 cytokines and the lack of elevated amounts of TNF-α and IFN-γ under inflammatory conditions in AD skin. These observations could explain the increased susceptibility of AD skin to microorganisms, and suggest a new fundamental rule that may explain the mechanism for frequent infection in other Th2 cytokine-mediated diseases.
Successful Allogeneic Neonatal Bone Marrow Transplantation Devoid of Myeloablation Requires Costimulatory Blockade
Authors: Soper Brian W. , Lessard Mark D. , Jude Craig D. , Schuldt Adam J. T. , Bunte Ralph M. , Barker Jane E.
Published: April 2014

A significant number of nonmalignant, progressive childhood disorders respond to bone marrow transplantation (BMT). Toxic myeloablative pretreatment regimens, graft failure, and graft-vs-host disease complicate the utility of BMT for neonatal treatment. We recently demonstrated high-dose BMT in neonatal animals enables chimeric engraftment without toxic myeloablation. Reagents that block T cell costimulation (anti-CD40L mAb and/or CTLA-4Ig) establish tolerant allogeneic engraftment in adult recipients. Donor lymphocyte infusion (DLI) re-establishes failing grafts and treats malignant relapse via a graft-vs-leukemia response. In this study, we tested the hypothesis that combining these approaches would allow tolerant allogeneic engraftment devoid of myeloablation in neonatal normal and mutant mice with lysosomal storage disease. Tolerant chimeric allogeneic engraftment was achieved before DLI only in the presence of both anti-CD40L mAb and CTLA-4Ig. DLI amplified allografts to full donor engraftment long-term. DLI-treated mice either maintained long-term tolerance or developed late-onset chronic graft-vs-host disease. This combinatorial approach provides a nontoxic method to establish tolerant allogeneic engraftment for treatment of progressive childhood diseases.
Betulinic Acid Suppresses Carcinogen-Induced NF-κB Activation Through Inhibition of IκBα Kinase and p65 Phosphorylation: Abrogation of Cyclooxygenase-2 and Matrix Metalloprotease-9
Authors: Takada Yasunari , Aggarwal Bharat B.
Published: April 2014

Betulinic acid (BA), a pentacyclic triterpene isolated from the bark of the white birch tree, has been reported to be a selective inducer of apoptosis in tumor cells. It also exhibits anti-inflammatory and immunomodulatory properties. How BA mediates these effects is not known. Because of the critical role of the transcription factor NF-κB in growth modulatory, inflammatory, and immune responses, we postulated that BA modulates the activity of this factor. In this study we investigated the effect of BA on NF-κB and NF-κB-regulated gene expression activated by a variety of inflammatory and carcinogenic agents. BA suppressed NF-κB activation induced by TNF, PMA, cigarette smoke, okadaic acid, IL-1, and H2O2. The suppression of NF-κB activation was not cell-type specific. BA suppressed the activation of IκBα kinase, thus abrogating the phosphorylation and degradation of IκBα. We found that BA inhibited NF-κB activated by TNFR 1, TNFR-associated death domain, TNFR-associated factor 2, NF-κB-inducing kinase, and IκBα kinase. Treatment of cells with this triterpinoid also suppressed NF-κB-dependent reporter gene expression and the production of NF-κB-regulated gene products such as cyclooxygenase-2 and matrix metaloproteinase-9 induced by inflammatory stimuli. Furthermore, BA enhanced TNF-induced apoptosis. Overall, our results indicated that BA inhibits activation of NF-κB and NF-κB-regulated gene expression induced by carcinogens and inflammatory stimuli. This may provide a molecular basis for the ability of BA to mediate apoptosis, suppress inflammation, and modulate the immune response.
High Efficiency TCR Gene Transfer into Primary Human Lymphocytes Affords Avid Recognition of Melanoma Tumor Antigen Glycoprotein 100 and Does Not Alter the Recognition of Autologous Melanoma Antigens
Authors: Morgan Richard A. , Dudley Mark E. , Yu Yik Y. L. , Zheng Zhili , Robbins Paul F. , Theoret Marc R. , Wunderlich John R. , Hughes Marybeth S. , Restifo Nicholas P. , Rosenberg Steven A.
Published: April 2014

The α- and β-chains of the TCR from a highly avid anti-gp100 CTL clone were isolated and used to construct retroviral vectors that can mediate high efficiency gene transfer into primary human lymphocytes. Expression of this TCR gene was confirmed by Western blot analysis, immunocytometric analysis, and HLA Ag tetramer staining. Gene transfer efficiencies of >50% into primary lymphocytes were obtained without selection for transduced cells using a method of prebinding retroviral vectors to cell culture vessels before the addition of lymphocytes. The biological activity of transduced cells was confirmed by cytokine production following coculture with stimulator cells pulsed with gp100 peptides, but not with unrelated peptides. The ability of this anti-gp100 TCR gene to transfer high avidity Ag recognition to engineered lymphocytes was confirmed in comparison with highly avid antimelanoma lymphocytes by the high levels of cytokine production (>200,000 pg/ml IFN-γ), by recognition of low levels of peptide (<200 pM), and by HLA class I-restricted recognition and lysis of melanoma tumor cell lines. CD4+ T cells engineered with this anti-gp100 TCR gene were Ag reactive, suggesting CD8-independent activity of the expressed TCR. Finally, nonmelanoma-reactive tumor-infiltrating lymphocyte cultures developed antimelanoma activity following anti-gp100 TCR gene transfer. In addition, tumor-infiltrating lymphocytes with reactivity against non-gp100 melanoma Ags acquired gp100 reactivity and did not lose the recognition of autologous melanoma Ags following gp100 TCR gene transfer. These results suggest that lymphocytes genetically engineered to express anti-gp100 TCR may be of value in the adoptive immunotherapy of patients with melanoma.
FcγRIIa Is Expressed on Natural IFN-α-Producing Cells (Plasmacytoid Dendritic Cells) and Is Required for the IFN-α Production Induced by Apoptotic Cells Combined with Lupus IgG
Authors: Båve Ullvi , Magnusson Mattias , Eloranta Maija-Leena , Perers Anders , Alm Gunnar V. , Rönnblom Lars
Published: April 2014

An ongoing production of IFN-α may be of etiopathogenic significance in systemic lupus erythematosus (SLE). It may be due to the natural IFN-producing cells (NIPC), also termed plasmacytoid dendritic cells (PDC), activated by immune complexes that contain nucleic acids derived from apoptotic cells. We here examined the role of FcγR in the IFN-α production in vitro by PBMC induced by the combination of apoptotic U937 cells and autoantibody-containing IgG from SLE patients (SLE-IgG). The Fc portion of the SLE-IgG was essential to induce IFN-α production, because Fab fragments or F(ab′)2 were ineffective. Normal, especially heat-aggregated, IgG inhibited the IFN-α production, suggesting a role for FcγR on PBMC. Using blocking anti-FcγR Abs, the FcγRIIa,c (CD32) but not FcγRI or FcγRIII were shown to be involved in the IFN-α induction by apoptotic cells combined with SLE-IgG, but not by HSV or CpG DNA. In contrast, the action of all of these inducers was inhibited by the anti-FcγRIIa,b,c mAb AT10 or heat-aggregated IgG. Flow cytometric analysis revealed that ∼50% of the BDCA-2-positive PBMC, i.e., NIPC/PDC, expressed low but significant levels of FcγRII, as did most of the actual IFN-α producers activated by HSV. RT-PCR applied to NIPC/PDC purified by FACS demonstrated expression of FcγRIIa, but not of FcγRIIb or FcγRIIc. We conclude that FcγRIIa on NIPC/PDC is involved in the activation of IFN-α production by interferogenic immune complexes, but may also mediate inhibitory signals. The FcγRIIa could therefore have a key function in NIPC/PDC and be a potential therapeutic target in SLE.
Murine CD8+ Recent Thymic Emigrants are αE Integrin-Positive and CC Chemokine Ligand 25 Responsive
Authors: Staton Tracy L. , Johnston Brent , Butcher Eugene C. , Campbell Daniel J.
Published: April 2014

Recent thymic emigrants (RTE) are an important subpopulation of naive CD8+ T cells because of their ability to reconstitute a diverse immune system after periods of T cell depletion. In neonatal mice, the majority of peripheral T lymphocytes are RTE, cells that have recently left the thymus to populate the periphery. Postulating that these cells could have unique trafficking mechanisms, we compared adhesion molecule and chemokine receptor expression of neonatal RTE with mature adult lymphocytes. Neonatal CD8+ splenocytes uniformly express αE integrin and exhibit a high responsiveness to CC chemokine ligand (CCL25) (as compared with adult CD8+ splenocytes). Mature CD8+ thymocytes have a similar αE integrin+ CCL25 responsive phenotype, as do adult CD8+ RTE identified by intrathymic FITC injection. With increasing age, the frequency of CD8+ αE integrin+ splenocytes decreases, roughly correlating with thymic involution. Moreover, halting thymic output by thymectomy accelerates the age-dependent decline in peripheral CD8+ αE integrin+ RTE phenotype cells. Low expression of CD44 distinguishes these CD8+ RTE from a population of memory phenotype αE integrin+ CD8+ cells that are CD44high. We conclude that CD8+ RTE have unique adhesive and chemotactic properties that distinguish them from naive CD8+ T cells. These properties may enable specialized microenvironmental and cell-cell interactions contributing to the fate of RTE in the periphery during the early post-thymic period. This phenotype will also facilitate the identification and isolation of RTE for further studies.
Initial Antigen Encounter Programs CD8+ T Cells Competent to Develop into Memory Cells That Are Activated in an Antigen-Free, IL-7- and IL-15-Rich Environment
Authors: Carrio Roberto , Bathe Oliver F. , Malek Thomas R.
Published: April 2014

Although much is known concerning the immunobiology of CD8+ T memory cells, the initial events favoring the generation of CD8+ T memory cells remain poorly defined. Using a culture system that yields memory-like CD8+ T cells, we show that 1 day after Ag encounter, Ag-activated T cells developed into memory-like T cells, but this optimally occurred 3 days after Ag encounter. Key phenotypic, functional, and molecular properties that typify central memory T cells were expressed within 48 h when the activated CD8+ T cells were cultured with IL-7 or IL-15 in the absence of Ag or following transfer into normal mice. These data support a model whereby Ag activation of naive CD8+ T cells not only programs effector cell expansion and contraction but the potential to develop into a memory cell which ensues in an Ag-free environment containing IL-7 or IL-15.
A Novel Role for p21-Activated Protein Kinase 2 in T Cell Activation
Authors: Chu Peter C. , Wu Jun , Liao X. Charlene , Pardo Jorge , Zhao Haoran , Li Congfen , Mendenhall Marcy K. , Pali Erlina , Shen Mary , Yu Simon , Taylor Vanessa C. , Aversa Gregorio , Molineaux Susan , Payan Donald G. , Masuda Esteban S.
Published: April 2014

To identify novel components of the TCR signaling pathway, a large-scale retroviral-based functional screen was performed using CD69 expression as a marker for T cell activation. In addition to known regulators, two truncated forms of p21-activated kinase 2 (PAK2), PAK2ΔL1–224 and PAK2ΔS1–113, both lacking the kinase domain, were isolated in the T cell screen. The PAK2 truncation, PAK2ΔL, blocked Ag receptor-induced NFAT activation and TCR-mediated calcium flux in Jurkat T cells. However, it had minimal effect on PMA/ionomycin-induced CD69 up-regulation in Jurkat cells, on anti-IgM-mediated CD69 up-regulation in B cells, or on the migratory responses of resting T cells to chemoattractants. We show that PAK2 kinase activity is increased in response to TCR stimulation. Furthermore, a full-length kinase-inactive form of PAK2 blocked both TCR-induced CD69 up-regulation and NFAT activity in Jurkat cells, demonstrating that kinase activity is required for PAK2 function downstream of the TCR. We also generated a GFP-fused PAK2 truncation lacking the Cdc42/Rac interactive binding region domain, GFP-PAK283–149. We show that this construct binds directly to the kinase domain of PAK2 and inhibits anti-TCR-stimulated T cell activation. Finally, we demonstrate that, in primary T cells, dominant-negative PAK2 prevented anti-CD3/CD28-induced IL-2 production, and TCR-induced CD40 ligand expression, both key functions of activated T cells. Taken together, these results suggest a novel role for PAK2 as a positive regulator of T cell activation.
Simvastatin Augments Lipopolysaccharide-Induced Proinflammatory Responses in Macrophages by Differential Regulation of the c-Fos and c-Jun Transcription Factors
Authors: Matsumoto Makoto , Einhaus Derek , Gold Elizabeth S. , Aderem Alan
Published: April 2014

The 3-hydroxyl-3-methylglutaryl-coenzyme A reductase inhibitors, or statins, are a widely used class of drugs for cholesterol reduction. The reduction in mortality and morbidity in statin-treated patients is incompletely explained by their effects on cholesterol, and an anti-inflammatory role for the drug has been proposed. We report in this work that, unexpectedly, simvastatin enhances LPS-induced IL-12p40 production by murine macrophages, and that it does so by activating the IL-12p40 promoter. Mutational analysis and dominant-negative expression studies indicate that both C/EBP and AP-1 transcription factors have a crucial role in promoter activation. This occurs via a c-Fos- and c-Jun-based mechanism; we demonstrate that ectopic expression of c-Jun activates the IL-12p40 promoter, whereas expression of c-Fos inhibits IL-12p40 promoter activity. Simvastatin prevents LPS-induced c-Fos expression, thereby relieving the inhibitory effect of c-Fos on the IL-12p40 promoter. Concomitantly, simvastatin induces the phosphorylation of c-Jun by the c-Jun N-terminal kinase, resulting in c-Jun-dependent activation of the IL-12p40 promoter. This appears to be a general mechanism because simvastatin also augments LPS-dependent activation of the TNF-α promoter, perhaps because the TNF-α promoter has C/EBP and AP-1 binding sites in a similar configuration to the IL-12p40 promoter. The fact that simvastatin potently augments LPS-induced IL-12p40 and TNF-α production has implications for the treatment of bacterial infections in statin-treated patients.
KIR2DL5 Can Inhibit Human NK Cell Activation Via Recruitment of Src Homology Region 2-Containing Protein Tyrosine Phosphatase-2 (SHP-2)
Authors: Yusa Sei-ichi , Catina Tracey L. , Campbell Kerry S.
Published: April 2014

Human NK cells use class I MHC-binding inhibitory receptors, such as the killer cell Ig-like receptor (KIR) family, to discriminate between normal and abnormal cells. Some tumors and virus-infected cells down-regulate class I MHC and thereby become targets of NK cells. Substantial evidence indicates that the mechanism of KIR-mediated inhibition involves recruitment of the protein tyrosine phosphatases, Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) and SHP-2, to two phosphorylated cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). KIR2DL5 is a type II member of the KIR2D family with an atypical extracellular domain and an intracytoplasmic domain containing one typical ITIM and one atypical ITIM sequence. Although KIR2DL5 structure is expressed by ∼50% of humans and is conserved among primate species, its function has not been determined. In the present study, we directly compared functional and biochemical properties of KIR2DL5, KIR3DL1 (a type I KIR with two ITIMs), and KIR2DL4 (the only other type II KIR, which has a single ITIM) in a human NK-like cell line. Our results show that KIR2DL5 is an inhibitory receptor that can recruit both SHP-1 and SHP-2, and its inhibitory capacity is more similar to that of the cytoplasmic domain of KIR2DL4 than KIR3DL1. Interestingly, inhibition of NK cell cytotoxicity by KIR2DL5 was blocked by dominant-negative SHP-2, but not dominant-negative SHP-1, whereas both dominant-negative phosphatases can block inhibition by KIR3DL1. Therefore, the cytoplasmic domains of type II KIRs (2DL4 and 2DL5) exhibit distinct inhibitory capacities when compared with type I KIRs (3DL1), due to alterations in the canonical ITIM sequences.
Host Dendritic Cells Alone Are Sufficient to Initiate Acute Graft-versus-Host Disease
Authors: Duffner Ulrich A. , Maeda Yoshinobu , Cooke Kenneth R. , Reddy Pavan , Ordemann Rainer , Liu Chen , Ferrara James L. M. , Teshima Takanori
Published: April 2014

Alloantigen expression on host APCs is essential to initiate graft-vs-host disease (GVHD); however, critical APC subset remains to be elucidated. We compared the ability of dendritic cells (DCs) and B cells to initiate acute GVHD by an add-back study of MHC class II-expressing APCs (II+/+) into MHC class II-deficient (II−/−) mice that were resistant to CD4-dependent GVHD. Injection of host-derived, but not donor-derived, II+/+ DCs or host-derived II+/+ B cells, was sufficient to break GVHD resistance of II−/− mice and induced lethal acute GVHD. By contrast, host-derived II+/+ B cells, both naive and LPS stimulated, failed to induce activation or tolerance of donor CD4+ T cells. Similarly, in a model of CD8-dependent GVHD across MHC class I mismatch injection of allogeneic DCs, but not B cells, induced robust proliferation of donor CD8+ T cells and broke GVHD resistance of chimeric recipients in which APCs were syngeneic to donors. These results demonstrate that host-derived DCs are critical in priming donor CD4+ and CD8+ T cells to cause GVHD, and selective targeting of host DCs may be a promising strategy to prevent GVHD.
Activation of Naive CD4+ T Cells In Vivo by a Self-Peptide Mimic: Mechanism of Tolerance Maintenance and Preservation of Immunity
Authors: Choi Jin-Young , Craft Joe
Published: April 2014

Intrathymic selection generates a peripheral repertoire of CD4+ T cells with receptors that retain low affinity for self-peptide MHC complexes. Despite self-recognition, T cells remain tolerant even in the setting of microbial challenge and resultant costimulatory signals. We demonstrate here a novel mechanism for tolerance maintenance under conditions of self-recognition and strong costimulation. TCR engagement in vivo with a low-avidity peptide, as a mimic of self, provided with poly(I:C) (dsRNA) led to division of naive T cells that was dependent upon costimulatory signals; however, the dividing cells rapidly underwent deletion. By contrast, the surviving cells that were activated as evidenced by up-regulation of CD69 did not become effectors upon restimulation with the same ligand and maintained an effective response against agonist peptide. We suggest TCR engagement with self-peptide MHC complexes promotes tolerance maintenance during pathogen challenge, while preserving efficient reactivity for subsequent encounter with foreign Ags.
Increased and Long-Term Generation of Dendritic Cells with Reduced Function from IL-6-Deficient Bone Marrow
Authors: Bleier Joshua I. , Pillarisetty Venu G. , Shah Alaap B. , DeMatteo Ronald P.
Published: April 2014

The importance of IL-6 in dendritic cell (DC) development and function has not been well defined. To establish the role of IL-6, we studied bone marrow-derived DC (BMDC) and freshly isolated splenic DC from IL-6−/−-transgenic mice. We found that although IL-6−/− bone marrow had a similar composition to that of wild-type (WT) mice, it generated up to 10 times more DC when cultured in GM-CSF. The difference persisted even when IL-6−/− and WT bone marrow were cultured together, excluding the possibility that the effects were simply due to different cytokine microenvironments. In comparison to WT BMDC, IL-6−/− BMDC captured at least as much Ag, had an equivalent surface phenotype, and matured similarly in response to LPS or CpG. However, IL-6−/− BMDC induced less T cell allostimulation and Ag-specific T cell activation, but only the former was related to their inability to generate IL-6. Although WT bone marrow cultures died within 4 wk, IL-6−/− cultures continued to generate BMDC for >120 days, although the BMDC became immature and less functional. In vivo, we found that IL-6−/− mice had similar numbers and types of splenic DC as WT mice, both normally and after treatment with either Flt-3 ligand or GM-CSF. These findings demonstrate that IL-6 has profound effects on DC development in vitro, although the number and subtype composition of DC are unaffected by the absence of IL-6 in vivo. Furthermore, secretion of IL-6 is critical to certain DC functions.
Transcriptional Regulation of Human CD5: Important Role of Ets Transcription Factors in CD5 Expression in T Cells
Authors: Arman Mònica , Calvo Javier , Trojanowska Maria E. , Cockerill Peter N. , Santana Mónica , López-Cabrera Manuel , Vives Jordi , Lozano Francisco
Published: April 2014

CD5 is a surface receptor constitutively expressed on thymocytes and mature T and B-1a cells. CD5 expression is tightly regulated during T and B cell development and activation processes. In this study we shown that the constitutive expression of CD5 on human T cells correlates with the presence of a DNase I-hypersensitive (DH) site at the 5′-flanking region of CD5. Human CD5 is a TATA-less gene for which 5′-RACE analysis shows multiple transcriptional start sites, the most frequent of which locates within an initiator sequence. Luciferase reporter assays indicate that a 282-bp region upstream of the initiation ATG displays full promoter activity in human T cells. Two conserved Ets-binding sites (at positions −239 and −185) were identified as functionally relevant to CD5 expression by site-directed mutagenesis, EMSAs, and cotransfection experiments. A possible contribution of Sp1 (−115 and −95), c-Myb (−177), and AP-1-like (−151) motifs was also detected. Further DH site analyses revealed an inducible DH site 10 kb upstream of the human CD5 gene in both T and B CD5+ cells. Interestingly, a 140-bp sequence showing high homology with a murine inducible enhancer is found within that site. The data presented indicate that the 5′-flanking region of human CD5 is transcriptionally active in T cells, and that Ets transcription factors in conjunction with other regulatory elements are responsible for constitutive and tissue-specific CD5 expression.
CD200 Is a Ligand for All Members of the CD200R Family of Immunoregulatory Molecules
Authors: Gorczynski Reginald , Chen Zhiqi , Kai Yu , Lee Lydia , Wong Simon , Marsden Philip A.
Published: April 2014

CD200Fc, a chimeric molecule including the extracellular domain of CD200 and a murine IgG2a Fc region, regulates immune responses following engagement of a cell surface receptor, CD200R, expressed on cells of the myeloid and T cell lineage. A recent report focused attention on a family of CD200Rs, but concluded that only one member used CD200 as its ligand. We have also cloned and sequenced a family of CD200Rs, but identify an amino terminus to two of the three isoforms not recognized by previous researchers. We show by FACS, using FITC-labeled CD200Fc, that COS7 cells transfected with all CD200R isoforms bind CD200 as ligand, although the functional consequences of this binding likely differs between the different isoforms. mAbs directed against the CD200 R1/R4 isoforms altered IL-2/IL-4 cytokine production and suppressed CTL responses in a fashion comparable to CD200Fc, with a significantly lesser effect seen following addition of anti-CD200 R2/R3.
Activation of Phosphoinositide 3-Kinases by the CCR4 Ligand Macrophage-Derived Chemokine Is a Dispensable Signal for T Lymphocyte Chemotaxis
Authors: Cronshaw Darran G. , Owen Charles , Brown Zarin , Ward Stephen G.
Published: April 2014

Macrophage-derived chemokine (MDC/CC chemokine ligand 22 (CCL22)) mediates its cellular effects principally by binding to its receptor CCR4, and together they constitute a multifunctional chemokine/receptor system with homeostatic and inflammatory roles in the body. We report the CCL22-induced accumulation of phosphatidylinositol-(3,4,5)-trisphosphate (PI(3,4,5)P3) in the leukemic T cell line CEM. CCL22 also had the ability to chemoattract human Th2 cells and CEM cells in a pertussis toxin-sensitive manner. Although the PI(3,4,5)P3 accumulation along with the pertussis toxin-susceptible phosphorylation of protein kinase B were sensitive to the two phosphoinositide 3-kinase inhibitors, LY294002 and wortmannin, cell migration was unaffected. However, cell migration was abrogated with the Rho-dependent kinase inhibitor, Y-27632. These data demonstrate that although there is PI(3,4,5)P3 accumulation downstream of CCR4, phosphoinositide 3-kinase activity is a dispensable signal for CCR4-stimulated chemotaxis of Th2 cells and the CEM T cell line.
Alterations in Lipid Raft Composition and Dynamics Contribute to Abnormal T Cell Responses in Systemic Lupus Erythematosus
Authors: Krishnan Sandeep , Nambiar Madhusoodana P. , Warke Vishal G. , Fisher Carolyn U. , Mitchell Jeanne , Delaney Nancy , Tsokos George C.
Published: April 2014

In response to appropriate stimulation, T lymphocytes from systemic lupus erythematosus (SLE) patients exhibit increased and faster intracellular tyrosine phosphorylation and free calcium responses. We have explored whether the composition and dynamics of lipid rafts are responsible for the abnormal T cell responses in SLE. SLE T cells generate and possess higher amounts of ganglioside-containing lipid rafts and, unlike normal T cells, SLE T cell lipid rafts include FcRγ and activated Syk kinase. IgM anti-CD3 Ab-mediated capping of TCR complexes occurs more rapidly in SLE T cells and concomitant with dramatic acceleration of actin polymerization kinetics. The significance of these findings is evident from the observation that cross-linking of lipid rafts evokes earlier and higher calcium responses in SLE T cells. Thus, we propose that alterations in the lipid raft signaling machinery represent an important mechanism that is responsible for the heightened and accelerated T cell responses in SLE.
Shortening the Infectious Period Does Not Alter Expansion of CD8 T Cells but Diminishes Their Capacity to Differentiate into Memory Cells
Authors: Williams Matthew A. , Bevan Michael J.
Published: April 2014

Following a primary immune response, a portion of effector T cells gives rise to long-lived memory cells. Although primary expansion and differentiation of effector CD8 T cells is dictated by a brief exposure to Ag, it is unclear whether full memory differentiation is also programmed within the same short window. By carefully modulating the kinetics of Listeria monocytogenes infection, we analyzed the requirements for the programming of effector and memory T cell development in vivo. We find that although limiting the infectious period to the first 24–48 h does not impact the size of the primary CD8 response, the ensuing memory population is significantly diminished. This effect is particularly pronounced in the development of tissue-homing memory cells and is inversely proportional to the initial infectious dose. In contrast to CD8 responses, the differentiation of primary CD4 responses was highly dependent on the continued presence of the infection. Shortening the duration of the infection greatly reduced the development of CD4 effector responses in the spleen and prevented their trafficking to peripheral sites of infection. We propose that the stimulus received by CD8 T cells during the early stages of infection largely contribute to the differentiation of CD8 effector cells, whereas continued or distinct signals received at later stages influence their ability to differentiate into memory cells.
Recruitment of Uterine NK Cells: Induction of CXC Chemokine Ligands 10 and 11 in Human Endometrium by Estradiol and Progesterone
Authors: Sentman Charles L. , Meadows Sarah K. , Wira Charles R. , Eriksson Mikael
Published: April 2014

Uterine NK (uNK) cells express a unique set of markers compared with blood NK cells. However, recent studies suggest that uNK cells may be derived from the recruitment of blood NK cells into the endometrium. In this study, we used an in vitro organ culture system to demonstrate that estradiol induces expression of chemokines CXCL10 and/or CXCL11 within human endometrium in 85% of patient samples tested. The average increase in gene expression after 10−9 M estradiol treatment was 8.5-fold for CXCL10 and 7.7-fold for CXCL11 compared with medium alone. We observed that a specific estrogen receptor antagonist (ICI182780) was able to prevent chemokine gene induction, indicating that the effect of estradiol was receptor mediated. Moreover, our study showed that progesterone induced CXCL10 and CXCL11 expression in 83% of endometrial samples tested. We have also found that uNK cells and blood NK cells express the receptor for CXCL10 and CXCL11, CXCR3, with the highest expression found on uNK cells and CD56bright blood NK cells. These data indicate that sex hormones induce specific chemokines in nonpregnant human endometrium that can activate NK cell migration, and suggest that this mechanism may account for the increased NK cell numbers in endometrium during the menstrual cycle.
Antibody Repertoire Development in Fetal and Neonatal Pigs. VII. Characterization of the Preimmune κ Light Chain Repertoire
Authors: Butler John E. , Wertz Nancy , Sun Jishan , Wang Huang , Chardon Patrick , Piumi Francois , Wells Kevin
Published: April 2014

Combinatorial diversity is highly restricted in the preimmune porcine H chain repertoire compared with that in humans and mice. This raised the question of whether similar restriction characterized the preimmune L chain repertoire. In this study we present evidence that >90% of all expressed Vκ genes in the porcine preimmune repertoire belong to three subfamilies of Vκ genes that share 87% sequence similarity with human IGKV2. This porcine Vκ family also shares sequence similarity with some, but not all, Vκ genes from sheep. Hybridization with sperm DNA and sequence analyses of polynucleotides from overlapping bacterial artificial chromosome clones suggest swine possess ∼60 IGVK2 genes. The latter method also revealed that certain IGKV2 subfamilies are not expressed in the preimmune repertoire. Six members of an IGVK1 family were also expressed as part of the preimmune repertoire, and these shared 87% sequence similarity with human IGVK1. Five Jκ segments, complete with recombination signal sequences and separated by ∼300 nt, were identified ∼3 kb upstream of a single Cκ. Surprisingly, Jκ2 accounted for >90% of all framework region 4 sequences in the preimmune repertoire. These findings show that swine use ∼10 IGVK2 genes from three of six subfamilies and preferentially one Jκ segment to generate their preimmune κ repertoire. These studies, like those of porcine Ig constant regions and MHC genes, also indicate unexpected high sequence similarity with their human counterparts despite differences in phylogeny and the mechanism of repertoire diversification.
Human CD8+ T Cells Recognize the 60-kDa Cysteine-Rich Outer Membrane Protein fromChlamydia trachomatis
Authors: Gervassi Ana L. , Grabstein Kenneth H. , Probst Peter , Hess Bruce , Alderson Mark R. , Fling Steven P.
Published: April 2014

The intracellular bacterial pathogen Chlamydia is sequestered from the host cell cytoplasm by remaining within an inclusion body during its replication cycle. Nevertheless, CD8+ T cells recognizing Chlamydia Ags in the context of MHC class I molecules are primed during infection. We have recently described derivation of Chlamydia-specific human CD8+ T cells by using infected dendritic cells as a surrogate system to reflect Chlamydia-specific CD8+ T cell responses in vivo. These CD8+ T cell clones recognize chlamydial Ags processed via the conventional class Ia processing pathway, as assessed by treatment of infected APC with lactacystin and brefeldin A, suggesting that the Ags are translocated from the chlamydial inclusion into the host cell cytosol. In this study, outer membrane protein 2 (OmcB) was identified as the Ag recognized by one of these Chlamydia-specific human CD8+ T cells, and we defined the HLA*A0101-restricted epitope from this Ag. CD8+ T cell responses to this epitope were present at high frequencies in the peripheral blood of both of two HLA*A0101 donors tested. In vitro chlamydial growth was completely inhibited by the OmcB-specific CD8+ T cell clone independently of lytic mechanisms. OmcB is a 60-kDa protein that has been postulated to be associated with the Chlamydia outer membrane complex. The subcellular localization of OmcB to the cytosol of infected cells, as determined by conventional MHC class I Ag processing and presentation, suggests the possibility of an additional, cytosolic-associated function for this protein.
CCR1 and CC Chemokine Ligand 5 Interactions Exacerbate Innate Immune Responses during Sepsis
Authors: Ness Traci L. , Carpenter Kristin J. , Ewing Jillian L. , Gerard Craig J. , Hogaboam Cory M. , Kunkel Steven L.
Published: April 2014

CCR1 has previously been shown to play important roles in leukocyte trafficking, pathogen clearance, and the type 1/type 2 cytokine balance, although very little is known about its role in the host response during sepsis. In a cecal ligation and puncture model of septic peritonitis, CCR1-deficient (CCR1−/−) mice were significantly protected from the lethal effects of sepsis when compared with wild-type (WT) controls. The peritoneal and systemic cytokine profile in CCR1−/− mice was characterized by a robust, but short-lived and regulated antibacterial response. CCR1 expression was not required for leukocyte recruitment, suggesting critical differences extant in the activation of WT and CCR1−/− resident or recruited peritoneal cells during sepsis. Peritoneal macrophages isolated from naive CCR1−/− mice clearly demonstrated enhanced cytokine/chemokine generation and antibacterial responses compared with similarly treated WT macrophages. CCR1 and CCL5 interactions markedly altered the inflammatory response in vivo and in vitro. Administration of CCL5 increased sepsis-induced lethality in WT mice, whereas neutralization of CCL5 improved survival. CCL5 acted in a CCR1-dependent manner to augment production of IFN-γ and MIP-2 to damaging levels. These data illustrate that the interaction between CCR1 and CCL5 modulates the innate immune response during sepsis, and both represent potential targets for therapeutic intervention.
Prostaglandin E2 Promotes the Survival of Bone Marrow-Derived Dendritic Cells
Authors: Vassiliou Evros , Sharma Vikas , Jing Huie , Sheibanie Farzad , Ganea Doina
Published: April 2014

Since dendritic cells (DC) participate in both innate and adaptive immunity, their survival and expansion is tightly controlled. Little is known about the mechanisms of DC apoptosis. PGE2, an arachidonic acid metabolite, plays an essential role in DC migration. We propose a novel function for PGE2 as a DC survival factor. Our studies demonstrate that PGE2 protects DC in vitro against apoptosis induced by withdrawal of growth factors or ceramide. DC matured in conditions that inhibit endogenous PGE2 release are highly susceptible to apoptosis and exogenous PGE2 re-establishes the more resistant phenotype. The antiapoptotic effect is mediated through EP-2/EP-4 receptors and involves the PI3K → Akt pathway. PGE2 leads to increased phosphorylation of Akt, protection against mitochondrial membrane compromise, and decreased caspase 3 activity. Macroarray data indicate that PGE2 leads to the down-regulation of a number of proapoptotic molecules, i.e., BAD, several caspases, and granzyme B. In vivo, higher numbers of immature and Ag-loaded CFSE-labeled DC are present in the draining lymph nodes of mice inoculated with PGE2 receptor agonists, compared with animals treated with ibuprofen or controls injected with PBS. This suggests that PGE2 acts as an endogenous antiapoptotic factor for DC and raises the possibility of using PGE2 agonists to increase the survival of Ag-loaded DC following in vivo administration.
Involvement of Eotaxin, Eosinophils, and Pancreatic Predisposition in Development of Type 1 Diabetes Mellitus in the BioBreeding Rat
Authors: Hessner Martin J. , Wang Xujing , Meyer Lisa , Geoffrey Rhonda , Jia Shuang , Fuller Jessica , Lernmark Ake , Ghosh Soumitra
Published: April 2014

Allergy and autoimmunity are both examples of deregulated immunity characterized by inflammation and injury of targeted tissues that have until recently been considered disparate disease processes. However, recent findings have implicated mast cells, in coordination with granulocytes and other immune effector cells, in the pathology of these two disorders. The BioBreeding (BB) DRlyp/lyp rat develops an autoimmune insulin-dependent diabetes similar to human type 1 diabetes mellitus (T1DM), whereas the BBDR+/+ rat does not. To better understand immune processes during development of T1DM, gene expression profiling at day (d) 40 (before insulitis) and d65 (before disease onset) was conducted on pancreatic lymph nodes of DRlyp/lyp, DR+/+, and Wistar-Furth (WF) rats. The eosinophil-recruiting chemokine, eotaxin, and the high-affinity IgE receptor (FcεRI) were up-regulated >5-fold in d65 DRlyp/lyp vs d65 DR+/+ pancreatic lymph nodes by microarray (p < 0.05) and quantitative RT-PCR studies (p < 0.05). DR+/+, WF, and d40 DRlyp/lyp animals possessed normal pancreatic histology; however, d65 DRlyp/lyp animals possessed eosinophilic insulitis. Therefore, immunohistochemistry for pancreatic eotaxin expression was conducted, revealing positive staining of d65 DRlyp/lyp islets. Islets of d65 DR+/+ rats also stained positively, consistent with underlying diabetic predisposition in the BB lineage, whereas WF islets did not. Other differentially expressed transcripts included those associated with eosinophils, mast cells, and lymphocytes. These data support an important role for these inflammatory mediators in BB rat T1DM and suggest that the lymphopenia due to the Ian5/(lyp) mutation may result in a deregulation of cells involved in insulitis and β cell destruction.
Notch Governing Mature T Cell Differentiation
Authors: Tsukumo Shin-ichi , Yasutomo Koji
Published: April 2014

The differentiation of naive T cells to effector/memory T cells is regulated by a variety of factors. The recent advance of the contribution of Notch signaling in this differentiation step has provided a new path to better understand the acquisition or persistence of effector function of mature T cells. In this review, we summarize emerging and, in some points, conflicting evidence for Notch signaling on mature T cell activation and differentiation.
Cutting Edge: TLR4 Activation Mediates Liver Ischemia/Reperfusion Inflammatory Response via IFN Regulatory Factor 3-Dependent MyD88-Independent Pathway
Authors: Zhai Yuan , Shen Xiu-da , O’Connell Ryan , Gao Feng , Lassman Charles , Busuttil Ronald W. , Cheng Genhong , Kupiec-Weglinski Jerzy W.
Published: April 2014

The triggering molecular mechanism of ischemia-reperfusion injury (IRI), which in clinical settings results in excessive and detrimental inflammatory responses, remains unclear. This study analyzes the role of the TLR system in an established murine model of liver warm ischemia followed by reperfusion. By contrasting in parallel TLR knockout mice with their wild-type counterparts, we found that TLR4, but not TLR2, was specifically required in initiating the IRI cascade, as manifested by liver function (serum alanine aminotransferase levels), pathology, and local induction of proinflammatory cytokines/chemokines (TNF-α, IL-6, IFN-inducible protein 10). We then investigated the downstream signaling pathway of TLR4 activation. Our results show that IFN regulatory factor 3, but not MyD88, mediated IRI-induced TLR4 activation leading to liver inflammation and hepatocellular damage. This study documents the selective usage of TLR in a clinically relevant noninfectious disease model, and identifies a triggering molecular mechanism in the pathophysiology of liver IRI.
Cutting Edge: CD28-Mediated Transcriptional and Posttranscriptional Regulation of IL-2 Expression Are Controlled through Different Signaling Pathways
Authors: Sanchez-Lockhart Mariano , Marin Elides , Graf Beth , Abe Ryo , Harada Yohsuke , Sedwick Caitlin E. , Miller Jim
Published: April 2014

Despite the clear functional importance of CD28 costimulation, the signaling pathways transduced through CD28 have remained controversial. PI3K was identified early as a candidate for CD28 signaling, but conflicting data during the past decade has left the role of PI3K unresolved. In this report, we have resolved this controversy. We show that mutation of the PI3K interaction site in the cytosolic tail of CD28 site disrupts the ability of CD28 to recruit protein kinase C-θ to the central supramolecular activation cluster (c-SMAC) region of the immunological synapse, promote NF-κB nuclear translocation, and enhance IL-2 gene transcription. In contrast, mutation of the PI3K interaction site had no effect on the ability of CD28 to enhance IL-2 mRNA stability. These results suggest that two distinct pathways mediate CD28-induced up-regulation of IL-2 expression, a PI3K-dependent pathway that may function through the immunological synapse to enhance IL-2 transcription and a PI3K-independent pathway that induces IL-2 mRNA stability.
Cutting Edge: Persistence of Transferred Lymphocyte Clonotypes Correlates with Cancer Regression in Patients Receiving Cell Transfer Therapy
Authors: Robbins Paul F. , Dudley Mark E. , Wunderlich John , El-Gamil Mona , Li Yong F. , Zhou Juhua , Huang Jianping , Powell Daniel J. , Rosenberg Steven A.
Published: April 2014

The lack of persistence of transferred autologous mature lymphocytes in humans has been a major limitation to the application of effective cell transfer therapies. The results of a pilot clinical trial in 13 patients with metastatic melanoma suggested that conditioning with nonmyeloablative chemotherapy before adoptive transfer of activated tumor-reactive T cells enhances tumor regression and increases the overall rates of objective clinical responses. The present report examines the relationship between T cell persistence and tumor regression through analysis of the TCR β-chain V region gene products expressed in samples obtained from 25 patients treated with this protocol. Sequence analysis demonstrated that there was a significant correlation between tumor regression and the degree of persistence in peripheral blood of adoptively transferred T cell clones, suggesting that inadequate T cell persistence may represent a major factor limiting responses to adoptive immunotherapy.
Cutting Edge: Expression Patterns of Surface and Soluble Triggering Receptor Expressed on Myeloid Cells-1 in Human Endotoxemia
Authors: Knapp Sylvia , Gibot Sébastien , de Vos Alex , Versteeg Henri H. , Colonna Marco , van der Poll Tom
Published: April 2014

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a recently identified molecule involved in the amplification of inflammation. To determine the regulation of TREM-1, we studied TREM-1 expression and soluble TREM-1 plasma levels upon i.v. LPS challenge in healthy humans in vivo and in vitro. Granulocyte TREM-1 expression was high at baseline and immediately down-regulated upon LPS exposure along with an increase in soluble TREM-1. Monocytes displayed a gradual up-regulation of TREM-1 upon LPS in vivo and in vitro. In vitro studies extended these findings to highly purified lipoteichoic acid and Streptococcus pneumoniae. Nonbacterial TLR ligands such as polyinosine-polycytidylic acid and imidazoquinoline, as well as the TLR9 ligand CpG, did not impact TREM-1 expression. The LPS-induced alterations in TREM-1 surface expression were not a result of increased TNF-α or IL-10. Inhibitor studies disclosed a PI3K-dependent pathway in LPS-induced up-regulation of TREM-1 on monocytes, whereas MAPK played a limited role.