bluuebunny/crossref_metadata_2025 huggingface dataset:
CD28 Costimulation Mediates T Cell Expansion Via IL-2-Independent and IL-2-Dependent Regulation of Cell Cycle Progression
Authors: Appleman Leonard J. , Berezovskaya Alla , Grass Isabelle , Boussiotis Vassiliki A.
Published: April 2014

In the presence of TCR ligation by Ag, CD28 pathway mediates the most potent costimulatory signal for T cell activation, cytokine secretion, and T cell expansion. Although CD28 costimulation promotes T cell expansion due to IL-2 secretion and subsequent signaling via the IL-2 receptor, recent studies indicate that the dramatic T cell expansion mediated through the unopposed CD28 stimulation in CTLA4-deficient mice is IL-2 independent. Therefore, we sought to dissect the effects of CD28 and IL-2 receptor pathways on cell cycle progression and determine the molecular mechanisms by which the CD28 pathway regulates T cell expansion. Here we show that CD28 costimulation directly regulates T cell cycle entry and progression through the G1 phase in an IL-2-independent manner resulting in activation of cyclin D2-associated cdk4/cdk6 and cyclin E-associated cdk2. Subsequent progression into the S phase is mediated via both IL-2-dependent and IL-2-independent mechanisms and, although in the absence of IL-2 the majority of T cells are arrested at the G1/S transition, a significant fraction of them progresses into the S phase. The key regulatory mechanism for the activation of cyclin-cdk complexes and cell cycle progression is the down-regulation of p27kip1 cdk inhibitor, which is mediated at the posttranscriptional level by its ubiquitin-dependent degradation in the proteasome pathway. Therefore, CD28 costimulation mediates T cell expansion in an IL-2-independent and IL-2 dependent manner and regulates cell cycle progression at two distinct points: at the early G1 phase and at the G1/S transition.
Glycine Inhibits Growth of T Lymphocytes by an IL-2-Independent Mechanism
Authors: Stachlewitz Robert F. , Li Xiangli , Smith Scott , Bunzendahl Hartwig , Graves Lee M. , Thurman Ronald G.
Published: April 2014

Previously, it was shown that glycine prevented increases in intracellular calcium ([Ca2+]i) in Kupffer cells. Since Kupffer cells and T lymphocytes are derived from the same pluripotent stem cell, it was hypothesized that glycine would prevent increases in [Ca2+]i in lymphocytes and inhibit cell proliferation. Lymphocyte proliferation was measured in one-way MLC with spleen cells from DA and Lewis rats and in enriched T lymphocyte preparations stimulated by immobilized anti-CD3 Ab. Glycine caused a dose-dependent decrease in cell proliferation to about 40% of control. Con A caused a dose-dependent increase in [Ca2+]i in Jurkat cells which was blunted maximally with 0.6 mM glycine. The effect of glycine was dependent on extracellular chloride and reversed by strychnine, an antagonist of the glycine-gated chloride channel. Similar results were obtained with rat T lymphocytes stimulated by anti-CD3 Ab. Surprisingly, glycine had no effect on IL-2 production in the mixed lymphocyte culture; therefore, the effect of glycine on IL-2-dependent proliferation was tested. Glycine and rapamycin caused dose-dependent decreases in IL-2-stimulated growth of Ctll-2 cells to about 60% and 40%, respectively, of control. Moreover, glycine also inhibited the IL-2-stimulated growth of rat splenic lymphocytes. It is concluded that glycine blunts proliferation in an IL-2-independent manner. This is consistent with the hypothesis that glycine activates a glycine-gated chloride channel and hyperpolarizes the cell membrane-blunting increases in [Ca2+]i that are required for transcription of factors necessary for cell proliferation.
Suppressor Effector Function of CD4+CD25+ Immunoregulatory T Cells Is Antigen Nonspecific
Authors: Thornton Angela M. , Shevach Ethan M.
Published: April 2014

CD4+CD25+ T cells represent a unique population of “professional” suppressor T cells that prevent induction of organ-specific autoimmune disease. In vitro, CD4+CD25+ cells were anergic to simulation via the TCR and when cultured with CD4+CD25− cells, markedly suppressed polyclonal T cell proliferation by specifically inhibiting the production of IL-2. Suppression was cytokine independent, cell contact dependent, and required activation of the suppressors via their TCR. Further characterization of the CD4+CD25+ population demonstrated that they do not contain memory or activated T cells and that they act through an APC-independent mechanism. CD4+CD25+ T cells isolated from TCR transgenic (Tg) mice inhibited responses of CD4+CD25− Tg T cells to the same Ag, but also inhibited the Ag-specific responses of Tg cells specific for a distinct Ag. Suppression required that both peptide/MHC complexes be present in the same culture, but the Ags could be presented by two distinct populations of APC. When CD4+CD25+ T cells were cultured with anti-CD3 and IL-2, they expanded, remained anergic, and in the absence of restimulation via their TCR, suppressed Ag-specific responses of CD4+CD25− T cells from multiple TCR transgenics. Collectively, these data demonstrate that CD4+CD25+ T cells require activation via their TCR to become suppressive, but once activated, their suppressor effector function is completely nonspecific. The cell surface molecules involved in this T-T interaction remain to be characterized.
IFN-γ-Dependent Delay of In Vivo Tumor Progression by Fas Overexpression on Murine Renal Cancer Cells
Authors: Lee Jong-Keuk , Sayers Thomas J. , Brooks Alan D. , Back Timothy C. , Young Howard A. , Komschlies Kristin L. , Wigginton Jon M. , Wiltrout Robert H.
Published: April 2014

The role of Fas in the regulation of solid tumor growth was investigated. Murine renal carcinoma (Renca) cells were constitutively resistant to Fas-mediated killing in vitro, but exhibited increased expression of Fas and sensitivity to Fas-mediated killing after exposure to IFN-γ and TNF. Transfected Renca cells overexpressing Fas were efficiently killed in vitro upon exposure to anti-Fas Ab (Jo2). When Fas-overexpressing Renca cells were injected into syngenic BALB/c mice, there was a consistent and significant delay in tumor progression, reduced metastasis, and prolonged survival that was not observed for Renca cells that overexpressed a truncated nonfunctional Fas receptor. The delay of in vivo tumor growth induced by Fas overexpression was not observed in IFN-γ−/− mice, indicating that IFN-γ is required for the delay of in vivo tumor growth. However, there was a significant increase of infiltrated T cells and in vivo apoptosis in Fas-overexpressing Renca tumors, and Fas-overexpressing Renca cells were also efficiently killed in vitro by T cells. In addition, a strong therapeutic effect was observed on Fas-overexpressing tumor cells by in vivo administration of anti-Fas Ab, confirming that overexpressed Fas provides a functional target in vivo for Fas-specific ligands. Therefore, our findings demonstrate that Fas overexpression on solid tumor cells can delay tumor growth and provides a rationale for therapeutic manipulation of Fas expression as a means of inducing tumor regression in vivo.
Characterization of Peripheral Regulatory CD4+ T Cells That Prevent Diabetes Onset in Nonobese Diabetic Mice
Authors: Lepault Françoise , Gagnerault Marie Claude
Published: April 2014

The period that precedes onset of insulin-dependent diabetes mellitus corresponds to an active dynamic state in which pathogenic autoreactive T cells are kept from destroying β cells by regulatory T cells. In prediabetic nonobese diabetic (NOD) mice, CD4+ splenocytes were shown to prevent diabetes transfer in immunodeficient NOD recipients. We now demonstrate that regulatory splenocytes belong to the CD4+ CD62Lhigh T cell subset that comprises a vast majority of naive cells producing low levels of IL-2 and IFN-γ and no IL-4 and IL-10 upon in vitro stimulation. Consistently, the inhibition of diabetes transfer was not mediated by IL-4 and IL-10. Regulatory cells homed to the pancreas and modified the migration of diabetogenic to the islets, which resulted in a decreased insulitis severity. The efficiency of CD62L+ T cells was dose dependent, independent of sex and disease prevalence. Protection mechanisms did not involve the CD62L molecule, an observation that may relate to the fact that CD4+ CD62Lhigh lymph node cells were less potent than their splenic counterparts. Regulatory T cells were detectable after weaning and persist until disease onset, sustaining the notion that diabetes is a late and abrupt event. Thus, the CD62L molecule appears as a unique marker that can discriminate diabetogenic (previously shown to be CD62L−) from regulatory T cells. The phenotypic and functional characteristics of protective CD4+ CD62L+ cells suggest they are different from Th2-, Tr1-, and NK T-type cells, reported to be implicated in the control of diabetes in NOD mice, and may represent a new immunoregulatory population.
Th2-Dependent B Cell Responses in the Absence of CD40-CD40 Ligand Interactions
Authors: Chirmule Narendra , Tazelaar John , Wilson James M.
Published: April 2014

CD40 is thought to play a central role in T cell-dependent humoral responses through two distinct mechanisms. CD4+ T helper cells are activated via CD40-dependent Ag presentation in which CD80/CD86 provides costimulation through CD28. In addition, engagement of CD40 on B cells provides a direct pathway for activation of humoral responses. We used a model of adenovirus-mediated gene transfer of β-galactosidase (lacZ) into murine lung to evaluate the specific CD40-dependent pathways required for humoral immunity at mucosal surfaces of the lung. Animals deficient in CD40L failed to develop T and B cell responses to vector. Activation of Th2 cells, which normally requires CD40-dependent stimulation of APCs, was selectively reconstituted in CD40 ligand-deficient mice by systemic administration of an Ab that is agonistic to CD28. Surprisingly, this resulted in the development of a functional humoral response to vector as evidenced by formation of germinal centers and production of antiadenovirus IgG1 and IgA that neutralized and prevented effective readministration of vector. The CD28-dependent B cell response required CD4+ T cells and was mediated via IL-4. These studies indicate that CD40 signals to the B cells are not necessary for CD4+ Th2 cell-dependent humoral responses to be generated.
Functional Responses and Costimulator Dependence of Memory CD4+ T Cells
Authors: London Cheryl A. , Lodge Michael P. , Abbas Abul K.
Published: April 2014

To examine the functional characteristics of memory CD4+ T cells, we used an adoptive transfer system to generate a stable population of Ag-specific memory cells in vivo and compared their responses to Ag with those of a similar population of Ag-specific naive cells. Memory cells localized to the spleen and lymph nodes of mice and exhibited extremely rapid recall responses to Ag in vivo, leaving the spleen within 3–5 days of Ag encounter. Unlike their naive counterparts, memory cells produced effector cytokines (IFN-γ, IL-4, IL-5) within 12–24 h of Ag exposure and did not require multiple cycles of cell division to do so. Memory cells proliferated at lower Ag concentrations than did naive cells, were less dependent on costimulation by B7 molecules, and independent of costimulation by CD40. Furthermore, effector cytokine production by memory cells also occurred in the absence of either B7 or CD40 costimulation. Lastly, memory cells were resistant to tolerance induction. Together, these findings suggest that the threshold for activation of memory CD4+ cells is lower than that of naive cells. This would permit memory cells to rapidly express their effector functions in vivo earlier in the course of a secondary immune response, when the levels of Ag and the availability of costimulation may be relatively low.
Conserved MHC Class I Peptide Binding Motif Between Humans and Rhesus Macaques
Authors: Dzuris John L. , Sidney John , Appella Ettore , Chesnut Robert W. , Watkins David I. , Sette Alessandro
Published: April 2014

Since the onset of the HIV pandemic, the use of nonhuman primate models of infection has increasingly become important. An excellent model to study HIV infection and immunological responses, in particular cell-mediated immune responses, is SIV infection of rhesus macaques. CTL epitopes have been mapped using SIV-infected rhesus macaques, but, to date, a peptide binding motif has been described for only one rhesus class I MHC molecule, Mamu-A*01. Herein, we have established peptide-live cell binding assays for four rhesus MHC class I molecules: Mamu-A*11, -B*03, -B*04, and -B*17. Using such assays, peptide binding motifs have been established for all four of these rhesus MHC class I molecules. With respect to the nature and spacing of crucial anchor positions, the motifs defined for Mamu-B*04 and -B*17 present unique features not previously observed for other primate species. The motifs identified for Mamu-A*11 and -B*03 are very similar to the peptide binding motifs previously described for human HLA-B*44 and -B*27, respectively. Accordingly, naturally processed peptides derived from HLA-B*44 and HLA-B*27 specifically bind Mamu-A*11 and Mamu-B*03, respectively, indicating that conserved MHC class I binding capabilities exist between rhesus macaques and humans. The definition of four rhesus MHC class I-specific motifs expands our ability to accurately detect and quantitate immune responses to MHC class I-restricted epitopes in rhesus macaques and to rationally design peptide epitope-based model vaccine constructs destined for use in nonhuman primates.
Protein Kinase Cε Is Required for the Induction of Mitogen-Activated Protein Kinase Phosphatase-1 in Lipopolysaccharide-Stimulated Macrophages
Authors: Valledor Annabel F. , Xaus Jordi , Comalada Mònica , Soler Concepció , Celada Antonio
Published: April 2014

LPS induces in bone marrow macrophages the transient expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1). Because MKP-1 plays a crucial role in the attenuation of different MAPK cascades, we were interested in the characterization of the signaling mechanisms involved in the control of MKP-1 expression in LPS-stimulated macrophages. The induction of MKP-1 was blocked by genistein, a tyrosine kinase inhibitor, and by two different protein kinase C (PKC) inhibitors (GF109203X and calphostin C). We had previously shown that bone marrow macrophages express the isoforms PKCβI, ε, and ζ. Of all these, only PKCβI and ε are inhibited by GF109203X. The following arguments suggest that PKCε is required selectively for the induction of MKP-1 by LPS. First, in macrophages exposed to prolonged treatment with PMA, MKP-1 induction by LPS correlates with the levels of expression of PKCε but not with that of PKCβI. Second, Gö6976, an inhibitor selective for conventional PKCs, including PKCβI, does not alter MKP-1 induction by LPS. Last, antisense oligonucleotides that block the expression of PKCε, but not those selective for PKCβI or PKCζ, inhibit MKP-1 induction and lead to an increase of extracellular-signal regulated kinase activity during the macrophage response to LPS. Finally, in macrophages stimulated with LPS we observed significant activation of PKCε. In conclusion, our results demonstrate an important role for PKCε in the induction of MKP-1 and the subsequent negative control of MAPK activity in macrophages.
Anergic T Lymphocytes Selectively Express an Integrin Regulatory Protein of the Cytohesin Family
Authors: Korthäuer Ulf , Nagel Wolfgang , Davis Elizabeth M. , Le Beau Michelle M. , Menon Raman S. , Mitchell Elizabeth O. , Kozak Christine A. , Kolanus Waldemar , Bluestone Jeffrey A.
Published: April 2014

It has been proposed that the maintenance of T cell anergy depends on the induction of negative regulatory factors. Differential display of reverse transcribed RNA was used to identify novel genes that might mediate this function in anergic Th1 clones. We report that anergic Th1 clones do indeed express a genetic program different from that of responsive T cells. Moreover, one gene, the general receptor of phosphoinositides 1 (GRP1), was selectively induced in anergic T cells. The GRP1, located in the plasma membrane, regulated integrin-mediated adhesion and was invariably associated with unresponsiveness in multiple models of anergy. T cells expressing retrovirally transduced GRP1 exhibited normal proliferation and cytokine production. However, GRP1-transduced T cells were not stable and rapidly lost GRP1 expression. Thus, although GRP1 may not directly mediate T cell anergy, it regulates cell expansion and survival, perhaps through its integrin-associated activities.
HLA-F Is a Predominantly Empty, Intracellular, TAP-Associated MHC Class Ib Protein with a Restricted Expression Pattern
Authors: Wainwright Shane D. , Biro P. Andrew , Holmes Christopher H.
Published: April 2014

HLA-F is currently the most enigmatic of the human MHC-encoded class Ib genes. We have investigated the expression of HLA-F using a specific Ab raised against a synthetic peptide corresponding to amino acids 61–84 in the α1 domain of the predicted HLA-F protein. HLA-F is expressed as a β2-microglobulin-associated, 42-kDa protein that shows a restricted tissue distribution. To date, we have detected this product only in peripheral blood B cells, B cell lines, and tissues containing B cells, in particular adult tonsil and fetal liver, a major site of B cell development. Thermostability assays suggest that HLA-F is expressed as an empty heterodimer devoid of peptide. Consistent with this, studies using endoglycosidase-H and cell surface immunoprecipitations also indicate that the overwhelming majority of HLA-F contains an immature oligosaccharide component and is expressed inside the cell. We have found that IFN-γ treatment induces expression of HLA-F mRNA and HLA-F protein, but that this does not result in concomitant cell surface expression. HLA-F associates with at least two components of the conventional class I assembly pathway, calreticulin and TAP. The unusual characteristics of the predicted peptide-binding groove together with the predominantly intracellular localization raise the possibility that HLA-F may be capable of binding only a restricted set of peptides.
Rescue of Defective T Cell Development and Function in Atm−/− Mice by a Functional TCRαβ Transgene
Authors: Chao Connie , Yang Eva Marie , Xu Yang
Published: April 2014

The Atm−/− mice recapitulate most of the defects observed in ataxia-telangiectasia (A-T) patients, including a high incidence of lymphoid tumors and immune defects characterized by defective T cell differentiation, thymus hypoplasia, and defective T-dependent immune responses. To understand the basis of the T cell developmental defects in Atm−/− mice, a functional TCRαβ transgene was introduced into these mutant mice. Analysis of the Atm−/−TCRαβ+ mice indicated that the transgenic TCRαβ can rescue the defective T cell differentiation and partially rescue the thymus hypoplasia in Atm−/− mice, indicating that thymocyte positive selection is normal in the Atm−/− mice. In addition, cell cycle analysis of the thymocytes derived from Atm−/−TCRαβ+ and control mice suggested that Atm is involved in the thymocyte expansion. Finally, evaluation of the T-dependent immune responses in Atm−/−TCRαβ+ mice indicated that Atm is dispensable for normal T cell function. Therefore, the defective T-dependent immune responses in Atm−/− mice must be secondary to greatly reduced T cell numbers in these mutant mice.
Convergence of Fcγ Receptor IIA and Fcγ Receptor IIIB Signaling Pathways in Human Neutrophils
Authors: Chuang Frank Y. S. , Sassaroli Massimo , Unkeless Jay C.
Published: April 2014

Human neutrophils (PMNs) express two receptors for the Fc domain of IgG: the transmembrane FcγRIIA, whose cytosolic sequence contains an immunoreceptor tyrosine-based activation motif, and the GPI-anchored FcγRIIIB. Cross-linking of FcγRIIIB induces cell activation, but the mechanism is still uncertain. We have used mAbs to cross-link selectively each of the two receptors and to assess their signaling phenotypes and functional relation. Cross-linking of FcγRIIIB induces intracellular Ca2+ release and receptor capping. The Ca2+ response is blocked by wortmannin and by N,N-dimethylsphingosine, inhibitors of phosphatidylinositol 3-kinase and sphingosine kinase, respectively. Identical dose-response curves are obtained for the Ca2+ release stimulated by cross-linking FcγRIIA, implicating these two enzymes in a common signaling pathway. Wortmannin also inhibits capping of both receptors, but not receptor endocytosis. Fluorescence microscopy in double-labeled PMNs demonstrates that FcγRIIA colocalizes with cross-linked FcγRIIIB. The signaling phenotypes of the two receptors diverge only under frustrated phagocytosis conditions, where FcγRIIIB bound to substrate-immobilized Ab does not elicit cell spreading. We propose that FcγRIIIB signaling is conducted by molecules of FcγRIIA that are recruited to protein/lipid domains induced by clustered FcγRIIIB and, thus, are brought into juxtaposition for immunoreceptor tyrosine-based activation motif phosphorylation and activation of PMNs.
IL-4 and IL-10 Antagonize IL-12-Mediated Protection Against Acute Vaccinia Virus Infection with a Limited Role of IFN-γ and Nitric Oxide Synthetase 2
Authors: van den Broek Maries , Bachmann Martin F. , Köhler Gabriele , Barner Marijke , Escher Rüdiger , Zinkernagel Rolf , Kopf Manfred
Published: April 2014

Resistance or susceptibility to most infectious diseases is strongly determined by the balance of type 1 vs type 2 cytokines produced during infection. However, for viruses, this scheme may be applicable only to infections with some cytopathic viruses, where IFN-γ is considered as mandatory for host defense with little if any participation of type 2 responses. We studied the role of signature Th1 (IL-12, IFN-γ) and Th2 (IL-4, IL-10) cytokines for immune responses against vaccinia virus (VV). IL-12−/− mice were far more susceptible than IFN-γ−/− mice, and primary CTL responses against VV were absent in IL-12−/− mice but remained intact in IFN-γ−/− mice. Both CD4+ and CD8+ T cells from IL-12−/− mice were unimpaired in IFN-γ production, although CD4+ T cells showed elevated Th2 cytokine responses. Virus replication was impaired in IL-4−/− mice and, even more strikingly, in IL-10−/− mice, which both produced elevated levels of the proinflammatory cytokines IL-1α and IL-6. Thus, IL-4 produced by Th2 cells and IL-10 produced by Th2 cells and probably also by macrophages counteract efficient anti-viral host defense. Surprisingly, NO production, which is considered as a major type 1 effector pathway inhibited by type 2 cytokines, appears to play a limited role against VV, because NO sythetase 2-deficient mice did not show increased viral replication. Thus, our results identify a new role for IL-12 in defense beyond the induction of IFN-γ and show that IL-4 and IL-10 modulate host protective responses to VV.
Macrophage Control of Mycobacterial Growth Induced by Picolinic Acid Is Dependent on Host Cell Apoptosis
Authors: Pais Teresa F. , Appelberg Rui
Published: April 2014

The effects of picolinic acid (PA) on the intramacrophagic growth of Mycobacterium avium were studied. PA reduced M. avium growth inside mouse macrophages and led to a complete control of mycobacterial growth when added together with IFN-γ. The mechanism involved did not require TNF-α, NO, or the respiratory burst, and was not dependent on either iron or zinc withholding. The mycobacteriostatic activity of the macrophages was associated with the induction of morphological changes that culminated in apoptosis at day 4 of treatment. PA alone induced apoptosis in macrophages, and this effect was increased by IFN-γ treatment. Apoptosis at day 4 of infection was reduced by inhibiting macrophage activation with the prostaglandin 15 deoxy-prostaglandin J2 or by treating the cells with the antioxidant N-acetylcysteine. Mycobacterial growth was partially restored in macrophages treated with PA and IFN-γ when 15 deoxy-prostaglandin J2 was added, concomitant with a delay in apoptosis. N-Acetylcysteine or glutathione could also completely revert the mycobacteriostatic effects of PA or PA plus IFN-γ.
Human Intestinal Mast Cells Are Capable of Producing Different Cytokine Profiles: Role of IgE Receptor Cross-Linking and IL-4
Authors: Lorentz Axel , Schwengberg Silke , Sellge Gernot , Manns Michael P. , Bischoff Stephan C.
Published: April 2014

Mast cells are recognized as a new type of immunoregulatory cells capable of producing different cytokines. So far, little is known about the cytokine profile of mature human mast cells isolated from intestinal tissue and cultured in the presence of stem cell factor (SCF). We observed that these cells express the proinflammatory cytokines TNF-α, IL-1β, IL-6, IL-8, IL-16, and IL-18 without further stimulation. Both IgE-dependent and IgE-independent agonists (e.g., Gram-negative bacteria) enhanced expression of TNF-α. Another set of cytokines consisting of IL-3, IL-5, IL-9, and IL-13 was expressed following activation by IgE receptor cross-linking. If mast cells were cultured in the presence of IL-4 and SCF, the production and release of IL-3, IL-5, and IL-13 was increased up to 4-fold compared with mast cells cultured with SCF alone. By contrast, IL-6 expression was completely blocked in response to culture with IL-4. In summary, our data show that mature human mast cells produce proinflammatory cytokines that may be up-regulated following triggering with IgE-independent agonists such as bacteria, whereas activation by IgE receptor cross-linking results in the expression of Th2-type cytokines. IL-4 enhances the expression of Th2-type cytokines but does not affect or even down-regulates proinflammatory cytokines.
Up-Regulation of IL-12 in Monocytes: A Fundamental Defect in Common Variable Immunodeficiency
Authors: Cambronero Rosario , Sewell W. A. Carrock , North Margaret E. , Webster A. David B. , Farrant John
Published: April 2014

We show that LPS-stimulated circulating CD14-positive monocytes from patients with common variable immunodeficiency (CVID) express a higher proportion of intracellular IL-12-positive cells than monocytes from patients with X-linked agammaglobulinemia or normal subjects. We used four-color flow cytometry and measured IL-12 with an Ab to the p40 subunit following stimulation with LPS. The raised IL-12 is associated with an increased frequency of IFN-γ-positive T cells, but not of IFN-γ-positive CD56+ NK cells. These increases in frequency of cytokine-positive cells are due to a decrease in the absolute numbers of circulating monocytes and T cells that are negative for IL-12 and IFN-γ, respectively. The increased frequency of IL-12-positive monocytes appears to be selective because TNF-α was not increased, and is thus unlikely to reflect a general activation. Chronic infection is also unlikely to explain our data since cells from X-linked agammaglobulinemia patients with a similar Ig deficiency do not show these changes. Our data suggest a fundamental abnormality in the IL-12/IFN-γ circuit in CVID, with up-regulation of IL-12 being the “primary” factor. This imbalance is likely to skew the immune response away from Ab production and also explains the failure of CVID T cells to make Ag-specific memory cells and the chronic inflammatory and granulomatous complications that are a feature of CVID. This disease appears to be a rare example of a polarized Th1-type response and may in part be due to a genetic defect in the control of IL-12 production.
Expansion of Tumor-T Cell Pairs from Fine Needle Aspirates of Melanoma Metastases
Authors: Panelli Monica C. , Riker Adam , Kammula Udai , Wang Ena , Lee Kang-Hun , Rosenberg Steven A. , Marincola Francesco M.
Published: April 2014

Lymphocytes expanded from excised specimens can be used to characterize intratumoral T cell responses. These analyses, however, are limited to one time point in the natural history of the removed tumor. The expansion of autologous tumor cells and tumor-infiltrating lymphocytes (TIL) from fine needle aspirates (FNA) of tumors potentially allows a dynamic evaluation of T cell responses within the same lesion at moments relevant to the disease course or response to therapy. Fourteen TIL cultures and 8 tumor cell lines were generated from 18 FNA (12 patients). Five of six TIL that could be tested against autologous tumor demonstrated specific reactivity. Two additional TIL for which no autologous tumor was available demonstrated recognition of HLA-matched melanoma cell lines. Serial FNA of the same lesions were performed in five HLA-A*0201 patients vaccinated with the emulsified melanoma Ag (MA) epitopes: MART-1:27–35; tyrosinase:368–376(370D); gp100:280–288(288V); and gp100:209–217 (210M). FNA material was separately cultured for a short time in IL-2 (300 IU/ml) after stimulation with irradiated autologous PBMC pulsed with each peptide or FluM1:58–66 (1 μmol/ml). No peptide-specific TIL could be expanded from prevaccination FNA. However, after vaccination, TIL specific for gp100:280(g280), gp100:209 (g209), and MART-1:27–35 (MART-1)-related epitopes were identified in three, three, and two patients, respectively. No Flu reactivity could be elicited in TIL, whereas it was consistently present in parallel PBMC cultures. This excluded PBMC contamination of the FNA material. This analysis suggests the feasibility of TIL expansion from minimal FNA material and localization of vaccine-specific T cells at the tumor site.
Protection of Murine Lupus by theEad Transgene Is MHC Haplotype-Dependent
Authors: Ibnou-Zekri Nabila , Iwamoto Masahiro , Gershwin M. Eric , Izui Shozo
Published: April 2014

A high-level expression of a transgene, Ead, encoding the I-Ed α-chain is very effective in protection against murine lupus. To investigate the specific contribution of select H-2 haplotypes on the Ead transgene-mediated disease-suppressing effect, we generated H-2 congenic (NZB × BXSB)F1 hybrid mice bearing either H-2b/b, H-2d/b, or H-2d/d haplotype, and compared the transgene-mediated protective effect on the clinical development (autoantibody production and glomerulonephritis) of lupus in these F1 hybrids. The level of protection was most remarkable in mice bearing the I-E− H-2b/b haplotype but was only minimal in I-E+ H-2d/d F1 hybrids. Additional analysis demonstrated a marked suppression of lupus in I-E+ H-2k/k (MRL × BXSB)F1 hybrid mice, indicating that the transgene is able to suppress autoimmune responses even in mice already expressing I-E molecules at a homozygous level. Our results indicate that the level of the transgene-mediated protection is dependent on the host H-2 haplotype. This suggests that the autoimmune suppressive activity of the Ead transgene is likely to be determined through the interaction of the transgene product with the host MHC class II molecules, providing new insight into the role of MHC in lupus-like autoimmunity.
Treatment of Allograft Recipients with Donor-Specific Transfusion and Anti-CD154 Antibody Leads to Deletion of Alloreactive CD8+ T Cells and Prolonged Graft Survival in a CTLA4-Dependent Manner
Authors: Iwakoshi Neal N. , Mordes John P. , Markees Thomas G. , Phillips Nancy E. , Rossini Aldo A. , Greiner Dale L.
Published: April 2014

A two-element protocol consisting of one donor-specific transfusion (DST) plus a brief course of anti-CD154 mAb greatly prolongs the survival of murine islet, skin, and cardiac allografts. To study the mechanism of allograft survival, we determined the fate of tracer populations of alloreactive transgenic CD8+ T cells in a normal microenvironment. We observed that DST plus anti-CD154 mAb prolonged allograft survival and deleted alloreactive transgenic CD8+ T cells. Neither component alone did so. Skin allograft survival was also prolonged in normal recipients treated with anti-CD154 mAb plus a depleting anti-CD8 mAb and in C57BL/6-CD8 knockout mice treated with anti-CD154 mAb monotherapy. We conclude that, in the presence of anti-CD154 mAb, DST leads to an allotolerant state, in part by deleting alloreactive CD8+ T cells. Consistent with this conclusion, blockade of CTLA4, which is known to abrogate the effects of DST and anti-CD154 mAb, prevented the deletion of alloreactive transgenic CD8+ T cells. These results document for the first time that peripheral deletion of alloantigen-specific CD8+ T cells is an important mechanism through which allograft survival can be prolonged by costimulatory blockade. We propose a unifying mechanism to explain allograft prolongation by DST and blockade of costimulation.
Immune Modulation in Pemphigus Vulgaris: Role of CD28 and IL-10
Authors: Toto Paola , Feliciani Claudio , Amerio Paolo , Suzuki Hirotake , Wang Binghe , Shivji Gulnar M. , Woodley David , Sauder Daniel N.
Published: April 2014

Pemphigus vulgaris (PV) is an autoimmune bullous skin disease characterized by Abs to the desmosomal cadherin desmoglein-3. Although the autoantibodies have been shown to be pathogenic, the role of the cellular immune system in the pathology of pemphigus-induced acantholysis is unclear. To further delineate the potential role of T cell-signaling pathways in the pathogenesis of PV, we performed passive transfer experiments with PV IgG in gene-targeted mutant mice. Our results demonstrated that CD28-deficient mice (lacking a costimulatory signal for T cell activation) are 5-fold more sensitive to the development of PV than wild-type mice. To evaluate whether the higher incidence of disease was due to an impairment in intercellular adhesion of keratinocytes, we performed an in vitro acantholysis, using CD28−/− mice keratinocytes. No alteration in in vitro adhesion was detected in CD28−/−-type keratinocytes. Because the CD28 molecule plays a pivotal role in the induction of Th2 cytokines, we examined the levels of a prototypic Th2 cytokine (IL-10) in CD28−/− mice. Lower levels of IL-10 mRNA were found in lesions from CD28−/− mice. To determine whether pemphigus susceptibility in CD28−/− was related to IL-10 deficiency, we performed passive transfer experiments in IL-10−/− mice that demonstrated increased blisters compared with controls. To confirm that IL-10 is involved in the pathogenesis, rIL-10 was given with PV IgG. IL-10 significantly suppressed the disease activity. These data suggest a potential role of IL-10 in PV.
Distinct Role of Follicular Dendritic Cells and T Cells in the Proliferation, Differentiation, and Apoptosis of a Centroblast Cell Line, L3055
Authors: Choe Jongseon , Li Li , Zhang Xin , Gregory Christopher D. , Choi Yong Sung
Published: April 2014

Germinal center (GC) B cells undergo complex interactions with follicular dendritic cells (FDC) and T cells in the course of differentiation into memory B and plasma cells. To delineate the individual roles of FDC and T cells at each stage of GC B cell differentiation at the clonal level and to analyze the signals involved, we adopted a unique experimental model using an FDC line, HK, and a lymphoma cell line, L3055, that resembles centroblasts. A detailed phenotypic analysis revealed L3055 cells to be a clonal population originating from the GC. Like freshly isolated centroblasts, L3055 cells underwent spontaneous apoptosis when cultured in the absence of fresh FDC or HK cells. L3055 cells proliferated continuously in the presence of HK cells, while they differentiated into a population with the phenotype of centrocytes after stimulation with CD40 ligand (CD40L) and IL-4. The CD40L-stimulated L3055 cells underwent CD95-mediated apoptosis, which was reminiscent of the feature of CD40L-stimulated tonsillar GC B cells. In contrast to HK cells that did not protect L3055 cells from anti-Ig killing, CD40L plus IL-2, IL-4, and IL-10 prevented anti-Ig-induced apoptosis. These experimental results demonstrate a distinct function of FDC and activated T cells, in that FDC provide signals for rapid proliferation of centroblasts, whereas T cells confer signals for differentiation of centroblasts into centrocytes and resistance to B cell receptor-mediated apoptosis. T cells collaborate with FDC in the protection and expansion of the Ag-specific GC B cells.
An Early Pseudorabies Virus Protein Down-Regulates Porcine MHC Class I Expression by Inhibition of Transporter Associated with Antigen Processing (TAP)
Authors: Ambagala Aruna P. N. , Hinkley Susanne , Srikumaran Subramaniam
Published: April 2014

The objectives of this study were to identify the mechanism(s) of pseudorabies virus (PrV)-induced down-regulation of porcine class I molecules and the viral protein(s) responsible for the effect. The ability of PrV to interfere with the peptide transport activity of TAP was determined by an in vitro transport assay. In this assay, porcine kidney (PK-15) cells were permeabilized with streptolysin-O and incubated with a library of 125I-labeled peptides having consensus motifs for glycosylation in the endoplasmic reticulum (ER). The efficiency of transport of peptides from the cytosol into the ER was determined by adsorbing the ER-glycosylated peptides onto Con A-coupled Sepharose beads. Dose-dependent inhibition of TAP activity was observed in PrV-infected PK-15 cells. This inhibition, which occurred as early as 2 h postinfection (h.p.i.), reached the maximum level by 6 h.p.i., indicating that TAP inhibition is one of the mechanisms by which PrV down-regulates porcine class I molecules. Infection of cells with PrV in the presence of metabolic inhibitors revealed that cycloheximide a protein synthesis inhibitor, but not phosphonoacetic acid a herpesvirus DNA synthesis inhibitor, could restore the cell surface expression of class I molecules, indicating that late proteins are not responsible for the down-regulation. Infection in the presence of cycloheximide followed by actinomycin-D, which results in accumulation of the immediate-early protein, failed to down-regulate class I, indicating that one or more early proteins are responsible for the down-regulation of class I molecules.
Murine Bone Marrow-Derived Mast Cells as Potent Producers of IL-9: Costimulatory Function of IL-10 andkitLigand in the Presence of IL-1
Authors: Stassen Michael , Arnold Martina , Hültner Lothar , Müller Christian , Neudörfl Christine , Reineke Tanja , Schmitt Edgar
Published: April 2014

Recently, the Th2-type cytokine IL-9 was identified by genetic mapping analyses as a key mediator that determines the susceptibility to asthma. This has been further supported by data from IL-9-transgenic mice in which the overexpression of IL-9 in the lung causes airway inflammation, mast cell hyperplasia, and bronchial hyperresponsiveness. In an accompanying paper, we demonstrate that murine bone marrow-derived mast cells (BMMC) after stimulation with either ionomycin, a combination of ionomycin and IL-1, or via IgE-Ag complexes and IL-1 are very potent producers of IL-9. Herein we show that a dramatic increase of IL-9 production is observed when BMMC activated with ionomycin/IL-1 or with IgE-Ag complexes/IL-1 are treated with either additional kit ligand (KL) or IL-10. Both KL and IL-10 considerably enhance the production of IL-9 mRNA and protein. We were also able to demonstrate that the production of endogenous IL-10 by activated mast cells acts on the production of IL-9. Half-life measurements of IL-9 mRNA revealed no significant effect by KL, but a 2-fold increase of mRNA stability under the influence of IL-10. Reporter gene assays of transfected BMMC showed an enhanced transcriptional activity of the IL-9 promoter in the presence of either IL-10 or KL compared with cells stimulated only with a combination of IL-1 and ionomycin. The influence of KL and IL-10 might be of physiological importance, because it is known that both cytokines are produced by bronchial epithelial cells.
In Activated Mast Cells, IL-1 Up-Regulates the Production of Several Th2-Related Cytokines Including IL-9
Authors: Hültner Lothar , Kölsch Stephan , Stassen Michael , Kaspers Uwe , Kremer Jean-Pierre , Mailhammer Reinhard , Moeller Jochen , Broszeit Hannelore , Schmitt Edgar
Published: April 2014

Mast cells can play detrimental roles in the pathophysiology and mortality observed in anaphylaxis and other Th2-dominated allergic diseases. In contrast, these cells contribute to protective host defense mechanisms against parasitic worm infections. After IgE/Ag activation, mast cells can produce multiple cytokines that may enhance allergic inflammations, while a similar panel of Th2-related cytokines may support immunological strategies against parasites. Here we report that in primary mouse bone marrow-derived mast cells activated by ionomycin or IgE/Ag, the proinflammatory mediator IL-1 (α or β) up-regulated production of IL-3, IL-5, IL-6, and IL-9 as well as TNF, i.e., cytokines implicated in many inflammatory processes including those associated with allergies and helminthic infections. IL-1 did not induce significant cytokine release in the absence of ionomycin or IgE/Ag, suggesting that Ca-dependent signaling was required. IL-1-mediated enhancement of cytokine expression was confirmed at the mRNA level by Northern blot and/or RT-PCR analysis. Our study reveals a role for IL-1 in the up-regulation of multiple mast cell-derived cytokines. Moreover, we identify mast cells as a novel source of IL-9. These results are of particular importance in the light of recent reports that strongly support a central role of IL-9 in allergic lung inflammation and in host defense against worm infections.
Inhibition of Lipopolysaccharide-Induced Signal Transduction in Endotoxin-Tolerized Mouse Macrophages: Dysregulation of Cytokine, Chemokine, and Toll-Like Receptor 2 and 4 Gene Expression
Authors: Medvedev Andrei E. , Kopydlowski Karen M. , Vogel Stefanie N.
Published: April 2014

In this study, the effect of in vitro endotoxin tolerance on LPS-induced mitogen-activated protein kinase activation, transcription factor induction, and cytokine, chemokine, and Toll-like receptor (TLR) 2 and 4 gene expression, as well as the involvement of TNF and IL-1 signaling pathways in tolerance, were examined. Pretreatment of mouse macrophages with LPS inhibited phosphorylation of the extracellular signal-regulated kinases, c-Jun NH2-terminal kinases, and p38 kinase; degradation of I-κBα (inhibitory protein that dissociates from NF-κB) and I-κBβ; and activation of the transcription factors NF-κB and AP-1 in response to subsequent LPS stimulation. These changes were accompanied by suppression of LPS-induced expression of mRNA for GM-CSF, IFN-γ-inducible protein-10, KC, JE/monocyte chemoattractant protein-1, macrophage-inflammatory protein-1β, and macrophage-inflammatory protein-2, with concurrent inhibition of chemokine secretion. In contrast to control cells, endotoxin-tolerant macrophages exhibited an increased basal level of TLR2 mRNA, and failed to increase levels of TLR2 mRNA or to down-regulate TLR4 gene expression upon restimulation with LPS. As judged by transcription factor activation, LPS and IL-1 were found to induce a state of cross-tolerance against each other, while no such reciprocal effect was seen for LPS and TNF-α. In addition, macrophages from TNFR I/II double knockout mice were LPS tolerizable, and blocking of endogenous TNF-α with TNFR-Fc fusion protein did not affect the capacity of LPS to tolerize macrophages. These data extend our understanding of LPS-signaling mechanisms that are inhibited in endotoxin-tolerized macrophages and suggest that endotoxin tolerance might result from impaired expression and/or functions of common signaling intermediates involved in LPS and IL-1 signaling.
Heterogeneity of Tonsillar Subepithelial B Lymphocytes, the Splenic Marginal Zone Equivalents
Authors: Dono Mariella , Zupo Simona , Leanza Nicolò , Melioli Giovanni , Fogli Manuela , Melagrana Andrea , Chiorazzi Nicholas , Ferrarini Manlio
Published: April 2014

The VH4 genes expressed by both resting and in vivo-activated subepithelial (SE) B cells from human tonsils were studied. Resting SE B cells were subdivided according to the presence (IgDlow) or absence (IgM-only) of surface IgD. CD27 was abundant on activated SE B cells and low on resting IgM-only B cells. Resting IgDlow SE B cells could be subdivided into CD27low and CD27high cell fractions. Resting IgDlow SE B cells displayed VH4 genes with a substantial number of mutations (13/29 of the molecular clones were mutated), whereas 25/26 of the clones from resting IgM-only SE B cells were unmutated. Moreover, mutated VH4 genes were detected mainly within the CD27high cell fraction of the IgDlow SE B cells. Several identical unmutated VH4DJH sequences (11/32) were found in different molecular clones from resting IgM-only SE B cells, suggesting local cellular expansion. Both unmutated (14/25) and mutated (11/25) sequences were found in μ transcripts of activated SE B cells. Extensive mutation was observed in the γ transcripts of activated SE B cells. Therefore, SE B cells are heterogeneous, being comprised of B cells with mutated Ig VH4 genes, that are Ag-experienced B cells, and a subset of B cells with unmutated VH4 genes that are either virgin cells or cells driven by Ags that did not induce or select for V gene mutations.
B Cell Responses to a Peptide Epitope. X. Epitope Selection in a Primary Response Is Thermodynamically Regulated
Authors: Nakra Pooja , Manivel Venkatasamy , Vishwakarma Ram A. , Rao Kanury V. S.
Published: April 2014

We examine the etiological basis of hierarchical immunodominance of B cell epitopes on a multideterminant Ag. A model T-dependant immunogen, containing a single immunodominant B cell epitope, was used. The primary IgM response to this peptide included Abs directed against diverse determinants presented by the peptide. Interestingly, affinity of individual monomeric IgM Abs segregated around epitope recognized and was independent of their clonal origins. Furthermore, affinity of Abs directed against the immunodominant epitope were markedly higher than that of the alternate specificities. These studies suggested that the affinity of an epitope-specific primary response, and variations therein, may be determined by the chemical composition of epitope. This inference was supported by thermodynamic analyses of monomer IgM binding to Ag, which revealed that this interaction occurs at the expense of unfavorable entropy changes. Permissible binding required compensation by net enthalpic changes. Finally, the correlation between chemical composition of an epitope, the resultant affinity of the early primary humoral response, and its eventual influence on relative immunogenicity could be experimentally verified. This was achieved by examining the effect of various amino-terminal substitutions on immunogenicity of a, hitherto cryptic, amino-terminal determinant. Such experiments permitted delineation of a hierarchy of individual amino acid residues based on their influence; which correlated well with calculated Gibbs-free energy changes that individual residue side chains were expected to contribute in a binding interaction. Thus, maturation of a T-dependant humoral response is initiated by a step that is under thermodynamic control.
Determination of the Relationship Between T Cell Responsiveness and the Number of MHC-Peptide Complexes Using Specific Monoclonal Antibodies
Authors: Reay Philip A. , Matsui Kiyoshi , Haase Katherine , Wulfing Christoph , Chien Yueh-Hsiu , Davis Mark M.
Published: April 2014

We describe the generation of three mAbs that recognize the complex of the class II MHC molecule IEk bound to a peptide derived from the carboxyl terminus of moth cytochrome c (residues 95–103). Reactivities of these mAbs are sensitive to single alterations in the sequence of both helices of the MHC molecule and to the bound peptide. The epitopes of these reagents are distinct but overlap substantially. One of these mAbs specifically blocks lymphokine release by T cells responsive to this complex but not others. We have used another to examine how the number of complexes on an APC is related to its ability to stimulate T cells. We find that 200–400 complexes per cell are necessary and sufficient to induce a degree of stimulation, whereas maximum stimulation is achieved only if more than 5000 complexes are present. The analysis indicates that T cell activation is a stochastic process.